U.S. patent application number 16/827164 was filed with the patent office on 2020-07-09 for automated instrumentation for production of t-cell receptor peptide libraries.
The applicant listed for this patent is Inscripta, Inc.. Invention is credited to Deanna Church, Stephen Federowicz, Michael Graige.
Application Number | 20200216977 16/827164 |
Document ID | / |
Family ID | 68236313 |
Filed Date | 2020-07-09 |
![](/patent/app/20200216977/US20200216977A1-20200709-D00000.png)
![](/patent/app/20200216977/US20200216977A1-20200709-D00001.png)
![](/patent/app/20200216977/US20200216977A1-20200709-D00002.png)
![](/patent/app/20200216977/US20200216977A1-20200709-D00003.png)
![](/patent/app/20200216977/US20200216977A1-20200709-D00004.png)
![](/patent/app/20200216977/US20200216977A1-20200709-D00005.png)
![](/patent/app/20200216977/US20200216977A1-20200709-D00006.png)
![](/patent/app/20200216977/US20200216977A1-20200709-D00007.png)
![](/patent/app/20200216977/US20200216977A1-20200709-D00008.png)
![](/patent/app/20200216977/US20200216977A1-20200709-D00009.png)
![](/patent/app/20200216977/US20200216977A1-20200709-D00010.png)
View All Diagrams
United States Patent
Application |
20200216977 |
Kind Code |
A1 |
Federowicz; Stephen ; et
al. |
July 9, 2020 |
AUTOMATED INSTRUMENTATION FOR PRODUCTION OF T-CELL RECEPTOR PEPTIDE
LIBRARIES
Abstract
The present disclosure provides instrumentation and automated
methods for creating cell surface display libraries, where the
cells of the library display engineered peptides on their cell
surfaces for identification of antigens that bind to T-cell
receptors. The engineered peptides may be putative antigens or
binding regions of the T-cell receptors.
Inventors: |
Federowicz; Stephen;
(Boulder, CO) ; Church; Deanna; (Boulder, CO)
; Graige; Michael; (Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Inscripta, Inc. |
Boulder |
CO |
US |
|
|
Family ID: |
68236313 |
Appl. No.: |
16/827164 |
Filed: |
March 23, 2020 |
Related U.S. Patent Documents
|
|
|
|
|
|
Application
Number |
Filing Date |
Patent Number |
|
|
16670340 |
Oct 31, 2019 |
|
|
|
16827164 |
|
|
|
|
16392605 |
Apr 23, 2019 |
10557216 |
|
|
16670340 |
|
|
|
|
62671266 |
May 14, 2018 |
|
|
|
62662126 |
Apr 24, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/22 20130101; C40B
30/04 20130101; C40B 20/04 20130101; C07K 16/005 20130101; C40B
50/06 20130101; C12N 15/102 20130101; C40B 70/00 20130101; C12N
2310/20 20170501; C07K 14/70539 20130101; C12N 15/81 20130101; A61K
39/00 20130101; C07K 2317/92 20130101; C40B 40/02 20130101; C12N
15/907 20130101; C40B 40/10 20130101; C12N 15/85 20130101; C07K
16/2809 20130101; C12N 15/1037 20130101 |
International
Class: |
C40B 50/06 20060101
C40B050/06; C12N 9/22 20060101 C12N009/22; C40B 30/04 20060101
C40B030/04; C40B 40/10 20060101 C40B040/10; C07K 16/28 20060101
C07K016/28; C07K 14/74 20060101 C07K014/74; C12N 15/10 20060101
C12N015/10; C12N 15/81 20060101 C12N015/81; C12N 15/85 20060101
C12N015/85; C12N 15/90 20060101 C12N015/90; C40B 20/04 20060101
C40B020/04; C40B 40/02 20060101 C40B040/02; C40B 70/00 20060101
C40B070/00 |
Claims
1. An automated method of creating a cell library co-expressing
engineered proteins and MHC molecules, the method comprising:
providing a population of cells; processing the population of cells
using an instrument for multiplexed nuclease-directed genome
editing using introduced nucleic acids and a nucleic acid-directed
nuclease, wherein the introduced nucleic acids comprise nucleic
acids that encode engineered proteins and MHC molecules configured
to be displayed on a surface of the cells, and wherein the proteins
are particular to a cellular pathway; incubating the processed
cells to facilitate nucleic acid editing in the cells, wherein the
edited cells co-express the engineered proteins and MHC molecules
in the cells; and allowing the cells to display the engineered
proteins and MHC molecules on the surface of the cells.
2. The method of claim 1, wherein the engineered proteins in the
cell library comprise all known proteins particular to a cellular
pathway.
3. The method of claim 1, wherein the engineered proteins in the
cell library comprise a subset of proteins particular to a cellular
pathway.
4. The method of claim 1, wherein the population of cells are yeast
cells.
5. The method of claim 1, wherein the population of cells are
mammalian cells.
6. The method of claim 1, wherein the nuclease comprises an
RNA-directed nuclease.
7. The method of claim 6, wherein the nuclease comprises Cas 9.
8. The method of claim 6, wherein the nuclease comprises Cas 12/Cpf
I.
9. The method of claim 6, wherein the RNA-directed nuclease
comprises MAD7.
10. A cell library produced using the method of claim 1.
11. An automated method of creating a cell library co-expressing
peptides and MHC molecules, the method comprising: providing a
population of cells; processing the population of cells using an
instrument for multiplexed nuclease-directed genome editing
targeting genes encoding proteins particular to a pathway using
introduced nucleic acids and a nucleic acid-directed nuclease,
wherein the introduced nucleic acids comprise nucleic acids that
encode peptides and MHC molecules configured to be displayed on a
surface of the cells, processing the population of cells using an
instrument for multiplexed nuclease-directed genome editing using
introduced nucleic acids and a nucleic acid-directed nuclease,
wherein the introduced nucleic acids comprise nucleic acids that
encode peptides and MHC molecules configured to be displayed on a
surface of the cells, and wherein the genome editing targets a
computationally determined set of peptides of interest; incubating
the processed cells to facilitate nucleic acid editing in the
cells, wherein the edited cells co-express the computationally
determined peptides and MHC molecules in the cells; and allowing
the cells to display the peptides and MHC molecules on the surface
of the cells.
12. The method of claim 11, wherein the computationally determined
peptides are from proteins that are disease related targets.
13. The method of claim 11, wherein the computationally determined
peptides comprises peptides from all possible cellular proteins in
a pathway.
14. The method of claim 11, wherein the population of cells are
yeast cells.
15. The method of claim 11, wherein the population of cells are
mammalian cells.
16. The method of claim 11, wherein the nuclease comprises an
RNA-directed nuclease.
17. The method of claim 16, wherein the nuclease comprises Cas
9.
18. The method of claim 16, wherein the nuclease comprises Cas
12/Cpf 1.
19. The method of claim 16, wherein the RNA-directed nuclease
comprises MAD7.
20. A cell library produced using the method of claim 11.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Ser. No.
16/670,340, filed 31 Oct. 2019; which is a continuation of U.S.
Ser. No. 16/392,605, filed Apr. 23, 2019, now U.S. Pat. No.
10,557,216 which claims priority to U.S. Provisional Patent
Application Ser. No. 62/671,266, entitled "MULTIPLEXED METHODS FOR
PRODUCTION AND USE OF CELL SURFACE DISPLAY LIBRARIES," filed May
14, 2018; and U.S. Patent Application Ser. No. 62/662,126, entitled
"MULTIPLEXED METHODS FOR PRODUCTION AND USE OF CELL SURFACE DISPLAY
LIBRARIES," filed Apr. 24, 2018, both of which are hereby
incorporated by reference in their entireties for all purposes.
FIELD OF THE INVENTION
[0002] The present disclosure relates to automated multi-module
instruments and multiplexed methods of making cell surface display
libraries using genomic editing technologies.
BACKGROUND OF THE INVENTION
[0003] In the following discussion certain articles and methods
will be described for background and introductory purposes. Nothing
contained herein is to be construed as an "admission" of prior art.
Applicant expressly reserves the right to demonstrate, where
appropriate, that the articles and methods referenced herein do not
constitute prior art under the applicable statutory provisions.
[0004] The binding and activation of a T-cell receptor (TCR) to its
specific antigen has been difficult to identify in high throughput
systems due to the diversity of major histocompatibility complexes,
the variety of potential antigens, and the diversity of T-cells in
humans and animals. Conventional techniques such as HPLC require a
priori information about the TCR target, and the identification
process can be both lengthy and cumbersome.
[0005] There is thus a need in the art for better and more robust
means for identifying specific antigens for TCRs in a high
throughput, multiplexed manner. The present invention addresses
this need.
SUMMARY OF THE INVENTION
[0006] This Summary is provided to introduce a selection of
concepts in a simplified form that are further described below in
the Detailed Description. This Summary is not intended to identify
key or essential features of the claimed subject matter, nor is it
intended to be used to limit the scope of the claimed subject
matter. Other features, details, utilities, and advantages of the
claimed subject matter will be apparent from the following written
Detailed Description including those aspects illustrated in the
accompanying drawings and defined in the appended claims.
[0007] The present disclosure provides compositions, instruments
and automated methods for providing multiplexed displays of
engineered peptides on the surface of a population of cells. The
engineered peptides are preferably expressed in the cells under
conditions that provide both secretion and display of the
engineered peptides on the cell surfaces, thus providing access of
the engineered peptides to potential binding targets. The cell
populations can be engineered using an automated editing system
that provides for one or more targeted edits per cell, allowing for
the rational design of a library of cells having engineered
peptides displayed on their respective surfaces. Accordingly, this
disclosure describes various automated methods for expressing and
displaying engineered peptides on cells.
[0008] In some embodiments, the disclosure provides a method of
producing a cell library expressing engineered peptides for
identification of T-cell receptor (TCR)-antigen binding, the method
comprising providing a population of cells, processing the
population of cells using an instrument for multiplexed
nuclease-directed genome editing using introduced nucleic acids and
a nucleic acid-directed nuclease to create cells comprising nucleic
acids that encode engineered peptides configured to be displayed on
a surface of the cells, incubating the processed cells to
facilitate nucleic acid editing in the cells, wherein the editing
provides nucleic acids that encode engineered peptide antigens in
the cells, and allowing the cells to express and display the
engineered peptides on the surface of the cells.
[0009] In some aspects, the engineered peptides are putative TCR
binding antigens. In other aspects, the engineered peptides
comprise predicted TCR binding regions. In some aspects the
engineered peptides derive from a target genomic sequence and
contain an inserted N-terminus or C-terminus cell surface display
conferring tag.
[0010] In other embodiments, the disclosure provides methods of
producing a cell library expressing engineered putative T-cell
receptor (TCR) antigens on the surface of the cells, the method
comprising providing a population of cells, processing the
population of cells using an instrument for multiplexed
nuclease-directed genome editing using introduced nucleic acids and
a nuclease to create cells comprising nucleic acids that encode
engineered peptide antigens configured to be displayed on a surface
of the cells, incubating the processed cells to facilitate nucleic
acid editing in the cells, wherein the editing provides nucleic
acids that encode engineered peptide antigens in the cells, and
allowing the cells to express and display the engineered peptide
antigens that are putative TCR antigens on the surface of the
cells.
[0011] The engineered peptide antigens in the population of edited
cells preferably comprise rationally designed peptides that can be
displayed on a cell surface in a manner by which the antigen is
available for binding to a TCR target, either known TCRs and/or
orphan TCRs. In some aspects, the engineered peptide antigens are
known antigens of one or more TCRs.
[0012] In specific embodiments the antigen is displayed on the cell
surface as part of an MHC (e.g. HLA) which includes the peptide
antigen thereby forming a TCR ligand. Accordingly, in some aspects,
the cells display the engineered peptide antigens as part of a
ligand. In some aspects, the cells co-express putative TCR antigens
and MHC molecules.
[0013] Peptide antigens for use with the systems and methods of the
disclosure include known antigens of one or more TCRs, predicted
antigens for one or more TCRs, or random peptides created using
nucleases in the automated cell editing instruments of the present
disclosure. In embodiments, the peptides that are displayed are
created using forward engineering to create peptide sequences based
on predictions of what antigens may be useful for specific
TCRs.
[0014] In some embodiments, the disclosure provides methods of
producing a cell library expressing engineered peptides derived
from the cells' genome(s) on the surface of cells, the method
comprising providing a population of cells, processing the
population of cells using an instrument for multiplexed
nuclease-directed genome editing using introduced nucleic acids and
a nuclease to create cells comprising nucleic acids that encode
engineered proteins configured with an N-terminus or C-terminus
cell surface display conferring tag to be displayed on a surface of
the cells, incubating the processed cells to facilitate nucleic
acid editing in the cells, wherein the editing provides nucleic
acids that encode cell surface display conferring tags at the
N-terminus or C-terminus of engineered proteins in the cells, and
allowing the cells to express and display the engineered proteins
on the surface of the cell.
[0015] In some embodiments, the disclosure provides multiplexed
method for identifying peptides that selectively bind one or more
TCRs, the method comprising providing a population of cells,
processing the population of cells using an automated system for
multiplexed nuclease-directed genome editing, wherein the system
comprises the steps of introducing nucleic acids that encode
engineered peptide antigens and a nuclease to a population of
cells, incubating the cells to facilitate nucleic acid editing in
the cells, allowing the edited cells to express and display the
engineered peptide antigens on the surface of the edited cells,
screening the edited cells displaying the engineered peptide
antigens against one or more TCRs, and identifying the edited cells
expressing engineered peptide antigens that selectively bind to one
or more TCRs.
[0016] In some aspects, the disclosure further provides isolating
the nucleic acids encoding the engineered peptide antigens that
selectively bind to one or more TCRs from the cells. In some
aspects, the disclosure further provides isolating the nucleic
acids encoding the engineered peptides that selectively bind to one
or more putative TCR antigens from the cells.
[0017] In some aspects, the cells encoding specific peptides are
identified by detection of a barcode associated with the engineered
peptides. In some aspects, the cells encoding specific are
identified by detection of a barcode associated with the engineered
peptide antigens that selectively bind to one or more TCRs. In some
embodiments, the barcode is used to isolate and/or further identify
or process the cells and nucleic acids encoding the peptides for
further analysis. In such embodiments, the barcode can be used as a
"handle" to pull out the cells of interest for further
analysis.
[0018] In specific aspects, the disclosure provides a method of
producing a cell library expressing engineered peptide antigens on
the surface of cells by providing a population of cells, editing
the population of cells using one or more introduced nucleic acids
comprising a guide RNA covalently linked to a donor DNA (e.g.,
homology arm) that selectively binds to a genomic region of
interest and a nuclease, incubating the cells to facilitate nucleic
acid editing in the cells, wherein the editing provides nucleic
acids that encode engineered peptide antigens in the cells, and
allowing the cells to express and display the engineered peptide
antigens on the surface of the edited cells.
[0019] In other specific aspects, the disclosure provides a method
of producing a cell library expressing engineered peptide antigens
on the surface of cells by providing a population of cells, editing
the population of cells employing an automated instrument for
multiplexed nuclease-directed genome editing using introduced
nucleic acids comprising the edits and a nuclease, incubating the
cells to facilitate nucleic acid editing in the cells, wherein the
editing provides nucleic acids that encode engineered peptide
antigens in the cells, and allowing the cells to express and
display the engineered peptide antigens on the surface of the
edited cells.
[0020] The engineered peptide antigens in the population of edited
cells preferably comprise rationally designed peptides that can be
displayed on a cell surface in a manner by which the antigen is
available for binding to a T-cell receptor ("TCR") target. In some
aspects of the disclosure, the engineered peptides are derived from
target genomic sequences.
[0021] Various nucleases may be used with the editing methods of
the present disclosure, including zinc finger nucleases,
meganucleases, TALENS, and nucleic acid-directed nucleases (e.g.,
RNA-directed nucleases). Preferably, the editing methods are
carried out using nucleic acid-directed nucleases, and more
preferably RNA-directed nucleases.
[0022] In specific embodiments, the disclosure provides multiplexed
methods for identifying cells expressing engineered putative TCR
antigens on their surface comprising providing a population of
cells, editing the population of cells using an automated
instrument for multiplexed nuclease-directed genome editing and
introduced nucleic acids and a nuclease to create nucleic acids
that encode putative TCR antigens in the cells, incubating the
cells to facilitate nucleic acid editing in the cells, allowing the
cells to express and display the engineered putative TCR antigens
on their surface, screening the cells displaying the engineered
putative TCR antigens against a target, and identifying the cells
expressing engineered putative TCR antigens that selectively bind
to the target.
[0023] In one embodiment, the disclosure provides multiplexed
methods for identifying cells expressing engineered putative TCR
antigens on their surface comprising providing a population of
cells; editing the population of cells using an automated
instrument for multiplexed nuclease-directed genome editing and
introduced nucleic acids and a nucleic acid-directed nuclease
thereby creating cells comprising nucleic acids that encode
engineered putative TCR antigens, incubating the cells to
facilitate nucleic acid editing in the cells, allowing the edited
cells to express and display the engineered putative TCR antigens
on their surface, screening the cells displaying the engineered
putative TCR antigens against a target, selecting the cells
expressing engineered putative TCR antigens that selectively bind
to one or more TCR targets, and detecting or isolating the nucleic
acid encoding the antigens. Alternatively, the conditions can be
varied to determine the selectivity under different conditions.
[0024] Detection of a specific peptide in a cell of interest can be
accomplished using various methods known in the art, e.g.,
sequencing, hybridization, identification of a barcode indicative
of an antigen sequence, and the like. Barcodes and other features
can also be used for further analysis, e.g., by providing a basis
for identifying and/or isolating cells of interest encoding
peptides identified for elucidation of TCR binding.
[0025] In one aspect, the disclosure provides methods for the
immobilization of one or more engineered peptide antigens on a cell
surface by providing fusion proteins for display of one or more
engineered peptide antigens on a yeast cell surface. In one
embodiment, the disclosure provides for methods for displaying an
engineered peptide antigen as part of an MHC antigen (e.g., HLA) on
the cell surface. In certain embodiments, the cells display
multiple copies of a single engineered antigen.
[0026] In specific embodiments, the disclosure provides methods for
providing receptors or binding regions thereof on the cell
[0027] In specific embodiments, the disclosure provides multiplexed
methods for identifying cells expressing engineered predicted TCR
binding regions (e.g., predicted binding regions of orphan TCRs) on
their surface comprising providing a population of cells, editing
the population of cells using an automated instrument for
multiplexed nuclease-directed genome editing and introduced nucleic
acids and a nuclease to create nucleic acids that encode TCR
binding regions in the cells, incubating the cells to facilitate
nucleic acid editing in the cells, allowing the cells to express
and display the engineered TCR binding regions on their surface,
screening the cells displaying the engineered TCR binding regions
against a target, and identifying the cells expressing engineered
TCR binding regions that selectively bind to putative antigens.
[0028] In one embodiment, the disclosure provides multiplexed
methods for identifying cells expressing engineered predicted
binding regions from TCRs (e.g., orphan TCRs) on their surface
comprising: providing a population of cells, editing the population
of cells using an automated instrument for multiplexed
nuclease-directed genome editing and introduced nucleic acids and a
nucleic acid-directed nuclease thereby creating cells comprising
nucleic acids that encode engineered TCR binding regions,
incubating the cells to facilitate nucleic acid editing in the
cells, allowing the edited cells to express and display the
engineered TCR binding regions on their surface, screening the
cells displaying the engineered TCR binding regions against a
target, and identifying the cells expressing engineered TCR binding
regions that selectively bind to one or more putative TCR binding
antigens. Alternatively, the conditions can be varied to determine
the selectivity under different conditions.
[0029] These aspects and other features and advantages of the
invention are described below in more detail.
BRIEF DESCRIPTION OF THE DRAWINGS
[0030] The foregoing and other features and advantages of the
present invention will be more fully understood from the following
detailed description of illustrative embodiments taken in
conjunction with the accompanying drawings in which:
[0031] FIG. 1 is a schematic showing the structure of the
TCR.alpha. and TCR.beta. loci.
[0032] FIG. 2 is a schematic showing how TCR gene segments
rearrange during T-cell development to form complete V-domain
exons.
[0033] FIG. 3 is a schematic showing the cluster of gene segments
encoding the .delta. chain within the TCR.alpha. locus.
[0034] FIGS. 4A-4D depict an automated multi-module instrument and
components thereof with which to generate the cell surface
libraries of the disclosure.
[0035] FIG. 5A depicts one embodiment of a rotating growth vial for
use with the cell growth module described herein. FIG. 5B
illustrates a perspective view of one embodiment of a rotating
growth vial in a cell growth module. FIG. 5C depicts a cut-away
view of the cell growth module from FIG. 5B. FIG. 5D illustrates
the cell growth module of FIG. 5B coupled to LED, detector, and
temperature regulating components.
[0036] FIG. 6A is a model of tangential flow filtration used in the
TFF device presented herein. FIG. 6B depicts a top view of a lower
member of one embodiment of an exemplary TFF device. FIG. 6C
depicts a top view of upper and lower members and a membrane of an
exemplary TFF device. FIG. 6D depicts a bottom view of upper and
lower members and a membrane of an exemplary TFF device. FIGS.
6E-6I depict various views of an embodiment of a TFF module
comprising a TFF device and having fluidically coupled reservoirs
for retentate, filtrate, and exchange buffer.
[0037] FIGS. 7A and 7B are top perspective and bottom perspective
views, respectively, of flow-through electroporation devices (here,
there are six such devices co-joined). FIG. 7C is a top view of one
embodiment of an exemplary flow-through electroporation device.
FIG. 7D depicts a top view of a cross section of the
electroporation device of FIG. 7C. FIG. 7E is a side view cross
section of a lower portion of the electroporation devices of FIGS.
7C and 7D.
[0038] FIG. 8A depicts a simplified graphic of a workflow for
singulating, editing and normalizing cells after nucleic
acid-guided nuclease genome editing in a solid wall device. FIG. 8B
is a photograph of one embodiment of a solid wall device. FIGS.
8C-8E are photographs of E. coli cells singulated (via Poisson
distribution) and grown into colonies in microwells in a solid wall
device with a permeable bottom at low, medium, and high
magnification, respectively. FIG. 8F is a simplified block diagram
of methods for enriching for live cells that have been edited via
nucleic acid-guided nuclease editing that do not involve
singulation or a singulation device and instead utilize cell growth
in liquid and induction of editing. FIG. 8G depicts a typical
growth curve for cells in culture. FIG. 8H is a graphic depiction
of methods for growing, inducing, editing, enriching, and screening
for edited cells in a population of cells.
[0039] FIGS. 9A and 9B depict an example reagent cartridge for use
in an automated multi-module cell editing instrument.
[0040] FIG. 10 is a flow chart of an example method for automated
multi-module cell editing to produce the cell libraries as
described herein.
[0041] FIG. 11 is a simplified flow chart of two exemplary methods
(1100a and 1100b) that may be performed by an automated
multi-module cell editing instrument comprising a singulation
device.
[0042] FIG. 12 is a simplified block diagram of an embodiment of an
exemplary automated multi-module cell processing instrument
comprising a solid wall singulation/growth/editing/normalization
module.
[0043] FIG. 13 is a simplified block diagram of an alternative
embodiment of an exemplary automated multi-module cell processing
instrument comprising a solid wall
singulation/growth/editing/normalization module.
[0044] FIG. 14 is a simplified process diagram of an embodiment of
an exemplary automated multi-module cell processing instrument.
[0045] FIG. 15 is a graph demonstrating the effectiveness of a
2-paddle rotating growth vial and cell growth device as described
herein for growing an EC23 cell culture vs. a conventional cell
shaker.
[0046] FIG. 16 is a graph demonstrating the effectiveness of a
3-paddle rotating growth vial and cell growth device as described
herein for growing an EC23 cell culture vs. a conventional cell
shaker.
[0047] FIG. 17 is a graph demonstrating the effectiveness of a
4-paddle rotating growth vial and cell growth device as described
herein for growing an EC138 cell culture vs. a conventional orbital
cell shaker.
[0048] FIG. 18 is a graph demonstrating the effectiveness of a
2-paddle rotating growth vial and cell growth device as described
herein for growing an EC138 cell culture vs. a conventional orbital
cell shaker.
[0049] FIG. 19 is a graph demonstrating real-time monitoring of
growth of an EC138 cell culture to OD600 employing the cell growth
device as described herein where a 2-paddle rotating growth vial
was used.
[0050] FIG. 20 is a graph demonstrating real-time monitoring of
growth of s288c yeast cell culture OD600 employing the cell growth
device as described herein where a 2-paddle rotating growth vial
was used.
[0051] FIG. 21A is a graph plotting filtrate conductivity against
filter processing time for an E. coli culture processed in the cell
concentration device/module described herein. FIG. 21B is a graph
plotting filtrate conductivity against filter processing time for a
yeast culture processed in the cell concentration device/module
described herein.
[0052] FIG. 22A is a bar graph showing the results of
electroporation of E. coli using a device of the disclosure and a
comparator electroporation device. FIG. 22B is a bar graph showing
uptake, cutting, and editing efficiencies of E. coli cells
transformed via an FTEP as described herein benchmarked against a
comparator electroporation device.
[0053] FIG. 23 is a bar graph showing the results of
electroporation of S. cerevisiae using an FTEP device of the
disclosure and a comparator electroporation method.
[0054] It should be understood that the drawings are not
necessarily to scale, and that like reference numbers refer to like
features.
DETAILED DESCRIPTION
[0055] All of the functionalities described in connection with one
embodiment of the methods, devices or instruments described herein
are intended to be applicable to the additional embodiments of the
methods, devices and instruments described herein except where
expressly stated or where the feature or function is incompatible
with the additional embodiments. For example, where a given feature
or function is expressly described in connection with one
embodiment but not expressly mentioned in connection with an
alternative embodiment, it should be understood that the feature or
function may be deployed, utilized, or implemented in connection
with the alternative embodiment unless the feature or function is
incompatible with the alternative embodiment.
[0056] The practice of the techniques described herein may employ,
unless otherwise indicated, conventional techniques and
descriptions of molecular biology (including recombinant
techniques), cell biology, biochemistry, and genetic engineering
technology, which are within the skill of those who practice in the
art. Such conventional techniques and descriptions can be found in
standard laboratory manuals such as Green and Sambrook, Molecular
Cloning: A Laboratory Manual. 4th, ed., Cold Spring Harbor
Laboratory Press, Cold Spring Harbor, N.Y., (2014); Current
Protocols in Molecular Biology, Ausubel, et al. eds., (2017);
Neumann, et al., Electroporation and Electrofusion in Cell Biology,
Plenum Press, New York, 1989; and Chang, et al., Guide to
Electroporation and Electrofusion, Academic Press, California
(1992), all of which are herein incorporated in their entirety by
reference for all purposes. Nucleic acid-guided nuclease techniques
can be found in, e.g., Genome Editing and Engineering from TALENs
and CRISPRs to Molecular Surgery, Appasani and Church (2018); and
CRISPR: Methods and Protocols, Lindgren and Charpentier (2015);
both of which are herein incorporated in their entirety by
reference for all purposes.
[0057] Note that as used herein and in the appended claims, the
singular forms "a," "an," and "the" include plural referents unless
the context clearly dictates otherwise. Thus, for example,
reference to "a cell" refers to one or more cells, and reference to
"the system" includes reference to equivalent steps, methods and
devices known to those skilled in the art, and so forth.
Additionally, it is to be understood that terms such as "left,"
"right," "top," "bottom," "front," "rear," "side," "height,"
"length," "width," "upper," "lower," "interior," "exterior,"
"inner," "outer" that may be used herein merely describe points of
reference and do not necessarily limit embodiments of the present
disclosure to any particular orientation or configuration.
Furthermore, terms such as "first," "second," "third," etc., merely
identify one of a number of portions, components, steps,
operations, functions, and/or points of reference as disclosed
herein, and likewise do not necessarily limit embodiments of the
present disclosure to any particular configuration or
orientation.
[0058] Unless defined otherwise, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention belongs. All
publications mentioned herein are incorporated by reference for the
purpose of describing and disclosing devices, formulations and
methodologies that may be used in connection with the presently
described invention.
[0059] Where a range of values is provided, it is understood that
each intervening value, between the upper and lower limit of that
range and any other stated or intervening value in that stated
range is encompassed within the invention. The upper and lower
limits of these smaller ranges may independently be included in
smaller ranges, and are also encompassed within the invention,
subject to any specifically excluded limit in the stated range.
Where the stated range includes one or both of the limits, ranges
excluding either or both of those included limits are also included
in the invention.
[0060] In the following description, numerous specific details are
set forth to provide a more thorough understanding of the present
invention. However, it will be apparent to one of skill in the art
that the present invention may be practiced without one or more of
these specific details. In other instances, features and procedures
well known to those skilled in the art have not been described in
order to avoid obscuring the invention. The terms used herein are
intended to have the plain and ordinary meaning as understood by
those of ordinary skill in the art.
[0061] The term "complementary" as used herein refers to
Watson-Crick base pairing between nucleotides and specifically
refers to nucleotides hydrogen bonded to one another with thymine
or uracil residues linked to adenine residues by two hydrogen bonds
and cytosine and guanine residues linked by three hydrogen bonds.
In general, a nucleic acid includes a nucleotide sequence described
as having a "percent complementarity" or "percent homology" to a
specified second nucleotide sequence. For example, a nucleotide
sequence may have 80%, 90%, or 100% complementarity to a specified
second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10
of 10 nucleotides of a sequence are complementary to the specified
second nucleotide sequence. For instance, the nucleotide sequence
3'-TCGA-5' is 100% complementary to the nucleotide sequence
5'-AGCT-3'; and the nucleotide sequence 3'-TCGA-5' is 100%
complementary to a region of the nucleotide sequence
5'-TTAGCTGG-3'.
