U.S. patent application number 16/892679 was filed with the patent office on 2020-12-10 for curing for recursive nucleic acid-guided cell editing.
The applicant listed for this patent is Inscripta, Inc.. Invention is credited to Charles Johnson, Eileen Spindler, Tian Tian.
Application Number | 20200385744 16/892679 |
Document ID | / |
Family ID | 1000005235032 |
Filed Date | 2020-12-10 |
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United States Patent
Application |
20200385744 |
Kind Code |
A1 |
Tian; Tian ; et al. |
December 10, 2020 |
CURING FOR RECURSIVE NUCLEIC ACID-GUIDED CELL EDITING
Abstract
The present disclosure provides automated multi-module
instrumentation and automated methods for performing recursive
editing of live cells with curing of editing vectors from prior
rounds of editing.
Inventors: |
Tian; Tian; (Boulder,
CO) ; Johnson; Charles; (Boulder, CO) ;
Spindler; Eileen; (Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Inscripta, Inc. |
Boulder |
CO |
US |
|
|
Family ID: |
1000005235032 |
Appl. No.: |
16/892679 |
Filed: |
June 4, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
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62857967 |
Jun 6, 2019 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C12N 9/22 20130101; C12N
15/113 20130101; C12N 15/70 20130101; C12N 2310/20 20170501; C12N
2800/80 20130101 |
International
Class: |
C12N 15/70 20060101
C12N015/70; C12N 15/113 20060101 C12N015/113; C12N 9/22 20060101
C12N009/22 |
Claims
1. A method for curing cells of choice during recursive nucleic
acid-directed nuclease editing comprising: designing and
synthesizing a first set of editing cassettes, wherein the first
set of editing cassettes comprises one or more editing gRNA and
donor DNA pairs wherein each editing gRNA and donor DNA pair is
under the control of a first inducible promoter; assembling the
first set of editing cassettes into a vector backbone thereby
forming a first set of editing vectors, wherein the vector backbone
comprises a first selectable marker, and a curing target sequence;
making cells of choice electrocompetent, wherein the cells of
choice comprise an engine vector and the engine vector comprises a
curing gRNA under the control of a second inducible promoter, a
nuclease under the control of a third inducible promoter; and a
second selectable marker; transforming the cells of choice with the
first set of editing vectors to produce first transformed cells;
selecting for the first transformed cells via the first and second
selectable markers thereby selecting for first selected cells;
inducing editing in the first selected cells by inducing the first
and third inducible promoters thereby inducing transcription of the
one or more editing gRNA and donor DNA pairs and the nuclease
producing first edited cells; growing the first edited cells until
the first edited cells reach a stationary phase of growth; curing
the first set of editing vectors in the first edited cells by
inducing the third and second inducible promoters thereby inducing
transcription of the nuclease and curing gRNA which cuts the curing
target sequence producing first cured cells; growing the first
cured cells; rendering the first cured cells electrocompetent; and
transforming the first cured cells with a second set of editing
vectors to produce second transformed cells, wherein the second set
of editing vectors comprises editing cassettes with one or more
editing gRNA and donor DNA pairs under the control of the first
inducible promoter, a third selectable marker, and the curing
target sequence.
2. The method of claim 1, wherein the first inducible promoter and
the third inducible promoters are the same inducible promoter.
3. The method of claim 1, wherein the first and third inducible
promoters are pL promoters and either the first set of editing
vectors or the engine vector comprises a c1857 gene under the
control of a constitutive promoter.
4. The method of claim 1, wherein the curing target sequence is a
pUC origin of replication.
5. The method of claim 4, wherein the curing gRNA is an anti-pUC
origin gRNA.
6. The method of claim 1, wherein the second inducible promoter is
a pPhIF promoter.
7. The method of claim 1, further comprising, after the second
transforming step, the steps of: selecting for the second
transformed cells via the second and third selectable markers
thereby selecting for second selected cells; inducing editing in
the second selected cells by inducing the first and third inducible
promoters thereby inducing transcription of the one or more editing
gRNA and donor DNA pairs and the nuclease producing second edited
cells; growing the second edited cells until the second edited
cells reach a stationary phase of growth; curing the second set of
editing vectors in the second edited cells by inducing the third
and second inducible promoters thereby inducing transcription of
the nuclease and curing gRNA which cuts the curing target sequence
producing second cured cells; growing the second cured cells;
rendering the second cured cells electrocompetent; and transforming
the second cured cells with a third set of the editing vectors to
produce third transformed cells, wherein the third set of editing
vectors comprises editing cassettes with one or more editing gRNAs
and donor DNA pairs under the control of the first inducible
promoter, a fourth selectable marker, and the curing target
sequence.
8. The method of claim 7, further comprising, after the third
transforming step, the steps of: selecting for the third
transformed cells via the second and fourth selectable markers
thereby selecting for third selected cells; inducing editing in the
third selected cells by inducing the first and third inducible
promoters thereby inducing transcription of the one or more editing
gRNA and donor DNA pairs and the nuclease producing third edited
cells; growing the third edited cells until the third edited cells
reach a stationary phase of growth; curing the third set of editing
vectors in the third edited cells by inducing the third and second
inducible promoters thereby inducing transcription of the nuclease
and curing gRNA which cuts the curing target sequence producing
third cured cells; growing the third cured cells; rendering the
third cured cells electrocompetent; and transforming the third
cured cells with a fourth set of the editing vectors to produce
fourth transformed cells, wherein the fourth set of editing vectors
comprises editing cassettes with one or more editing gRNAs and
donor DNA pairs under the control of the first inducible promoter,
a fifth selectable marker, and the curing target sequence.
9. The method of claim 8, wherein the first, second, third and
fourth sets of editing cassettes each comprises a library of
editing gRNA and donor DNA pairs.
10. The method of claim 9, wherein each library of editing vectors
comprises at least 1000 different editing gRNA and donor DNA
pairs.
11. A method for curing cells of choice during recursive nucleic
acid-directed nuclease editing comprising: designing and
synthesizing a first set of editing cassettes, wherein the first
set of editing cassettes comprises one or more editing gRNA and
donor DNA pairs wherein each editing gRNA and donor DNA pair is
under the control of a first inducible promoter; assembling the
first set of editing cassettes into a vector backbone thereby
forming a first set of editing vectors, wherein the vector backbone
comprises a first selectable marker, a curing target sequence, and
a curing gRNA under the control of a second inducible promoter;
making cells of choice electrocompetent, wherein the cells of
choice comprise an engine vector and the engine vector comprises a
nuclease under the control of a third inducible promoter, and a
second selectable marker; transforming the cells of choice with the
first set of editing vectors to produce first transformed cells;
selecting for the first transformed cells via the first and second
selectable markers thereby selecting for first selected cells;
inducing editing in the first selected cells by inducing the first
and third inducible promoters thereby inducing transcription of the
one or more editing gRNA and donor DNA pairs and nuclease producing
first edited cells; growing the first edited cells until the first
edited cells reach a stationary phase of growth; curing the first
set of editing vectors in the first edited cells by inducing the
third and second inducible promoters thereby inducing transcription
of the nuclease and curing gRNA which cuts the curing target
sequence producing first cured cells; growing the first cured
cells; rendering the first cured cells electrocompetent; and
transforming the first cured cells with a second set of editing
vectors to produce second transformed cells, wherein the second set
of editing vectors comprises editing cassettes with one or more
editing gRNA and donor DNA pairs under the control of the first
inducible promoter, a third selectable marker, the curing target
sequence, and the curing gRNA under the control of the second
inducible promoter.