[0062] The term DNA "control sequences" refers collectively to
promoter sequences, polyadenylation signals, transcription
termination sequences, upstream regulatory domains, origins of
replication, internal ribosome entry sites, nuclear localization
sequences, enhancers, and the like, which collectively provide for
the replication, transcription and translation of a coding sequence
in a recipient cell. Not all of these types of control sequences
need to be present so long as a selected coding sequence is capable
of being replicated, transcribed and--for some
components--translated in an appropriate host cell.
[0063] As used herein the term "donor DNA" or "donor nucleic acid"
refers to nucleic acid that is designed to introduce a DNA sequence
modification (insertion, deletion, substitution) into a locus by
homologous recombination using nucleic acid-guided nucleases. For
homology-directed repair, the donor DNA must have sufficient
homology to the regions flanking the "cut site" or site to be
edited in the genomic target sequence. The length of the homology
arm(s) will depend on, e.g., the type and size of the modification
being made. In many instances and preferably, the donor DNA will
have two regions of sequence homology (e.g., two homology arms) to
the genomic target locus. Preferably, an "insert" region or "DNA
sequence modification" region--the nucleic acid modification that
one desires to be introduced into a genome target locus in a
cell-will be located between two regions of homology. The DNA
sequence modification may change one or more bases of the target
genomic DNA sequence at one specific site or multiple specific
sites. A change may include changing 1, 2, 3, 4, 5, 10, 15, 20, 25,
30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more base
pairs of the target sequence. A deletion or insertion may be a
deletion or insertion of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50,
75, 100, 150, 200, 300, 400, or 500 or more base pairs of the
target sequence.
[0064] The term "engineered peptide antigen" encompasses naturally
occurring and synthetic polypeptides and protein constructs that
comprise a synthetic polypeptide or naturally occurring peptide
associated with different elements, like, for instance, peptides
for MHC display of the peptide, an immobilization peptide, reporter
peptide or secretion peptide. engineered peptide antigens are
encoded and/or expressed from a recombinant nucleic acid that may
be engineered to include sequence variants, recombinant promoters,
transcriptional control elements, fusion peptides, other
modifications, or any combination of two or more thereof. The
peptide presentation may include presentation of all or a portion
of a protein of interest. In some embodiments, engineered peptide
antigens comprise a binding motif that is modified by a coupling
enzyme, resulting in the coupling of a second binding target to the
binding motif. In some embodiments, the second binding target is
coupled to the engineered peptide antigen intracellularly.
[0065] As used herein, "enrichment" refers to enriching for edited
cells by singulation, optionally inducing editing, and growth of
singulated cells into terminal-sized colonies (e.g., saturation or
normalization of colony growth).
[0066] The terms "guide nucleic acid" or "guide RNA" or "gRNA"
refer to a polynucleotide comprising 1) a guide sequence capable of
hybridizing to a genomic target locus, and 2) a scaffold sequence
capable of interacting or complexing with a nucleic acid-guided
nuclease.
[0067] "Homology" or "identity" or "similarity" refers to sequence
similarity between two peptides or, more often in the context of
the present disclosure, between two nucleic acid molecules. The
term "homologous region" or "homology arm" refers to a region on
the donor DNA with a certain degree of homology with the target
genomic DNA sequence. Homology can be determined by comparing a
position in each sequence which may be aligned for purposes of
comparison. When a position in the compared sequence is occupied by
the same base or amino acid, then the molecules are homologous at
that position. A degree of homology between sequences is a function
of the number of matching or homologous positions shared by the
sequences.
[0068] As used herein, the terms "leader peptide", "secretion
peptide" or secretion leader peptide refers to any signaling
sequence that directs a synthesized fusion protein away from the
translation site, including signaling sequences that will result in
the fusion peptide crossing the cell membrane and being
secreted.
[0069] "Operably linked" refers to an arrangement of elements where
the components so described are configured so as to perform their
usual function. Thus, control sequences operably linked to a coding
sequence are capable of effecting the transcription, and in some
cases, the translation, of a coding sequence. The control sequences
need not be contiguous with the coding sequence so long as they
function to direct the expression of the coding sequence. Thus, for
example, intervening untranslated yet transcribed sequences can be
present between a promoter sequence and the coding sequence and the
promoter sequence can still be considered "operably linked" to the
coding sequence. In fact, such sequences need not reside on the
same contiguous DNA molecule (i.e. chromosome) and may still have
interactions resulting in altered regulation.
[0070] As used herein, the terms "protein" and "polypeptide" are
used interchangeably. Proteins may or may not be made up entirely
of amino acids.
[0071] A "promoter" or "promoter sequence" is a DNA regulatory
region capable of binding RNA polymerase and initiating
transcription of a polynucleotide or polypeptide coding sequence
such as messenger RNA, ribosomal RNA, small nuclear or nucleolar
RNA, guide RNA, or any kind of RNA transcribed by any class of any
RNA polymerase I, II or III. Promoters may be constitutive or
inducible, and in some embodiments--particularly many embodiments
in which selection is employed--the transcription of at least one
component of the nucleic acid-guided nuclease editing system is
under the control of an inducible promoter.
[0072] As used herein the term "selectable marker" refers to a gene
introduced into a cell, which confers a trait suitable for
artificial selection. General use selectable markers are well-known
to those of ordinary skill in the art. Drug selectable markers such
as ampicillin/carbenicillin, kanamycin, chloramphenicol,
erythromycin, tetracycline, gentamicin, bleomycin, streptomycin,
rifampicin, puromycin, hygromycin, blasticidin, and G418 may be
employed. In other embodiments, selectable markers include, but are
not limited to sugars such as rhamnose. human nerve growth factor
receptor (detected with a MAb, such as described in U.S. Pat. No.
6,365,373); truncated human growth factor receptor (detected with
MAb); mutant human dihydrofolate reductase (DHFR; fluorescent MTX
substrate available); secreted alkaline phosphatase (SEAP;
fluorescent substrate available); human thymidylate synthase (TS;
confers resistance to anti-cancer agent fluorodeoxyuridine); human
glutathione S-transferase alpha (GSTA1; conjugates glutathione to
the stem cell selective alkylator busulfan; chemoprotective
selectable marker in CD34+cells); CD24 cell surface antigen in
hematopoietic stem cells; human CAD gene to confer resistance to
N-phosphonacetyl-L-aspartate (PALA); human multi-drug resistance-1
(MDR-1; P-glycoprotein surface protein selectable by increased drug
resistance or enriched by FACS); human CD25 (IL-2.alpha.;
detectable by Mab-FITC); Methylguanine-DNA methyltransferase (MGMT;
selectable by carmustine); and Cytidine deaminase (CD; selectable
by Ara-C). "Selective medium" as used herein refers to cell growth
medium to which has been added a chemical compound or biological
moiety that selects for or against selectable markers.
[0073] The term "specifically binds" as used herein includes an
interaction between two molecules, e.g., an engineered peptide
antigen and a binding target, with a binding affinity represented
by a dissociation constant of about 10.sup.-7M, about 10.sup.-8M,
about 10.sup.-9 M, about 10.sup.-10 M, about 10.sup.-11M, about
10.sup.-12M, about 10.sup.-13M, about 10.sup.-14M or about
10.sup.-15M.
[0074] The terms "target genomic DNA sequence", "target sequence",
or "genomic target locus" refer to any locus in vitro or in vivo,
or in a nucleic acid (e.g., genome) of a cell or population of
cells, in which a change of at least one nucleotide is desired
using a nucleic acid-guided nuclease editing system. The target
sequence can be a genomic locus or extrachromosomal locus.
[0075] The term "variant" may refer to a polypeptide or
polynucleotide that differs from a reference polypeptide or
polynucleotide, but retains essential properties. A typical variant
of a polypeptide differs in amino acid sequence from another
reference polypeptide. Generally, differences are limited so that
the sequences of the reference polypeptide and the variant are
closely similar overall and, in many regions, identical. A variant
and reference polypeptide may differ in amino acid sequence by one
or more modifications (e.g., substitutions, additions, and/or
deletions). A variant of a polypeptide may be a conservatively
modified variant. A substituted or inserted amino acid residue may
or may not be one encoded by the genetic code (e.g., a non-natural
amino acid). A variant of a polypeptide may be naturally occurring,
such as an allelic variant, or it may be a variant that is not
known to occur naturally.
[0076] A "vector" is any of a variety of nucleic acids that
comprise a desired sequence or sequences to be delivered to and/or
expressed in a cell. Vectors are typically composed of DNA,
although RNA vectors are also available. Vectors include, but are
not limited to, plasmids, fosmids, phagemids, virus genomes,
synthetic chromosomes, and the like. As used herein, the phrase
"engine vector" comprises a coding sequence for a nuclease to be
used in the nucleic acid-guided nuclease systems and methods of the
present disclosure. The engine vector may also comprise, in a
bacterial system, the .lamda. Red recombineering system or an
equivalent thereto. Engine vectors also typically comprise a
selectable marker. As used herein the phrase "editing vector"
comprises a donor nucleic acid, optionally including an alteration
to the target sequence that prevents nuclease binding at a PAM or
spacer in the target sequence after editing has taken place, and a
coding sequence for a gRNA. The editing vector may also comprise a
selectable marker and/or a barcode. In some embodiments, the engine
vector and editing vector may be combined; that is, all editing and
selection components may be found on a single vector. Further, the
engine and editing vectors comprise control sequences operably
linked to, e.g., the nuclease coding sequence, recombineering
system coding sequences (if present), donor nucleic acid, guide
nucleic acid, and selectable marker(s).
Cell Libraries, Screening and Editing Methods
[0077] The present disclosure provides multiplexed methods and
automated instruments for creating cell populations with cell
surface displays where the methods employ editing technologies. The
cell populations edited using the multiplexed and automated
instrumentation of the disclosure comprise one or more putative
receptor antigens displayed on a cell's surface and available for
binding to a binding target. The cells that may be edited and used
according to the disclosure include, but are not limited to,
bacterial cells, yeast cells and mammalian cells. In addition, the
cells that are edited may include sequences that are heterologous
to the host (e.g., editing of mammalian sequences inserted into a
yeast or bacterial genome).
[0078] In particular the methods and automated instruments used to
create the cells are useful in identifying antigens that
specifically bind to T-cell receptors (TCRs). The ability to
quickly and easily identify antigens, e.g. putative antigen targets
of orphan TCRs, can be extremely useful in immunology, e.g.,
immunotherapy research and development.
[0079] The disclosure also provides methods for multiplexed display
and screening of antigens (e.g., as components of ligands) that
bind to a TCR target. In some embodiments, the antigens are
displayed on a cell surface using any of the cell display methods
described herein. In some embodiments the antigens are complexed in
an MHC complex and displayed on the cell population surfaces.
[0080] Antigens that specifically bind to T-cell receptors (TCRs)
can be identified using various detection methods, including
isolation of the cells and sequencing of the introduced antigen
sequences or identification by hybridization, e.g., on an array. In
other aspects, the barcodes associated with a specific displayed
antigen may be identified and used to identify the antigens that
selectively bind to a TCR. The barcodes may be identified, e.g.,
using sequencing and/or array hybridization.
[0081] In some embodiments, the cells that encode engineered
peptide antigens that selectively bind to one or more targets of
interest from the cells are identified and/or isolated using a
barcode associated with the peptide. In specific embodiments, the
barcode is used to further isolate and/or analyze the cells
expressing the peptides identified as potentially elucidating the
binding of an antigen to a TCR. In such embodiments, the barcode
can be used as a "handle" to pull out the cells of interest for
further analysis.
[0082] In some embodiments, the method comprises producing via
genome editing a population or library of edited cells each
displaying a single engineered peptide antigen on its surface,
wherein the different engineered peptide antigens are created using
nuclease editing and are subsequently displayed on the surface of
different cells. In other embodiments, the editing method results
in a population or library of edited cells, where each edited cell
displays a plurality of different engineered peptide antigens on
its surface. The cells thus can express one or more engineered
peptide antigens that are displayed on the cell surface of a single
cell of the population, optionally in one or more MHCs (e.g.,
HLAs)
[0083] In some embodiments, the disclosure provides a method for
displaying an engineered peptide antigen on a cell surface, the
method comprising editing a cell using a nucleic acid-directed
nuclease to create a nucleic acid encoding an engineered putative
HLA and incubating an edited cell under conditions sufficient for
expressing the engineered HLA.
[0084] In some embodiments, the cells of the library display at
least 10.sup.2 engineered peptide antigens. In some embodiments,
the cell displays at least 10.sup.3 engineered peptide antigens. In
some embodiments, the cell displays at least 10.sup.4 engineered
peptide antigens. In some embodiments, the cell displays at least
10.sup.5 engineered peptide antigens, at least 10.sup.6 engineered
peptide antigens or more. In some embodiments, the disclosure
provides a library of any of the cells described herein. In some
embodiments, the library has at least 10.sup.8 different members.
In some embodiments, the library has at least 2, at least 5, at
least 10, at least 50, at least 100, at least 1000, at least
10,000, at least 100,000, at least 1,000,000, at least 10.sup.7, at
least 10.sup.8, at least 10.sup.9, at least 10.sup.10 or at least
10.sup.11 cells.
[0085] In some embodiments, the disclosure provides populations or
libraries of edited cells, wherein the cells encode different
engineered peptide antigens and variants thereof, and wherein the
variants also comprise a binding motif capable of coupling a
binding target. In some embodiments, the binding motif is a
biotinylation motif. In some embodiments, the library has at least
10.sup.8 different members. In some embodiments, the library has at
least 2, at least 5, at least 10, at least 50, at least 100, at
least 1000, at least 10,000, at least 100,000, at least 1,000,000,
at least 10.sup.7, at least 10.sup.8, at least 10.sup.9, at least
10.sup.10 or at least 10.sup.11 members.
[0086] Methods of editing that may be used to generate the
libraries or populations of cells are described in detail below, as
are the cell processing modules and instruments used to perform the
nuclease-directed genome editing.
[0087] The antigens displayed on the edited cells in the libraries
can be any length between 3-50 amino acids and are preferably
between 5-20 amino acids. In specific aspects, the amino acid
peptides are displayed in a manner that allows the appropriate
presentation of the antigenic region of a peptide, e.g., 8-11 amino
acids that are known to be available in an MHC on the cell
surface.
T-Cell Receptors
[0088] T-cell receptors (TCRs) are structurally similar to
immunoglobulins, are encoded by homologous genes, and are assembled
by somatic recombination from sets of gene segments similar to
recombination of immunoglobulin genes. TCR loci have roughly the
same number of V gene segments but more J gene segments, and there
is greater diversification of the junctions between gene segments
during gene rearrangement. Moreover, functional TCRs are not known
to diversify their V genes after rearrangement through somatic
hypermutation. This leads to a TCR in which the highest diversity
is in the central part of the receptor, which contacts the bound
antigen of the ligand.
[0089] TCR .alpha. and .beta. chains each consist of a variable (V)
amino-terminal region and a constant (C) region. The organization
of the TCR.alpha. and TCR.beta. loci is shown in FIG. 1. The
TCR.alpha. locus, like those for the immunoglobulin light chains,
contains V and J gene segments (V.sub..alpha. and J.sub..alpha.).
The TCR.beta. locus, like that for the immunoglobulin heavy-chain,
contains D gene segments in addition to V.sub..beta. and
J.sub..beta. gene segments.
[0090] The TCR gene segments rearrange during T-cell development to
form complete V-domain exons (FIG. 2). The TCR gene segments are
flanked by heptamer and nonamer recombination signal sequences
(RSSs) that are homologous to those flanking immunoglobulin
gene
[0091] A further shared feature of immunoglobulin and TCR gene
rearrangement is the presence of P- and N-nucleotides in the
junctions between the V, D, and J gene segments of the rearranged
TCR.beta. gene. In T cells, P- and N-nucleotides are also added
between the V and J gene segments of all rearranged TCR.alpha.
genes, whereas only about half the V-J joints in immunoglobulin
light-chain genes are modified by N-nucleotide addition and these
are often left without any P-nucleotides as well.
[0092] The ligand for the TCR is usually a peptide bound to an MHC
molecule. Most of the variability of the TCR ligand is thus in the
bound antigenic peptide occupying the center of the surface in
contact with the receptor. In fact, the three-dimensional structure
of the antigen-recognition site of a TCR looks much like that of an
antibody molecule.
[0093] The structural diversity of TCRs is mainly attributable to
combinatorial and junctional diversity generated during the process
of gene rearrangement. The variability in TCR chains is focused on
the junctional region encoded by V, D, and J gene segments and
modified by P- and N-nucleotides. The TCR.alpha. locus contains
many more J gene segments than either of the immunoglobulin
light-chain loci: in humans, 61 J.sub..alpha. gene segments are
distributed over about 80 kb of DNA, whereas immunoglobulin
light-chain loci have only five J gene segments at most. Because
the TCR.alpha. locus has so many J gene segments, the variability
generated in this region is even greater for TCRs than for
immunoglobulins. This region encodes the CDR3 loops in
immunoglobulins and TCRs that form the center of the
antigen-binding site. Thus, the center of the TCR will be highly
variable, whereas the periphery will be subject to relatively
little variation.
[0094] A minority of T cells bear TCRs composed of .gamma. and
.delta. chains. The cluster of gene segments encoding the .delta.
chain is found entirely within the TCR.alpha. locus, between the
V.sub..alpha. and the J.sub..alpha. gene segments. See FIG. 3.
Because all V.sub..alpha. gene segments are oriented such that
rearrangement will delete the intervening DNA, any rearrangement at
the .alpha. locus results in the loss of the .delta. locus. There
are substantially fewer V gene segments at the TCR.gamma. and
TCR.delta. loci than at either the TCR.alpha. or TCR.beta. loci or
at any of the immunoglobulin loci. Increased junctional variability
in the .delta. chains may compensate for the small number of V gene
segments and has the effect of focusing almost all of the
variability in the .gamma.:.delta. receptor in the junctional
region. As we have seen, the amino acids encoded by the junctional
regions lie at the center of the TCR binding site. In humans, the
TCR.gamma. and TCR.delta. loci, like the TCR.alpha. and TCR.beta.
loci, have discrete V, D, and J gene segments, and C genes.
[0095] T cells bearing .gamma.:.delta. receptors are a distinct
lineage of T cells whose functions are at present unknown. The
ligands for these receptors are also largely unknown. Some
.gamma.:6 TCRs appear to be able to recognize antigen directly,
much as antibodies do, without the requirement for presentation by
an MHC molecule or processing of the antigen. Accordingly, the
co-expression of an MHC molecule with a putative antigen is
optional.
Cell Surface Display
[0096] Various display technologies can be used with the cell
libraries and populations generated by the methods and
instrumentation described herein, including yeast surface display
technologies, mammalian cell surface display technologies, and
bacterial surface display technologies. Cell surface display
technologies include, but are not limited to, those disclosed in
U.S. Pat. Nos. 8,883,692; 8,685,893; and 6,699,658; U.S. Pat. Pub.
Nos. 20170218382; 20170088611; 20150307560; 20150203834;
20140221621; 20140031292; 20140235476, 20140221621; 20130184177;
20110008883; No. 20100233195; 20100210473; 20100216659;
20090280560; 20090111126; and 20040146976. Bacterial cells, yeast
cells and mammalian cells can all be used for cell surface
display.
[0097] In certain embodiments, immobilization of an engineered
peptide antigen to a cell surface may involve specific interactions
between the engineered peptide antigen and a binding motif on the
engineered peptide antigen.
[0098] The engineered peptide antigens of the invention can be
expressed in any cell amenable to editing and surface display, and
the invention embraces any prokaryotic or eukaryotic cell,
including bacterial cells, yeast cells (e.g., Saccharomyces and/or
Picchia species), insect cells, Xenopus cells, and mammalian cells.
Cells that are particularly suited for expression of the fusion
proteins of the invention are E. Coli., S. cerevisiae, CHO and 293T
cells. The cells may be `wild type` cells or the cells may be
optimized for a particular characteristic or for a particular
enzyme function that may aid in protein expression. Optimized or
engineered cells include cells that have an optimized capability to
take up and maintain nucleic acids, cells that have increased
protein synthesis capability, and/or cells that have increased
protein secretion capability. Cells that maintain the integrity of
the edited nucleic acid and the synthesized proteins are
particularly useful.
[0099] In specific aspects, the edited cells comprise a binding
target on their surface, and the cells are incubated under
conditions resulting in secretion of the engineered peptide
antigen, wherein the engineered peptide antigen binds to a binding
target, thereby displaying the engineered peptide antigen on the
cell surface.
[0100] A commonly used organism for protein display is yeast. Yeast
display offers the advantage over bacteria-based technologies in
that yeast can process proteins that require endoplasmic reticulum
(ER)-specific post-translational processing for efficient folding
and activity. While mammalian cell display also facilitates
post-translational processing, yeast offers the advantage of ease
of generation of nucleic acid libraries as the vectors can be
simpler, and yeast allow for an easier introduction of editing
machinery (e.g., editing vectors) into the cells. Most yeast
expression fusion proteins are based on GPI
(Glycosyl-Phosphatidyl-Inositol) anchor proteins which play
important roles in the surface expression of cell-surface proteins
and are essential for the viability of the yeast. One such anchor
protein--alpha-agglutinin--consists of a core subunit encoded by
AGA1 and is linked through disulfide bridges to a small binding
subunit encoded by AGA2. Proteins encoded by the nucleic acid
libraries described herein can be introduced on the N-terminal
region of AGA1 or on the C terminal or N-terminal region of AGA2.
These fusion patterns will result in the display of the polypeptide
on the yeast cell surface.
[0101] In some embodiments, fusion proteins for yeast display
include an engineered peptide antigen fused to the N-terminal or
C-terminal part of a protein capable of anchoring in a eukaryotic
cell wall (e.g., a-agglutinin, AGA1, Flo1 or major cell wall
protein of lower eukaryotes; see U.S. Pat. Nos. 6,027,910 and
6,114,147 which are hereby incorporated by reference), for example,
proteins fused with the GPI fragment of Flol or to the Flol
functional domain (Kondo et al., Appl. MicroBiol. Biotech., 64:
28-40 (2004)).
[0102] In addition to surface display methods based on established
fusion proteins comprising a GPI anchor motif, the invention also
embraces display methods based on novel fusion proteins comprising
a modified GPI anchor motif. Fusion proteins of the invention may
comprise a protein to be displayed (e.g., one or more engineered
peptide antigens, binding targets, molecular targets, substrates,
etc., or any combination thereof), a GPI anchor and appropriate
signaling sequences, which may be post-translationally modified
when the fusion protein is expressed in yeast. As a protein
containing the GPI anchor and C-terminal signaling sequence is
trafficked through the ER, a hydrophobic region on the C-terminal
signal sequence adjacent to the GPI anchor becomes embedded in the
ER membrane, where it is cleaved by an ER protease. As the ER
protease cleaves this C-terminal signal sequence, it simultaneously
attaches a preformed GPI anchor to the new C-terminus of the
engineered peptide antigen (e.g., binding target, molecular target,
substrate, etc., or any combination thereof) ultimately resulting
in the display of the protein (e.g., binding target, molecular
target, substrate, etc., or any combination thereof) on the cell
surface (See, e.g., Kondo et al., cited above). The invention
embraces C-terminal sequences with improved processing properties
resulting in the improved display of fusion proteins comprising the
GPI-anchor proteins. Improved display comprises an increase in the
number of displayed proteins and/or an increase in the number of
correctly expressed proteins. In some embodiments, C-terminal
sequences with improved processing properties are evolved by
screening libraries containing variant C-terminal sequences
according to techniques known in the art.
[0103] In some embodiments, the disclosure provides a method for
displaying an engineered peptide antigen on a cell, the method
comprising incubating an edited cell comprising a first nucleic
acid under conditions sufficient for expressing an engineered
peptide antigen encoded by the first nucleic acid, wherein the cell
displays a first binding target, wherein the engineered peptide
antigen comprises a binding motif and a second binding target is
coupled to the binding motif when the engineered peptide antigen is
expressed, and, wherein the expressed engineered peptide antigen is
secreted from the cell and displayed on the cell surface via
binding of the second binding target to the first binding target.
In some embodiments, the first binding target is an avidin-like
protein. In some embodiments, the second binding target is biotin.
In some embodiments the binding motif is a biotinylation peptide.
In some embodiments, coupling of the second binding target is done
by a coupling enzyme. In some embodiments, the coupling enzyme is a
biotin ligase.
[0104] In some embodiments, the disclosure provides a method for
generating a library of edited cells comprising engineered (edited)
peptide antigens displayed on the cell surfaces of the cells, the
method comprising introducing a plurality of editing vectors into a
population of cells, creating conditions to allow the editing
vectors to edit nucleic acids in the cells; and creating conditions
where the edited cells express the engineered peptide antigens and
display the engineered peptide antigens on the cell surfaces,
wherein the vectors comprise a nuclease, and a donor nucleic acid
sequence comprising an edit in the coding region of the antigen to
be engineered. In specific aspects, the encoded engineered peptide
antigens comprise a unique polypeptide linked to an immobilization
peptide, wherein the immobilization peptide comprises a first
binding motif that selectively binds to a second binding motif
present on the cell surface of the edited cells, and the engineered
peptide antigens are expressed under conditions sufficient for
binding of the first binding motif to the second binding motif on
the cell surface. The immobilization peptide may also or
alternatively comprise, for example, a transmembrane polypeptide, a
polypeptide membrane anchor, a GPI-linked polypeptide or a natural
surface polypeptide.
[0105] In some embodiments, the disclosure provides a method for
generating a library of edited cells expressing engineered peptide
antigens displayed on a cell surface, the method comprising
introducing a plurality of vectors into a population of cells,
wherein the vectors comprise a nucleic acid-guided nuclease, a
guide RNA, and a donor nucleic acid comprising an edit in the
coding region of the protein to be engineered. In specific aspects,
the antigens to be edited are encoded engineered peptide antigens
that comprise a unique polypeptide linked to an immobilization
peptide, wherein the immobilization peptide comprises a first
binding motif that selectively binds to a second binding motif
present on the cell surface of the edited cells, and the engineered
peptide antigens are expressed under conditions sufficient for
binding of the first binding motif to the second binding motif on
the cell surface.
[0106] In the aspects that comprise the use of an immobilization
peptide or other moiety comprising a binding motif, the peptide or
motif can be linked to the C-terminus or the N-terminus of the
engineered peptide antigen.
[0107] In some embodiments, the engineered peptide antigen further
comprises a leader peptide. The leader peptide or secretion peptide
may be proteolytically removed from the mature protein concomitant
or immediately following export of the protein into the lumen of
intracellular compartment along the secretory pathway. The leader
peptide may be a naturally occurring sequence or a synthetic
sequence.
[0108] The edited cell library can have at least 2, at least 5, at
least 10, at least 50, at least 100, at least 1000, at least
10,000, at least 100,000, at least 1,000,000, at least at least
10.sup.7, at least 10.sup.8, at least 10.sup.9, at least 10.sup.10
or at least 10.sup.11 cells comprising one or more engineered
peptide antigens.
[0109] In some embodiments the expression of the engineered peptide
antigens in the cells is inducible or transient. In some
embodiments, no induction step is necessary and incubating the cell
results in the expression of the engineered peptide antigen. In
some embodiments, engineered peptide antigens comprising a first
binding motif are secreted and bind to a second binding motif
present on the cell surface, thereby displaying the engineered
peptide antigen on the cell surface. In some embodiments, the first
binding motif is avidin, streptavidin or neutravidin and the second
binding motif is biotin. In some embodiments, avidin is covalently
conjugated to the cell surface (e.g., directly or indirectly). Yet
in some embodiments, the first binding target is expressed by the
cell and displayed at the cell surface. For example, one of the
binding targets may be expressed by the cell as a fusion protein
such as a cell wall or a membrane fusion protein and displayed at
the surface of the cell.
Screening Methods
[0110] The methods of the disclosure may be useful to identify one
or more peptides that selectively bind to a TCR. By providing a
system that creates a cell library with engineered peptide antigens
displayed on the surface of the cells in which they are expressed,
cells that express engineered peptide antigens can be identified
using any assay that can be performed on a cell surface (e.g.,
performed on a cellular preparation to detect one or more molecules
that are displayed on the cell surface). The methods of the
disclosure can be used to screen libraries expressing engineered
peptide antigen variants to identify one or more TCRs that
selectively bind to the antigen(s).
[0111] An embodiment of the disclosure provides a method for
selecting cells displaying engineered peptide antigens with
desirable affinity or specificity for a target TCR, e.g., a known
TCR or an orphan TCR. Some aspects of the invention relate to
methods to screen for cells expressing an antigen that can interact
with a specific target molecule (e.g., a known TCR or orphan TCR)
with a desired specificity.
[0112] In some embodiments, the disclosure provides an antigen
screening method comprising expressing an engineered peptide
antigen in a cell edited using a nuclease, wherein the expressed
engineered peptide antigen is secreted and displayed on the cell
surface as a component of a ligand specific for a TCR and
evaluating the binding of the ligand to one or more TCRs. Upon
identification of a particular TCR and/or peptide, the sequences
can be sequenced, e.g., using next-generation sequencing such as
Illumina HiSeq or MiSeq. In other aspects, the specific TCR and/or
peptide can be identified through the detection of a barcode that
is associated with a particular TCR and/or peptide.
[0113] In some embodiments, the disclosure provides an antigen
screening method comprising expressing an engineered peptide
antigen in a cell edited using a nucleic-acid directed nuclease
(e.g., an RNA-directed nuclease such as a CRISPR nuclease). The
expressed engineered peptide antigens are secreted and displayed on
the cell surface as a component of a ligand specific for a TCR and
evaluating the binding of the ligand to the one or more TCRs.