12. The method of claim 11, wherein the first inducible promoter
and the third inducible promoters are the same inducible
promoter.
13. The method of claim 11, wherein the first and third inducible
promoters are pL promoters and either the first set of editing
vectors or the engine vector comprises a c1857 gene under the
control of a constitutive promoter.
14. The method of claim 11, wherein the curing target sequence is a
pUC origin of replication.
15. The method of claim 14, wherein the curing gRNA is an anti-pUC
origin gRNA.
16. The method of claim 11 wherein the second inducible promoter is
a pPhIF promoter.
17. The method of claim 11, further comprising, after the second
transforming step, the steps of: selecting for the second
transformed cells via the second and third selectable markers
thereby selecting for third selected cells; inducing editing in the
third selected cells by inducing the first and third inducible
promoters thereby inducing transcription of the one or more editing
gRNA and donor DNA pairs and the nuclease producing second edited
cells; growing the second edited cells until the second edited
cells reach a stationary phase of growth; curing the second set of
editing vectors in the second edited cells by inducing the third
and second inducible promoters thereby inducing transcription of
the nuclease and curing gRNA which cuts the curing target sequence
producing second cured cells; growing the second cured cells;
rendering the second cured cells electrocompetent; and transforming
the second cured cells with a third set of editing vectors to
produce third transformed cells, wherein the third set of editing
vectors comprises editing cassettes with one or more editing gRNAs
and donor DNA pairs under the control of the first inducible
promoter, a fourth selectable marker, the curing target sequence,
and the curing gRNA under the control of the second inducible
promoter.
18. The method of claim 17, further comprising, after the third
transforming step, the steps of: selecting for the third
transformed cells via the second and fourth selectable markers
thereby selecting for third selected cells; inducing editing in the
third selected cells by inducing the first and third inducible
promoters thereby inducing transcription of the one or more editing
gRNA and donor DNA pairs and the nuclease producing third edited
cells; growing the third edited cells until the third edited cells
reach a stationary phase of growth; curing the third set of editing
vectors in the third edited cells by inducing the third and second
inducible promoters thereby inducing transcription of the nuclease
and curing gRNA which cuts the curing target sequence producing
third cured cells; growing the third cured cells; rendering the
third cured cells electrocompetent; and transforming the third
cured cells with a fourth set of editing vectors to produce fourth
transformed cells, wherein the fourth set of editing vectors
comprises editing cassettes with one or more editing gRNAs and
donor DNA pairs under the control of the first inducible promoter,
a fifth selectable marker, the curing target sequence, and a curing
gRNA under the control of a second inducible promoter.
19. The method of claim 18, wherein the first, second, third and
fourth sets of editing cassettes each comprises a library of
editing gRNA and donor DNA pairs.
20. The method of claim 19, wherein each library of editing vectors
comprises at least 1000 different editing gRNA and donor DNA pairs.
Description
RELATED CASES
[0001] The present application claims priority to U.S. Ser. No.
62/857,967, entitled "Curing for Recursive Nucleic Acid-Guided Cell
Editing", filed 6 Jun. 2019.
FIELD OF THE INVENTION
[0002] The present disclosure relates to automated multi-module
instruments, compositions and methods for performing recursive
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 ability to make precise, targeted changes to the genome
of living cells has been a long-standing goal in biomedical
research and development. Recently, various nucleases have been
identified that allow for manipulation of gene sequences, and hence
gene function. The nucleases include nucleic acid-guided nucleases
and nuclease fusions, which enable researchers to generate
permanent edits in live cells. It is desirable to be able to
perform two to many rounds of nucleic acid-guided nuclease editing
sequentially (e.g., perform recursive editing), but in doing so it
is also desirable to clear or "cure" a prior editing nucleic acid
from the cells before transforming or transfecting the cells with a
subsequent editing nucleic acid. Curing is a way to eliminate the
prior editing vector--including the attendant gRNA and donor DNA
sequences (e.g., editing or CREATE cassette) contained on an
editing vector--and also selection genes and other sequences
contained on the editing vector. Further, eliminating the editing
vector from a prior round of editing permits a new editing vector
to propagate within a cell without competition from the prior
editing vector.
[0005] There is thus a need in the art of nucleic acid-guided
nuclease gene editing for improved methods, compositions, modules
and instruments for curing editing vectors used in prior rounds of
editing. The present invention satisfies 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, automated
methods and multi-module automated instrumentation for performing
curing of editing vectors in recursive editing protocols.
[0008] Thus, in some embodiments there is provided a method for
curing cells during recursive nucleic acid-directed nuclease
editing comprising: designing and synthesizing sets of editing
cassettes, wherein the sets of editing cassettes comprise one or
more editing gRNA and donor DNA pairs wherein each editing gRNA and
donor DNA pair is under the control of a first inducible promoter;
assembling the editing cassettes into a vector backbone thereby
forming editing vectors, wherein the vector backbone comprises a
first selectable marker, and a curing target sequence; making cells
of choice electrocompetent, wherein the cells of choice comprise an
engine vector and the engine vector comprises a curing gRNA under
the control of a second inducible promoter, a nuclease under the
control of the third inducible promoter; and a second selectable
marker; transforming the cells of choice with a first set editing
vectors to produce transformed cells; selecting for transformed
cells via the first and second selectable markers; inducing editing
in the selected cells by inducing the first and third inducible
promoters thereby inducing transcription of the one or more editing
gRNA and donor DNA pairs and the nuclease; growing the cells until
the cells reach a stationary phase of growth; curing the editing
vector by inducing the third and second inducible promoters thereby
inducing transcription of the nuclease and curing gRNA; growing the
cells; rendering the cells electrocompetent; and transforming the
cells with a second set of the editing vectors to produce second
transformed cells, wherein the second set of editing vectors
comprises editing cassettes with one or more gRNA and donor DNA
pairs under the control of the first inducible promoter, a third
selectable marker, and the curing target sequence.
[0009] In some aspects of this embodiment, the first inducible
promoter and the third inducible promoters are the same inducible
promoter, and in some aspects, the first and third inducible
promoters are pL promoters and either the editing vector or the
engine vector comprises a c1857 gene under the control of a
constitutive promoter.
[0010] In some aspects, the target curing sequence is a pUC origin
of replication, and the curing gRNA is an anti-pUC origin gRNA.
[0011] In some aspects, the second inducible promoter is a pPhIF
promoter.