Expression of Edited Proteins
[0114] The engineered peptide antigens in the edited cells of the
invention can be expressed from the edited nucleic acids using
methods known in the art. In some embodiments, protein expression
is constitutive. Constitutive expression covers both expression
from nucleic acids that have been integrated into the genome and
expression from nucleic acids that are located on episomal vectors.
In some embodiments, expression is initiated by an inducible event.
In some embodiments, edited nucleic acids that encode the
engineered peptide antigens are operably connected to an initiator
sequence that regulates expression of the engineered peptide
antigen. Initiator sequences that can induce expression are known
in the art and include inducible promoters. In some embodiments
protein expression is induced. In some embodiments, protein
expression occurs when the cell comprising a nucleic acid encoding
the protein is incubated and no separate induction step is
required.
Cell Libraries
[0115] Libraries of the invention include libraries of edited cells
expressing unique engineered peptide antigens. The cells of the
libraries are preferably edited using a nuclease, and more
preferably using one or more nucleases (e.g., a nucleic
acid-directed nuclease) in an automated multi-module cell editing
instrument as described in more detail herein.
[0116] In some embodiments, the library provides edited cells with
a high density of engineered peptide antigens immobilized on the
cell surface. In some embodiments, the high density is accomplished
by binding multiple engineered polypeptides expressed in a cell to
a cell-surface binding target. In some embodiments, the number of
engineered peptide antigens that are displayed per cell is greater
than 10.sup.3, greater than 10.sup.4, greater than 10.sup.5,
greater than 10.sup.6, greater than 10.sup.7, or greater than
10.sup.8 engineered peptide antigens per cell. In some embodiments,
the immobilization peptide is a biotinylation peptide. The antigens
displayed may be a single peptide antigen or two or more peptide
antigens depending on the display strategy for the cells. In some
embodiments, the immobilization peptide is a transmembrane protein.
In some embodiments, the immobilization peptide comprises a GPI
anchor. In some embodiments, the immobilization peptide is a
peptide that is naturally present on the cell surface. In some
embodiments, the immobilization peptide is a peptide that binds one
or more molecules naturally present on the cell surface (e.g.,
surface carbohydrates or proteins on the cell surface).
[0117] In some embodiments, libraries of binding proteins may be
evaluated or screened to identify and/or isolate variants that bind
to one or more TCR targets. Methods of the invention may be
designed to identify engineered peptide antigens that have
affinities for a particular TCR greater than a binding affinity
represented by a dissociation constant of about 10.sup.-7 M, about
10.sup.-8 M, about 10.sup.-9 M, about 10.sup.-10 M, about
10.sup.-11 M, about 10.sup.-12 M, about 10.sup.-13 M, about
10.sup.-14 M or about 10.sup.-15 M. In some embodiments, methods of
the invention may be designed to identify target peptide sequences
that have affinities for a TCR greater than a binding affinity
represented by a dissociation constant of about 10.sup.-7 M, about
10.sup.-8 M, about 10.sup.-9 M, about 10.sup.-10 M, about
10.sup.-11 M, about 10.sup.-12 M, about 10.sup.-13 M, about
10.sup.-14 M or about 10.sup.-15M.
Nuclease-Directed Genome Editing
[0118] In embodiments, the automated instrument described herein
utilizes a nuclease-directed genome editing system for introducing
edits to a population of cells allowing the engineering of proteins
for cell surface display. Multiple different nuclease-based systems
exist for providing edits into an organism's genome, and each can
be used in either single editing systems, sequential editing
systems (e.g., using different nuclease-directed systems
sequentially to provide two or more genome edits in a cell) and/or
recursive editing systems, (e.g., utilizing a single
nuclease-directed system to introduce two or more genome edits in a
cell). Exemplary nuclease-directed genome editing systems are
described herein, although a person of skill in the art would
recognize upon reading the present disclosure that other such
editing instruments are also useful in the creation of populations
of cells for cell surface display of engineered peptide
antigens.
[0119] It should be noted that the automated editing instruments as
set forth herein can use the nucleases for cleaving the genome,
introduction of an edit into a target region, or both.
[0120] In particular aspects of the invention, the nuclease editing
system is an inducible system that allows control of the timing of
the editing. The ability to modulate nuclease activity can reduce
off-target cleavage and facilitate precise genome engineering.
Numerous different inducible systems can be used with the
instrument and systems of the disclosure, as will be apparent to
one skilled in the art upon reading the present disclosure.
[0121] In certain aspects, cleavage by a nuclease can be used with
the instruments and systems of the invention to select cells with a
genomic edit at a target region. For example, cells that have been
subjected to a genomic edit that removes a particular nuclease
recognition site (e.g., via homologous recombination) can be
selected using the instruments described herein by exposing the
cells to the nuclease following the edit. The DNA in the cells
without the genome edit will be cleaved and subsequently will have
limited growth and/or perish, whereas the cells that received the
genome edit removing the nuclease recognition site will not be
affected by the subsequent exposure to the nuclease.
[0122] In other aspects, cells for editing may be treated in some
fashion to cleave the genome prior to introduction of the cells to
the instrument, and the instrument used for automated introduction
of desired genome edits in such cells. The initial cleavage can be
performed by the same or a different enzyme than the one used for
the initial cleavage event.
[0123] When the cell or population of cells comprising nucleic
acid-guided nuclease encoding DNA is in the presence of the inducer
molecule, expression of the nuclease can occur. For example,
CRISPR-nuclease expression can be repressed in the presence of a
repressor molecule. When the cell or population of cells comprising
nucleic acid-guided nuclease encoding DNA is in the absence of a
molecule that represses expression of the CRISPR-nuclease,
expression of the CRISPR-nuclease can occur.
[0124] For example, inducible systems for editing using RNA-guided
nuclease have been described, which use chemical induction to limit
the temporal exposure of the cells to the RNA-guided nuclease. Dow,
et al., Nature Biotechnology, 33:390-394 (2015); see also inducible
lentiviral expression vectors available at Dharmacon, GE Life
Sciences, Lafayette, Colo. For additional techniques, see e.g.,
Campbell, Biochem J., 473(17): 2573-89 (2010).
[0125] In other examples, a virus-inducible nuclease can be used to
induce gene editing in cells. See, e.g., Don, Antiviral Res.,
130:50-57 (2016). In another example, for inducible expression of
nucleic acid directed nucleases, variants can be switched on and
off in human cells with 4-hydroxytamoxifen (4-HT) by fusing the
nuclease with the hormone-binding domain of the estrogen receptor
(ERT2). Liu, et al., Nature Chemical Biology, 12:980-87 (2016).
[0126] Zinc-finger nucleases (ZFNs) are artificial restriction
enzymes generated by fusing a zinc finger DNA-binding domain to a
DNA-cleavage domain. Zinc finger domains can be engineered to
target specific target regions in an organism's genome. See, e.g.,
Urnov, et al., Nature Reviews Genetics 11,636-646 (2010). Using the
endogenous DNA repair machinery of an organism, ZFNs can be used to
precisely alter a target region of the genome. ZFNs can be used to
disable dominant mutations in heterozygous individuals by producing
double-strand breaks ("DSBs") in the DNA in the mutant allele,
which will, in the absence of a homologous template, be repaired by
non-homologous end-joining (NHEJ). NHEJ repairs DSBs by joining the
two ends together and usually produces no mutations, provided that
the cut is clean and uncomplicated. Dural, et al., Nucleic Acids
Res. 33 (18): 5978-90 (2005). This repair mechanism can be used to
induce errors in the genome via indels or chromosomal
rearrangement, often rendering the gene products coded at that
location non-functional.
[0127] Alternatively, DNA can be introduced into a genome in the
presence of exogenous double-stranded DNA fragments using homology
dependent repair (HDR). The dependency of HDR on a homologous
sequence to repair DSBs can be exploited by inserting a desired
sequence within a sequence that is homologous to the flanking
sequences of a DSB which, when used as a template by HDR system,
would lead to the creation of the desired change within the genomic
region of interest.
[0128] Multiple pairs of ZFNs can also be used to completely remove
entire large segments of genomic sequence (Lee. et al., Genome Res.
20(1): 81-89 (2009). Expanded CAG/CTG repeat tracts are the genetic
basis for more than a dozen inherited neurological disorders
including Huntington's disease, myotonic dystrophy, and several
spinocerebellar ataxias. It has been demonstrated in human cells
that ZFNs can direct DSBs to CAG repeats and shrink the repeat from
long pathological lengths to short, less toxic lengths (Mittelman,
et al., PNAS USA, 106(24): 9607-12 (2009)).
[0129] Meganucleases were identified in the 1990s, and subsequent
work has shown that they are particularly promising tools for
genome editing, as they are able to efficiently induce homologous
recombination, generate mutations in coding or non-coding regions
of the genome, and alter reading frames of the coding regions of
genomes. See, e.g., Epinat, et al., Nucleic Acids Research,
31(11):2952-62 (2003). The high specificity of meganucleases gives
them a high degree of precision and much lower cell toxicity than
other naturally occurring restriction enzymes.
[0130] Transcription activator-like effector nucleases (TALENs) are
restriction enzymes that can be engineered to cut specific
sequences of DNA. They are made by fusing a TAL effector
DNA-binding domain to a DNA cleavage domain (a nuclease which cuts
DNA strands). Transcription activator-like effectors (TALEs) can be
engineered to bind to practically any desired DNA sequence, so when
combined with a nuclease, DNA can be cut at specific locations.
(See, e.g., Miller, et al., Nature Biotechnology, 29(2): 143-48
(2011); Boch, Nature Biotechnology, 29(2): 135-36 (2011)).
[0131] Like ZFNs, TALEN can edit genomes by inducing DSBs. The
TALEN-created site-specific DSBs at target regions are repaired
through NHEJ or HDR, resulting in targeted genome edits. TALENs can
be used to introduce indels, rearrangements, or to introduce DNA
into a genome through NHEJ in the presence of exogenous
double-stranded DNA fragments.
[0132] A recent discovery for editing live cells involves nucleic
acid-guided nuclease (e.g., RNA-guided nuclease) editing. A nucleic
acid-guided nuclease complexed with an appropriate synthetic guide
nucleic acid in a cell can cut the genome of the cell at a desired
location. The guide nucleic acid helps the nucleic acid-guided
nuclease recognize and cut the DNA at a specific target sequence.
By manipulating the nucleotide sequence of the guide nucleic acid,
the nucleic acid-guided nuclease may be programmed to target any
DNA sequence for cleavage as long as an appropriate protospacer
adjacent motif (PAM) is nearby. In certain aspects, the nucleic
acid-guided nuclease editing system may use two separate guide
nucleic acid molecules that combine to function as a guide nucleic
acid, e.g., a CRISPR RNA (crRNA) and trans-activating CRISPR RNA
(tracrRNA). In other aspects, the guide nucleic acid may be a
single guide nucleic acid that includes both the crRNA and tracrRNA
sequences.
[0133] In general, a guide nucleic acid (e.g., gRNA) complexes with
a compatible nucleic acid-guided nuclease and can then hybridize
with a target sequence, thereby directing the nuclease to the
target sequence. A guide nucleic acid can be DNA or RNA;
alternatively, a guide nucleic acid may comprise both DNA and RNA.
In some embodiments, a guide nucleic acid may comprise modified or
non-naturally occurring nucleotides. In cases where the guide
nucleic acid comprises RNA, the gRNA may be encoded by a DNA
sequence on a polynucleotide molecule such as a plasmid, linear
construct, or the coding sequence may reside within an editing
cassette and is under the control of a constitutive promoter, or,
in some embodiments and preferably, an inducible promoter as
described below.
[0134] A guide nucleic acid comprises a guide sequence, where the
guide sequence is a polynucleotide sequence having sufficient
complementarity with a target sequence to hybridize with the target
sequence and direct sequence-specific binding of a complexed
nucleic acid-guided nuclease to the target sequence. The degree of
complementarity between a guide sequence and the corresponding
target sequence, when optimally aligned using a suitable alignment
algorithm, is about or more than about 50%, 60%, 75%, 80%, 85%,
90%, 95%, 97.5%, 99%, or more. Optimal alignment may be determined
with the use of any suitable algorithm for aligning sequences. In
some embodiments, a guide sequence is about or more than about 10,
11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27,
28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length. In
some embodiments, a guide sequence is less than about 75, 50, 45,
40, 35, 30, 25, 20 nucleotides in length. Preferably the guide
sequence is 10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19,
or 20 nucleotides in length.
[0135] In the present methods and compositions, the guide nucleic
acid is provided as a sequence to be expressed from a plasmid or
vector and comprises both the guide sequence and the scaffold
sequence as a single transcript under the control of a promoter,
and in some embodiments, an inducible promoter. The guide nucleic
acid can be engineered to target a desired target sequence by
altering the guide sequence so that the guide sequence is
complementary to a desired target sequence, thereby allowing
hybridization between the guide sequence and the target sequence.
In general, to generate an edit in the target sequence, the
gRNA/nuclease complex binds to a target sequence as determined by
the guide RNA, and the nuclease recognizes a proto spacer adjacent
motif (PAM) sequence adjacent to the target sequence. The target
sequence can be any polynucleotide endogenous or exogenous to a
prokaryotic or eukaryotic cell, or in vitro. For example, the
target sequence can be a polynucleotide residing in the nucleus of
a eukaryotic cell. A target sequence can be a sequence encoding a
gene product (e.g., a protein) or a non-coding sequence (e.g., a
regulatory polynucleotide, an intron, a PAM, or "junk" DNA).
[0136] The guide nucleic acid may be part of an editing cassette
that encodes the donor nucleic acid. Alternatively, the guide
nucleic acid may not be part of the editing cassette and instead
may be encoded on the engine or editing vector backbone. For
example, a sequence coding for a guide nucleic acid can be
assembled or inserted into a vector backbone first, followed by
insertion of the donor nucleic acid in, e.g., the editing cassette.
In other cases, the donor nucleic acid in, e.g., an editing
cassette can be inserted or assembled into a vector backbone first,
followed by insertion of the sequence coding for the guide nucleic
acid. In yet other cases, the sequence encoding the guide nucleic
acid and the donor nucleic acid (inserted, for example, in an
editing cassette) are simultaneously but separately inserted or
assembled into a vector. In yet other embodiments, the sequence
encoding the guide nucleic acid and the sequence encoding the donor
nucleic acid are both included in the editing cassette.
[0137] The target sequence is associated with a protos-spacer
mutation (PAM), which is a short nucleotide sequence recognized by
the gRNA/nuclease complex. The precise preferred PAM sequence and
length requirements for different nucleic acid-guided nucleases
vary; however, PAMs typically are 2-7 base-pair sequences adjacent
or in proximity to the target sequence and, depending on the
nuclease, can be 5' or 3' to the target sequence. Engineering of
the PAM-interacting domain of a nucleic acid-guided nuclease may
allow for alteration of PAM specificity, improve target site
recognition fidelity, decrease target site recognition fidelity, or
increase the versatility of a nucleic acid-guided nuclease. In
certain embodiments, the genome editing of a target sequence both
introduces a desired DNA change to a target sequence, e.g., the
genomic DNA of a cell, and removes, mutates, or renders inactive a
proto-spacer mutation (PAM) region in the target sequence.
Rendering the PAM at the target sequence inactive precludes
additional editing of the cell genome at that target sequence,
e.g., upon subsequent exposure to a nucleic acid-guided nuclease
complexed with a synthetic guide nucleic acid in later rounds of
editing. Thus, cells having the desired target sequence edit and an
altered PAM can be selected using a nucleic acid-guided nuclease
complexed with a synthetic guide nucleic acid complementary to the
target sequence. Cells that did not undergo the first editing event
will be cut rendering a double-stranded DNA break, and thus will
not continue to be viable. The cells containing the desired target
sequence edit and PAM alteration will not be cut, as these edited
cells no longer contain the necessary PAM site and will continue to
grow and propagate.
[0138] The range of target sequences that nucleic acid-guided
nucleases can recognize is constrained by the need for a specific
PAM to be located near the desired target sequence. As a result, it
often can be difficult to target edits with the precision that is
necessary for genome editing. It has been found that nucleases can
recognize some PAMs very well (e.g., canonical PAMs), and other
PAMs less well or poorly (e.g., non-canonical PAMs). Because the
methods disclosed herein allow for identification of edited cells
in a background of unedited cells, the methods allow for
identification of edited cells where the PAM is less than optimal;
that is, the methods for identifying edited cells herein allow for
identification of edited cells even if editing efficiency is very
low. Additionally, the present methods expand the scope of target
sequences that may be edited since edits are more readily
identified, including cells where the genome edits are associated
with less functional PAMs.
[0139] As for the nuclease component of the nucleic acid-guided
nuclease editing system, a polynucleotide sequence encoding the
nucleic acid-guided nuclease can be codon optimized for expression
in particular cell types, such as archaeal, prokaryotic or
eukaryotic cells. Eukaryotic cells can be yeast, fungi, algae,
plant, animal, or human cells. Eukaryotic cells may be those of or
derived from a particular organism, such as a mammal, including but
not limited to human, mouse, rat, rabbit, dog, or non-human mammals
including non-human primates. The choice of nucleic acid-guided
nuclease to be employed depends on many factors, such as what type
of edit is to be made in the target sequence and whether an
appropriate PAM is located close to the desired target sequence.
Nucleases of use in the methods described herein include but are
not limited to Cas 9, Cas 12/CpfI, MAD2, or MAD7 or other MADzymes.
As with the guide nucleic acid, the nuclease may be encoded by a
DNA sequence on a vector (e.g., the engine vector) and be under the
control of a constitutive or inducible promoter. In some
embodiments, the sequence encoding the nuclease is under the
control of an inducible promoter, and the inducible promoter may be
separate from but the same as the inducible promoter controlling
transcription of the guide nucleic acid; that is, a separate
inducible promoter drives the transcription of the nuclease and
guide nucleic acid sequences but the two inducible promoters may be
the same type of inducible promoter (e.g., both are pL promoters).
Alternatively, the inducible promoter controlling expression of the
nuclease may be different from the inducible promoter controlling
transcription of the guide nucleic acid; that is, e.g., the
nuclease may be under the control of the pBAD inducible promoter,
and the guide nucleic acid may be under the control of the pL
inducible promoter.
[0140] Another component of the nucleic acid-guided nuclease system
is the donor nucleic acid. In some embodiments, the donor nucleic
acid is on the same polynucleotide (e.g., editing vector or editing
cassette) as the guide nucleic acid and may be (but not
necessarily) under the control of the same promoter as the guide
nucleic acid (e.g., a single promoter driving the transcription of
both the guide nucleic acid and the donor nucleic acid). The donor
nucleic acid is designed to serve as a template for homologous
recombination with a target sequence nicked or cleaved by the
nucleic acid-guided nuclease as a part of the gRNA/nuclease
complex. A donor nucleic acid polynucleotide may be of any suitable
length, such as about or more than about 20, 25, 50, 75, 100, 150,
200, 500, or 1000 nucleotides in length. In certain preferred
aspects, the donor nucleic acid can be provided as an
oligonucleotide of between 20-300 nucleotides, more preferably
between 50-250 nucleotides. The donor nucleic acid comprises a
region that is complementary to a portion of the target sequence
(e.g., a homology arm). When optimally aligned, the donor nucleic
acid overlaps with (is complementary to) the target sequence by,
e.g., about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more
nucleotides. In many embodiments, the donor nucleic acid comprises
two homology arms (regions complementary to the target sequence)
flanking the mutation or difference between the donor nucleic acid
and the target template. The donor nucleic acid comprises at least
one mutation or alteration compared to the target sequence, such as
an insertion, deletion, modification, or any combination thereof
compared to the target sequence.
[0141] Often the donor nucleic acid is provided as an editing
cassette, which is inserted into a vector backbone where the vector
backbone may comprise a promoter driving transcription of the gRNA
and the coding sequence of the gRNA, or the vector backbone may
comprise a promoter driving the transcription of the gRNA but not
the gRNA itself. Moreover, there may be more than one, e.g., two,
three, four, or more guide nucleic acid/donor nucleic acid
cassettes inserted into an engine vector, where each guide nucleic
acid is under the control of separate different promoters, separate
like promoters, or where all guide nucleic acid/donor nucleic acid
pairs are under the control of a single promoter. In some
embodiments the promoter driving transcription of the gRNA and the
donor nucleic acid (or driving more than one gRNA/donor nucleic
acid pair) is an inducible promoter and the promoter driving
transcription of the nuclease is an inducible promoter as well. For
additional information regarding editing cassettes, see U.S. Pat.
No. 9,982,278, and Ser. No. 15/948,789; 15/116,616; 15/948,785;
16/056,310; 16,275,439; and Ser. No. 16/275,465.
[0142] Inducible editing is advantageous in that singulated cells
can be grown for several to many cell doublings before editing is
initiated, which increases the likelihood that cells with edits
will survive, as the double-strand cuts caused by active editing
are largely toxic to the cells. This toxicity results both in cell
death in the edited colonies, as well as possibly a lag in growth
for the edited cells that do survive but must repair and recover
following editing. However, once the edited cells have a chance to
recover, the size of the colonies of the edited cells will
eventually catch up to the size of the colonies of unedited cells.
Further, a guide nucleic acid may be efficacious directing the edit
of more than one donor nucleic acid in an editing cassette; e.g.,
if the desired edits are close to one another in a target
sequence.
[0143] In addition to the donor nucleic acid, an editing cassette
may comprise one or more primer sites. The primer sites can be used
to amplify the editing cassette by using oligonucleotide primers;
for example, if the primer sites flank one or more of the other
components of the editing cassette.
[0144] Also, as described above, the donor nucleic acid may
comprise--in addition to the at least one mutation relative to a
target sequence--one or more PAM sequence alterations that mutate,
delete or render inactive the PAM site in the target sequence. The
PAM sequence alteration in the target sequence renders the PAM site
"immune" to the nucleic acid-guided nuclease and protects the
target sequence from further editing in subsequent rounds of
editing if the same nuclease is used.
[0145] In addition, the editing cassette may comprise a barcode. A
barcode is a unique DNA sequence that corresponds to the donor DNA
sequence such that the barcode can identify the edit made to the
corresponding target sequence. The barcode typically comprises four
or more nucleotides. In some embodiments, the editing cassettes
comprise a collection of donor nucleic acids representing, e.g.,
gene-wide or genome-wide libraries of donor nucleic acids. The
library of editing cassettes is cloned into vector backbones where,
e.g., each different donor nucleic acid is associated with a
different barcode.
[0146] Additionally, in some embodiments, an expression vector or
cassette encoding components of the nucleic acid-guided nuclease
system further encodes a nucleic acid-guided nuclease comprising
one or more nuclear localization sequences (NLSs), such as about or
more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more NLSs. In
some embodiments, the engineered nuclease comprises NLSs at or near
the amino-terminus, NLSs at or near the carboxy-terminus, or a
combination.
[0147] The engine and editing vectors comprise control sequences
operably linked to the component sequences to be transcribed. As
stated above, the promoters driving transcription of one or more
components of the nucleic acid-guided nuclease editing system may
be inducible. A number of gene regulation control systems have been
developed for the controlled expression of genes in plant, microbe,
and animal cells, including mammalian cells, including the pL
promoter (induced by heat inactivation of the CI857 repressor), the
pBAD promoter (induced by the addition of arabinose to the cell
growth medium), and the rhamnose inducible promoter (induced by the
addition of rhamnose to the cell growth medium). Other systems
include the tetracycline-controlled transcriptional activation
system (Tet-On/Tet-Off, Clontech, Inc. (Palo Alto, Calif.); Bujard
and Gossen, PNAS, 89(12):5547-5551 (1992)), the Lac Switch
Inducible system (Wyborski et al., Environ Mol Mutagen,
28(4):447-58 (1996); DuCoeur et al., Strategies 5(3):70-72 (1992);
U.S. Pat. No. 4,833,080), the ecdysone-inducible gene expression
system (No et al., PNAS, 93(8):3346-3351 (1996)), the cumate
gene-switch system (Mullick et al., BMC Biotechnology, 6:43
(2006)), and the tamoxifen-inducible gene expression (Zhang et al.,
Nucleic Acids Research, 24:543-548 (1996)) as well as others. In
the present methods used in the modules and instruments described
herein, it is preferred that at least one of the nucleic
acid-guided nuclease editing components (e.g., the nuclease and/or
the gRNA) is under the control of a promoter that is activated by a
rise in temperature, as such a promoter allows for the promoter to
be activated by an increase in temperature, and de-activated by a
decrease in temperature, thereby "turning off" the editing process.
Thus, in the scenario of a promoter that is de-activated by a
decrease in temperature, editing in the cell can be turned off
without having to change media; to remove, e.g., an inducible
biochemical in the medium that is used to induce editing.
Automated Cell Editing Instruments and Modules to Create Cell
Surface Display Libraries
Automated Cell Editing Instruments
[0148] FIG. 4A depicts an exemplary automated multi-module cell
processing instrument 400 to, e.g., perform one of the exemplary
workflows described above, as well as additional exemplary modules.
The instrument 400, for example, may be and preferably is designed
as a desktop instrument for use within a laboratory environment.
The instrument 400 may incorporate a mixture of reusable and
disposable elements for performing various staged processes in
conducting automated genome cleavage and/or editing in cells.
Illustrated is a gantry 402, providing an automated mechanical
motion system (actuator) (not shown) that supplies XYZ axis motion
control to, e.g., an automated liquid handling system 458
including, e.g., an air displacement pipette as well as modules of
the automated multi-module cell processing instrument 400. In some
automated multi-module cell processing instruments, the air
displacement pipettor 432 is moved by gantry 402 and the various
modules and reagent cartridges remain stationary; however, in other
embodiments, the liquid handling system may stay stationary while
the various modules are moved. Also included in the automated
multi-module cell processing instrument 400 is reagent cartridge
410 comprising reservoirs 412 and transformation module 430, as
well as a wash cartridge 404 comprising reservoirs 406. The wash
cartridge 404 may be configured to accommodate large tubes, for
example, wash solutions, or solutions that are used often
throughout an iterative process. In one example, wash cartridge 404
may be configured to remain in place when two or more reagent
cartridges 410 are sequentially used and replaced. Although reagent
cartridge 410 and wash cartridge 404 are shown in FIG. 4A as
separate cartridges, the contents of wash cartridge 404 may be
incorporated into reagent cartridge 410. Note in this embodiment
transformation module 430 is contained within reagent cartridge
410; however, in alternative embodiments transformation module 430
is contained within its own module or may be part of another
module, such as a growth module.
[0149] The wash and reagent cartridges 404 and 410 in some
implementations, are disposable kits provided for use in the
automated multi-module cell editing instrument 400. For example, a
user may open and position each of the reagent cartridge 410 and
the wash cartridge 404 within a chassis of the automated
multi-module cell editing instrument prior to activating cell
processing.
[0150] Also illustrated is the robotic handling system 458
including the gantry 402 and air displacement pipettor 432. In some
examples, the robotic handling system 458 may include an automated
liquid handling system such as those manufactured by Tecan Group
Ltd. of Mannedorf, Switzerland, Hamilton Company of Reno, Nev.
(see, e.g., WO2018015544A1), or Beckman Coulter, Inc. of Fort
Collins, Colo. (see, e.g., US20160018427A1). Pipette tips may be
provided in a pipette transfer tip supply (not shown) for use with
the air displacement pipettor 432.
[0151] Components of the cartridges 404, 410, in some
implementations, are marked with machine-readable indicia (not
shown), such as bar codes, for recognition by the robotic handling
system 458. For example, the robotic handling system 458 may scan
containers within each of the cartridges 404, 410 to confirm
contents. In other implementations, machine-readable indicia may be
marked upon each cartridge 404, 410, and the processing system 426
(shown in FIG. 4D) of the automated multi-module cell editing
instrument 400 may identify a stored materials map based upon the
machine-readable indicia. The exemplary automated multi-module cell
processing instrument 400 of FIG. 4A further comprises a cell
growth module 434. (Note, all modules recited briefly here are
described in detail below.) In the embodiment illustrated in FIG.
4A, the cell growth module 434 comprises two cell growth vials 418,
420 (described in greater detail below in relation to FIGS. 5A-5D)
as well as a cell concentration module 422 (described in detail in
relation to FIGS. 6A-6F). In alternative embodiments, the cell
concentration module 422 may be separate from cell growth module
434, e.g., in a separate, dedicated module. Also illustrated as
part of the automated multi-module cell processing instrument 400
of FIG. 4A is an optional enrichment module 440, served by, e.g.,
robotic handing system 458 and air displacement pipettor 432. Also
seen are an optional nucleic acid assembly/desalting module 414
comprising a reaction chamber or tube receptacle (not shown) and a
magnet 416 to allow for purification of nucleic acids using, e.g.,
magnetic solid phase reversible immobilization (SPRI) beads
(Applied Biological Materials Inc., Richmond, BC). The cell growth
module, cell concentration module, transformation module,
enrichment module, reagent cartridge, and nucleic acid assembly
module are described in greater detail below.
[0152] FIG. 4B is a plan view of the front of the exemplary
multi-module cell processing instrument 400 depicted in FIG. 4A.
Cartridge-based source materials (such as in reagent cartridge
410), for example, may be positioned in designated areas on a deck
402 of the instrument 400 for access by a robotic handling
instrument (not shoen in this figure). As illustrated in FIG. 4B,
the deck 402 may include a protection sink such that contaminants
spilling, dripping, or overflowing from any of the modules of the
instrument 400 are contained within a lip of the protection sink.
In addition to reagent cartridge 410, also seen in FIG. 4B is wash
cartridge 404, optional enrichment module 440, and a portion of
growth module 434. Also seen in this view is touch screen display
450, transformation module controls 438, electronics rack 436, and
processing system 426.