[0012] In some aspects, the method further comprises, after the
second transforming step, the additional steps of: selecting for
the second transformed cells via the second and third selectable
markers; inducing editing in the selected cells by inducing the
first and third inducible promoter thereby inducing transcription
of the one or more editing gRNA and donor DNA pairs and the
nuclease; growing the induced cells until the cells reach a
stationary phase of growth; curing the editing vectors from the
second set of editing vectors in the induced cells by inducing the
third and second inducible promoters thereby inducing transcription
of the nuclease and curing gRNA; growing the cells; rendering the
cells electrocompetent; and transforming the cells with a third set
of the editing vectors to produce third transformed cells, wherein
the third set of editing vectors comprises editing cassettes with
one or more gRNA and donor DNA pairs under the control of the first
inducible promoter, a fourth selectable marker, and the curing
target sequence.
[0013] In other aspects, the method further comprises after the
third transforming step, the steps of: selecting for the third
transformed cells via the second and fourth selectable markers;
inducing editing in the selected cells by inducing the first and
third inducible promoter thereby inducing transcription of the one
or more editing gRNA and donor DNA pairs and the nuclease; growing
the induced cells until the cells reach a stationary phase of
growth; curing the editing vectors from the third set of editing
vectors in the induced cells by inducing the third and second
inducible promoters thereby inducing transcription of the nuclease
and curing gRNA; growing the cells; rendering the cells
electrocompetent; and transforming the cells with a fourth set of
the editing vectors to produce fourth transformed cells, wherein
the fourth set of editing vectors comprises editing cassettes with
one or more gRNAs and donor DNA pairs under the control of the
first inducible promoter, a fifth selectable marker, and the curing
target sequence.
[0014] In some aspects, the first, second, third and fourth sets of
editing cassettes each comprise a library of editing gRNA and donor
DNA pairs; and in some aspects, the libraries of editing vectors
each comprises at least 1000 different editing gRNA and donor DNA
pairs.
[0015] Other embodiments provide a method for curing cells during
recursive nucleic acid-directed nuclease editing comprising:
designing and synthesizing sets of editing cassettes, wherein the
sets of editing cassettes comprise one or more editing gRNA and
donor DNA pairs wherein each editing gRNA and donor DNA pair is
under the control of a first inducible promoter; assembling the
editing cassettes into a vector backbone thereby forming editing
vectors, wherein the vector backbone comprises a first selectable
marker, a curing target sequence, and a curing gRNA under the
control of a second inducible promoter; making cells of choice
electrocompetent, wherein the cells of choice comprise an engine
vector and the engine vector comprises a nuclease under the control
of the third inducible promoter, and a second selectable marker;
transforming the cells of choice with a first set editing vectors
to produce transformed cells; selecting for transformed cells via
the first and second selectable markers; inducing editing in the
selected cells by inducing the first and third inducible promoters
thereby inducing transcription of the one or more editing gRNA and
donor DNA pairs and nuclease; growing the cells until the cells
reach a stationary phase of growth; curing the editing vector by
inducing the third and second inducible promoters thereby inducing
transcription of the nuclease and curing gRNA; growing the cells;
rendering the cells electrocompetent; and transforming the cells
with a second set of the editing vectors to produce second
transformed cells, wherein the second set of editing vectors
comprises editing cassettes with one or more gRNA and donor DNA
pairs under the control of the first inducible promoter, a third
selectable marker, the curing target sequence, and the curing gRNA
under the control of the second inducible promoter.
[0016] In some aspects of this embodiment, the first inducible
promoter and the third inducible promoters are the same inducible
promoter, and in some aspects, the first and third inducible
promoters are pL promoters and either the editing vector or the
engine vector comprises a c1857 gene under the control of a
constitutive promoter.
[0017] In some aspects, the target curing sequence is a pUC origin
of replication, and the curing gRNA is an anti-pUC origin gRNA.
[0018] In some aspects, the second inducible promoter is a pPhIF
promoter.
[0019] In some aspects of this embodiment, the method further
comprises, after the second transforming step, the additional steps
of: selecting for the second transformed cells via the second and
third selectable markers; inducing editing in the selected cells by
inducing the first and third inducible promoter thereby inducing
transcription of the one or more editing gRNA and donor DNA pairs
and the nuclease; growing the induced cells until the cells reach a
stationary phase of growth; curing the editing vectors from the
second set of editing vectors in the induced cells by inducing the
third and second inducible promoters thereby inducing transcription
of the nuclease and curing gRNA; growing the cells; rendering the
cells electrocompetent; and transforming the cells with a third set
of the editing vectors to produce third transformed cells, wherein
the third set of editing vectors comprises editing cassettes with
one or more gRNA and donor DNA pairs under the control of the first
inducible promoter, a fourth selectable marker, the curing target
sequence, and the curing gRNA under the control of the second
inducible promoter.
[0020] In yet another aspect, the method may further comprise the
steps of, after the third transforming step: selecting for the
third transformed cells via the second and fourth selectable
markers; inducing editing in the selected cells by inducing the
first and third inducible promoter thereby inducing transcription
of the one or more editing gRNA and donor DNA pairs and the
nuclease; growing the induced cells until the cells reach a
stationary phase of growth; curing the editing vectors from the
third set of editing vectors in the induced cells by inducing the
third and second inducible promoters thereby inducing transcription
of the nuclease and curing gRNA; growing the cells; rendering the
cells electrocompetent; and transforming the cells with a fourth
set of the editing vectors to produce fourth transformed cells,
wherein the fourth set of editing vectors comprises editing
cassettes with one or more gRNA and donor DNA pairs under the
control of the first inducible promoter, a fifth selectable marker,
the curing target sequence, and the curing gRNA under the control
of the second inducible promoter.
[0021] In some aspects of any of the methods, the method further
comprises between the transforming step and inducing step,
singulating the cells in a SWIIN, and wherein the selecting,
inducing, growing, and curing steps are performed in the SWIIN.
[0022] In some aspects, the first, second, third and fourth sets of
editing cassettes each comprise a library of editing gRNA and donor
DNA pairs; and in some aspects, the libraries of editing vectors
each comprises at least 1000 different editing gRNA and donor DNA
pairs.
[0023] These aspects and other features and advantages of the
invention are described below in more detail.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] 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:
[0025] FIG. 1A is a flow chart showing steps for an exemplary
curing method according to the present disclosure. FIG. 1B is an
exemplary growth curve for cells. FIG. 1C depicts an exemplary
plasmid architecture for engine vector curing of an editing vector
and FIG. 1D depicts an exemplary plasmid architecture for
self-curing of an editing vector. FIG. 1E depicts an exemplary
recursive method using a standard plating protocol. FIG. 1F depicts
the exemplary recursive method using the standard plating protocol
of FIG. 1E. FIG. 1G depicts an exemplary recursive method using a
bulk liquid editing protocol. FIG. 1H depicts the exemplary
recursive method using the bulk liquid editing protocol of FIG. 1G.
FIG. 1I depicts an exemplary recursive method using a solid wall
isolation device.
[0026] FIGS. 2A-2C depict three different views of an exemplary
automated multi-module cell processing instrument for performing
nucleic acid-guided nuclease editing.
[0027] FIG. 3A depicts one embodiment of a rotating growth vial for
use with the cell growth module described herein and in relation to
FIGS. 3B-3D. FIG. 3B illustrates a perspective view of one
embodiment of a rotating growth vial in a cell growth module
housing. FIG. 3C depicts a cut-away view of the cell growth module
from FIG. 3B. FIG. 3D illustrates the cell growth module of FIG. 3B
coupled to LED, detector, and temperature regulating
components.
[0028] FIG. 4A depicts retentate (top) and permeate (middle)
members for use in a tangential flow filtration module (e.g., cell
growth and/or concentration module), as well as the retentate and
permeate members assembled into a tangential flow assembly
(bottom). FIG. 4B depicts two side perspective views of a reservoir
assembly of a tangential flow filtration module. FIGS. 4C-4E depict
an exemplary top, with fluidic and pneumatic ports and gasket
suitable for the reservoir assemblies shown in FIG. 4B.
[0029] FIGS. 5A and 5B depict the structure and components of an
embodiment of a reagent cartridge. FIG. 5C is a top perspective
view of one embodiment of an exemplary flow-through electroporation
device that may be part of a reagent cartridge. FIG. 5D depicts a
bottom perspective view of one embodiment of an exemplary
flow-through electroporation device that may be part of a reagent
cartridge. FIGS. 5E-5G depict a top perspective view, a top view of
a cross section, and a side perspective view of a cross section of
an FTEP device useful in a multi-module automated cell processing
instrument such as that shown in FIGS. 2A-2C.
[0030] FIG. 6A depicts a simplified graphic of a workflow for
singulating, editing and normalizing cells in a solid wall device.
FIGS. 6B-6D depict an embodiment of a solid wall isolation
incubation and normalization (SWIIN) module. FIG. 6E depicts the
embodiment of the SWIIN module in FIGS. 6B-6D further comprising a
heater and a heated cover.
[0031] FIG. 7 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 recursive cell editing.
[0032] FIG. 8 is a simplified process diagram of an alternative
embodiment of an exemplary automated multi-module cell processing
instrument useful for recursive cell editing.
[0033] FIG. 9 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.
[0034] FIG. 10 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.
[0035] FIG. 11 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.
[0036] FIG. 12 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.
[0037] FIG. 13 is a graph demonstrating real-time monitoring of
growth of an EC138 cell culture to OD.sub.600 employing the cell
growth device as described herein where a 2-paddle rotating growth
vial was used.
[0038] FIG. 14 is a graph demonstrating real-time monitoring of
growth of s288c yeast cell culture OD.sub.600 employing the cell
growth device as described herein where a 2-paddle rotating growth
vial was used.
[0039] FIG. 15A is a graph plotting filtrate conductivity against
filter processing time for an E. coli culture processed in the cell
concentration device/module described herein.
[0040] FIG. 15B is a graph plotting filtrate conductivity against
filter processing time for a yeast culture processed in the cell
concentration device/module described herein.
[0041] FIG. 16A is a bar graph showing the results of
electroporation of E. coli using a device of the disclosure and a
comparator electroporation device. FIG. 16B 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.
[0042] FIG. 17 is a bar graph showing the results of
electroporation of S. cerevisiae using an FTEP device of the
disclosure and a comparator electroporation method.
[0043] FIG. 18 is a graph showing the editing results obtained via
the liquid bulk method for increasing observed editing in live
cells.
[0044] FIG. 19 is a graph comparing the percentage of editing
obtained for a standard plating protocol (SPP), and replicate
samples using two different conditions in a solid wall isolation,
induction, and normalization device (SWIIN): the first with
LB+arabinose; and the second with SOB followed by
SOB+arabinose.
[0045] FIGS. 20A, 20B, and 20C are graphs of results obtained in
experiments testing editing efficiency and curing efficiency in
engine vector-driven recursive experiments performed by a standard
plating protocol, a bulk liquid protocol, and a SWIIN protocol.
[0046] It should be understood that the drawings are not
necessarily to scale, and that like reference numbers refer to like
features.
DETAILED DESCRIPTION
[0047] 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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] 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.
[0052] 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.
[0053] 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'-TAGCTG-3'.
[0054] 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.
[0055] 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
(e.g., a target genomic DNA sequence or cellular target sequence)
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. 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 genomic 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 genomic target sequence.
[0056] As used herein, "enrichment" refers to enriching for edited
cells by singulation, inducing editing, and growth of singulated
cells into terminal-sized colonies (e.g., saturation or
normalization of colony growth).
[0057] 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. The term "editing gRNA" refers to the gRNA used to edit a
target sequence in a cell, typically a sequence endogenous to the
cell. The term "curing gRNA" refers to the gRNA used to target the
curing target sequence on the editing vector.
[0058] "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.
[0059] "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.
[0060] As used herein, the terms "protein" and "polypeptide" are
used interchangeably. Proteins may or may not be made up entirely
of amino acids.
[0061] 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
such as those described herein--the transcription of at least one
component of the nucleic acid-guided nuclease editing system--and
typically at least three components of the nucleic acid-guided
nuclease editing system--is under the control of an inducible
promoter.
[0062] 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, nourseothricin N-acetyl
transferase, 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-2a; 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.
[0063] 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.-7 M, about 10.sup.-8 M,
about 10.sup.-9M, 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.
[0064] The terms "target genomic DNA sequence", "cellular 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
cellular target sequence can be a genomic locus or extrachromosomal
locus. The term "curing target sequence" refers to a sequence in
the editing vector that is cleaved or cut to cure or clear the
editing vector. The term "target sequence" refers to either or both
of a cellular target sequence and a curing target sequence.
[0065] 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.
[0066] 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 thereof. Engine vectors also typically comprise a
selectable marker. As used herein the phrase "editing vector"
comprises a donor nucleic acid, including an alteration to the
cellular target sequence that prevents nuclease binding at a PAM or
spacer in the cellular target sequence after editing has taken
place, and a coding sequence for a gRNA. The editing vector may
also and preferably does 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(s), and
selectable marker(s).
Nuclease-Directed Genome Editing Generally
[0067] In preferred embodiments, the automated instrument described
herein performs recursive nuclease-directed genome editing methods
for introducing edits to a population of cells, where editing
vectors from previous rounds of editing are cured (e.g., cleared)
before a subsequent editing vector is introduced into the
population of cells. 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 (either a cellular target sequence or a
curing 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.
[0068] 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 and preferably does reside
within an editing cassette and is under the control of an inducible
promoter as described below. For additional information regarding
"CREATE" editing cassettes, see U.S. Pat. Nos. 9,982,278;
10,266,849; 10,240,167; 10,351,877; 10,364,442; 10,435,715; and
10,465,207 and U.S. Ser. Nos. 16/551,517; 16,773,618; and
16,773,712, all of which are incorporated by reference herein.
[0069] 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.
[0070] In the present methods and compositions, the guide nucleic
acids are 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 an inducible
promoter. The guide nucleic acids are engineered to target a
desired target sequence (either cellular target sequence or curing
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 protospacer
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) or a
curing target sequence in an editing vector. In the present
description, the target sequence for one of the gRNAs, the curing
gRNA, is on the editing vector.