[0153] FIGS. 4C through 4D illustrate multi-module cell processing
instruments 480 comprising chassis 490 for use in desktop versions
the cell editing instrument 480. For example, the chassis 490 may
have a width of about 24-48 inches, a height of about 24-48 inches
and a depth of about 24-48 inches. Chassis 490 may be and
preferably is designed to hold multiple modules and disposable
supplies used in automated cell processing. Further, chassis 490
may mount a robotic handling system 458 for moving materials
between modules.
[0154] As illustrated, the chassis 490 includes a cover having a
handle 454 and hinges 456a-456c for lifting the cover and accessing
the interior of the chassis 490. A cooling grate 464 allows for air
flow via an internal fan (not shown). Further, the chassis 490 is
lifted by adjustable feet 470 (feet 470 a-c are shown). The feet
470a-470c, for example, may provide additional air flow beneath the
chassis 490. A control button 466, in some embodiments, allows for
single-button automated start and/or stop of cell processing within
the chassis 490.
[0155] Inside the chassis 490, in some implementations, a robotic
handling system 458 is disposed along a gantry 402 above materials
cartridges 404 and 410. Control circuitry, liquid handling tubes,
air pump controls, valves, thermal units (e.g., heating and cooling
units) and other control mechanisms, in some embodiments, are
disposed below a deck of the chassis 490, in a control box region
468. Also seen in FIG. 4D is enrichment module 440 and nucleic acid
assembly module 414 comprising a magnet 416
[0156] Although not illustrated, in some embodiments a display
screen may be positioned on the front face of the chassis 490, for
example covering a portion of the cover (e.g., see FIG. 4B). The
display screen may provide information to the user regarding the
processing status of the automated multi-module cell editing
instrument. In another example, the display screen may accept
inputs from the user for conducting the cell processing.
The Rotating Cell Growth Module
[0157] FIG. 5A shows one embodiment of a rotating growth vial 500
for use with the cell growth device described herein. The rotating
growth vial is an optically-transparent container having an open
end 504 for receiving liquid media and cells, a central vial region
506 that defines the primary container for growing cells, a
tapered-to-constricted region 518 defining at least one light path
510, a closed end 516, and a drive engagement mechanism 512. The
rotating growth vial has a central longitudinal axis 520 around
which the vial rotates, and the light path 510 is generally
perpendicular to the longitudinal axis of the vial. The first light
path 510 is positioned in the lower constricted portion of the
tapered-to-constricted region 518. Optionally, some embodiments of
the rotating growth vial 500 have a second light path 508 in the
tapered region of the tapered-to-constricted region 518. Both light
paths in this embodiment are positioned in a region of the rotating
growth vial that is constantly filled with the cell culture
(cells+growth media) and is not affected by the rotational speed of
the growth vial. The first light path 510 is shorter than the
second light path 508 allowing for sensitive measurement of OD
values when the OD values of the cell culture in the vial are at a
high level (e.g., later in the cell growth process), whereas the
second light path 508 allows for sensitive measurement of OD values
when the OD values of the cell culture in the vial are at a lower
level (e.g., earlier in the cell growth process). Also shown is lip
502, which allows the rotating growth vial to be seated in a growth
module (not shown) and further allows for easy handling for the
user.
[0158] In some configurations of the rotating growth vial, the
rotating growth vial has two or more "paddles" or interior features
disposed within the rotating growth vial, extending from the inner
wall of the rotating growth vial toward the center of the central
vial region. In some aspects, the width of the paddles or features
varies with the size or volume of the rotating growth vial, and may
range from 1/20 to just over 1/3 the diameter of the rotating
growth vial, or from 1/15 to 1/4 the diameter of the rotating
growth vial, or from 1/10 to 1/5 the diameter of the rotating
growth vial. In some aspects, the length of the paddles varies with
the size or volume of the rotating growth vial, and may range from
4/5 to 1/4 the length of the main body of the rotating growth vial,
or from 3/4 to 1/3 the length of the main body of the rotating
growth vial, or from 1/2 to 1/3 the length of the main body of the
rotating growth vial. In other aspects, there may be concentric
rows of raised features disposed on the inner surface of the main
body of the rotating growth vial arranged horizontally or
vertically; and in other aspects, there may be a spiral
configuration of raised features disposed on the inner surface of
the main body of the rotating growth vial. In alternative aspects,
the concentric rows of raised features or spiral configuration may
be disposed upon a post or center structure of the rotating growth
vial. Though described above as having two paddles, the rotating
growth vial may comprise 3, 4, 5, 6 or more paddles, and up to 20
paddles. The number of paddles will depend upon, e.g., the size or
volume of the rotating growth vial. The paddles may be arranged
symmetrically as single paddles extending from the inner wall of
the vial into the interior of the vial, or the paddles may be
symmetrically arranged in groups of 2, 3, 4 or more paddles in a
group (for example, a pair of paddles opposite another pair of
paddles) extending from the inner wall of the vial into the
interior of the vial. In another embodiment, the paddles may extend
from the middle of the rotating growth vial out toward the wall of
the rotating growth vial, from, e.g., a post or other support
structure in the interior of the rotating growth vial.
[0159] The drive engagement mechanism 512 engages with a motor (not
shown) to rotate the vial. In some embodiments, the motor drives
the drive engagement mechanism 512 such that the rotating growth
vial is rotated in one direction only, and in other embodiments,
the rotating growth vial is rotated in a first direction for a
first amount of time or periodicity, rotated in a second direction
(i.e., the opposite direction) for a second amount of time or
periodicity, and this process may be repeated so that the rotating
growth vial (and the cell culture contents) are subjected to an
oscillating motion. The first amount of time and the second amount
of time may be the same or may be different. The amount of time may
be 1, 2, 3, 4, 5, or more seconds, or may be 1, 2, 3, 4 or more
minutes. In another embodiment, in an early stage of cell growth
the rotating growth vial may be oscillated at a first periodicity
(e.g., every 60 seconds), and then a later stage of cell growth the
rotating growth vial may be oscillated at a second periodicity
(e.g., every one second) different from the first periodicity.
[0160] The rotating growth vial 500 may be reusable or, preferably,
the rotating growth vial is consumable. In some embodiments, the
rotating growth vial is consumable and is presented to the user
pre-filled with growth medium, where the vial is hermetically
sealed at the open end 504 with a foil seal. A medium-filled
rotating growth vial packaged in such a manner may be part of a kit
for use with a stand-alone cell growth device or with a cell growth
module that is part of an automated multi-module cell processing
instrument. To introduce cells into the vial, a user need only
pipette up a desired volume of cells and use the pipette tip to
punch through the foil seal of the vial. Open end 504 may
optionally include an extended lip 502 to overlap and engage with
the cell growth device (not shown). In automated systems, the
rotating growth vial 500 may be tagged with a barcode or other
identifying means that can be read by a scanner or camera that is
part of the automated system (not shown).
[0161] The volume of the rotating growth vial 500 and the volume of
the cell culture (including growth medium) may vary greatly, but
the volume of the rotating growth vial 500 must be large enough for
the cell culture in the growth vial to get proper aeration while
the vial is rotating. In practice, the volume of the rotating
growth vial 500 may range from 1-250 ml, 2-100 ml, from 5-80 ml,
10-50 ml, or from 12-35 ml. Likewise, the volume of the cell
culture (cells+growth media) should be appropriate to allow proper
aeration in the rotating growth vial. Thus, the volume of the cell
culture should be approximately 10-85% of the volume of the growth
vial or from 20-60% of the volume of the growth vial. For example,
for a 35 ml growth vial, the volume of the cell culture would be
from about 4 ml to about 27 ml, or from 7 ml to about 21 ml.
[0162] The rotating growth vial 500 preferably is fabricated from a
bio-compatible optically transparent material--or at least the
portion of the vial comprising the light path(s) is transparent.
Additionally, material from which the rotating growth vial is
fabricated should be able to be cooled to about 4.degree. C. or
lower and heated to about 55.degree. C. or higher to accommodate
both temperature-based cell assays and long-term storage at low
temperatures. Further, the material that is used to fabricate the
vial must be able to withstand temperatures up to 55.degree. C.
without deformation while spinning. Suitable materials include
glass, polyvinyl chloride, polyethylene, polyamide, polyethylene,
polypropylene, polycarbonate, poly(methyl methacrylate (PMMA),
polysulfone, polyurethane, and co-polymers of these and other
polymers. Preferred materials include polypropylene, polycarbonate,
or polystyrene. In some embodiments, the rotating growth vial is
inexpensively fabricated by, e.g., injection molding or
extrusion.
[0163] FIGS. 5B-5D show an embodiment of a cell growth module 550
comprising a rotating growth vial 500. FIG. 5B is a perspective
view of one embodiment of a cell growth device 550. FIG. 5C depicts
a cut-away view of the cell growth device 550 from FIG. 5B. In both
figures, the rotating growth vial 500 is seen positioned inside a
main housing 526 with the extended lip 502 of the rotating growth
vial 500 extending above the main housing 526. Additionally, end
housings 522, a lower housing 532, and flanges 524 are indicated in
both figures. Flanges 524 are used to attach the cell growth device
to heating/cooling means or other structure (not shown). FIG. 5C
depicts additional detail. In FIG. 5C, upper bearing 542 and lower
bearing 530 are shown positioned in main housing 526. Upper bearing
542 and lower bearing 530 support the vertical load of rotating
growth vial 500. Lower housing 532 contains the drive motor 536.
The cell growth device of FIG. 5C comprises two light paths: a
primary light path 534, and a secondary light path 530. Light path
534 corresponds to light path 510 positioned in the constricted
portion of the tapered-to-constricted portion of the rotating
growth vial, and light path 530 corresponds to light path 508 in
the tapered portion of the tapered-to-constricted portion of the
rotating growth vial. Light paths 510 and 508 are not shown in FIG.
5C but may be seen in, e.g., FIG. 5A. In addition to light paths
534 and 530, there is an emission board 528 to illuminate the light
path(s), and detector board 546 to detect the light after the light
travels through the cell culture liquid in the rotating growth
vial.
[0164] The motor 536 used to rotate the rotating growth vial 500 in
some embodiments is a brushless DC type drive motor with built-in
drive controls that can be set to hold a constant revolution per
minute (RPM) between 0 and about 3000 RPM. Alternatively, other
motor types such as a stepper, servo, brushed DC, and the like can
be used. Optionally, the motor 506 may also have direction control
to allow reversing of the rotational direction, and a tachometer to
sense and report actual RPM. The motor is controlled by a processor
(not shown) according to, e.g., standard protocols programmed into
the processor and/or user input, and the motor may be configured to
vary RPM to cause axial precession of the cell culture thereby
enhancing mixing, e.g., to prevent cell aggregation, increase
aeration, and optimize cellular respiration.
[0165] Main housing 526, end housings 522 and lower housing 532 of
the cell growth device 550 may be fabricated from any suitable,
robust material including aluminum, stainless steel, and other
thermally conductive materials, including plastics. These
structures or portions thereof can be created through various
techniques, e.g., metal fabrication, injection molding, creation of
structural layers that are fused, etc. Whereas the rotating growth
vial is envisioned in some embodiments to be reusable but
preferably is consumable, the other components of the cell growth
device 550 are preferably reusable and can function as a
stand-alone benchtop device or, as here, as a module in a
multi-module cell processing system.
[0166] The processor (not shown) of the cell growth system may be
programmed with information to be used as a "blank" or control for
the growing cell culture. A "blank" or control is a vessel
containing cell growth medium only, which yields 100% transmittance
and 0 OD, while the cell sample will deflect light rays and will
have a lower percent transmittance and higher OD. As the cells grow
in the media and become denser, transmittance will decrease and OD
will increase. The processor of the cell growth system may be
programmed to use wavelength values for blanks commensurate with
the growth media typically used in cell culture (whether, e.g.,
mammalian cells, bacterial cells, animal cells, yeast cells, etc.).
Alternatively, a second spectrophotometer and vessel may be
included in the cell growth system, where the second
spectrophotometer is used to read a blank at designated
intervals.
[0167] FIG. 5D illustrates a cell growth device as part of an
assembly comprising the cell growth device of FIG. 5B coupled to
light source 590, detector 592, and thermal components 594. The
rotating growth vial 500 is inserted into the cell growth device.
Components of the light source 590 and detector 592 (e.g., such as
a photodiode with gain control to cover 5-log) are coupled to the
main housing of the cell growth device. The lower housing 532 that
houses the motor that rotates the rotating growth vial is
illustrated, as is one of the flanges 524 that secures the cell
growth device to the assembly. Also illustrated is a Peltier device
or thermoelectric cooler 594. In this embodiment, thermal control
is accomplished by attachment and electrical integration of the
cell growth device 500 to the thermal device 594 via the flange 504
on the base of the lower housing 532. Thermoelectric coolers are
capable of "pumping" heat to either side of a junction, either
cooling a surface or heating a surface depending on the direction
of current flow. In one embodiment, a thermistor is used to measure
the temperature of the main housing and then, through a standard
electronic proportional-integral-derivative (PID) controller loop,
the rotating growth vial 500 is controlled to approximately
+/-0.5.degree. C.
[0168] In certain embodiments, a rear-mounted power entry module
contains the safety fuses and the on-off switch, which when
switched on powers the internal AC and DC power supplies (not
shown) activating the processor. Measurements of optical densities
(OD) at programmed time intervals are accomplished using a 600 nm
Light Emitting Diode (LED) (not shown) that has been columnated
through an optic into the lower constricted portion of the rotating
growth vial which contains the cells of interest. The light
continues through a collection optic to the detection system which
consists of a (digital) gain-controlled silicone photodiode.
Generally, optical density is normally shown as the absolute value
of the logarithm with base 10 of the power transmission factors of
an optical attenuator: OD=-log 10 (Power out/Power in). Since OD is
the measure of optical attenuation--that is, the sum of absorption,
scattering, and reflection--the cell growth device OD measurement
records the overall power transmission, so as the cells grow and
become denser in population the OD (the loss of signal) increases.
The OD system is pre-calibrated against OD standards with these
values stored in an on-board memory accessible by the measurement
program.
[0169] In use, cells are inoculated (cells can be pipetted, e.g.,
from an automated liquid handling system or by a user) into
pre-filled growth media of a rotating growth vial by piercing
though the foil seal. The programmed software of the cell growth
device sets the control temperature for growth, typically
30.degree. C., then slowly starts the rotation of the rotating
growth vial. The cell/growth media mixture slowly moves vertically
up the wall due to centrifugal force allowing the rotating growth
vial to expose a large surface area of the mixture to a normal
oxygen environment. The growth monitoring system takes either
continuous readings of the OD or OD measurements at pre-set or
pre-programmed time intervals. These measurements are stored in
internal memory and if requested the software plots the
measurements versus time to display a growth curve. If enhanced
mixing is required, e.g., to optimize growth conditions, the speed
of the vial rotation can be varied to cause an axial precession of
the liquid, and/or a complete directional change can be performed
at programmed intervals. The growth monitoring can be programmed to
automatically terminate the growth stage at a pre-determined OD,
and then quickly cool the mixture to a lower temperature to inhibit
further growth.
[0170] One application for the cell growth device 550 is to
constantly measure the optical density of a growing cell culture.
One advantage of the described cell growth device is that optical
density can be measured continuously (kinetic monitoring) or at
specific time intervals; e.g., every 5, 10, 15, 20, 30 45, or 60
seconds, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 on minutes.
While the cell growth device has been described in the context of
measuring the optical density (OD) of a growing cell culture, it
should, however, be understood by a skilled artisan given the
teachings of the present specification that other cell growth
parameters can be measured in addition to or instead of cell
culture OD. For example, spectroscopy using visible, UV, or near
infrared (NIR) light allows monitoring the concentration of
nutrients and/or wastes in the cell culture. Additionally,
spectroscopic measurements may be used to quantify multiple
chemical species simultaneously. Nonsymmetric chemical species may
be quantified by identification of characteristic absorbance
features in the NIR. Conversely, symmetric chemical species can be
readily quantified using Raman spectroscopy. Many critical
metabolites, such as glucose, glutamine, ammonia, and lactate have
distinct spectral features in the IR, such that they may be easily
quantified. The amount and frequencies of light absorbed by the
sample can be correlated to the type and concentration of chemical
species present in the sample. Each of these measurement types
provides specific advantages. FT-NIR provides the greatest light
penetration depth and can be used for thicker sample. FT-mid-IR
(MIR) provides information that is more easily discernible as being
specific for certain analytes as these wavelengths are closer to
the fundamental IR absorptions. FT-Raman is advantageous when
interference due to water is to be minimized. Other spectral
properties can be measured via, e.g., dielectric impedence
spectroscopy, visible fluorescence, fluorescence polarization, or
luminescence. Additionally, the cell growth device may include
additional sensors for measuring, e.g., dissolved oxygen, carbon
dioxide, pH, conductivity, and the like.
The Cell Concentration Module
[0171] FIGS. 6A-6I depict variations on one embodiment of a cell
concentration/buffer exchange cassette and module that utilizes
tangential flow filtration. One embodiment of a cell concentration
device described herein operates using tangential flow filtration
(TFF), also known as crossflow filtration, in which the majority of
the feed flows tangentially over the surface of the filter thereby
reducing cake (retentate) formation as compared to dead-end
filtration, in which the feed flows into the filter. Secondary
flows relative to the main feed are also exploited to generate
shear forces that prevent filter cake formation and membrane
fouling thus maximizing particle recovery, as described below.
[0172] The TFF device described herein was designed to take into
account two primary design considerations. First, the geometry of
the TFF device leads to filtering the cell culture over a large
surface area so as to minimize processing time. Second, the design
of the TFF device is configured to minimize filter fouling. FIG. 6A
is a general model 150 of tangential flow filtration. The TFF
device operates using tangential flow filtration, also known as
cross-flow filtration. FIG. 6A shows cells flowing over a membrane
124, where the feed flow of the cells 152 in medium or buffer is
parallel to the membrane 124. TFF is different from dead-end
filtration where both the feed flow and the pressure drop are
perpendicular to a membrane or filter.
[0173] FIG. 6B depicts a top view of the lower member of one
embodiment of a TFF device/module providing tangential flow
filtration. As can be seen in the embodiment of the TFF device of
FIG. 6B, TFF device 600 comprises a channel structure 616
comprising a flow channel 602b through which a cell culture is
flowed. The channel structure 616 comprises a single flow channel
602b that is horizontally bifurcated by a membrane (not shown)
through which buffer or medium may flow, but cells cannot. This
particular embodiment comprises an undulating serpentine geometry
614 (i.e., the small "wiggles" in the flow channel 602) and a
serpentine "zig-zag" pattern where the flow channel 602
crisscrosses the device from one end at the left of the device to
the other end at the right of the device. The serpentine pattern
allows for filtration over a high surface area relative to the
device size and total channel volume, while the undulating
contribution creates a secondary inertial flow to enable effective
membrane regeneration preventing membrane fouling. Although an
undulating geometry and serpentine pattern are exemplified here,
other channel configurations may be used as long as the channel can
be bifurcated by a membrane, and as long as the channel
configuration provides for flow through the TFF module in
alternating directions. In addition to the flow channel 602b,
portals 604 and 606 as part of the channel structure 616 can be
seen, as well as recesses 608. Portals 604 collect cells passing
through the channel on one side of a membrane (not shown) (the
"retentate"), and portals 606 collect the medium ("filtrate" or
"permeate") passing through the channel on the opposite side of the
membrane (not shown). In this embodiment, recesses 608 accommodate
screws or other fasteners (not shown) that allow the components of
the TFF device to be secured to one another.
[0174] The length 610 and width 612 of the channel structure 616
may vary depending on the volume of the cell culture to be grown
and the optical density of the cell culture to be concentrated. The
length 610 of the channel structure 616 typically is from 1 mm to
300 mm, or from 50 mm to 250 mm, or from 60 mm to 200 mm, or from
70 mm to 150 mm, or from 80 mm to 100 mm. The width of the channel
structure 616 typically is from 1 mm to 120 mm, or from 20 mm to
100 mm, or from 30 mm to 80 mm, or from 40 mm to 70 mm, or from 50
mm to 60 mm. The cross-section configuration of the flow channel
102 may be round, elliptical, oval, square, rectangular,
trapezoidal, or irregular. If square, rectangular, or another shape
with generally straight sides, the cross section may be from about
10 .mu.m to 1000 .mu.m wide, or from 200 .mu.m to 800 .mu.m wide,
or from 300 .mu.m to 700 .mu.m wide, or from 400 .mu.m to 600 .mu.m
wide; and from about 10 .mu.m to 1000 .mu.m high, or from 200 .mu.m
to 800 .mu.m high, or from 300 .mu.m to 700 .mu.m high, or from 400
.mu.m to 600 .mu.m high. If the cross section of the flow channel
602 is generally round, oval or elliptical, the radius of the
channel may be from about 50 .mu.m to 1000 .mu.m in hydraulic
radius, or from 5 .mu.m to 800 .mu.m in hydraulic radius, or from
200 .mu.m to 700 .mu.m in hydraulic radius, or from 300 .mu.m to
600 .mu.m wide in hydraulic radius, or from about 200 to 500 .mu.m
in hydraulic radius.
[0175] When looking at the top view of the TFF device/module of
FIG. 6B, note that there are two retentate portals 604 and two
filtrate portals 606, where there is one of each type portal at
both ends (e.g., the narrow edge) of the device 600. In other
embodiments, retentate and filtrate portals can on the same surface
of the same member (e.g., upper or lower member), or they can be
arranged on the side surfaces of the assembly. Unlike other TFF
devices that operate continuously, the TFF device/module described
herein uses an alternating method for concentrating cells. The
overall work flow for cell concentration using the TFF
device/module involves flowing a cell culture or cell sample
tangentially through the channel structure. The membrane
bifurcating the flow channels retains the cells on one side of the
membrane and allows unwanted medium or buffer to flow across the
membrane into a filtrate side (e.g, lower member 620) of the
device. In this process, a fixed volume of cells in medium or
buffer is driven through the device until the cell sample is
collected into one of the retentate portals 604, and the
medium/buffer that has passed through the membrane is collected
through one or both of the filtrate portals 606. All types of
prokaryotic and eukaryotic cells--both adherent and non-adherent
cells--can be grown in the TFF device. Adherent cells may be grown
on beads or other cell scaffolds suspended in medium that flow
through the TFF device.
[0176] In the cell concentration process, passing the cell sample
through the TFF device and collecting the cells in one of the
retentate portals 604 while collecting the medium in one of the
filtrate portals 606 is considered "one pass" of the cell sample.
The transfer between retentate reservoirs "flips" the culture, The
retentate and filtrate portals collecting the cells and medium,
respectively, for a given pass reside on the same end of TFF
device/module 600 with fluidic connections arranged so that there
are two distinct flow layers for the retentate and filtrate sides,
but if the retentate portal 604 resides on the upper member of
device/module 600 (that is, the cells are driven through the
channel above the membrane and the filtrate (medium) passes to the
portion of the channel below the membrane), the filtrate portal 606
will reside on the lower member of device/module 100 and vice versa
(that is, if the cell sample is driven through the channel below
the membrane, the filtrate (medium) passes to the portion of the
channel above the membrane). This configuration can be seen more
clearly in FIGS. 6C-6D, where the retentate flows 660 from the
retentate portals 604 and the filtrate flows 670 from the filtrate
portals 606.
[0177] At the conclusion of a "pass" in the growth concentration
process, the cell sample is collected by passing through the
retentate portal 604 and into the retentate reservoir (not shown).
To initiate another "pass", the cell sample is passed again through
the TFF device, this time in a flow direction that is reversed from
the first pass. The cell sample is collected by passing through the
retentate portal 604 and into retentate reservoir (not shown) on
the opposite end of the device/module from the retentate portal 604
that was used to collect cells during the first pass. Likewise, the
medium/buffer that passes through the membrane on the second pass
is collected through the filtrate portal 606 on the opposite end of
the device/module from the filtrate portal 606 that was used to
collect the filtrate during the first pass, or through both
portals. This alternating process of passing the retentate (the
concentrated cell sample) through the device/module is repeated
until the cells have been concentrated to a desired volume, and
both filtrate portals can be open during the passes to reduce
operating time. In addition, buffer exchange may be effected by
adding a desired buffer (or fresh medium) to the cell sample in the
retentate reservoir, before initiating another "pass", and
repeating this process until the old medium or buffer is diluted
and filtered out and the cells reside in fresh medium or buffer.
Note that buffer exchange and cell concentration may (and typically
do) take place simultaneously.
[0178] FIG. 6C depicts a top view of upper (622) and lower (620)
members of an exemplary TFF module. Again, portals 604 and 606 are
seen. As noted above, recesses--such as the recesses 608 seen in
FIG. 6B--provide a means to secure the components (upper member
622, lower member 620, and membrane 624) of the TFF device/membrane
to one another during operation via, e.g., screws or other like
fasteners. However, in alterative embodiments an adhesive, such as
a pressure sensitive adhesive, or ultrasonic welding, or solvent
bonding, may be used to couple the upper member 622, lower member
620, and membrane 624 together. Indeed, one of ordinary skill in
the art given the guidance of the present disclosure can find yet
other configurations for coupling the components of the TFF device,
such as e.g., clamps; mated fittings disposed on the upper and
lower members; combination of adhesives, welding, solvent bonding,
and mated fittings; and other such fasteners and couplings.
[0179] Note that there is one retentate portal and one filtrate
portal on each "end" (e.g., the narrow edges) of the TFF
device/module. The retentate and filtrate portals on the left side
of the device/module will collect cells (flow path at 660) and
medium (flow path at 670), respectively, for the same pass.
Likewise, the retentate and filtrate portals on the right side of
the device/module will collect cells (flow path at 660) and medium
(flow path at 670), respectively, for the same pass. In this
embodiment, the retentate is collected from portals 604 on the top
surface of the TFF device, and filtrate is collected from portals
606 on the bottom surface of the device. The cells are maintained
in the TFF flow channel above the membrane 624, while the filtrate
(medium) flows through membrane 624 and then through portals 606;
thus, the top/retentate portals and bottom/filtrate portals
configuration is practical. It should be recognized, however, that
other configurations of retentate and filtrate portals may be
implemented such as positioning both the retentate and filtrate
portals on the side (as opposed to the top and bottom surfaces) of
the TFF device. In FIG. 6C, the channel structure 602b can be seen
on the bottom member 620 of the TFF device 600. However, in other
embodiments, retentate and filtrate portals can reside on the same
of the TFF device.
[0180] Also seen in FIG. 6C is membrane or filter 624. Filters or
membranes appropriate for use in the TFF device/module are those
that are solvent resistant, are contamination free during
filtration, and are able to retain the types and sizes of cells of
interest. For example, in order to retain small cell types such as
bacterial cells, pore sizes can be as low as 0.2 .mu.m, however for
other cell types, the pore sizes can be as high as 5 .mu.m. Indeed,
the pore sizes useful in the TFF device/module include filters with
sizes from 0.20 .mu.m, 0.21 .mu.m, 0.22 .mu.m, 0.23 .mu.m, 0.24
.mu.m, 0.25 .mu.m, 0.26 .mu.m, 0.27 .mu.m, 0.28 .mu.m, 0.29 .mu.m,
0.30 .mu.m, 0.31 .mu.m, 0.32 .mu.m, 0.33 .mu.m, 0.34 .mu.m, 0.35
.mu.m, 0.36 .mu.m, 0.37 .mu.m, 0.38 .mu.m, 0.39 .mu.m, 0.40 .mu.m,
0.41 .mu.m, 0.42 .mu.m, 0.43 .mu.m, 0.44 .mu.m, 0.45 .mu.m, 0.46
.mu.m, 0.47 .mu.m, 0.48 .mu.m, 0.49 .mu.m, 0.50 .mu.m and larger.
The filters may be fabricated from any suitable non-reactive
material including cellulose mixed ester (cellulose nitrate and
acetate) (CME), polycarbonate (PC), polyvinylidene fluoride (PVDF),
polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon,
glass fiber, or metal substrates as in the case of laser or
electrochemical etching. The TFF device shown in FIGS. 6C and 6D do
not show a seat in the upper 612 and lower 620 members where the
filter 624 can be seated or secured (for example, a seat half the
thickness of the filter in each of upper 612 and lower 620
members); however, such a seat is contemplated in some
embodiments.
[0181] FIG. 6D depicts a bottom view of upper and lower components
of the exemplary TFF module shown in FIG. 6C. FIG. 6D depicts a
bottom view of upper (622) and lower (620) components of an
exemplary TFF module. Again portals 604 and 606 are seen. Note
again that there is one retentate portal and one filtrate portal on
each end of the device/module. The retentate and filtrate portals
on the left side of the device/module will collect cells (flow path
at 660) and medium (flow path at 670), respectively, for the same
pass. Likewise, the retentate and filtrate portals on the right
side of the device/module will collect cells (flow path at 660) and
medium (flow path at 670), respectively, for the same pass. In FIG.
6D, the channel structure 602a can be seen on the upper member 622
of the TFF device 600. Thus, looking at FIGS. 6C and 6D, note that
there is a channel structure 602 (602a and 602b) in both the upper
and lower members, with a membrane 624 between the upper and lower
portions of the channel structure. The channel structure 602 of the
upper 622 and lower 620 members (602a and 602b, respectively) mate
to create the flow channel with the membrane 624 positioned
horizontally between the upper and lower members of the flow
channel thereby bifurcating the flow channel.
[0182] Medium exchange (during cell growth) or buffer exchange
(during cell concentration or rendering the cells competent) is
performed on the TFF device/module by adding fresh medium to
growing cells or a desired buffer to the cells concentrated to a
desired volume; for example, after the cells have been concentrated
at least 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold,
80-fold, 90-fold, 100-fold, 150-fold, 200-fold or more. A desired
exchange medium or exchange buffer is added to the cells either by
addition to the retentate reservoir or thorough the membrane from
the filtrate side and the process of passing the cells through the
TFF device 600 is repeated until the cells have been grown to a
desired optical density or concentrated to a desired volume in the
exchange medium or buffer. This process can be repeated any number
of desired times so as to achieve a desired level of exchange of
the buffer and a desired volume of cells. The exchange buffer may
comprise, e.g., glycerol or sorbitol thereby rendering the cells
competent for transformation in addition to decreasing the overall
volume of the cell sample.
[0183] The TFF device 600 may be fabricated from any robust
material in which channels (and channel branches) may be milled
including stainless steel, silicon, glass, aluminum, or plastics
including cyclic-olefin copolymer (COC), cyclo-olefin polymer
(COP), polystyrene, polyvinyl chloride, polyethylene, polyamide,
polyethylene, polypropylene, acrylonitrile butadiene,
polycarbonate, polyetheretheketone (PEEK), poly(methyl
methylacrylate) (PMMA), polysulfone, and polyurethane, and
co-polymers of these and other polymers. If the TFF device/module
is disposable, preferably it is made of plastic. In some
embodiments, the material used to fabricate the TFF device/module
is thermally-conductive so that the cell culture may be heated or
cooled to a desired temperature. In certain embodiments, the TFF
device is formed by precision mechanical machining, laser
machining, electro discharge machining (for metal devices); wet or
dry etching (for silicon devices); dry or wet etching, powder or
sandblasting, photostructuring (for glass devices); or
thermoforming, injection molding, hot embossing, or laser machining
(for plastic devices) using the materials mentioned above that are
amenable to this mass production techniques.
[0184] FIG. 6E depicts an exemplary configuration of an assembled
TFF device, where, like the other configurations, the upper member
and lower member in combination form a channel structure with a
membrane disposed between the upper and lower members; however, in
this configuration in addition to the retentate reservoirs, there
is in addition an optional buffer or medium reservoir positioned
between the retentate reservoirs, and a lower filtrate or permeate
reservoir. In the TFF device 6000 configuration shown in FIG. 6E,
6044 is the top or cover of the TFF device 6000, having three ports
6046, where there is a pipette tip 6048 disposed in the right-most
port 6046. The top 6044 of the TFF device 6000 is adjacent to and
in operation is coupled with a combined reservoir and upper member
structure 6050. Combined reservoir and upper member structure 6050
comprises a top surface that is adjacent the top or cover 6044 of
the TFF device, a bottom surface which comprises the upper member
6022 of the TFF device, where the upper member 6022 of the TFF
device defines the upper portion of the flow channel (not shown)
disposed on the bottom surface of the upper member 6022 of the
combined reservoir and upper member structure 6050. Additionally,
combined reservoir and upper member structure 6050 comprises two
retentate reservoirs 6080 and an optional buffer or medium
reservoir 6082. The retentate reservoirs are fluidically coupled to
the upper portion of the flow channel, and the buffer or medium
reservoir is fluidically coupled to the retentate reservoirs. Also
seen in this assembled view of TFF device 6000 is membrane 6024,
lower member 6020 which, as described previously comprises on its
top surface the lower portion of the tangential flow channel (not
shown), where the channel structures of the upper member 6022 and
lower member 6020 (neither shown in this view) mate to form a
single flow channel. Beneath and adjacent to lower member 6020 is a
gasket 6040, which is interposed between lower member 6020 and an
optional filtrate (or permeate) reservoir 6042. The filtrate
reservoir 6042 is in fluid connection with the lower portion of the
flow channel, as a receptacle for the filtrate or permeate that is
removed from the cell culture. In operation, top 6044, combined
reservoir and upper member structure 6050, membrane 6024, lower
member 6020, gasket 6040, and filtrate reservoir 6042 are coupled
and secured together to be fluid- and air-tight. The assembled TFF
device 1100 typically is from 4 to 25 cm in height, or from 5 to 20
cm in height, or from 7 to 15 cm in height; from 5 to 30 cm in
length, or from 8 to 25 cm in length, or from 10 to 20 cm in
length; and is from 3 to 15 cm in depth, or from 5 to 10 cm in
depth. An exemplary TFF device is 11 cm in height, 12 cm in length,
and 8 cm in depth. The retentate reservoirs, buffer or medium
reservoir, and tangential flow channel-forming structures may be
configured to be cooled to 4.degree. C. for cell maintenance. The
dimensions for the serpentine channel recited above, as well as the
specifications and materials for the filter and the TFF device
apply to the embodiment of the device shown in FIGS. 6E-6I. In
embodiments including the present embodiment, up to 120 mL of cell
culture can be grown and/or filtered, or up to 100 mL, 90 mL, 80
mL, 70 mL, 60 mL, 50 mL, 40 mL, 30 mL or 20 mL of cell culture can
be grown and/or filtered.
[0185] FIG. 6F depicts an exploded perspective view of TFF device
6000. In this configuration, 6044 is the top or cover of the TFF
device 6000, having three ports 6046, where there is a pipette tip
6048 disposed in the left-most port 6046. The top 6044 of the TFF
device 6000 is, in operation, coupled with a combined reservoir and
upper member structure 6050. Combined reservoir and upper member
structure 6050 comprises a top surface that, in operation, is
adjacent the top or cover 6044 of the TFF device, a bottom surface
which comprises the upper member 6022 of the TFF device, where the
upper member 6022 of the TFF device defines the upper portion of
the tangential flow channel (not shown). Combined reservoir and
upper member structure 6050 comprises two retentate reservoirs 6080
and an optional buffer or medium reservoir 6082. The retentate
reservoirs are fluidically coupled to the upper portion of the flow
channel, and the optional buffer or medium reservoir is fluidically
coupled to the retentate reservoirs. Also seen in this exploded
view of TFF device 6000 is lower member 6020 which, as described
previously comprises on its top surface the lower portion of the
tangential flow channel 6002b (seen on the top surface of lower
member 6020), where the upper and lower portions of the channel
structures of the upper member 6022 and lower member 6020,
respectively, when coupled mate to form a single flow channel (the
membrane that is interposed between the upper member 6022 and lower
member 6020 in operation is not shown). Beneath lower member 6020
is gasket 6040, which in operation is interposed between lower
member 6020 and a filtrate (or permeate) reservoir 6042. In
operation, top 6044, combined reservoir and upper member structure
6050, membrane (not shown), lower member 6020, gasket 6040, and
filtrate reservoir 6042 are coupled and secured together to be
fluid- and air-tight. In FIG. 6F, fasteners are shown that can be
used to couple the various structures (top 6044, combined reservoir
and upper member structure 6050, membrane (not shown), lower member
6020, gasket 6040, and filtrate reservoir 6042) together. However,
as an alternative to screws or other like fasteners, the various
structures of TFF device 6000 can be coupled using an adhesive,
such as a pressure sensitive adhesive; ultrasonic welding; or
solvent bonding. Further, a combination of fasteners, adhesives,
and/or welding types may be employed to couple the various
structures of the TFF device. One of ordinary skill in the art
given the guidance of the present disclosure could find yet other
configurations for coupling the components of TFF device 6000, such
as e.g., clamps, mated fittings, and other such fasteners.
[0186] FIG. 6G depicts combined reservoir and upper member
structure 6050, comprising two retentate reservoirs 6080 and an
optional buffer or medium reservoir 6082, as well as upper member
6020, which is disposed on the bottom of combined reservoir and
upper member structure 6050. Upper member 6022 of the TFF device
defines the upper portion of the tangential flow channel (not
shown) disposed on the bottom surface of the combined reservoir and
upper member structure 6050. FIG. 6H is a top-down view of the
upper surface of combined reservoir and upper member structure
6050, depicting the top of retentate reservoirs 6080 and buffer or
medium reservoir 6082, as well as fluid or vacuum ports 6046. The
retentate reservoirs are fluidically coupled to the upper portion
of the flow channel, and the buffer or medium reservoir is
fluidically coupled to the retentate reservoirs. FIG. 6I is a
bottom-up view of the lower surface of combined reservoir and upper
member structure 6050, showing the upper member 6020 with the upper
portion of the tangential flow channel 6002a disposed on the bottom
surface of upper member 6020. The flow channel 6002a disposed on
the bottom surface of upper member 6020 in operation is mated to
the bottom portion of the tangential flow channel disposed on the
top surface of the lower member (not shown in this view, but see
FIG. 6F), where the upper and lower portions of the flow channel
structure mate to form a single flow channel.
[0187] As an alternative to the TFF module described above, a cell
concentration module comprising a hollow filter may be employed.
Examples of filters suitable for use in the present invention
include membrane filters, ceramic filters and metal filters. The
filter may be used in any shape; the filter may for example be
cylindrical or essentially flat. Preferably, the filter used is a
membrane filter, preferably a hollow fiber filter. The term "hollow
fiber" is meant a tubular membrane. The internal diameter of the
tube is at least 0.1 mm, more preferably at least 0.5 mm, most
preferably at least 0.75 mm and preferably the internal diameter of
the tube is at most 10 mm, more preferably at most 6 mm, most
preferably at most 1 mm. Filter modules comprising hollow fibers
are commercially available from various companies, including G.E.
Life Sciences (Marlborough, Mass.) and InnovaPrep (Drexel, Mo.).
Specific examples of hollow fiber filter systems that can be used,
modified or adapted for use in the present methods and systems
include, but are not limited to, Ser. U.S. Pat. Nos. 9,738,918;
9,593,359; 9,574,977; 9,534,989; 9,446,354; 9,295,824; 8,956,880;
8,758,623; 8,726,744; 8,677,839; 8,677,840; 8,584,536; 8,584,535;
and 8,110,112.
Nucleic Acid Assembly Module
[0188] Certain embodiments of the automated multi-module cell
editing instruments of the present disclosure optionally include a
nucleic acid assembly module. The nucleic acid assembly module is
configured to accept and assemble the nucleic acids necessary to
facilitate the desired genome editing events. In general, the term
"vector" refers to a nucleic acid molecule capable of transporting
a desired nucleic acid to which it has been linked into a cell.
Vectors include, but are not limited to, nucleic acid molecules
that are single-stranded, double-stranded, or partially
double-stranded; nucleic acid molecules that include one or more
free ends, no free ends (e.g., circular); nucleic acid molecules
that include DNA, RNA, or both; and other varieties of
polynucleotides known in the art. One type of vector is a
"plasmid," which refers to a circular double stranded DNA loop into
which additional DNA segments can be inserted, such as by standard
molecular cloning techniques. Another type of vector is a viral
vector, where virally-derived DNA or RNA sequences are present in
the vector for packaging into a virus (e.g. retroviruses,
replication defective retroviruses, adenoviruses, replication
defective adenoviruses, and adeno-associated viruses). Viral
vectors also include polynucleotides carried by a virus for
transfection into a host cell. Certain vectors are capable of
autonomous replication in a host cell into which they are
introduced (e.g. bacterial vectors having a bacterial origin of
replication and episomal mammalian vectors). Other vectors (e.g.,
non-episomal mammalian vectors) are integrated into the genome of a
host cell upon introduction into the host cell, and thereby are
replicated along with the host genome. Moreover, certain vectors
are capable of directing the expression of genes to which they are
operatively-linked. Such vectors are referred to herein as
"expression vectors" or "editing vectors." Common expression
vectors of utility in recombinant DNA techniques are often in the
form of plasmids. Additional vectors include fosmids, phagemids,
and synthetic chromosomes.
[0189] Recombinant expression vectors can include a nucleic acid in
a form suitable for transcription, and for some nucleic acid
sequences, translation and expression of the nucleic acid in a host
cell, which means that the recombinant expression vectors include
one or more regulatory elements--which may be selected on the basis
of the host cells to be used for expression--that are
operatively-linked to the nucleic acid sequence to be expressed.
Within a recombinant expression vector, "operably linked" is
intended to mean that the nucleotide sequence of interest is linked
to the regulatory element(s) in a manner that allows for
transcription, and for some nucleic acid sequences, translation and
expression of the nucleotide sequence (e.g. in an in vitro
transcription/translation system or in a host cell when the vector
is introduced into the host cell). Appropriate recombination and
cloning methods are disclosed in US Pub. No. 2004/0171156, the
contents of which are herein incorporated by reference in their
entirety for all purposes.
[0190] In some embodiments, a regulatory element is operably linked
to one or more elements of a targetable nuclease system so as to
drive transcription, and for some nucleic acid sequences,
translation and expression of the one or more components of the
targetable nuclease system.
[0191] In addition, the polynucleotide sequence encoding the
nucleic acid-guided nuclease can be codon optimized for expression
in particular cells, such as prokaryotic or eukaryotic cells.
Eukaryotic cells can be yeast, fungi, algae, plant, animal, or
human cells. Eukaryotic cells may be those of or derived from a
particular organism, such as a mammal, including but not limited to
human, mouse, rat, rabbit, dog, or non-human mammal including
non-human primate. In addition or alternatively, a vector may
include a regulatory element operably liked to a polynucleotide
sequence, which, when transcribed, forms a guide RNA.
[0192] The nucleic acid assembly module can be configured to
perform a wide variety of different nucleic acid assembly
techniques in an automated fashion. Nucleic acid assembly
techniques that can be performed in the nucleic acid assembly
module of the disclosed automated multi-module cell editing
instruments include, but are not limited to, those assembly methods
that use restriction endonucleases, including PCR, BioBrick
assembly (U.S. Pat. No. 9,361,427), Type IIS cloning (e.g.,
GoldenGate assembly, European Patent Application Publication EP 2
395 087 A1), and Ligase Cycling Reaction (de Kok, ACS Synth Biol.,
3(2):97-106 (2014); Engler, et al., PLoS One, 3(11):e3647 (2008);
and U.S. Pat. No. 6,143,527). In other embodiments, the nucleic
acid assembly techniques performed by the disclosed automated
multi-module cell editing instruments are based on overlaps between
adjacent parts of the nucleic acids, such as Gibson Assembly.RTM.,
CPEC, SLIC, Ligase Cycling etc. Additional assembly methods include
gap repair in yeast (Bessa, Yeast, 29(10):419-23 (2012)), gateway
cloning (Ohtsuka, Curr Pharm Biotechnol, 10(2):244-51 (2009)); U.S.
Pat. No. 5,888,732; and 6,277,608), and topoisomerase-mediated
cloning (Udo, PLoS One, 10(9):e0139349 (2015); and U.S. Pat. No.
6,916,632). These and other nucleic acid assembly techniques are
described, e.g., in Sands and Brent, Curr Protoc Mol Biol.,
113:3.26.1-3.26.20 (2016).
[0193] The nucleic acid assembly module is temperature controlled
depending upon the type of nucleic acid assembly used in the
automated multi-module cell editing instrument. For example, when
PCR is utilized in the nucleic acid assembly module, the module
includes a thermocycling capability allowing the temperatures to
cycle between denaturation, annealing and extension steps. When
single temperature assembly methods (e.g., isothermal assembly
methods) are utilized in the nucleic acid assembly module, the
module provides the ability to reach and hold at the temperature
that optimizes the specific assembly process being performed. These
temperatures and the duration for maintaining these temperatures
can be determined by a preprogrammed set of parameters executed by
a script, or manually controlled by the user using the processing
system of the automated multi-module cell editing instrument.
[0194] In one embodiment, the nucleic acid assembly module is a
module to perform assembly using a single, isothermal reaction.
Certain isothermal assembly methods can combine simultaneously up
to 15 nucleic acid fragments based on sequence identity. The
assembly method provides, in some embodiments, nucleic acids to be
assembled which include an approximate 20-40 base overlap with
adjacent nucleic acid fragments. The fragments are mixed with a
cocktail of three enzymes--an exonuclease, a polymerase, and a
ligase-along with buffer components. Because the process is
isothermal and can be performed in a 1-step or 2-step method using
a single reaction vessel, isothermal assembly reactions are ideal
for use in an automated multi-module cell editing instrument. The
1-step method allows for the assembly of up to five different
fragments using a single step isothermal process. The fragments and
the master mix of enzymes are combined and incubated at 50.degree.
C. for up to one hour. For the creation of more complex constructs
with up to fifteen fragments or for incorporating fragments from
100 bp up to 10 kb, typically the 2-step is used, where the 2-step
reaction requires two separate additions of master mix; one for the
exonuclease and annealing step and a second for the polymerase and
ligation steps.
The Cell Transformation Module
[0195] In addition to the modules for cell growth, cell
concentration, and nucleic acid assembly, FIGS. 7A-7E depict
variations on one embodiment of a cell transformation module (in
this case, a flow through electroporation device) that may be
included in a cell growth/concentration/transformation instrument.
FIGS. 7A and 7B are top perspective and bottom perspective views,
respectively, of six co-joined flow-through electroporation devices
750. FIG. 7A depicts six flow-through electroporation units 750
arranged on a single substrate 756. Each of the six flow-through
electroporation units 750 have wells 752 that define cell sample
inlets and wells 754 that define cell sample outlets. FIG. 7B is a
bottom perspective view of the six co-joined flow-through
electroporation devices of FIG. 7A also depicting six flow-through
electroporation units 750 arranged on a single substrate 4156. Six
inlet wells 4152 can be seen, one for each flow-through
electroporation unit 750, and one outlet well 754 can be seen (the
outlet well of the left-most flow-through electroporation unit
750). Additionally seen in FIG. 7B are an inlet 702, outlet 704,
flow channel 706 and two electrodes 708 on either side of a
constriction in flow channel 706 in each flow-through
electroporation unit 750. Once the six flow-through electroporation
units 750 are fabricated, they can be separated from one another
(e.g., "snapped apart") and used one at a time, or alternatively in
embodiments two or more flow-through electroporation units 750 can
be used in parallel without separation.
[0196] The flow-through electroporation devices achieve high
efficiency cell electroporation with low toxicity. The flow-through
electroporation devices of the disclosure allow for particularly
easy integration with robotic liquid handling instrumentation that
is typically used in automated systems such as air displacement
pipettors. Such automated instrumentation includes, but is not
limited to, off-the-shelf automated liquid handling systems from
Tecan (Mannedorf, Switzerland), Hamilton (Reno, Nev.), Beckman
Coulter (Fort Collins, Colo.), etc.
[0197] Generally speaking, microfluidic electroporation--using cell
suspension volumes of less than approximately 10 ml and as low as 1
.mu.l--allows more precise control over a transfection or
transformation process and permits flexible integration with other
cell processing tools compared to bench-scale electroporation
devices. Microfluidic electroporation thus provides unique
advantages for, e.g., single cell transformation, processing and
analysis; multi-unit electroporation device configurations; and
integrated, automatic, multi-module cell processing and
analysis.
[0198] In specific embodiments of the flow-through electroporation
devices of the disclosure the toxicity level of the transformation
results in greater than 10% viable cells after electroporation,
preferably greater than 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 70%, 75%, 80%, 85%, 90%, or even 95% viable cells
following transformation, depending on the cell type and the
nucleic acids being introduced into the cells.
[0199] The flow-through electroporation device described in
relation to FIGS. 7A-7E comprises a housing with an electroporation
chamber, a first electrode and a second electrode configured to
engage with an electric pulse generator, by which electrical
contacts engage with the electrodes of the electroporation device.
In certain embodiments, the electroporation devices are
autoclavable and/or disposable, and may be packaged with reagents
in a reagent cartridge. The electroporation device may be
configured to electroporate cell sample volumes between 1 .mu.l to
2 ml, 10 .mu.l to 1 ml, 25 .mu.l to 750 .mu.l, or 50 .mu.l to 500
.mu.l. The cells that may be electroporated with the disclosed
electroporation devices include mammalian cells (including human
cells), plant cells, yeasts, other eukaryotic cells, bacteria,
archaea, and other cell types.
[0200] In one exemplary embodiment, FIG. 7C depicts a top view of a
flow-through electroporation device 750 having an inlet 702 for
introduction of cells and an exogenous reagent to be electroporated
into the cells ("cell sample") and an outlet 704 for the cell
sample following electroporation. Electrodes 708 are introduced
through electrode channels (not shown) in the device. FIG. 7D shows
a cutaway view from the top of flow-through electroporation device
750, with the inlet 702, outlet 704, and electrodes 708 positioned
with respect to a constriction in flow channel 706. A side cutaway
view of the bottom portion of flow-through electroporation device
750 in FIG. 7E illustrates that electrodes 708 in this embodiment
are positioned in electrode channels 710 and perpendicular to flow
channel 706 such that the cell sample flows from the inlet channel
712 through the flow channel 706 to the outlet channel 714, and in
the process the cell sample flows into the electrode channels 710
to be in contact with electrodes 708. In this aspect, the inlet
channel, outlet channel and electrode channels all originate from
the top planar side of the device; however, the flow-through
electroporation architecture depicted in FIGS. 7C-7E is but one
architecture useful with the reagent cartridges described herein.
Additional electrode architectures are described, e.g., in Ser. No.
16/147,120, filed 24 Sep. 2018; Ser. No. 16/147,865, filed 30 Sep.
2018; and Ser. No. 16/147,871, filed 30 Sep. 2018.
The Cell Enrichment Module
[0201] One optional aspect provides automated modules and
instruments for nucleic acid-guided nuclease genome editing that
implement enrichment techniques for cells whose genomes have been
properly edited. The enrichment modules perform methods that use
cell singulation and normalization to reduce growth competition
between edited and unedited cells. Singulation overcomes growth
bias from unedited cells or cells containing edits conferring
growth advantages or disadvantages. The methods, modules and
instruments may be applied to all cell types including, archaeal,
prokaryotic, and eukaryotic (e.g., yeast, fungal, plant and animal)
cells.
[0202] Singulating, optional induction of editing, and
normalization of cell colonies leads to 2-250.times.,
1.0-225.times., 25-200.times., 40-175.times., 50-150.times.,
60-100.times.. or 5-100.times. gains in identifying edited cells
over prior art methods and provides new approaches for generating
arrayed or pooled edited cells comprising genome libraries.
Additionally, the methods, modules, and instruments may be
leveraged to create iterative editing systems to generate
combinatorial libraries, identify rare cell edits, and enable
high-throughput enrichment applications to identify editing
activity.
[0203] The compositions and methods described herein improve
nucleic acid-guided nuclease editing systems in which nucleic
acid-guided nucleases (e.g., RNA-guided nucleases) are used to edit
specific target regions in an organism's genome. FIG. 8A depicts a
solid wall device 850 and a workflow for singulating cells in
microwells in the solid wall device, where in this workflow one or
both of the gRNA and nuclease are under the control of an inducible
promoter. At the top left of the figure (i), there is depicted
solid wall device 850 with microwells 852. A section 854 of
substrate 850 is shown at (ii), also depicting microwells 852. At
(iii), a side cross-section of solid wall device 850 is shown, and
microwells 852 have been loaded, where, in this embodiment, Poisson
loading has taken place; that is, each microwell has one or no
cells, and the likelihood that any one microwell has more than one
cell is low. At (iv), workflow 840 is illustrated where substrate
850 having microwells 852 shows microwells 856 with one cell per
microwell, microwells 857 with no cells in the microwells, and one
microwell 260 with two cells in the microwell. In step 851, and the
cells in the microwells are allowed to double approximately 2-50
times to form clonal colonies (v), then editing is induced 853 by
heating the substrate (e.g., for temperature-induced editing) or
flowing chemicals under or over the substrate (e.g., sugars,
antibiotics for chemical-induced editing) or by moving the solid
wall device to a different medium; particularly facile if the solid
wall device is placed on a fluid permeable membrane which forms the
bottom of microwells 852. After induction of editing 853, many
cells in the colonies of cells that have been edited die as a
result of the double-strand cuts caused by active editing, and
there is possibly a lag in growth for the edited cells that do
survive but must repair and recover following editing (microwells
858), where cells that do not undergo editing thrive (microwells
859) (vi). All cells are allowed to grow to continue to establish
colonies and normalize, where the colonies of edited cells in
microwells 858 catch up in size and/or cell number with the cells
in microwells 859 that do not undergo editing (vii) due to cell
senescence as the unedited cells reach stationary phase. Once the
cell colonies are normalized, either pooling of all cells in the
microwells can take place, in which case the cells are enriched for
edited cells by eliminating the bias from non-editing cells and
fitness effects from editing; alternatively, colony growth in the
microwells is monitored after editing, and slow growing colonies
(e.g., the cells in microwells 858) are identified and selected
(e.g., "cherry picked") resulting in even greater enrichment of
edited cells.
[0204] In growing the cells, the medium used will depend, of
course, on the type of cells being edited--e.g., bacterial, yeast
or mammalian. For example, medium for bacterial growth includes LB,
SOC, M9 Minimal medium, and Magic medium; medium for yeast cell
growth includes TPD, YPG, YPAD, and synthetic minimal medium; and
medium for mammalian cell growth includes MEM, DMEM, IMDM, RPMI,
and Hanks.
[0205] FIG. 8B is a photograph of one embodiment of a solid wall
device comprising microwells for singulating cells. As can be seen
from the photo, the solid wall device is approximately 2 inches
(.about.47 mm) in diameter. The solid device seen in this
photograph is essentially a perforated disk of 816 stainless steel,
where the perforations form the walls of the microwells, and a
filter or membrane is used to form the bottom of the microwells.
Use of a filter or membrane (such as a 0.22.mu. PVDF Duropore.TM.
woven membrane filter) allows for medium and/or nutrients to enter
the microwells but prevents the cells from flowing down and out of
the microwells. Filter or membrane members that may be used in the
solid wall singulation/growth/editing/normalization devices and
modules are those that are solvent resistant, are contamination
free during filtration, and are able to retain the types and sizes
of cells of interest. For example, in order to retain small cell
types such as bacterial cells, pore sizes can be as low as 0.2
.mu.m, however for other cell types, the pore sizes can be as high
as 0.5 .mu.m. Indeed, the pore sizes useful in the cell
concentration device/module include filters with sizes from 0.20
.mu.m, 0.21 .mu.m, 0.22 .mu.m, 0.23 .mu.m, 0.24 .mu.m, 0.25 .mu.m,
0.26 .mu.m, 0.27 .mu.m, 0.28 .mu.m, 0.29 .mu.m, 0.30 .mu.m, 0.31
.mu.m, 0.32 .mu.m, 0.33 .mu.m, 0.34 .mu.m, 0.35 .mu.m, 0.36 .mu.m,
0.37 .mu.m, 0.38 .mu.m, 0.39 .mu.m, 0.40 .mu.m, 0.41 .mu.m, 0.42
.mu.m, 0.43 .mu.m, 0.44 .mu.m, 0.45 .mu.m, 0.46 .mu.m, 0.47 .mu.m,
0.48 .mu.m, 0.49 .mu.m, 0.50 .mu.m and larger. The filters may be
fabricated from any suitable material including cellulose mixed
ester (cellulose nitrate and acetate) (CME), polycarbonate (PC),
polyvinylidene fluoride (PVDF), polyethersulfone (PES),
polytetrafluoroethylene (PTFE), nylon, or glass fiber.
[0206] In the photograph shown in FIG. 8B, the perforations are
approximately 152 nM in diameter, resulting in the microwells
having a volume of approximately 2.5 nL, with a total of
approximately 30,000 wells. The distance between the microwells is
approximately 279 nM center-to-center. Though here the microwells
have a volume of approximately 2.5 nL, the volume of the microwells
may be from 1 to 25 nL, or preferably from 2 to 10 nL, and even
more preferably from 2 to 4 nL. The preferred size/volume of the
microwells will depend of cell type (e.g., bacterial, yeast,
mammalian). The perforated disk shown here is made of 316 stainless
steel; however other bio-compatible metals and materials may be
used. The solid wall device may be disposable or it may be
reusable. The solid wall device shown in FIG. 8B is round, but can
be of any shape, for example, square, rectangular, oval, etc. Round
solid wall devices are useful if petri dishes are used to supply
the solid wall device with nutrients via solid medium. The filters
used to form the bottom of the wells of the solid wall device
include 0.22.mu. PVDF Duropore.TM. woven membrane filters. Further,
though a 2-inch (.about.47 mm) diameter solid wall device is shown,
the solid wall devices may be smaller or larger as desired and the
configuration of the solid wall device will depend on how nutrients
are supplied to the solid wall device, and how media exchange is
performed. Although a round solid wall device is described here,
the solid wall devices can be of any shape and size, including
rectangular solid wall devices with 100K, 200K or more wells, in
addition to configurations of solid wall devices and cassettes that
are multiplexed, e.g., stacked.
[0207] FIGS. 8C-8E are photographs of E. coli cells at low, medium
and high magnification, respectively, singulated via Poisson
distribution in microwells in a solid wall device with a membrane
bottom. FIG. 8C shows digital growth at low magnification where the
darker microwells are microwells where cells are growing. FIG. 8D
is a top view of microwells in a solid wall device where the darker
microwells are microwells where cells are growing. FIG. 8E is a
photograph of microwells where the membrane (e.g., the permeable
membrane that forms the bottom of the microwells) has been removed,
where unpatterned (smooth) microwells are microwells where cells
are not growing, and microwells with irregular pigment/patterned
are microwells where cells are growing, and, in this photograph,
have filled the microwells in which they are growing. In these
photographs, a 0.2 .mu.m filter (membrane) was pressed against the
perforated metal sold wall device such as the round solid wall
device depicted in FIG. 8B. The perforated metal solid wall device
formed the walls of the microwells, and the 0.2 .mu.m filter formed
the bottom of the microwells. To load the solid wall device, the E.
coli cells were pulled into the microwells using a vacuum. The
solid wall device+filter was then placed on an LB agar plate
membrane-side down, and the cells were grown overnight at
30.degree. C., then two days at room temperature. The membrane was
removed and the bottomless microwells were photographed by light
microscopy. Note the ease with which different selective media can
be used to select for certain cell phenotypes; that is, one need
only transfer the solid wall device+filter to a different plate or
petri dish comprising a desired selective medium or flow a desired
selective medium into a substrate onto which the solid wall device
and coupled membrane are positioned.