[0071] The editing guide nucleic acid may be and preferably is part
of an editing cassette that encodes the donor nucleic acid that
targets a cellular target sequence. Alternatively, the editing
guide nucleic acid may not be part of the editing cassette and
instead may be encoded on the editing vector backbone. For example,
a sequence coding for an editing guide nucleic acid can be
assembled or inserted into a vector backbone first, followed by
insertion of the donor nucleic acid in, e.g., an 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 editing guide
nucleic acid. Preferably, the sequence encoding the editing guide
nucleic acid and the donor nucleic acid are located together in a
rationally-designed editing cassette and are simultaneously
inserted or assembled into a vector backbone to create an editing
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.
[0072] The target sequence--both the cellular target sequence and
the curing target sequence--is associated with a proto-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.
[0073] In certain embodiments, the genome editing of a cellular
target sequence both introduces a desired DNA change to a cellular
target sequence, e.g., the genomic DNA of a cell, and removes,
mutates, or renders inactive a proto-spacer mutation (PAM) region
in the cellular target sequence. Rendering the PAM at the cellular
target sequence inactive precludes additional editing of the cell
genome at that cellular 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 cellular target sequence edit and an
altered PAM can be selected for by using a nucleic acid-guided
nuclease complexed with a synthetic guide nucleic acid
complementary to the cellular 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 cellular 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.
[0074] The range of target sequences (both cellular target
sequences and curing 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.
[0075] 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
and nuclease fusions thereof. Nuclease fusion enzymes typically
comprise a CRISPR nucleic acid-guided nuclease engineered to cut
one DNA strand in the target DNA rather than making a
double-stranded cut, and the nuclease portion is fused to a reverse
transcriptase. For more information on nickases and nuclease fusion
editing see U.S. Ser. Nos. 16/740,418; 16/740,420 and 16/740,421,
all filed 11 Jan. 2020. As with the guide nucleic acid, the
nuclease is encoded by a DNA sequence on a vector (e.g., the engine
vector) and be under the control of an inducible promoter. In some
embodiments, 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 or nuclease fusion 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.
[0076] Another component of the nucleic acid-guided nuclease system
is the donor nucleic acid comprising homology to the cellular
target sequence. 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 preferably is (but not necessarily
is) under the control of the same promoter as the editing gRNA
(e.g., a single promoter driving the transcription of both the
editing gRNA and the donor nucleic acid). The donor nucleic acid is
designed to serve as a template for homologous recombination with a
cellular 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 cellular target sequence (e.g., a
homology arm). When optimally aligned, the donor nucleic acid
overlaps with (is complementary to) the cellular target sequence
by, e.g., about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more
nucleotides. The donor nucleic acid comprises two homology arms
(regions complementary to the cellular target sequence) flanking
the mutation or difference between the donor nucleic acid and the
cellular target sequence. The donor nucleic acid comprises at least
one mutation or alteration compared to the cellular target
sequence, such as an insertion, deletion, modification, or any
combination thereof compared to the cellular target sequence.
[0077] Again, the donor nucleic acid is preferably provided as part
of a rationally-designed editing cassette, which is inserted into
an editing vector backbone where the editing vector backbone may
comprise a promoter driving transcription of the editing gRNA and
the donor DNA, and also comprise a selectable marker different from
the selectable marker contained on the engine vector, as well as a
curing target sequence that is cut or cleaved during curing.
Moreover, there may be more than one, e.g., two, three, four, or
more editing gRNA/donor nucleic acid rationally-designed editing
cassettes inserted into an editing vector (alternatively, a single
rationally-designed editing cassette may comprise two to several
editing gRNA/donor DNA pairs), where each editing gRNA is under the
control of separate different promoters, separate like promoters,
or where all gRNAs/donor nucleic acid pairs are under the control
of a single promoter. In preferred embodiments the promoter driving
transcription of the editing gRNA and the donor nucleic acid (or
driving more than one editing gRNA/donor nucleic acid pair) is an
inducible promoter and the promoter driving transcription of the
nuclease or nuclease fusion is an inducible promoter as well. In
some embodiments and preferably, the nuclease and editing
gRNA/donor DNA are under the control of the same inducible
promoter.
[0078] 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.
It is this toxicity, however, that is exploited herein to perform
curing.
[0079] In addition to the donor nucleic acid, an editing cassette
may comprise and preferably does 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.
[0080] Also, as described above, the donor nucleic acid may
comprise--in addition to the at least one mutation relative to a
cellular target sequence-one or more PAM sequence alterations that
mutate, delete or render inactive the PAM site in the cellular
target sequence. The PAM sequence alteration in the cellular target
sequence renders the PAM site "immune" to the nucleic acid-guided
nuclease and protects the cellular target sequence from further
editing in subsequent rounds of editing if the same nuclease is
used.
[0081] 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 cellular target sequence. The barcode typically
comprises four or more nucleotides. In some embodiments, the
editing cassettes comprise a collection or library editing gRNAs
and of donor nucleic acids representing, e.g., gene-wide or
genome-wide libraries of editing gRNAs and 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.
[0082] 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.
[0083] 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
preferably are 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 pPhIF promoter (induced by the addition of
2,4 diacetylphloroglucinol (DAPG)), 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.
Curing
[0084] "Curing" is a process in which a vector-here, the editing
vector used in a prior round of editing or an engine vector after
the final round of editing--is eliminated from the cells being
edited. Curing can be accomplished by 1) cleaving the editing
vector using a curing gRNA on the engine or editing vectors thereby
rendering the editing vector nonfunctional; 2) diluting the editing
vector in the cell population via cell growth--that is, the more
growth cycles the cells go through in medium without the antibiotic
that selects for the editing vector the fewer daughter cells will
retain the editing or engine vector(s)); or 3) by utilizing a
heat-sensitive origin of replication on the editing vector. The
present disclosure is drawn to transcribing a curing gRNA located
on either the editing vector ("self cure") or the engine vector
("engine cure") to cut or cleave a locus located in a curing target
sequence in the editing vector after a round of editing and before
a next round of editing in a recursive editing process.
[0085] FIG. 1A is a flow chart for the curing methods 100 according
to the present disclosure. In a first step, a library of
rationally-designed editing cassettes is synthesized 102. Methods
and compositions particularly favored for designing and
synthesizing editing cassettes are described in U.S. Pat. Nos.
9,982,278; 10,266,849; 10,240,167; 10,351,877; 10,364,442;
10,435,715; and 10,465,207 and U.S. Ser. Nos. 16/551,517;
16,773,618; and 16,773,712, all of which are incorporated by
reference herein.
[0086] Once designed and synthesized, the editing cassettes are
amplified, purified and assembled into a vector backbone 104 to
create editing cassettes. A number of methods may be used to
assemble the editing cassettes including 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 to Hartley et al.; U.S. Pat. No. 6,277,608 to
Hartley et al.; and topoisomerase-mediated cloning (Udo, PLoS One,
10(9):e0139349 (2015)); U.S. Pat. No. 6,916,632 B2 to Chestnut et
al. 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); Casini et al., Nat Rev Mol Cell Biol., (9):568-76 (2015);
and Patron, Curr Opinion Plant Biol., 19:14-9 (2014)).
[0087] In addition to preparing editing cassettes, cells of choice
are made electrocompetent 120 for transformation. The cells that
can be edited include any prokaryotic, archaeal or eukaryotic cell.
For example, prokaryotic cells for use with the present
illustrative embodiments can be gram positive bacterial cells,
e.g., Bacillus subtilis, or gram-negative bacterial cells, e.g., E.
coli cells. Eukaryotic cells for use with the automated
multi-module cell editing instruments of the illustrative
embodiments include any plant cells and any animal cells, e.g.
fungal cells, insect cells, amphibian cells nematode cells, or
mammalian cells.
[0088] Once the cells of choice are rendered electrocompetent 120,
the cells and editing vectors are combined and the editing vectors
are transformed into (e.g., electroporated into) the cells 106. The
cells may be also transformed simultaneously with a separate engine
vector expressing an editing nuclease; alternatively and
preferably, the cells may already have been transformed with an
engine vector configured to express the nuclease; that is, 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.
[0089] Transformation is intended to include to a variety of
art-recognized techniques for introducing an exogenous nucleic acid
sequence (e.g., DNA) into a target cell, and the term
"transformation" as used herein includes all transformation and
transfection techniques. Such methods include, but are not limited
to, electroporation, lipofection, optoporation, injection,
microprecipitation, microinjection, liposomes, particle
bombardment, sonoporation, laser-induced poration, bead
transfection, calcium phosphate or calcium chloride
co-precipitation, or DEAE-dextran-mediated transfection. Cells can
also be prepared for vector uptake using, e.g., a sucrose or
glycerol wash. Additionally, hybrid techniques that exploit the
capabilities of mechanical and chemical transfection methods can be
used, e.g., magnetofection, a transfection methodology that
combines chemical transfection with mechanical methods. In another
example, cationic lipids may be deployed in combination with gene
guns or electroporators. Suitable materials and methods for
transforming or transfecting target cells can be found, e.g., in
Green and Sambrook, Molecular Cloning: A Laboratory Manual, 4th,
ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.,
2014). The present automated methods using the automated
multi-module cell processing instrument utilize flow-through
electroporation such as the exemplary device shown in FIGS.
5C-5G.
[0090] Once transformed, the cells are allowed to recover and
selection is performed 108 to select for cells transformed with the
editing vector, which in addition to an editing cassette comprises
an appropriate selectable marker. As described above, drug
selectable markers such as ampilcillin/carbenicillin, kanamycin,
chloramphenicol, nourseothricin N-acetyl transferase, erythromycin,
tetracycline, gentamicin, bleomycin, streptomycin, puromycin,
hygromycin, blasticidin, and G418 or other selectable markers may
be employed.
[0091] Following selection for properly transformed cells, editing
is induced 110 in the cells by induction of transcription of one or
both-preferably both--of the nuclease and gRNA. Induction of
transcription of one, or, preferably both, of the nuclease and gRNA
is prompted 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. 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, in addition to the pL
promoter, the pPhIF promoter (induced by the addition of 2,4
diacetylphloroglucinol (DAPG)), 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.
[0092] The present compositions and methods preferably make use of
rationally-designed editing cassettes such as CREATE cassettes, as
described above. Each editing cassette comprises an editing gRNA, a
donor DNA comprising an intended edit and a PAM or spacer mutation;
thus, e.g., a two-cassette multiplex editing cassette comprises a
first editing gRNA, a first editing donor DNA, and a first intended
edit and a first PAM or spacer mutation, and at least a second
editing gRNA, at least a second donor DNA, and at least a second
intended edit and a second PAM or spacer mutation. In some
embodiments, a single promoter may drive transcription of both the
first and second editing gRNAs and both the first and second donor
DNAs, and in some embodiments, separate promoters may drive
transcription of the first editing gRNA and first donor DNA, and
transcription of the second editing gRNA and second donor DNA. In
addition, multiplex editing cassettes may comprise nucleic acid
elements between the editing cassettes with, e.g., primer
sequences, bridging oligonucleotides, and other
"cassette-connecting" sequence elements that allow for the assembly
of the multiplex editing cassettes.
[0093] Once editing is induced 110, the cells are grown until the
cells enter (or are close to entering) the stationary phase of
growth 112, followed by inducing curing of the editing vector 114
by activating an inducible promoter driving transcription of the
curing gRNA and inducing the inducible promoter driving
transcription of the nuclease. It has been found that curing is
particularly effective if the edited cells are in the stationary
phase of growth. In yet some aspects, the cells are grown for at
least 75% of log phase, 80% of log phase, 85% of log phase, 90% of
log phase, 95% of log phase, or are in a stationary phase of growth
before inducing curing. Exemplary genetic and inducing components
for inducing curing are described in more detail in relation to
FIGS. 1C and 1D. Once the editing vector has been cured 114, the
cells are allowed to recover and grow, and then the cells are made
electrocompetent 116 once again, ready for another round of editing
118.
[0094] FIG. 1B depicts a typical growth curve 150 for cells in
culture (optical density versus time). Initially there is a lag
phase 151, then the cells enter log phase 152 where they grow
quickly, and finally the cells reach stationary phase 154 where the
cells are no longer dividing. The present methods employ inducing
curing at timepoint 153 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.
[0095] FIG. 1C depicts an exemplary plasmid architecture for engine
curing of an editing vector comprising an engine vector (on left)
and an editing vector (on right); that is, the sequence for the
curing gRNA that targets the curing target sequence on the editing
vector is located on the engine vector. The engine vector comprises
a pBAD inducible promoter 5' of and driving a lambda Red
recombineering system. The .lamda. Red recombineering system works
as a "band aid" or repair system for double-strand breaks in
bacteria, and in some species of bacteria the .lamda. Red
recombineering system (or some other recombineering system) must be
present for the double-strand breaks that occur during editing to
resolve. In yeast and other eukaryotic cells, however,
recombineering systems are not required. The inducible promoter (in
this case pBAD, but other inducible promoters may be used) driving
transcription of the .lamda. Red recombineering system components
is most preferably a different inducible promoter than that driving
transcription of the nuclease and the editing gRNA, as it is
preferred that the recombineering system be active before the
nuclease is induced. That is, it is preferred that the "band aid"
double-strand break repair machinery be active before the nuclease
starts cutting the cellular genome.