[0208] In addition to the solid wall cell singulation device
described in relation to FIGS. 8A-8E, other cell singulation
devices may be employed in the multi-module cell processing
instrument, such as those described in U.S. Ser. No. 62/735,365,
entitled "Detection of Nuclease Edited Sequences in Automated
Modules and Systems", filed 24 Sep. 2018, and U.S. Ser. No.
62/781,112, entitled "Improved Detection of Nuclease Edited
Sequences in Automated Modules and Systems", filed 18 Dec. 2018,
including singulation by plating on agar, singulation by isolating
cells on functionalized islands, singulation within aqueous
droplets carried in a hydrophobic carrier fluid, or singulation
within a polymerized alginate scaffold (for this embodiment of
singulation, also see U.S. Ser. No. 62/769,805, entitled "Improved
Detection of Nuclease Edited Sequences in Automated Modules and
Instruments via Bulk Cell Culture", filed 20 Nov. 2018.
[0209] As an alternative to singulation, inducing editing via an
inducible promoter driving one or both of the gRNA and the nuclease
at a specific time in the cell growth cycle may be employed. FIG.
8F shows a simplified flow chart for exemplary methods 8000 for
enriching for edited cells. Looking at FIG. 8F, method 8000 begins
by designing and synthesizing editing cassettes 8002. As described
in relation to nucleic acid-guided editing above, each editing
cassette typically comprises a gRNA, a donor DNA, and a PAM or
spacer mutation. Once the individual editing cassettes have been
synthesized, the individual editing cassettes may be "linked" or
"assembled" together and are amplified and assembled into editing
vector backbones 8004. The editing vectors comprising the editing
cassettes are then used to transform cells 8006 thereby creating a
library of transformed cells. In addition to the vectors comprising
the assembled editing cassettes, the cells may be transformed
simultaneously with a separate engine vector comprising a coding
sequence for a nuclease. Alternatively, the cells may already be
expressing the nuclease (e.g., the cells may have already been
transformed with an engine vector or the coding sequence for the
nuclease may be stably integrated into the cellular genome) such
that only the editing vector needs to be transformed into the
cells; or the cells may be transformed with a single vector
comprising all components required to perform nucleic acid-guided
nuclease genome editing (e.g., all of the nuclease and an editing
cassette), which is advantageous when employing curing and
recursive rounds of editing.
[0210] A variety of delivery systems may be used to introduce
(e.g., transform or transfect) nucleic acid-guided nuclease editing
system components into a host cell 8008. These delivery systems
include the use of yeast systems, lipofection systems,
microinjection systems, biolistic systems, virosomes, liposomes,
immunoliposomes, polycations, lipid:nucleic acid conjugates,
virions, artificial virions, viral vectors, electroporation, cell
permeable peptides, nanoparticles, nanowires, exosomes.
Alternatively, molecular trojan horse liposomes may be used to
deliver nucleic acid-guided nuclease components across the blood
brain barrier. Of particular interest is the use of
electroporation, particularly flow-through electroporation (either
as a stand-alone instrument or as a module in an automated
multi-module system) as described in, e.g., Ser. No. 16/024,831
filed 30 Jun. 2018; Ser. No. 16/024,816 filed 30 Jun. 2018; Ser.
No. 16/147,353 filed 28 Sep. 2018; Ser. No. 16/147,865 filed 30
Sep. 2018; and Ser. No. 16/147,871 filed 30 Jun. 2018. If the
screening/selection module is one module in an automated
multi-module cell editing system, the cells are likely transformed
in an automated cell transformation module.
[0211] Once transformed 8006, the cells can then be subjected to
selection using a selectable marker 8008. Selectable markers are
employed to select for cells that have received both the engine and
editing vectors, or for cells that have been transformed with a
single, combined engine and editing vector. Commonly used
selectable markers include drug selectable markers such as
ampicillin/carbenicillin, kanamycin, chloramphenicol, erythromycin,
tetracycline, gentamicin, bleomycin, streptomycin, rhamnose,
puromycin, hygromycin, blasticidin, and G418.
[0212] Once cells that have been properly transformed are selected
8008, the next step in method 8000 is to grow cells in liquid
medium until the cells enter (or are close to entering) the
stationary phase of growth. Once the cells are in stationary phase
8010 (or nearly so), editing is induced 8012 in the cells by
induction of transcription of one or both of the nuclease and gRNA.
Once editing is induced 8012, the cells can be grown, rendered
electrocompetent, and subjected to another round of editing
8014.
[0213] FIG. 8G depicts a typical growth curve 8020 for cells in
culture (optical density versus time). Initially there is a lag
phase 8022, then the cells enter log phase 8024 where they grow
quickly, and finally the cells reach stationary phase 8028 where
the cells are no longer dividing. The present methods employ
inducing transcription of either or both the nuclease and/or gRNA
at timepoint 8026 or later when the cells are in the stationary
phase of growth or nearly so; that is, the cells are induced at a
timepoint at least 60% into the log phase of growth, or at least
65% into the log phase of growth, or at least 70% into the log
phase of growth, or at least 71, 72, 73, 74, 75, 76, 77, 78, 79,
80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96,
79, 98, or 99% into the log phase of growth, and at any time during
the stationary phase of growth.
[0214] FIG. 8H depicts an exemplary protocol 8050 for performing
nucleic acid-guided nuclease genome editing. FIG. 8H depicts the
protocols shown in FIG. 8F for editing cells. First, a library or
collection of editing vectors 8052 (editing vectors each comprising
an editing cassette) is introduced 8053 (e.g., electroporated) into
cultured cells 8054 that comprise a coding sequence for a nuclease
under the control of a constitutive or inducible promoter
(preferably an inducible promoter), contained 1) on an "engine
plasmid" (most often along with a selectable marker) that has
already been transformed into the cells; 2) integrated into the
genome of the cells being transformed; or 3) the coding sequence
for the nuclease may be located on the editing vector. The editing
vectors 8052 comprise a donor DNA, a PAM or spacer-altering
sequence (most often a sequence that disables the PAM at the target
site in the genome), a coding sequence for a gRNA under the control
of an inducible promoter, and a selectable marker.
[0215] At step 8059, cells are grown until they reach stationary
phase, or nearly so. Once the cells reach the stationary phase,
editing is induced 8067 (e.g., where transcription of the nuclease,
gRNA or both is induced) and the cells in the culture 8082 are
edited and then allowed to recover from editing. Once recovered,
the cells can be plated 8069, grown and pooled 8084. Alternatively,
the cells from culture 8082 can be plated 8081, and slow-growing
colonies are selected 8086 thereby cherry picking edited colonies.
In yet another alternative, the cells can be retained in liquid
culture, grown to an appropriate OD, rendered electrocompetent, and
subjected to another round of editing 8088. This method of
enrichment of edited cells is particularly desirable as may be
performed in a high throughput manner and does not require plating
cells and is automatable. Induction at step 8067 can take place by,
e.g., using a pL promoter system where the pL promoter is induced
by raising the temperature of the cells in the medium to 42.degree.
C. for, e.g., one to many hours to induce expression of the
nuclease and gRNA for cutting and editing. Once editing has been
induced, the temperature of the culture 8082 is returned to
30.degree. C.
[0216] In one method 8081, the cells from the bulk liquid culture
are plated and the slow-growing colonies are selected 8086. In
edited cells, cell viability is compromised in the period after
editing is induced. The selection method shown in FIG. 8H (e.g.,
selecting slow growing colonies 8081) takes advantage of the growth
lag in colonies of edited cells to identify edited cells. In some
embodiments, the colony size of the edited cells is 20% smaller
than colonies of non-edited cells. In some aspects the colony size
of the edited cells is 30%, 40%, 50%, 60%, 70%, 80% or 90% smaller
than the colonies of non-edited cells. In many embodiments, the
colony size of the edited cells is 30-80% smaller than colonies of
non-edited cells, and in some embodiments, the colony size of the
edited cells is 40-70% smaller than colonies of non-edited
cells.
The Reagent Cartridge
[0217] FIG. 9A depicts a reagent cartridge 922 including a set of
eighteen tubes or vials 940. One or more of the tubes or vials 940,
in some embodiments, is sealed with pierceable foil for access by
an automated liquid handling system, such as a sipper or pipettor.
In other embodiments, one or more of the tubes or vials may include
a sealable access gasket. The top of each of the small tubes or
vials, in some embodiments, is marked with machine-readable indicia
(not illustrated) for automated identification of the contents. The
machine-readable indicia may include a bar code, QR code, or other
machine-readable coding. Other automated means for identifying a
particular container can include color coding, symbol recognition
(e.g., text, image, icon, etc.), and/or shape recognition (e.g., a
relative shape of the container). Rather than being marked upon the
vessel itself, in some embodiments, an upper surface of the
cartridge body and/or the cartridge cover may contain
machine-readable indicia for identifying contents. The small tubes
or vials may each be of a same size. Alternatively, multiple
volumes of tubes or vials may be provided in the reagent cartridge
922. In an illustrative example, each tube or vial may be designed
to hold between 2 and 20 mL, between 4 and 10 mL, or about 5 mL. In
some embodiments where only small volumes of some reagents are
required, tube inserts may be used to accommodate small (e.g.,
microfuge) tubes in a larger receptacle (not shown).
[0218] In an illustrative example, the tubes or vials may each hold
one the following materials: a vector backbone, oligonucleotides,
reagents for nucleic acid assembly, a user-supplied cell sample, an
inducer agent, magnetic beads in buffer, ethanol, an antibiotic for
cell selection, reagents for eluting cells and nucleic acids, an
oil overlay, other reagents, and cell growth and/or recovery media.
In addition, the cell transformation module such as the
flow-through electroporation device described above optionally may
be part of the reagent cartridge.
[0219] In some implementations, a cover 924 as seen in FIG. 9B
secures the tubes or vials 940 within the cartridge body 922 of
FIG. 9A. Turning to FIG. 9B, the cover 924 may include apertures
for access to each of the small tubes or vials 940. Three large
apertures 932 are outlined in a bold band to indicate positions to
add user-supplied materials. The user-supplied materials, for
example, may include a vector backbone, oligonucleotides, and a
cell sample. Further, the cover 924 may include machine-readable
indicia 930 for identifying the type of cartridge (e.g., accessing
a map of the cartridge contents). Alternatively, each aperture may
be marked separately with the individual contents. In some
implementations, to ensure positioning of user-supplied materials,
the vials or tubes provided for filling in the lab environment may
have unique shapes or sizes such that the cell sample vial or tube
only fits in the cell sample aperture, the oligonucleotides vial or
tube only fits in the oligonucleotides aperture, and so on.
Use of the Cell Growth Device
[0220] FIG. 10 is a flow chart of an example method 1000 for using
an automated multi-module cell editing instrument such as the
systems illustrated in FIGS. 4A-4D. A processing system, for
example, directs the processing stage of the method 1000. For
example, a software script may identify settings for each
processing stage and instructions for movement of a robotic
handling system to perform the actions of the method 1000. In some
embodiments, a software instruction script may be identified by a
cartridge supplied to the automated multi-module cell editing
instrument. For example, the cartridge may include machine-readable
indicia, such as a bar code or QR code, including identification of
a script stored in a memory of the automated multi-module cell
editing instrument. In another example, the cartridge may contain a
downloadable script embedded in machine-readable indicia such as a
radio frequency (RF) tag. In other embodiments, the user may
identify a script, for example through downloading the script via a
wired or wireless connection to the processing system of the
automated multi-module cell editing instrument or through selecting
a stored script through a user interface of the automated
multi-module cell editing instrument. In a particular example, the
automated multi-module cell editing instrument may include a touch
screen interface for submitting user settings and activating cell
processing.
[0221] In some implementations, the method 1000 begins with
transferring cells to a cell growth module (1002). The growth
module may be any growth module amendable to automation such as,
for example, the cell growth module 550 described in relation to
FIGS. 5B-5D. In a particular example, the processing system may
direct the robotic handling system to transfer cells to the growth
module. In another example, the cells may be transferred from one a
reagent cartridge to the growth module by the robotic handling
system. In some embodiments, the growth vial may contain growth
media and be supplied, e.g., as part of a kit. In other
embodiments, the growth vial may be filled with medium transferred,
e.g., via the liquid handling device, from a reagent container.
[0222] In some embodiments, prior to transferring the cells (e.g.,
from the reagent cartridge or from a vial added to the instrument),
machine-readable indicia may be scanned upon the vial or other
container situated in a position designated for cells to confirm
that the vial or container is marked as containing cells. Further,
the machine-readable indicia may indicate a type of cells provided
to the instrument. The type of cells, in some embodiments, may
cause the instrument to select a particular processing script
(e.g., series of instructions for the robotic handling system and
settings and activation of the various modules).
[0223] In some implementations, the cells are grown in the growth
module to a desired optical density (1004). For example, the
processing system may manage a temperature setting of the growth
module for incubating the cells during the growth cycle. The
processing system may further receive sensor signals from the
growth module indicative of optical density and analyze the sensor
signals to monitor growth of the cells. In some embodiments, a user
may set growth parameters for managing growth of the cells. For
example, temperature, and the degree of agitation of the cells.
Further, in some embodiments, the user may be updated regarding
growth process. The updates, in some examples, may include a
message presented on a user interface of the automated multi-module
cell editing instrument, a text message to a user's cell phone
number, an email message to an email account, or a message
transmitted to an app executing upon a portable electronic device
(e.g., cell phone, tablet, etc.). Responsive to the messages, in
some embodiments, the user may modify parameters, such as
temperature, to adjust cell growth. For example, the user may
submit updated parameters through a user interface of the automated
multi-module cell editing instrument or through a portable
computing device application in communication with the automated
multi-module cell editing instrument, such as a user interface (see
element 450 of FIG. 4B).
[0224] Although described in relation to optical density, in other
implementations cell growth within the growth module may be
monitored using a different measure of cell density and
physiological state such as, in some examples, pH, dissolved
oxygen, released enzymes, acoustic properties, and electrical
properties.
[0225] In some implementations, upon reaching the desired optical
density (1004), the cells are transferred from the growth module to
a filtration module or cell wash and concentration module (1006).
The robotic handling system, for example, may transfer the cells
from the growth module to the cell concentration module. The cell
concentration module, for example, may be (and typically is)
designed to render the cells electrocompetent. See FIG. 6A-6I in
relation to the TFF device, above. The cells are rendered
electrocompetent and eluted in the filtration module or cell wash
and concentration module (1008). The cells may be eluted using a
wash solution. For example, the cells may be eluted using reagents
from a reagent supply.
[0226] Once the cells have been rendered electrocompetent and
suspended in an appropriate volume such as 50 .mu.L to 10 mL, or
100 .mu.L to 80 mL, or 150 .mu.L to 8 mL, or 250 .mu.L to 7 mL, or
500 .mu.L to 6 mL, or 750 .mu.L to 5 mL for transformation (1006),
the cells are transferred to, e.g., an FTEP module (1018). The
robotic handling system, for example, may transfer the cells from
the filtration module to the FTEP. The filtration module may be
physically coupled to the FTEP device, or these modules may be
separate.
[0227] In some implementations, nucleic acids are prepared outside
of the automated multi-module cell editing instrument. For example,
an assembled vector or other nucleic acid assembly may be included
as a reagent by a user prior to running the transformation process
and other processes in the method 1000.
[0228] However, in other implementations, nucleic acids are
prepared by the automated multi-module cell editing instrument. A
portion of the following steps 1010 through 1016, in some
embodiments, are performed in parallel with a portion of steps 1002
through 1008. At least a portion of the following steps, in some
embodiments, are performed before and/or after steps 1002 through
1008.
[0229] In some implementations, nucleic acids such as an editing
oligonucleotide and a vector backbone, as well as in some examples,
enzymes and other reaction components are transferred to a nucleic
acid assembly module (1010). The nucleic acid assembly module may
be configured to perform one or more of a wide variety of different
nucleic acid assembly techniques in an automated fashion. Nucleic
acid assembly techniques that can be performed in the nucleic acid
assembly module may include, but are not limited to, those assembly
methods that use restriction endonucleases, including PCR, BioBrick
assembly, Type IIS cloning, GoldenGate assembly, and Ligase Cycling
Reaction. In other examples, the nucleic acid assembly module may
perform an assembly technique based on overlaps between adjacent
parts of the nucleic acids, such as Gibson Assembly.RTM., CPEC,
SLIC, Ligase Cycling, etc., as described above. Additional example
assembly methods that may be performed by the nucleic acid assembly
module include gap repair in yeast, gateway cloning and
topoisomerase-mediated cloning. In a particular example, the
processing system may direct the robotic handling system to
transfer nucleic acids to the nucleic acid assembly module. In
another example, the nucleic acids may be transferred from a
reagent cartridge to a nucleic acid assembly module by the robotic
handling system.
[0230] In some embodiments--prior to transferring each of the
nucleic acid samples, the enzymes, and other reaction
components--machine-readable indicia may be scanned upon the vials
or other containers situated in positions designated for these
materials to confirm that the vials or containers are marked as
containing the anticipated material. Further, the machine-readable
indicia may indicate a type of one or more of the nucleic acid
samples, the enzymes, and other reaction components provided to the
instrument. The type(s) of materials, in some embodiments, may
cause the instrument to select a particular processing script
(e.g., series of instructions for the robotic handling system to
identify further materials and/or settings and activation of the
nucleic acid assembly module).
[0231] In some embodiments, the nucleic acid assembly module is
temperature controlled depending upon the type of nucleic acid
assembly used. For example, when PCR is utilized in the nucleic
acid assembly module, the module can have a thermocycling
capability allowing the temperatures to cycle between denaturation,
annealing and extension steps. When single temperature assembly
methods are utilized in the nucleic acid assembly module, the
module can have the ability to reach and hold at the temperature
that optimizes the specific assembly process being performed.
[0232] Temperature control, in some embodiments, is managed by a
processing system of the automated multi-module cell editing
instrument, such as the processing system. These temperatures and
the duration of maintaining the temperatures can be determined by a
preprogrammed set of parameters (e.g., identified within the
processing script or in another memory space accessible by the
processing system), or manually controlled by the user through
interfacing with the processing system.
[0233] Once sufficient time has elapsed for the assembly reaction
to take place, in some implementations, the nucleic acid assembly
may be transferred to a purification module (1014). The processing
system, for example, may monitor timing of the assembly reaction
based upon one or more of the type of reaction, the type of
materials, and user settings provided to the automated multi-module
cell editing instrument. The robotic handling system, for example,
may transfer the nucleic acid assembly to the purification module
through a sipper or pipettor interface. In another example, the
robotic handling system may transfer a vial containing the nucleic
acid assembly from a chamber of the nucleic acid assembly module to
a chamber of the de-salt/purification module.
[0234] In some implementations, the nucleic acid assembly is
de-salted and eluted at the purification module (1016). The
purification module, for example, may remove unwanted components of
the nucleic acid assembly mixture (e.g., salts, minerals, etc.). In
some embodiments, the purification module concentrates the
assembled nucleic acids into a smaller volume that the nucleic acid
assembly volume. Examples of methods for exchanging liquid
following nucleic acid assembly include magnetic beads (e.g., SPRI
or Dynal (Dynabeads) by Invitrogen Corp. of Carlsbad, Calif.),
silica beads, silica spin columns, glass beads, precipitation
(e.g., using ethanol or isopropanol), alkaline lysis, osmotic
purification, extraction with butanol, membrane-based separation
techniques, filtration etc. For example, one or more
micro-concentrators fitted with anisotropic, hydrophilic-generated
cellulose membranes of varying porosities may be used. In another
example, the de-salt % purification module may process a liquid
sample including a nucleic acid and an ionic salt by contacting the
mixture with an ion exchanger including an insoluble phosphate
salt, removing the liquid, and eluting nucleic acid from the ion
exchanger.
[0235] In an illustrative embodiment, the nucleic acid assembly may
be combined with magnetic beads, such as SPRI beads, in a chamber
of a purification module. The nucleic acid assembly may be
incubated at a set temperature for sufficient time for the
assembled nucleic acids to bind to the magnetic beads. After
incubation, a magnet may be engaged proximate to the chamber so
that the nucleic acid assembly can be washed and eluted. Once the
nucleic acid assembly has been eluted, the nucleic acid assembly is
transferred to the transformation module (1018). The robotic
handling system, for example, may transfer the assembled nucleic
acids to the transformation module through a sipper or pipettor
interface to the FTEP as described above. For example, the
de-salted assembled nucleic acids, during the transfer, may be
combined with the electrocompetent cells from step 108. In other
embodiments, the transformation module may accept each of the
electrocompetent cells and the nucleic acid assembly separately and
enable the mixing (e.g., open one or more channels to combine the
materials in a shared chamber).
[0236] The cells are transformed in the FTEP module (1020). A
buffer or medium may be transferred to the transformation module
and added to the cells so that the cells may be suspended in a
buffer or medium that is favorable for cell survival during
electroporation. Prior to transferring the buffer or medium,
machine-readable indicia may be scanned upon the vial or other
container or reservoir situated in the position designated for the
buffer or medium to confirm the contents of the vial, container, or
reservoir. Further, the machine-readable indicia may indicate a
type of buffer or medium provided to the instrument. The type of
buffer or medium, in some embodiments, may cause the instrument to
select a particular processing script (e.g., settings and
activation of the transformation module appropriate for the
particular buffer or medium). For bacterial cell electroporation,
low conductance mediums, such as water or glycerol solutions, may
be used to reduce the heat production by transient high current.
For yeast cells a sorbitol solution may be used. For mammalian cell
electroporation, cells may be suspended in a highly conductive
medium or buffer, such as MEM, DMEM, IMDM, RPMI, Hanks', PBS, HBSS,
HeBS and Ringer's solution. In a particular example, the robotic
handling system may transfer a buffer solution to FTEP module from
the reagent cartridge. As described in relation to FIGS. 7A-7E, the
FTEP device may be a disposable FTEP device and/or the FTEP device
may be provided as part of the reagent cartridge. Alternatively, as
shown in FIG. 4A, the FTEP device may a separate module.
[0237] Once transformed, the cells are transferred to a second
growth/recovery/editing module (1022) such as the cell growth
module described in relation to FIGS. 5A-5D. The robotic handling
system, for example, may transfer the transformed cells to the
second growth module through a sipper or pipettor interface. In
another example, the robotic handling system may transfer a vial
containing the transformed cells from a chamber of the
transformation module to a chamber of the second growth module.
[0238] The second growth module, in some embodiments, acts as a
recovery module, allowing the cells to recover from the
transformation process. In other embodiments, the cells may be
provided to a separate recovery module prior to being transported
to the second growth module. During recovery, the second growth
module allows the transformed cells to uptake and, in certain
aspects, integrate the introduced nucleic acids into the genome of
the cell. The second growth module may be configured to incubate
the cells at any user-defined temperature optimal for cell growth,
preferably 25.degree., 30.degree., or 37.degree. C.
[0239] In some embodiments, the second growth module behaves as a
selection module, selecting the transformed cells based on an
antibiotic or other reagent. In one example, the RNA-guided
nuclease (RGN) protein system is used for selection to cleave the
genomes of cells that have not received the desired edit. In the
example of an antibiotic selection agent, the antibiotic may be
added to the second growth module to enact selection. Suitable
antibiotic resistance genes include, but are not limited to, genes
such as ampicillin-resistance gene, tetracycline-resistance gene,
kanamycin-resistance gene, neomycin-resistance gene,
canavanine-resistance gene, blasticidin-resistance gene,
hygromycin-resistance gene, puromycin-resistance gene, or
chloramphenicol-resistance gene. The robotic handling system, for
example, may transfer the antibiotic to the second growth module
through a sipper or pipettor interface. In some embodiments,
removing dead cell background is aided using lytic enhancers such
as detergents, osmotic stress by hyponic wash, temperature,
enzymes, proteases, bacteriophage, reducing agents, or chaotropes.
The processing system, for example, may alter environmental
variables, such as temperature, to induce selection, while the
robotic handling system may deliver additional materials (e.g.,
detergents, enzymes, reducing agents, etc.) to aid in selection. In
other embodiments, cell removal and/or media exchange by filtration
is used to reduce dead cell background.
[0240] In further embodiments, in addition to or as an alternative
to applying selection, the second growth module serves as an
editing module, allowing for genome editing in the transformed
cells. Alternatively, in other embodiments the cells post-recovery
and selection (if performed) are transferred to a separate editing
module. As an editing module, the second growth module induces
editing of the cells' genomes, e.g., through facilitating
expression of the introduced nucleic acids. Expression of the
nuclease and/or editing cassette nucleic acids may involve one or
more of chemical, light, viral, or temperature induction methods.
The second growth module, for example, may be configured to heat or
cool the cells during a temperature induction process. In a
particular illustration, the cells may be induced by heating at
42.degree. C.-50.degree. C. Further to the illustration, the cells
may then be are cooled to 0-10.degree. C. after induction. In the
example of chemical or viral induction, an inducing agent may be
transferred to the second growth module to induce editing. If an
inducible nuclease and/or editing cassette was introduced to the
cells during editing, it can be induced through introduction of an
inducer molecule. The inducing agent or inducer molecule, in some
implementations, is transferred to the second growth module by the
robotic handling system, e.g., through a pipettor or sipper
interface.
[0241] In some implementations, if no additional cell editing is
desired (1024), the cells may be transferred from the cell growth
module to a storage unit for later removal from the automated
multi-module cell editing instrument (1026). The robotic handling
system, for example, may transfer the cells to a storage unit
through a sipper or pipettor interface. In another example, the
robotic handling system may transfer a vial containing the cells
from a chamber of the second growth module to a vial or tube within
the storage unit.
[0242] In some implementations, if additional cell editing is
desired (1024), the cells may be transferred to the same or a
different filtration module and rendered electrocompetent (1008).
Further, in some embodiments, a new assembled nucleic acid sample
may be prepared by the nucleic acid assembly module at this time,
or, alternatively, a second fully assembled nucleic acid may be
directly introduced to the cells. Prior to recursive editing, in
some embodiments, the automated multi-module cell editing
instrument may require additional materials be supplied by the
user, e.g., through the introduction of one or more separate
reagents vails or cartridge.
[0243] The steps may be the same or different during the second
round of editing. For example, in some embodiments, upon a
subsequent execution of step 1004, a selective growth medium is
transferred to the growth module to enable selection of edited
cells from the first round of editing. The robotic handling system
may transfer the selective growth medium from a vial or container
in a reagent cartridge situated in a position designated for
selective growth medium. Prior to transferring the selective growth
medium, machine-readable indicia may be scanned upon the vial or
other container or reservoir situated in the position designated
for the selective growth medium to confirm the contents of the
vial, container, or reservoir. Further, the machine-readable
indicia may indicate a type of selective growth medium provided to
the instrument. The type of selective growth medium, in some
embodiments, may cause the instrument to select a particular
processing script (e.g., settings and activation of the growth
module appropriate for the particular selective growth medium).
Particular examples of recursive editing workflows are described in
relation to FIG. 13.
[0244] In some implementations, the method 1000 can be timed to
introduce materials and/or complete the editing cycle or growth
cycle in coordination with a user's schedule. For example, the
automated multi-module cell editing instrument may provide the user
the ability to schedule completion of one or more cell processing
cycles (e.g., one or more recursive edits) such that the method
1000 is enacted with a goal of completion at the user's preferred
time. The time scheduling, for example, may be set through a user
interface. For illustration only, a user may set completion of a
first cycle to 4:00 PM so that the user can supply additional
cartridges of materials to the automated multi-module cell editing
instrument to enable overnight processing of another round of cell
editing. Thus a user may time the programs so that two or more
cycles may be programmed in a specific time period, e.g., a 24-hour
period.
[0245] In some implementations, throughout the method 1000, the
automated multi-module cell editing instrument may alert the user
to its current status. For example, the user interface may present
a graphical indication of the present stage of processing. In a
particular example, a front face of the automated multi-module call
processing instrument may be overlaid with a user interface (e.g.,
touch screen) that presents an animated graphic depicting present
status of the cell processing. The user interface may further
present any user and/or default settings associated with the
current processing stage (e.g., temperature setting, time setting,
etc.). In certain implementations, the status may be communicated
to a user via wireless communications controller.
[0246] Although illustrated as a particular series of operations,
in other embodiments, more or fewer steps may be included in the
method 1000. For example, in some embodiments, prior to engaging in
each round of editing, the contents of reservoirs, cartridges,
and/or vials may be screened to confirm appropriate materials are
available to proceed with processing. For example, in some
embodiments, one or more imaging sensors (e.g., barcode scanners,
cameras, etc.) may confirm contents at various locations within the
housing of the automated multi-module cell editing instrument. In
one example, multiple imaging sensors may be disposed within the
housing of the automated multi-module cell editing instrument, each
imaging sensor configured to detect one or more materials (e.g.,
machine-readable indicia such as barcodes or QR codes, shapes/sizes
of materials, etc.). In another example, at least one imaging
sensor may be moved by the robotic handling system to multiple
locations to detect one or more materials. In further embodiments,
one or more weight sensors may detect presence or absence of
disposable or replaceable materials. In an illustrative example,
the transfer tip supply holder may include a weight sensor to
detect whether or not tips have been loaded into the region. In
another illustrative example, an optical sensor may detect that a
level of liquid waste has reached a threshold level, requiring
disposal prior to continuation of cell processing or addition of
liquid if the minimum level has not been reached to proceed.