[0096] In addition to the .lamda. Red recombineering system, the
engine vector also comprises an origin of replication (here an
SC101 origin, which may be temperature sensitive, but need not be)
3' of the .lamda. Red recombineering system, followed by a pPhIF
inducible primer 3' of the SC101 origin driving transcription of
the curing gRNA, in this case the curing gRNA is a gRNA that
renders inactive the pUC origin of replication on the editing
vector (e.g., the curing target sequence). Next, 3' of the curing
gRNA sequence is a promoter (typically a constitutive promoter)
driving transcription of the c1857 repressor gene, which actively
represses the pL promoter at 30.degree. C. and degrades at
42.degree. C. thereby activating the pL promoter. Three prime of
the c1857 coding sequence is a promoter (also typically a
constitutive promoter) driving transcription of an antibiotic
resistance gene-here, a carbenicillin resistance gene-followed by a
pL inducible promoter driving transcription of MAD7, the
nuclease.
[0097] The editing vector on the right in FIG. 1C comprises a pL
promoter driving transcription of an editing cassette, where the
editing cassette includes a coding sequence for an editing gRNA and
a donor DNA sequence (e.g., a homology arm or "HA"). The donor DNA
sequence--in addition to a sequence for a desired edit in a nucleic
acid sequence endogenous to the cell (e.g., the cellular target
sequence)--often further comprises a PAM-altering sequence, which
is most often a sequence that disables the PAM at the cellular
target sequence in the genome. The editing vector further comprises
a promoter (typically a constitutive promoter) driving
transcription of an antibiotic resistance gene (e.g., kanamycin or
chloramphenicol), followed by a pUC origin of replication. The
anti-pUC gRNA (e.g., the curing gRNA), whose transcription is
controlled by the pPhIF inducible promoter on the engine, comprises
a gRNA that disables the pUC origin on the editing vector
(represented by an arrow and scissors in FIG. 1C).
[0098] FIG. 1D depicts an exemplary plasmid architecture for
self-curing of an editing vector, comprising an engine vector (on
left) and an editing vector (on right); that is, the sequence for
the curing gRNA that targets the curing target sequence on the
editing vector is located on the editing vector itself. The engine
vector comprises a pBAD inducible promoter 5' of and driving a
lambda red recombineering system. As described above, the .lamda.
Red recombineering system works as a "band aid" or repair system
for double-strand breaks in bacteria. The inducible promoter (in
this case pBAD, but other inducible promoters may be used) driving
transcription of the .lamda. Red recombineering system components
is most preferably a different inducible promoter than that driving
transcription of the nuclease and the editing gRNA, as it is
preferred that the recombineering system be active before the
nuclease is induced. In addition to the .lamda. Red recombineering
system, the engine vector also comprises an origin of replication
(here an SC101 origin, which may be temperature sensitive, but need
not be) 3' of the .lamda. Red recombineering system, followed by a
promoter (typically a constitutive promoter) driving transcription
of the c1857 repressor gene, which actively represses the pL
promoter at 30.degree. C. and degrades at 42.degree. C. thereby
activating the pL promoter. Three prime of the c1857 coding
sequence is a promoter (also typically a constitutive promoter)
driving transcription of a carbenicillin resistance gene, followed
by a pL inducible promoter driving transcription of MAD7, the
nuclease.
[0099] The editing vector on the right in FIG. 1D comprises a pL
promoter driving transcription of an editing cassette, where the
editing cassette includes a coding sequence for an editing gRNA and
a donor DNA sequence (e.g., a homology arm or "HA"). The donor DNA
sequence--in addition to a sequence for a desired, intended edit in
a nucleic acid sequence endogenous to the cell-further comprises a
PAM-altering sequence, a sequence that disables the PAM at the
cellular target sequence in the genome. The editing vector further
comprises a pPhIF inducible primer 3' of the editing cassette,
where the pPhIF promoter drives transcription of the curing gRNA.
As in FIG. 1C, the curing gRNA is a gRNA that renders inactive the
pUC origin located on the editing vector; however, in this
architecture the curing gRNA is located on the editing vector,
hence the editing vector is "self-curing." Three prime of the
curing gRNA sequence is a promoter, typically a constitutive
promoter, driving transcription of an antibiotic resistance gene
(e.g., kanamycin or chloramphenicol, that is an antibiotic
resistance gene different from the antibiotic resistance gene
located on the engine vector), followed by the pUC origin of
replication. The anti-pUC curing gRNA, whose transcription is
controlled by the pPhIF inducible promoter, comprises a curing gRNA
that cures the editing vector by disabling (e.g., cleaving or
cutting) the pUC origin on the editing vector (represented by an
arrow and scissors in FIG. 1D). Thus, in the system in FIG. 1D, the
editing vector self cures, as the curing gRNA (e.g., the anti-pUC
gRNA) is transcribed from the editing vector and disables the pUC
origin of replication on the editing vector.
[0100] FIG. 1E depicts an exemplary recursive method 160a using a
standard plating protocol (SPP) employing, e.g., the exemplary
engine and editing vectors shown in FIG. 1C or 1D. The recursive
method 160a begins with competent cells 162, for example, E. coli
cells that have been previously transformed with an engine vector
expressing a nucleic acid-guided nuclease such as MAD7 and a
selective marker, such as a chloramphenicol resistance gene. At
step 163, the competent cells are transformed with a library of
editing vectors, where each editing vector in the library of
editing vectors comprises an editing cassette with a sequence
coding for an editing gRNA and a donor DNA, and each editing vector
also comprises an antibiotic resistance gene such as a gene
conferring resistance to carbenicillin and a curing target
sequence. Following transformation, the cells are allowed to
recover 164 in medium without antibiotic, and then at step 165, the
cells are diluted if necessary, or transferred into medium
containing, e.g., chloramphenicol and, e.g., carbenicillin to
select for both the engine and editing vectors. The cells are then
plated 166a on solid medium containing, e.g., arabinose, which
induces the lambda red recombination system encoded by, e.g., the
engine vector (see, e.g., FIGS. 1C and 1D). Once plated the cells
are allowed to grow at, e.g., 30.degree. C. for 9 hours, at
42.degree. C. for 2 hours-thereby inducing the pL promoters driving
transcription of the nuclease on the engine vector and the editing
cassette (e.g., the editing gRNA and donor DNA) on the editing
vector-then at 30.degree. C. for at least 9 more hours.
[0101] At step 167, colonies formed by the transformed cells are
removed from the solid medium by, e.g., scraping the colonies from
the medium or by picking colonies and then the harvested cells are
placed in medium, washed to remove the carbenicillin and
resuspended in medium containing chloramphenicol (continuing to
select for the engine vector) and allowed to grow until the cells
reach the stationary phase of growth or nearly so. Again, it has
been found that curing is particularly effective if the edited
cells are in the stationary phase of growth when curing is induced.
For example, the cells are grown until they have grown for at least
75% of log phase, 80% of log phase, 85% of log phase, 90% of log
phase, 95% of log phase, or are in a stationary phase of growth
before inducing curing.