Requests for additional materials, removal of waste supplies, or
other user interventions (e.g., manual cleaning of one or more
elements, etc.), in some implementations, are presented on a
graphical user interface of the automated multi-module cell editing
instrument. The automated multi-module cell editing instrument, in
some implementations, contacts the user with requests for new
materials or other manual interventions, for example through a
software app, email, or text message.
[0247] FIG. 11 shows simplified flow charts for two alternative
exemplary methods 1100a and 1100b for singulating cells for
enrichment (1100a) and for cherry picking (1100b). Looking at FIG.
11, method 1100a begins by transforming cells 1110 with the
components necessary to perform nucleic acid-guided nuclease
editing. For example, the cells may be transformed simultaneously
with separate engine and editing vectors; the cells may already be
expressing the nuclease (e.g., the cells may have already been
transformed with an engine vector or the coding sequence for the
nuclease may be stably integrated into the cellular genome) such
that only the editing vector needs to be transformed into the
cells; or the cells may be transformed with a single vector
comprising all components required to perform nucleic acid-guided
nuclease genome editing.
[0248] As described above, a variety of delivery systems can be
used to introduce (e.g., transform or transfect) nucleic
acid-guided nuclease editing system components into a host cell
1110. These delivery systems include the use of yeast systems,
lipofection systems, microinjection systems, biolistic systems,
virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic
acid conjugates, virions, artificial virions, viral vectors,
electroporation, cell permeable peptides, nanoparticles, nanowires,
exosomes. Alternatively, molecular trojan horse liposomes may be
used to deliver nucleic acid-guided nuclease components across the
blood brain barrier. Of interest, particularly in the context of a
multi-module cell editing instrument is the use of electroporation,
particularly flow-through electroporation (either as a stand-alone
instrument or as a module in an automated multi-module system) as
described in, e.g., Ser. No. 16/147,120, filed 28 Sep. 2018; Ser.
No. 16/147,353, filed 28 Sep. 2018; Ser. No. 16/147,865, filed 30
Sep. 2018; and Ser. No. 16/147,871, filed 30 Sep. 2018. If the
solid wall singulation/growth/editing/normalization module is one
module in an automated multi-module cell editing instrument, the
cells are likely transformed in an automated cell transformation
module.
[0249] After the cells are transformed with the components
necessary to perform nucleic acid-guided nuclease editing, the
cells are singulated in microwells in a, e.g., solid wall device
1120; that is, the cells are diluted (if necessary) in a liquid
culture medium (in some embodiments, including Tween, at a
concentration of 0.1% or less to effect a good distribution) so
that the cells, when delivered to the solid wall device, fill the
microwells of the solid wall device in a Poisson or substantial
Poisson distribution. Singulation is accomplished when an average
of 1/2 cell is delivered to each microwell; that is, where some
microwells contain one cell and other microwells contain no
cells.
[0250] Once the cells in this embodiment have been singulated in
1100a, the cells are actively editing, as the editing "machinery"
is under the control of a constitutive promoter. As the cells are
editing, they are grown into colonies of terminal size 1130; that
is, the colonies arising from the singulated cells are grown into
colonies to a point where cell growth has peaked and is normalized
or saturated for both edited and unedited cells. Normalization
occurs as the nutrients in the medium around a growing cell colony
are depleted and/or cell growth fills the microwells and further
growth is constrained. Again, in the embodiment 1100a shown in FIG.
11, the editing components are under the control of a constitutive
promoter; thus, editing begins immediately (or almost immediately)
upon transformation. However, in other embodiments such as the
embodiment shown in 1100b described below, one or both of the
nuclease and the guide nucleic acid (as well as, e.g., the .lamda.
red recombination system components in bacterial systems) may be
under the control of an inducible promoter, in which case editing
may be induced after, e.g., a desired number of cell doublings.
Turning back to method 1100a, the terminal-size colonies are pooled
1140 by flushing the clonal cell colonies from the microwells to
mix the cells from the normalized cell colonies. Again, because
singulation overcomes growth bias from unedited cells or cells
exhibiting fitness effects as the result of edits made,
singulation/normalization alone enriches the total population of
cells with cells that have been edited; that is, singulation
combined with normalization (e.g., growing colonies to terminal
size) allows for high-throughput enrichment of edited cells.
[0251] The method 1100b shown in FIG. 11 is similar to the method
1100a in that cells of interest are transformed 1110 with the
components necessary to perform nucleic acid-guided nuclease
editing. As described above, the cells may be transformed
simultaneously with both the engine and editing vectors, the cells
may already be expressing the nuclease (e.g., the cells may have
already been transformed with an engine vector or the coding
sequence for the nuclease may be stably integrated into the
cellular genome) such that only the editing vector needs to be
transformed into the cells, or the cells may be transformed with a
single vector comprising all components required to perform nucleic
acid-guided nuclease genome editing. Further, if the
singulation/growth/editing/normalization solid wall module is one
module in an automated multi-module cell editing instrument, cell
transformation may be performed in an automated transformation
module as described above.
[0252] After the cells are transformed with the components
necessary to perform nucleic acid-guided nuclease editing, the
cells are diluted (if necessary) in liquid medium so that the
cells, when delivered to the solid wall device, fill the microwells
of the solid wall device in a Poisson or substantial Poisson
distribution.
[0253] Once the cells have been singulated in the microwells of the
solid wall device 1120, the cells are allowed to grow to, e.g.,
between 2 and 150, or between 5 and 120, or between 10 and 100
doublings, establishing clonal colonies 1150. After colonies are
established, in this embodiment 1100b editing is induced 1160 by,
e.g., activating inducible promoters that control transcription of
one or more of the components needed for nucleic acid-guided
nuclease editing, such as, e.g., transcription of the gRNA,
nuclease, or, in the case of bacteria, a recombineering system.
Once editing is induced 1160, many of the edited cells in the
clonal colonies die due to the double-strand DNA breaks that occur
during the editing process; however, in a percentage of edited
cells, the genome is edited and the double strand break is properly
repaired. These edited cells then start growing and re-establish
colonies; however, the growth of edited colonies tends to lag
behind the growth of clonal colonies where an edit has not taken
place. The small or slow-growing colonies (edited cells) are cherry
picked 1170.
[0254] FIG. 12 is a simplified block diagram of an embodiment of an
exemplary automated multi-module cell processing instrument
comprising a solid wall singulation/growth/editing/normalization
module for enrichment for edited cells. The cell processing
instrument 1200 may include a housing 1244, a reservoir of cells to
be transformed or transfected 1202, and a growth module (a cell
growth device) 1204. The cells to be transformed are transferred
from a reservoir to the growth module to be cultured until the
cells hit a target OD. Once the cells hit the target OD, the growth
module may cool or freeze the cells for later processing, or the
cells may be transferred to a filtration module 1230 where the
cells are rendered electrocompetent and concentrated to a volume
optimal for cell transformation. Once concentrated, the cells are
then transferred to the electroporation device 1208 (e.g.,
transformation/transfection module). Exemplary electroporation
devices of use in the automated multi-module cell processing
instruments for use in the multi-module cell processing instrument
include flow-through electroporation devices such as those
described in Ser. No. 16/147,120, filed 28 Sep. 2018; Ser. No.
16/147,353, filed 28 Sep. 2018; Ser. No. 16/147,865, filed 30 Sep.
2018; and Ser. No. 16/147,871, filed 30 Sep. 2018 all of which are
herein incorporated by reference in their entirety.
[0255] In addition to the reservoir for storing the cells, the
system 1200 may include a reservoir for storing editing
oligonucleotide cassettes 1216 and a reservoir for storing an
expression vector backbone 1218. Both the editing oligonucleotide
cassettes and the expression vector backbone are transferred from
the reagent cartridge to a nucleic acid assembly module 1220, where
the editing oligonucleotide cassettes are inserted into the
expression vector backbone. The assembled nucleic acids may be
transferred into an optional purification module 1222 for desalting
and/or other purification and/or concentration procedures needed to
prepare the assembled nucleic acids for transformation.
Alternatively, pre-assembled nucleic acids, e.g., an editing
vector, may be stored within reservoir 1216 or 1218. Once the
processes carried out by the purification module 1222 are complete,
the assembled nucleic acids are transferred to, e.g., an
electroporation device 1205, which already contains the cell
culture grown to a target OD and rendered electrocompetent via
filtration module 1230. In electroporation device 1208, the
assembled nucleic acids are introduced into the cells. Following
electroporation, the cells are transferred into a combined
recovery/selection module 1210.
[0256] Following recovery, and, optionally, selection, the cells
are transferred to a singulation, editing, and growth module 1240,
where the cells are diluted and compartmentalized such that there
is an average of one cell per compartment. Once singulated, the
cells are allowed to grow for a pre-determined number of doublings.
Once these initial colonies are established, editing is induced and
the edited cells are allowed to establish colonies, which are grown
to terminal size (e.g., the colonies are normalized). In some
embodiments, editing is induced by one or more of the editing
components being under the control of an inducible promoter. In
some embodiments, the inducible promoter is activated by a rise in
temperature and "deactivated" by lowering the temperature.
Alternatively, in embodiments where the singulation device is a
solid wall device comprising a filter forming the bottom of the
microwell, the solid wall device can be transferred to a plate
(e.g., agar plate or even to liquid medium) comprising a medium
with a component that activates or induced editing, then
transferred to a medium that deactivates editing. Once the colonies
are grown to terminal size, the colonies are pooled. Again,
singulation overcomes growth bias from unedited cells and growth
bias resulting from fitness effects of different edits.
[0257] The recovery, selection, singulation, induction, editing and
growth modules may all be separate, may be arranged and combined as
shown in FIG. 12, or may be arranged or combined in other
configurations. In certain embodiments, all of recovery, selection,
singulation, growth, editing, and normalization are performed in a
solid wall device. Alternatively, recovery, selection, and
dilution, if necessary, are performed in liquid medium in a
separate vessel (module), then transferred to the solid wall
singulation/growth/induction/editing/normalization module.
[0258] Once the normalized cell colonies are pooled, the cells may
be stored, e.g., in a storage module 1212, where the cells can be
kept at, e.g., 4.degree. C. until the cells are retrieved for
further study. Alternatively, the cells may be used in another
round of editing. The multi-module cell processing instrument is
controlled by a processor 1242 configured to operate the instrument
based on user input, as directed by one or more scripts, or as a
combination of user input or a script. The processor 1242 may
control the timing, duration, temperature, and operations of the
various modules of the system 500 and the dispensing of reagents.
For example, the processor 1242 may cool the cells
post-transformation until editing is desired, upon which time the
temperature may be raised to a temperature conducive of genome
editing and cell growth. The processor may be programmed with
standard protocol parameters from which a user may select, a user
may specify one or more parameters manually or one or more scripts
associated with the reagent cartridge may specify one or more
operations and/or reaction parameters. In addition, the processor
may notify the user (e.g., via an application to a smart phone or
other device) that the cells have reached the target OD as well as
update the user as to the progress of the cells in the various
modules in the multi-module system.
[0259] The automated multi-module cell processing instrument 1200
is a nuclease-directed genome editing system and can be used in
single editing systems (e.g., introducing one or more edits to a
cellular genome in a single editing process). The system of FIG.
13, described below, is configured to perform sequential editing,
e.g., using different nuclease-directed systems sequentially to
provide two or more genome edits in a cell; and/or recursive
editing, e.g. utilizing a single nuclease-directed system to
introduce sequentially two or more genome edits in a cell.
[0260] FIG. 13 illustrates another embodiment of a multi-module
cell processing instrument. This embodiment depicts an exemplary
system that performs recursive gene editing on a cell population.
As with the embodiment shown in FIG. 12, the cell processing
instrument 1300 may include a housing 1344, a reservoir for storing
cells to be transformed or transfected 1302, and a cell growth
module (comprising, e.g., a rotating growth vial) 1304. The cells
to be transformed are transferred from a reservoir to the cell
growth module to be cultured until the cells hit a target OD. Once
the cells hit the target OD, the growth module may cool or freeze
the cells for later processing or transfer the cells to a
filtration module 1360 where the cells are subjected to buffer
exchange and rendered electrocompetent, and the volume of the cells
may be reduced substantially. Once the cells have been concentrated
to an appropriate volume, the cells are transferred to
electroporation device 1308. In addition to the reservoir for
storing cells, the multi-module cell processing instrument includes
a reservoir for storing the vector pre-assembled with editing
oligonucleotide cassettes 1352. The pre-assembled nucleic acid
vectors are transferred to the electroporation device 1308, which
already contains the cell culture grown to a target OD. In the
electroporation device 1308, the nucleic acids are electroporated
into the cells. Following electroporation, the cells are
transferred into an optional recovery module 1356, where the cells
are allowed to recover briefly post-transformation.
[0261] After recovery, the cells may be transferred to a storage
module 1312, where the cells can be stored at, e.g., 4.degree. C.
for later processing, or the cells may be diluted and transferred
to a selection/singulation/growth/induction/editing/normalization
module 1358. In the singulation/edit/growth module 1358, the cells
are arrayed such that there is an average of one cell per
microwell. The arrayed cells may be in selection medium to select
for cells that have been transformed or transfected with the
editing vector(s). Once singulated, the cells grow through 2-50
doublings and establish colonies. Once colonies are established,
editing is induced by providing conditions (e.g., temperature,
addition of an inducing or repressing chemical) to induce editing.
Once editing is initiated and allowed to proceed, the cells are
allowed to grow to terminal size (e.g., normalization of the
colonies) in the microwells and then can be flushed out of the
microwells and pooled, then transferred to the storage (or
recovery) unit 1314 or can be transferred to a growth module 1304
for another round of editing. In between pooling and transfer to a
growth module, there may be one or more additional steps, such as
cell recovery, medium exchange, cells concentration, etc., by,
e.g., filtration. Note that the
selection/singulation/growth/induction/editing and normalization
modules may be the same module, where all processes are performed
in the solid wall device, or selection and/or dilution may take
place in a separate vessel before the cells are transferred to the
solid wall singulation/growth/induction/editing/normalization
module (solid wall device). As an alternative to singulation in,
e.g., a solid wall device, the transformed cells may be grown
in--and editing can be induced in--bulk liquid as described above
in relation to FIGS. 8F-8H above. Once the putatively-edited cells
are pooled, they may be subjected to another round of editing,
beginning with growth, cell concentration and treatment to render
electrocompetent, and transformation by yet another donor nucleic
acid in another editing cassette via the electroporation module
1308.
[0262] In electroporation device 1308, the cells selected from the
first round of editing are transformed by a second set of editing
oligos (or other type of oligos) and the cycle is repeated until
the cells have been transformed and edited by a desired number of,
e.g., editing cassettes. The multi-module cell processing
instrument exemplified in FIG. 13 is controlled by a processor 1342
configured to operate the instrument based on user input or is
controlled by one or more scripts including at least one script
associated with the reagent cartridge. The processor 1342 may
control the timing, duration, and temperature of various processes,
the dispensing of reagents, and other operations of the various
modules of the instrument 1300. For example, a script or the
processor may control the dispensing of cells, reagents, vectors,
and editing oligonucleotides; which editing oligonucleotides are
used for cell editing and in what order; the time, temperature and
other conditions used in the recovery and expression module, the
wavelength at which OD is read in the cell growth module, the
target OD to which the cells are grown, and the target time at
which the cells will reach the target OD. In addition, the
processor may be programmed to notify a user (e.g., via an
application) as to the progress of the cells in the automated
multi-module cell processing instrument.
[0263] It should be apparent to one of ordinary skill in the art
given the present disclosure that the process described may be
recursive and multiplexed; that is, cells may go through the
workflow described in relation to FIG. 13, then the resulting
edited culture may go through another (or several or many) rounds
of additional editing (e.g., recursive editing) with different
editing vectors. For example, the cells from round 1 of editing may
be diluted and an aliquot of the edited cells edited by editing
vector A may be combined with editing vector B, an aliquot of the
edited cells edited by editing vector A may be combined with
editing vector C, an aliquot of the edited cells edited by editing
vector A may be combined with editing vector D, and so on for a
second round of editing. After round two, an aliquot of each of the
double-edited cells may be subjected to a third round of editing,
where, e.g., aliquots of each of the AB-, AC-, AD-edited cells are
combined with additional editing vectors, such as editing vectors
X, Y, and Z. That is that double-edited cells AB may be combined
with and edited by vectors X, Y, and Z to produce triple-edited
edited cells ABX, ABY, and ABZ; double-edited cells AC may be
combined with and edited by vectors X, Y, and Z to produce
triple-edited cells ACX, ACY, and ACZ; and double-edited cells AD
may be combined with and edited by vectors X, Y, and Z to produce
triple-edited cells ADX, ADY, and ADZ, and so on. In this process,
many permutations and combinations of edits can be executed,
leading to very diverse cell populations and cell libraries. In any
recursive process, it is advantageous to "cure" the previous engine
and editing vectors (or single engine+editing vector in a single
vector system). "Curing" is a process in which one or more vectors
used in the prior round of editing is eliminated from the
transformed cells. Curing can be accomplished by, e.g., cleaving
the vector(s) using a curing plasmid thereby rendering the editing
and/or engine vector (or single, combined vector) nonfunctional;
diluting the vector(s) in the cell population via cell growth (that
is, the more growth cycles the cells go through, the fewer daughter
cells will retain the editing or engine vector(s)), or by, e.g.,
utilizing a heat-sensitive origin of replication on the editing or
engine vector (or combined engine+editing vector). The conditions
for curing will depend on the mechanism used for curing; that is,
in this example, how the curing plasmid cleaves the editing and/or
engine plasmid.
[0264] FIG. 14 is a simplified block diagram of an embodiment of an
exemplary automated multi-module cell processing instrument
comprising a bulk liquid growth module for induced editing and
enrichment for edited cells as described above in relation to FIGS.
8H-8F. The cell processing instrument 1400 may include a housing
1444, a reservoir of cells to be transformed or transfected 1402,
and a growth module (a cell growth device) 1404. The cells to be
transformed are transferred from a reservoir to the growth module
to be cultured until the cells hit a target OD. Once the cells hit
the target OD, the growth module may cool or freeze the cells for
later processing, or the cells may be transferred to a filtration
module 1430 where the cells are rendered electrocompetent and
concentrated to a volume optimal for cell transformation. Once
concentrated, the cells are then transferred to an electroporation
device 1408 (e.g., transformation/transfection module). Exemplary
electroporation devices of use in the automated multi-module cell
processing instruments for use in the multi-module cell processing
instrument include flow-through electroporation devices such as
those described in Ser. No. 16/147,120, filed 28 Sep. 2018; Ser.
No. 16/147,353, filed 28 Sep. 2018; Ser. No. 16/147,865, filed 30
Sep. 2018; and Ser. No. 16/147,871, filed 30 Sep. 2018 all of which
are herein incorporated by reference in their entirety.
[0265] In addition to the reservoir for storing the cells, the
system 1400 may include a reservoir for storing editing cassettes
1416 and a reservoir for storing an expression vector backbone
1418. Both the editing oligonucleotide cassettes and the expression
vector backbone are transferred from the reagent cartridge to a
nucleic acid assembly module 1420, where the editing
oligonucleotide cassettes are inserted into the expression vector
backbone. The assembled nucleic acids may be transferred into an
optional purification module 1422 for desalting and/or other
purification and/or concentration procedures needed to prepare the
assembled nucleic acids for transformation. Alternatively,
pre-assembled nucleic acids, e.g., an editing vector, may be stored
within reservoir 1416 or 1418. Once the processes carried out by
the purification module 1422 are complete, the assembled nucleic
acids are transferred to, e.g., an electroporation device 1408,
which already contains the cell culture grown to a target OD and
rendered electrocompetent via filtration module 1430. In
electroporation device 1408, the assembled nucleic acids are
introduced into the cells. Following electroporation, the cells are
transferred into a combined recovery/selection module 1410. For
examples of multi-module cell editing instruments, see Ser. Nos.
16/024,816 and 16/024,831, filed 30 Jun. 2018, both of which are
herein incorporated by reference in their entirety.
[0266] Following recovery, and, optionally, selection, the cells
are transferred to a growth, induction, and editing module (bulk
liquid culture) 1440. The cells are allowed to grow until the cells
reach the stationary growth phase (or nearly so), then editing is
induced by induction of transcription of one or both of the
nuclease and gRNA. In some embodiments, editing is induced by
transcription of one or both of the nuclease and the gRNA being
under the control of an inducible promoter. In some embodiments,
the inducible promoter is a pL promoter where the promoter is
activated by a rise in temperature and "deactivated" by lowering
the temperature.
[0267] The recovery, selection, growth, induction, editing and
storage modules may all be separate, may be arranged and combined
as shown in FIG. 14, or may be arranged or combined in other
configurations. In certain embodiments, recovery and selection are
performed in one module, and growth, editing, and re-growth are
performed in a separate module. Alternatively, recovery, selection,
growth, editing, and re-growth are performed in a single
module.
[0268] Once the cells are edited and re-grown (e.g., recovered from
editing), the cells may be stored, e.g., in a storage module 1412,
where the cells can be kept at, e.g., 4.degree. C. until the cells
are retrieved for further study. Alternatively, the cells may be
used in another round of editing. The multi-module cell processing
instrument is controlled by a processor 1442 configured to operate
the instrument based on user input, as directed by one or more
scripts, or as a combination of user input or a script. The
processor 1442 may control the timing, duration, temperature, and
operations of the various modules of the system 1400 and the
dispensing of reagents. For example, the processor 1442 may cool
the cells post-transformation until editing is desired, upon which
time the temperature may be raised to a temperature conducive of
genome editing and cell growth. The processor may be programmed
with standard protocol parameters from which a user may select, a
user may specify one or more parameters manually or one or more
scripts associated with the reagent cartridge may specify one or
more operations and/or reaction parameters. In addition, the
processor may notify the user (e.g., via an application to a smart
phone or other device) that the cells have reached the target OD as
well as update the user as to the progress of the cells in the
various modules in the multi-module system.
EXAMPLES
[0269] The following examples are put forth so as to provide those
of ordinary skill in the art with a complete disclosure and
description of how to make and use the present invention, and are
not intended to limit the scope of what the inventors regard as
their invention, nor are they intended to represent or imply that
the experiments below are all of or the only experiments performed.
It will be appreciated by persons skilled in the art that numerous
variations and/or modifications may be made to the invention as
shown in the specific aspects without departing from the spirit or
scope of the invention as broadly described. The present aspects
are, therefore, to be considered in all respects as illustrative
and not restrictive.
Example 1: Growth in the Cell Growth Module
[0270] One embodiment of the cell growth device as described herein
was tested against a conventional cell shaker shaking a 5 ml tube
and an orbital shaker shaking a 125 ml baffled flask to evaluate
cell growth in bacterial and yeast cells. Additionally, growth of a
bacterial cell culture and a yeast cell culture was monitored in
real time using an embodiment of the cell growth device described
herein.
[0271] In a first example, 20 ml EC23 cells (E. coli cells) in LB
were grown in a 35 ml rotating growth vial with a 2-paddle
configuration at 30.degree. C. using the cell growth device as
described herein. The rotating growth vial was spun at 600 rpm and
oscillated (i.e., the rotation direction was changed) every 1
second. In parallel, 5 ml EC23 cells in LB were grown in a 5 ml
tube at 30.degree. C. and were shaken at 750 rpm. OD600 was
measured at intervals using a NanoDrop.TM. spectrophotometer
(Thermo Fisher Scientific). The results are shown in FIG. 15. The
rotating growth vial/cell growth device performed better than the
cell shaker in growing the cells to OD.sub.600 2.6 in slightly over
4 hours. Another experiment was performed with the same conditions
(volumes, cells, oscillation) the only difference being a 3-paddle
rotating growth vial was employed with the cell growth device, and
the results are shown in FIG. 16. Again, the rotating growth
vial/cell growth device performed better than the cell shaker in
growing the cells to OD.sub.600 1.9.
[0272] Two additional experiments were performed, this time
comparing the rotating growth vial/cell growth device to a baffled
flask and an orbital shaker. In one experiment, 20 ml EC138 cells
(E. coli cells) in LB were grown in a 35 ml rotating growth vial
with a 4-paddle configuration at 30.degree. C. The rotating growth
vial was spun at 600 rpm and oscillated (i.e., the rotation
direction was changed) every 1 second. In parallel, 20 ml EC138
cells in LB were grown in a 125 ml baffled flask at 30.degree. C.
using an orbital shaker. OD.sub.600 was measured at intervals using
a NanoDrop.TM. spectrophotometer (Thermo Fisher Scientific). The
results are shown in FIG. 17, demonstrating that the rotating
growth vial/cell growth device performed as well as the orbital
shaker in growing the cells to OD.sub.600 1.0. In a second
experiment 20 ml EC138 cells (E. coli cells) in LB were grown in a
35 ml rotating growth vial with a 2-paddle configuration at
30.degree. C. using the cell growth device as described herein. The
rotating growth vial was spun at 600 rpm and oscillated (i.e., the
rotation direction was changed) every 1 second. In parallel, 20 ml
EC138 cells in LB were grown in a 125 ml baffled flask at
30.degree. C. using an orbital shaker. OD600 was measured at
intervals using a NanoDrop.TM. spectrophotometer (Thermo Fisher
Scientific). The results are shown in FIG. 18, demonstrating that
the rotating growth vial/cell growth device performed as well--or
better--as the orbital shaker in growing the cells to OD.sub.600
1.2.
[0273] In yet another experiment, the rotating growth vial/cell
growth device was used to measure OD.sub.600 in real time. FIG. 19
is a graph showing the results of real time measurement of growth
of an EC138 cell culture at 30.degree. C. using oscillating
rotation and employing a 2-paddle rotating growth vial. Note that
OD.sub.600 2.6 was reached in 4.4 hours.
[0274] In another experiment, the rotating growth vial/cell growth
device was used to measure OD.sub.600 in real time of yeast s288c
cells in YPAD. The cells were grown at 30.degree. C. using
oscillating rotation and employing a 2-paddle rotating growth vial.
FIG. 20 is a graph showing the results. Note that OD.sub.600 6.0
was reached in 14 hours.
Example 2: Cell Concentration
[0275] The TFF module as described above in relation to FIGS. 6A-6I
has been used successfully to process and perform buffer exchange
on both E. coli and yeast cultures. In concentrating an E. coli
culture, the following steps were performed:
[0276] First, a 20 ml culture of E. coli in LB grown to OD 0.5-0.62
was passed through the TFF device in one direction, then passed
through the TFF device in the opposite direction. At this point the
cells were concentrated to a volume of approximately 5 ml. Next, 50
ml of 10% glycerol was added to the concentrated cells, and the
cells were passed through the TFF device in one direction, in the
opposite direction, and back in the first direction for a total of
three passes. Again the cells were concentrated to a volume of
approximately 5 ml. Again, 50 ml of 10% glycerol was added to the 5
ml of cells and the cells were passed through the TFF device for
three passes. This process was repeated; that is, again 50 ml 10%
glycerol was added to cells concentrated to 5 ml, and the cells
were passed three times through the TFF device. At the end of the
third pass of the three 50 ml 10% glycerol washes, the cells were
again concentrated to approximately 5 ml of 10% glycerol. The cells
were then passed in alternating directions through the TFF device
three more times, wherein the cells were concentrated into a volume
of approximately 400 .mu.l.
[0277] Filtrate conductivity and filter processing time was
measured for E. coli with the results shown in FIG. 21A. Filter
performance was quantified by measuring the time and number of
filter passes required to obtain a target solution electrical
conductivity. Cell retention was determined by comparing the
optical density (OD600) of the cell culture both before and after
filtration. Filter health was monitored by measuring the
transmembrane flow rate during each filter pass. Target
conductivity (.about.16 .mu.S/cm) was achieved in approximately 30
minutes utilizing three 50 ml 10% glycerol washes and three passes
of the cells through the device for each wash. The volume of the
cells was reduced from 20 ml to 400 .mu.l, and recovery of
approximately 90% of the cells has been achieved.
[0278] The same process was repeated with yeast cell cultures. A
yeast culture was initially concentrated to approximately 5 ml
using two passes through the TFF device in opposite directions. The
cells were washed with 50 ml of 1M sorbitol three times, with three
passes through the TFF device after each wash. After the third pass
of the cells following the last wash with 1M sorbitol, the cells
were passed through the TFF device two times, wherein the yeast
cell culture was concentrated to approximately 525 .mu.l. FIG. 21B
presents the filter buffer exchange performance for yeast cells
determined by measuring filtrate conductivity and filter processing
time. Target conductivity (.about.10 .mu.S/cm) was achieved in
approximately 23 minutes utilizing three 50 ml 1M sorbitol washes
and three passes through the TFF device for each wash. The volume
of the cells was reduced from 20 ml to 525 .mu.l. Recovery of
approximately 90% of the cells has been achieved.
Example 3: Production and Transformation of Electrocompetent E.
coli and S. Cerevisiae
[0279] For testing transformation of the FTEP device,
electrocompetent E. coli cells were created. To create a starter
culture, 6 ml volumes of LB chlor-25 (LB with 25 .mu.g/ml
chloramphenicol) were transferred to 14 ml culture tubes. A 25
.mu.l aliquot of E. coli was used to inoculate the LB chlor-25
tubes. Following inoculation, the tubes were placed at a 45.degree.
angle in the shaking incubator set to 250 RPM and 30.degree. C. for
overnight growth, between 12-16 hrs. The OD600 value should be
between 2.0 and 4.0. A 1:100 inoculum volume of the 250 ml LB
chlor-25 tubes were transferred to four sterile 500 ml baffled
shake flasks, i.e., 2.5 ml per 250 ml volume shake flask. The
flasks were placed in a shaking incubator set to 250 RPM and
30.degree. C. The growth was monitored by measuring OD600 every 1
to 2 hr. When the OD600 of the culture was between 0.5-0.6 (approx.
3-4 hrs), the flasks were removed from the incubator. The cells
were centrifuged at 4300 RPM, 10 min, 4.degree. C. The supernatant
was removed, and 100 ml of ice-cold 10% glycerol was transferred to
each sample. The cells were gently resuspended, and the wash
procedure performed three times, each time with the cells
resuspended in 10% glycerol. After the fourth centrifugation, the
cell resuspension was transferred to a 50 ml conical Falcon tube
and additional ice-cold 10% glycerol added to bring the volume up
to 30 ml. The cells were again centrifuged at 4300 RPM, 10 min,
4.degree. C., the supernatant removed, and the cell pellet
resuspended in 10 ml ice-cold glycerol. The cells are aliquoted in
1:100 dilutions of cell suspension and ice-cold glycerol.
[0280] The comparative electroporation experiment was performed to
determine the efficiency of transformation of the electrocompetent
E. coli using the FTEP device described. The flow rate was
controlled with a pressure control system. The suspension of cells
with DNA was loaded into the FTEP inlet reservoir. The transformed
cells flowed directly from the inlet and inlet channel, through the
flow channel, through the outlet channel, and into the outlet
containing recovery medium. The cells were transferred into a tube
containing additional recovery medium, placed in an incubator
shaker at 30.degree. C. shaking at 250 rpm for 3 hours. The cells
were plated to determine the colony forming units (CFUs) that
survived electroporation and failed to take up a plasmid and the
CFUs that survived electroporation and took up a plasmid. Plates
were incubated at 30.degree. C.; E. coli colonies were counted
after 24 hrs.
[0281] The flow-through electroporation experiments were
benchmarked against 2 mm electroporation cuvettes (Bull dog Bio)
using an in vitro high voltage electroporator (NEPAGENE.TM.
ELEPO21). Stock tubes of cell suspensions with DNA were prepared
and used for side-to-side experiments with the NEPAGENE.TM. and the
flow-through electroporation. The results are shown in FIG. 22A. In
FIG. 22A, the left-most bars hatched///denote cell input, the bars
to the left bars hatched\\\denote the number of cells that survived
transformation, and the right bars hatched/// denote the number of
cells that were actually transformed. The FTEP device showed
equivalent transformation of electrocompetent E. coli cells at
various voltages as compared to the NEPAGENE.TM. electroporator. As
can be seen, the transformation survival rate is at least 90% and
in some embodiments is at least 95%, 96%, 97%, 98%, or 99%. The
recovery ratio (the fraction of introduced cells which are
successfully transformed and recovered) is in certain embodiments
at least 0.001 and preferably between 0.00001 and 0.01. In FIG. 25A
the recovery ratio is approximately 0.0001.
[0282] Additionally, a comparison of the NEPAGENE.TM. ELEPO21 and
the FTEP device was made for efficiencies of transformation
(uptake), cutting, and editing. In FIG. 22B, triplicate experiments
were performed where the bars hatched///denote the number of cells
input for transformation, and the bars hatched\\\denote the number
of cells that were transformed (uptake), the number of cells where
the genome of the cells was cut by a nuclease transcribed and
translated from a vector transformed into the cells (cutting), and
the number of cells where editing was effected (cutting and repair
using a nuclease transcribed and translated from a vector
transformed into the cells, and using a guide RNA and a donor DNA
sequence both of which were transcribed from a vector transformed
into the cells). Again, it can be seen that the FTEP showed
equivalent transformation, cutting, and editing efficiencies as the
NEPAGENE.TM. electroporator. The recovery rate in FIG. 22B for the
FTEP is treater than 0.001.
[0283] For testing transformation of the FTEP device in yeast, S.
Cerevisiae cells were created using the methods as generally set
forth in Bergkessel and Guthrie, Methods Enzymol., 529:311-20
(2013). Briefly, YFAP media was inoculated for overnight growth,
with 3 ml inoculate to produce 100 ml of cells. Every 100 ml of
culture processed resulted in approximately 1 ml of competent
cells. Cells were incubated at 30.degree. C. in a shaking incubator
until they reached an OD600 of 1.5+/-0.1.
[0284] A conditioning buffer was prepared using 100 mM lithium
acetate, 10 mM dithiothreitol, and 50 mL of buffer for every 100 mL
of cells grown and kept at room temperature. Cells were harvested
in 250 ml bottles at 4300 rpm for 3 minutes, and the supernatant
removed. The cell pellets were suspended in 100 ml of cold 1 M
sorbitol, spun at 4300 rpm for 3 minutes and the supernatant once
again removed. The cells were suspended in conditioning buffer,
then the suspension transferred into an appropriate flask and
shaken at 200 RPM and 30.degree. C. for 30 minutes. The suspensions
were transferred to 50 ml conical vials and spun at 4300 rpm for 3
minutes. The supernatant was removed and the pellet resuspended in
cold 1 M sorbitol. These steps were repeated three times for a
total of three wash-spin-decant steps. The pellet was suspended in
sorbitol to a final OD of 150+/-20.
[0285] A comparative electroporation experiment was performed to
determine the efficiency of transformation of the electrocompetent
S. Cerevisiae using the FTEP device. The flow rate was controlled
with a syringe pump (Harvard apparatus PHD ULTRA.TM. 4400). The
suspension of cells with DNA was loaded into a 1 mL glass syringe
(Hamilton 81320 Syringe, PTFE Luer Lock) before mounting on the
pump. The output from the function generator was turned on
immediately after starting the flow. The processed cells flowed
directly into a tube with 1M sorbitol with carbenicillin. Cells
were collected until the same volume electroporated in the
NEPAGENE.TM. had been processed, at which point the flow and the
output from the function generator were stopped. After a 3-hour
recovery in an incubator shaker at 30.degree. C. and 250 rpm, cells
were plated to determine the colony forming units (CFUs) that
survived electroporation and failed to take up a plasmid and the
CFUs that survived electroporation and took up a plasmid. Plates
were incubated at 30.degree. C. Yeast colonies are counted after
48-76 hrs.
[0286] The flow-through electroporation experiments were
benchmarked against 2 mm electroporation cuvettes (Bull dog Bio)
using an in vitro high voltage electroporator (NEPAGENE.TM.
ELEPO21). Stock tubes of cell suspensions with DNA were prepared
and used for side-to-side experiments with the NEPAGENE.TM. and the
flow-through electroporation. The results are shown in FIG. 23. The
device showed better transformation and survival of
electrocompetent S. Cerevisiae at 2.5 kV voltages as compared to
the NEPAGENE.TM. method. Input is total number of cells that were
processed.
Example 4: Fully-Automated Singleplex RGN-Directed Editing Run
[0287] Singleplex automated genomic editing using MAD7 nuclease was
successfully performed with an automated multi-module instrument of
the disclosure. See U.S. Pat. No. 9,982,279; and Ser. No.
16/024,831 filed 30 Jun. 2018; Ser. No. 16/024,816 filed 30 Jun.
2018; Ser. No. 16/147,353 filed 28 Sep. 2018; Ser. No. 16/147,865
filed 30 Sep. 2018; and Ser. No. 16/147,871 filed 30 Jun. 2018.
[0288] An ampR plasmid backbone and a lacZ_F172* editing cassette
were assembled via Gibson Assembly.RTM. into an "editing vector" in
an isothermal nucleic acid assembly module included in the
automated instrument. lacZ_F172 functionally knocks out the lacZ
gene. "lacZ_F172*" indicates that the edit happens at the 172nd
residue in the lacZ amino acid sequence. Following assembly, the
product was de-salted in the isothermal nucleic acid assembly
module using AMPure beads, washed with 80% ethanol, and eluted in
buffer. The assembled editing vector and recombineering-ready,
electrocompetent E. Coli cells were transferred into a
transformation module for electroporation. The cells and nucleic
acids were combined and allowed to mix for 1 minute, and
electroporation was performed for 30 seconds. The parameters for
the poring pulse were: voltage, 2400 V; length, 5 ms; interval, 50
ms; number of pulses, 1; polarity, +. The parameters for the
transfer pulses were: Voltage, 150 V; length, 50 ms; interval, 50
ms; number of pulses, 20; polarity, +/-. Following electroporation,
the cells were transferred to a recovery module (another growth
module), and allowed to recover in SOC medium containing
chloramphenicol. Carbenicillin was added to the medium after 1
hour, and the cells were allowed to recover for another 2 hours.
After recovery, the cells were held at 4.degree. C. until recovered
by the user.
[0289] After the automated process and recovery, an aliquot of
cells was plated on MacConkey agar base supplemented with lactose
(as the sugar substrate), chloramphenicol and carbenicillin and
grown until colonies appeared. White colonies represented
functionally edited cells, purple colonies represented un-edited
cells. All liquid transfers were performed by the automated liquid
handling device of the automated multi-module cell processing
instrument.
[0290] The result of the automated processing was that
approximately 1.0E.sup.-03 total cells were transformed (comparable
to conventional benchtop results), and the editing efficiency was
83.5%. The lacZ_172 edit in the white colonies was confirmed by
sequencing of the edited region of the genome of the cells.
Further, steps of the automated cell processing were observed
remotely by webcam and text messages were sent to update the status
of the automated processing procedure.
Example 5: Fully-Automated Recursive Editing Run
[0291] Recursive editing was successfully achieved using the
automated multi-module cell processing system. An ampR plasmid
backbone and a lacZ_V10* editing cassette were assembled via Gibson
Assembly.RTM. into an "editing vector" in an isothermal nucleic
acid assembly module included in the automated system. Similar to
the lacZ_F172 edit, the lacZ_V10 edit functionally knocks out the
lacZ gene. "lacZ_V10" indicates that the edit happens at amino acid
position 10 in the lacZ amino acid sequence. Following assembly,
the product was de-salted in the isothermal nucleic acid assembly
module using AMPure beads, washed with 80% ethanol, and eluted in
buffer. The first assembled editing vector and the
recombineering-ready electrocompetent E. Coli cells were
transferred into a transformation module for electroporation. The
cells and nucleic acids were combined and allowed to mix for 1
minute, and electroporation was performed for 30 seconds. The
parameters for the poring pulse were: voltage, 2400 V; length, 5
ms; interval, 50 ms; number of pulses, 1; polarity, +. The
parameters for the transfer pulses were: Voltage, 150 V; length, 50
ms; interval, 50 ms; number of pulses, 20; polarity, +/-. Following
electroporation, the cells were transferred to a recovery module
(another growth module) allowed to recover in SOC medium containing
chloramphenicol. Carbenicillin was added to the medium after 1
hour, and the cells were grown for another 2 hours. The cells were
then transferred to a centrifuge module and a media exchange was
then performed. Cells were resuspended in TB containing
chloramphenicol and carbenicillin where the cells were grown to
OD600 of 2.7, then concentrated and rendered electrocompetent.
[0292] During cell growth, a second editing vector was prepared in
the isothermal nucleic acid assembly module. The second editing
vector comprised a kanamycin resistance gene, and the editing
cassette comprised a galK Y145* edit. If successful, the galK Y145*
edit confers on the cells the ability to uptake and metabolize
galactose. The edit generated by the galK Y154* cassette introduces
a stop codon at the 154th amino acid reside, changing the tyrosine
amino acid to a stop codon. This edit makes the galK gene product
non-functional and inhibits the cells from being able to metabolize
galactose. Following assembly, the second editing vector product
was de-salted in the isothermal nucleic acid assembly module using
AMPure beads, washed with 80% ethanol, and eluted in buffer. The
assembled second editing vector and the electrocompetent E. Coli
cells (that were transformed with and selected for the first
editing vector) were transferred into a transformation module for
electroporation, using the same parameters as detailed above.
Following electroporation, the cells were transferred to a recovery
module (another growth module), allowed to recover in SOC medium
containing carbenicillin. After recovery, the cells were held at
4.degree. C. until retrieved, after which an aliquot of cells were
plated on LB agar supplemented with chloramphenicol, and kanamycin.
To quantify both lacZ and galK edits, replica patch plates were
generated on two media types: 1) MacConkey agar base supplemented
with lactose (as the sugar substrate), chloramphenicol, and
kanamycin, and 2) MacConkey agar base supplemented with galactose
(as the sugar substrate), chloramphenicol, and kanamycin. All
liquid transfers were performed by the automated liquid handling
device of the automated multi-module cell processing system.
[0293] In this recursive editing experiment, 41% of the colonies
screened had both the lacZ and galK edits, the results of which
were comparable to the double editing efficiencies obtained using a
"benchtop" or manual approach.
Example 6: Design and Creation of a Yeast Display Library of
Putative TCR Antigens
[0294] The binding motifs for peptides presented by human MHC
allele HLA-A*02 have been well characterized (Falk, K., et al.,
Nature, 1991. 351(6324): p. 290-296; Glanville, J., et al., Nature,
2017. 547(7661): p. 94-98) and a number of restricted clinically
relevant TCRs identified (Johnson, L. A., et al., Blood, 2009.
114(3): p. 535-546). A yeast-display library for screening
potential HLA-A*02:01 restricted TCRs is created as follows. A
library of approximately 10,000 oligonucleotide editing cassettes
for introduction of synthetic pMHC (Glanville, J, supra) peptides
of different sequence into the genome of S. Cerevisiae are designed
and ordered from Agilent (Santa Clara, Calif.).
[0295] Briefly, the structural elements of each of the oligo
cassettes is as follows: a promoter region, a CRISPR guide RNA
region, an optional spacer region, a homology arm and optionally
other sequences (e.g., barcodes) helpful for further analysis based
on the functional assay to be used in the selection and/or
confirmation of the specific edits. The cassettes range in length
from 180 nt to 230 nt, depending on the edit to be introduced and
the overall design of the oligos. The design of the homology arm
includes a synonymous codon change (if necessary) to generate a
restriction site which is used to insert constant regions of the
cassette. These constant regions include the HLA-A*02:01 heavy
chain and the AGA2P cell surface display conferring protein. The
constant region may also contain an epitope tag for ease of
downstream use in selections.
[0296] Briefly, the structural elements of each of the oligo
cassettes is as follows: a promoter region, a CRISPR guide RNA
region, an optional spacer region, a homology arm and optionally
other sequences (e.g., barcodes) helpful for further analysis based
on the functional assay to be used in the selection and/or
confirmation of the specific edits. The cassettes range in length
from 180 nt to 230 nt, depending on the edit to be introduced and
the overall design of the oligos. The design of the homology arm
includes a synonymous codon change (if necessary) to generate a
restriction site which is used to insert constant regions of the
cassette. These constant regions include the HLA-A*02:01 heavy
chain and the AGA2P cell surface display conferring protein. The
constant region may also contain an epitope tag or barcode "handle"
for ease of downstream use in selections and further analysis. In
addition or alternatively, the cassette design may include the
addition of a "landing. pad" for the future addition of sequences.
The CRISPR guide RNA region may also be targeted to a high
efficiency cut and integration site.
[0297] Optionally, the oligonucleotide editing cassettes can be
further processed with degenerate PCR reactions to generate
10.sup.7-10.sup.8 permutations of the original TCR antigen
sequence. Such degenerate PCR can be performed either before or
after introduction into the genome of the cells. Degenerate PCR
reactions are performed with primers positioned over the portions
of the intended edit representing the peptide displayed on the pMHC
construct (See, e.g., Boder, E. T. and K. D. Wittrup, Nature
Biotechnology, 1997. 15(6): p. 553-557; McMahon, C., et al., Nature
Structual & Molecular Biology, 2018. 25(3): p. 289-296).
[0298] Importantly, combinatorial sequence diversity could be
created anywhere along the heavy chain construct representing the
HLA-A allele as well as in the peptide region. Individual yeast
then express a random peptide tethered to the constant HLA
molecule. HLA-A*02:01 typically presents peptides 8 to 11 amino
acids in length (Hassan, C., et al., The Journal of Biological
Chemistry, 2015. 290(5): p. 2593-2603 and peptide length libraries
are generated using peptides of lengths within these ranges. The
library has a theoretical nucleotide diversity dictated by the
library composition and length but is designed to result in one or
more libraries representing millions of unique peptides ranging
from 8 to 11 amino acids. After incubating the cells and going
through the editing process, a pool of edited cells exists with the
pMHC complex displayed on the surface of the cell attached to the
AGA2P protein. An optional initial selection for edited cells
displaying the pMHC complex can be performed via the displayed
epitope tag.
Example 7: Validation of the Proper Identification of TCR Antigens
Using a Yeast Display Library
[0299] A validation study is performed to determine whether the
HLA-A*02:01 complex on the surface of the cells in the library of
Example 1 is properly folded to present peptides. The validation
uses the identification of cells displaying target antigens of TCRs
with known specificities. Briefly, a system is designed using the
libraries generated as in Example 1 to validate the libraries for
proper expression of the antigens. In this system, yeast cells
displaying the pMHC conjugates are exposed to a population of
expanded T-cells from a single T-cell with known TCR. Using this
system, a user can correctly match TCRs to a known predicted
antigen target. Selections are performed using TCRs with known
antigen sequences. Following selection, the selected samples are
determined, e.g. using sequencing of barcodes associated with the
selected antigens in the cells of the library. The top peptide
antigens identified using the system of the disclosure are able to
stimulate TCR-transduced T cells, despite sequence differences from
the actual epitope.
Example 8: Identification of New TCR Antigens Using a Yeast Display
Library
[0300] To test whether the automated system would work to identify
novel antigen targets, known TCRs and/or orphan TCRs are used to
identify antigens using the methods of the disclosure. These
identified antigens can then be used by bioinformatic methods to
query the universe of expected or potential peptide antigens. These
bioinformatics methods will attempt to determine common peptides
derived from known protein sequences that will also bind the
representative TCRs. These predicted peptide sequences can then be
designed into one of the libraries of Example 1 or directly tested
with other assays. These libraries which are then displaying the
predicted peptide pMHC molecules can then be exposed to one or more
orphan TCRs to find antigens that specifically bind to the orphan
TCRs. These peptides are then identified as probable antigen
targets for the TCRs.
Example 9: Identification of Genome-Wide Protein-Protein
Interactions
[0301] Protein-protein interactions have been traditionally studied
in high-throughput using yeast two hybrid (Y2H) based approaches
(Rolland, T., et al, Cell, 2014. 159(5): p. 1212-1226; Huttlin, E.
L., et al., 2017. 545(7655): p. 505-509) or mass-spectrometry based
assays (Hein, Marco Y., et al., Cell, 2015. 163(3): p. 712-723.
Flow cytometry has also been used heavily to enable yeast surface
display applications and has been extended to facilitate studies of
protein-protein interactions and enzymatic properties (Lim, S., et
al., Biotechnology Journal, 2017. 12(5): p. 10; Cherf, G. M. and J.
R. Cochran, Methods in molecular biology (Clifton, N.J.), 2015.
1319: p. 155-175.
[0302] CREATE display can be used to facilitate rapid screening of
one vs. all or all vs. all protein-protein interactions. First, a
genome-wide CREATE display library is generated by ordering a set
of approximately 6,000 oligonucleotide editing cassettes from
Agilent (Santa Clara, Calif.). These oligonucleotide editing
cassettes are configured as described in previous examples with a
crRNA, spacer region, and homology arm. These particular
oligonucleotide cassettes can also optionally contain optimally
positioned restriction enzyme sites if they contain repetitive
sequence to aid in the addition of a surface display conferring tag
via standard cloning methods. Many surface display conferring tags
exist. McMahon, C., et al., supra; Cherf, G. M. and J. R. Cochran,
Methods in molecular biology (Clifton, N.J.), 2015. 1319: p.
155-175; Ucha ski, T., et al., Scientific Reports, 2019. 9(1): p.
382. These may include and extend upon the original method of using
the yeast AGA2P mating protein that is typically fused to the
N-terminus of the displayed protein or peptide of interest (Boder,
E. T. and K. D. Wittrup, supra). To facilitate display of essential
proteins critical to cellular function a non-optimal cleavage site
could optionally be designed in-between the surface display
conferring tag and the protein of interest. Many cleavage
conferring sequences exist but one exemplar is tobacco etch virus
(TEV) cleavage site which could be modified to result in
sub-optimal cleavage (Ioannou, M., et al., Mammalian expression
vectors for metabolic biotinylation tandem affinity tagging by
co-expression in cis of a mammalian codon-optimized BirA biotin
ligase. BMC research notes, 2018. 11(1): p. 390-390) and hence
simultaneous surface display of the desired protein while
maintaining a viable intracellular concentration of the native
protein. Once oligonucleotide cassettes have been designed,
ordered, and subsequently modified to include the standard parts
conferring display to the surface of the cell, the CREATE process
can proceed. Briefly, as described previously, a population of
cells is transformed with the oligonucleotide cassettes and
incubated using an automated machine that results in a population
of edited cells. This population of cells is such that each
individual cell contains one or more edits that have resulted in
insertion of the cell surface display conferring tag at a designed
location of interest around the genome. To create a genome-wide
library displaying all proteins in the yeast genome approximately
6,572 edits would be made to insert surface display conferring tags
at the N-terminus of all 6,572 annotated proteins in the yeast
genome (https://www.yeastgenome.org/genomesnapshot). This would
result in a library of 6,572 distinct cells each displaying one of
the 6,572 proteins on its surface. This library of cells could then
be split into two populations and one of the populations
transformed with a construct expressing green-fluorescent-protein
(GFP). The two populations could then be incubated together and run
through a flow-cytometer to detect doublet formation (Wersto, R.
P., et al., Cytometry, 2001. 46(5): p. 296-306) indicative of a
positive protein-protein interaction. Doublets can then be placed
into individual partitions of a standard 96 or 384 well plate and
the DNA sequence barcodes read off of the cassettes present in each
cell of the doublet to determine a protein-protein interaction.
Notably, this technique can be performed in an all-by-all manner in
which all 6,572 GFP expressing surface displayed proteins are
incubated with all 6,572 non-GFP expressing surface displayed
proteins. However, it can also be performed in a one-vs-all or
many-vs-all configuration in which isolates of a protein of
specific interest are incubated and sorted using flow cytometry as
described above. This one-vs-all or many-vs-all could offer
additional specificity or clarity to determination of an individual
proteins binary interaction partners. It should also be noted that
this same procedure can be done throughout multiple rounds of
screening as is traditionally done in phage or yeast display
(Bradbury, A. R. M., et al., Nature Biotechnology, 2011. 29: p.
245) to selectively enrich for the highest affinity binding
partners and to lower false positive rates. It can also be used on
a previously edited genome containing pathogenic or other variants
of interest edited into the cell population before introduction of
the cell surface display conferring tags. The previously edited
genomes could also contain sets of variants specifically designed
to disrupt protein-protein interactions. Notably, CREATE display
can also be used to display more than one protein on the surface of
a single cell via introduction of multiple tags at multiple loci
throughout a cell.
Example 10: Identifying Druggable Targets
[0303] Identifying targets of drugs and subsequent mechanism of
action remains a challenging endeavor. Schenone, M., et al., Nature
Chemical Biology, 2013. 9: p. 232; Stockwell, B. R., Exploring
biology with small organic molecules. Nature, 2004. 432(7019): p.
846-854; Xie, L., L. Xie, and P. E. Bourne, Structure-based systems
biology for analyzing off-target binding. Current opinion in
structural biology, 2011. 21(2): p. 189-199.
[0304] Reverse genetic screens tend to use computational or other
rational methods to pre-select a list of likely disease related
targets. Biochemical screens are then performed using a library of
chemical compounds against one or more of these disease related
targets. However, biochemical assays are often costly or time
consuming and subsequently are generally limited to a small number
of potential targets. 17. Wyatt, P. G., et al., Target validation:
linking target and chemical properties to desired product profile.
Current topics in medicinal chemistry, 2011. 11(10): p.
1275-1283.
[0305] The small number of feasible targets in biochemical screens
often translates into an inability to identify potential
off-targets which can then result in difficult to understand side
effects and a necessary "deconvolution" step whilst determining
mechanism of action. Knight, Z. A., H. Lin, and K. M. Shokat,
Nature reviews. Cancer, 2010. 10(2): p. 130-137.
[0306] In contrast, forward genetic screens generally start with a
phenotype of interest and then screen a large number of molecules
against the model system to see if the phenotype can be disrupted.
Stockwell, B. R., Exploring biology with small organic molecules.
Nature, 2004. 432(7019): p. 846-854.
[0307] This however can often result in not knowing what protein or
pathway the molecule is targeting and can also lead to unintended
side-effects when administered in further studies or in patients.
Xie, L., et. Al., supra.
[0308] Both forward and reverse genetic screens could greatly
benefit from the ability to uniformly assess the binding of a drug
to all intracellular proteins in a simple cost-effective assay. For
forward screens it can identify the actual targets and for reverse
screens it can identify off-targets. Using the CREATE display
methods described here, one can efficiently generate a library
displaying all possible cellular proteins on the surface of a
population of cells and then expose that population to a small
molecule with an attached fluorophore or other detection handle to
determine all protein-drug binding events. First a CREATE display
library is generated as described in Example 5. This display
library can optionally display all proteins in a genome or a subset
of proteins particular to a pathway or computationally determined
set of interest. This display library can also be created in a
population of cells that already harbors one or more pathogenic
variants identified a priori and programmed into the cell
population via previous rounds of CREATE. This library can then be
exposed to a single molecule of interest with an attached organic
fluorophore. Incubation of the CREATE display library with the
small molecule of interest results in complexes of small molecule
bound to the cells displaying a protein in the case in which the
small molecule can bind that protein. Using flow cytometry, the
cells displaying protein with bound ligand can be sorted and
barcodes on the CREATE cassettes used to determine which proteins
are bound by a given small molecule. This results in a binary
mapping of small molecule to protein and can uniquely identify all
possible binding partners of a given small molecule. Optionally,
using a DNA encoded chemical library or other combinatorial
screening approaches (Zimmermann, G. and D. Neri, Drug discovery
today, 2016. 21(11): p. 1828-1834; Szymanski, P., M. Markowicz, and
E. Mikiciuk-Olasik, International journal of molecular sciences,
2011. 13(1): p. 427-452) one could perform an all-by-all screen of
a library of chemical compounds against a CREATE display library of
surface displayed proteins.
Example 11: Affinity Maturation of Biological Binders to a Pathway
of Interest
[0309] Traditional antibody drug development has focused on cell
surface or other extracellular targets that can be readily accessed
by an antibody. However, of the approximately 700 protein molecular
targets approved for drugs, more than half are intracellular
proteins. See, e.g., Carter, P. J. and G. A. Lazar, Nature Reviews
Drug Discovery, 2017. 17: p. 197; Santos, R., et al., Drug
discovery, 2017. 16(1): p. 19-34; Wang, X., et al., Genome biology
and evolution, 2013. 5(7): p. 1291-1297.
[0310] Significant efforts are underway to develop delivery systems
for antibodies or small peptide therapeutic molecules. Stewart, M.
P., et al., Nature, 2016. 538: p. 183. If the promise of
intracellular antibody or peptide delivery comes to fruition, then
a method to systematically affinity mature antibodies that bind to
one or more intracellular proteins would be of tremendous value.
Using CREATE display, a large library of intracellular proteins can
be displayed on the surface of a population of cells and
systematically exposed to yeast or phage display libraries to
select for mono, bi, or poly-specific binders to a set of targets.
First, a yeast display library would be created via the methods
described here or as described elsewhere (McMahon, C., et al.,
supra; Lim S. et al., supra; Cherf, G. M. and J. R. Cochran, supra)
in which many combinatorically encoded proteins are encoded into a
population of yeast cells for display on the surface. At this
point, the workflow would proceed in the same fashion as laid out
in Example 5. In particular, the library of cells with
combinatorically encoded peptides displayed on the surface would
also be transformed with DNA sequences conferring expression of
green-fluorescent-protein. This library of cells with up
10{circumflex over ( )}10 distinct displayed antibodies,
nanobodies, or peptide fragments would then be incubated with the
CREATE displayed library of all intracellular proteins. Using flow
cytometry and selecting for doublets would then enable
determination of any pairwise binding interaction between the
engineered peptide(s) and one or more surface displayed cellular
proteins. This procedure could also be carried out iteratively in
the same manner that traditional affinity maturation of antibodies
is done via yeast display (Cherf, G. M. and J. R. Cochran, supra).
Carrying it out iteratively on a library of surface displayed
cellular proteins that represented a given pathway or subset of
genomic proteins would result in identification of high-affinity
binders to an entire pathway of proteins. In this manner
poly-specific binders could be determined to inhibit or identify
the mechanism of action for entire pathways. Importantly, in a
genome-wide CREATE display library there is a built-in negative
control for off-target affects via the presence in solution of all
other intracellular proteins. Thus while selecting for binders to
only a subset of proteins in a pathway, one can find the pareto
optimum between strong binding to one or more desired intracellular
proteins while simultaneously minimizing binding to non-desired
intracellular proteins. Thus, throughout successive rounds of
CREATE display one can affinity mature antibodies for binding to
specific targets while also selecting for minimization of
off-target binding to other intracellular proteins.
[0311] While this invention is satisfied by embodiments in many
different forms, as described in detail in connection with
embodiments of the invention, it is understood that the present
disclosure is to be considered as exemplary of the principles of
the invention and is not intended to limit the invention to the
specific embodiments illustrated and described herein. Numerous
variations may be made by persons skilled in the art without
departure from the spirit of the invention. The scope of the
invention will be measured by the appended claims and their
equivalents. The abstract and the title are not to be construed as
limiting the scope of the present invention, as their purpose is to
enable the appropriate authorities, as well as the general public,
to quickly determine the general nature of the invention. In the
claims that follow, unless the term "means" is used, none of the
features or elements recited therein should be construed as
means-plus-function limitations pursuant to 35 U.S.C. .sctn. 112,
916.
* * * * *
References