[0102] The editing vectors in the cells are then cured 168 by
inducing transcription of the curing gRNA that cuts the pUC origin
of replication on the editing vector. Induction of the curing gRNA
is accomplished by first raising the temperature of the culture to
42.degree. C. for 2 hours, thereby inducing the pL promoter driving
transcription of the nuclease, and second by the addition of 2,4
diacetylphloroglucinol (DAPG) to induce the pPhIF promoter driving
transcription of the anti-pUC gRNA. After 2 hours at 42.degree. C.,
the temperature of the cell culture is lowered to 30.degree. C.
thereby halting transcription of the nuclease, and the cells are
allowed to recover and grow for 6 additional hours. The
co-expression of the nuclease and the anti-pUC gRNA permits
targeting of the pUC origin of expression on the editing vector
(e.g., the curing target sequence). As an alternative protocol to
increasing the temperature of the culture to 42.degree. C. combined
with addition of DAPG, one can induce the pL promoter driving
transcription of the nuclease by increasing the temperature of the
culture to 42.degree. C. for two hours, then lower the temperature
of the culture to 30.degree. C. and add DAPG to induce
transcription of the anti-pUC gRNA; that is, the induction of the
nuclease and the anti-pUC gRNA may be sequential rather than
simultaneous.
[0103] At step 169, the cells are washed with medium containing
chloramphenicol (again to select for cells comprising the engine
vector), and the cells are allowed to recover. At this point, the
edited cells can be grown to, e.g., OD=0.5, made electrocompetent
once more 170, and be subjected to a second round of editing
171.
[0104] FIG. 1F depicts the exemplary recursive editing and curing
method using the standard plating protocol (SPP) of FIG. 1E. FIG.
1F depicts the exemplary standard plating protocol embodiment of an
improved protocol 180 for performing nucleic acid-guided nuclease
genome editing and curing using inducible promoters to drive
expression of the editing gRNA, the nuclease, and the curing gRNA.
In FIG. 1F as in FIG. 1E, a library or collection of editing
vectors 182 is introduced 183 (e.g., electroporated) into cultured
cells 184 that already comprise a coding sequence for a nuclease
under the control of an inducible promoter. Like FIG. 1E (and as
depicted in FIGS. 1C and 1D), the coding sequence for the nuclease
is contained on an "engine vector" with a selectable marker that
has already been transformed into the cells, although in other
embodiments, the coding sequence for the nuclease may be integrated
into the genome of the cells. In yet other embodiments, the coding
sequence for the nuclease may be located on the editing vector
(that is, a combined engine and editing vector).
[0105] The editing vectors 182 comprise a donor DNA with a desired,
intended edit vis-a-vis a cellular target sequence with a
PAM-altering sequence which is most often a sequence that disables
the PAM at the target site in the genome, a coding sequence for an
editing gRNA under the control of an inducible promoter, and a
selectable marker. Depending on whether the system is an engine
curing system or a self-curing system, there is also a coding
sequence for a curing gRNA under the control of an inducible
promoter located on the engine vector or editing vector,
respectively.
[0106] Once the cells 184 have been transformed with the editing
vectors, the cells are plated 185 on selective medium on substrate
or plate 186 to select for cells that have both the engine and the
editing vectors. The cells are diluted before plating such that the
cells are substantially or largely singulated-separated enough so
that they and the colonies they form are separated from other cell
colonies--and the cells are then grown 187 on plate or substrate
186 until colonies 188 begin to form. The cells are allowed to grow
at, e.g., 30.degree. C. for, e.g., between 2 and 300, or between 5
and 150, or between 10 and 50 doublings, establishing clonal
colonies. This initial growth of cells is to accumulate enough
clonal cells in a colony to survive induction of editing.
[0107] Once colonies are established, cutting and editing of the
cellular genome is induced by first inducing the promoter driving
transcription of the .lamda. Red recombineering system, and second
by inducing or activating the promoters driving the gRNA and
nuclease. The inducible promoter driving expression of the .lamda.
Red recombineering system preferably is different from the
inducible promoter driving transcription of the gRNA and nuclease
and preferably the .lamda. Red recombineering system is induced
before induction of the nuclease and gRNA, as the .lamda. Red
recombineering system works as the "band aid" or repair system for
double-strand breaks in bacteria, and in some species of bacteria
(but not in other cell types such as yeast or other eukaryotic
cells) must be present for the double-strand breaks that occur
during editing to resolve. The .lamda. Red recombineering system
may be under the control of, e.g., a pBAD promoter. The pBAD
promoter is regulated (induced) by the addition of arabinose to the
growth medium. Thus, if there is arabinose contained in the
selective medium of substrate or plate 326, the .lamda. Red
recombineering system will be activated when the cells are grown
327. As for induction of editing, if transcription of the gRNA and
nuclease are both under control of the pL promoter, transcription
of the gRNA and nuclease is induced by increasing the temperature
to 42.degree. C. for, e.g., a half-hour to two hours (or more,
depending on the cell type), which activates the pL inducible
promoter. Following induction of cutting and editing and a two-hour
42.degree. C. incubation, the temperature is returned to 30.degree.
C. to allow the cells to recover and to disable the pL promoter
system.
[0108] Once the cells have been edited and have been grown at
30.degree. C. for several hours, the colonies are then pooled 189
by, e.g., scraping the colonies off the substrate or plate to pool
190 the cells from the cell colonies. Once the colonies are pooled,
curing 191 can take place. As described in relation to FIG. 1E, the
editing vectors in the cells are cured by growing the cells until
they are in the stationary phase of growth or nearly so, and by
inducing transcription of the curing gRNA that cuts the pUC origin
of replication on the editing vector. Induction of the anti-pUC
gRNA is accomplished by first raising the temperature of the
culture to 42.degree. C. for 2 hours, thereby inducing the pL
promoter driving transcription of the nuclease, and second by the
addition of 2,4 diacetylphloroglucinol (DAPG) to induce the pPhIF
promoter driving transcription of the anti-pUC gRNA. After 2 hours
at 42.degree. C., the temperature of the cell culture is lowered to
30.degree. C., and the cells are allowed to recover and grow for 6
additional hours. The co-expression of the nuclease and the
anti-pUC gRNA permits targeting of the pUC origin of expression on
the editing vector thereby producing edited cells in which the
editing vector has been cured 192. Additionally, growing the cells
performs "passive" curing as well, where the editing vector is
"diluted out" of the growing cell population, as removing from the
medium the antibiotic that selects for the editing vector removes
pressure on cells to retain the editing vector. Finally, at step
193 the edited cells are allowed to recover, then are grown to,
e.g., OD=0.5, and rendered electrocompetent 193 so that the cells
194 are ready for another round of editing.
[0109] FIG. 1G depicts an exemplary recursive method 160b using a
bulk liquid editing protocol. The recursive method 160b--as with
the recursive method 160a--begins with competent cells 162, for
example E. coli cells that have been previously transformed with an
engine vector expressing a nucleic acid-guided nuclease such as
MAD7 and a selective marker, such as a chloramphenicol resistance
gene. At step 163, the competent cells are transformed with a
library of editing vectors, where each editing vector in the
library of editing vectors comprises an editing cassette with a
sequence coding for an editing gRNA and a donor DNA, and each
editing vector also comprises an antibiotic resistance gene such as
a gene conferring resistance to, e.g., carbenicillin, where the
antibiotic resistance gene located on the editing vector is
different from the antibiotic resistance gene located on the engine
vector. Following transformation, the cells are allowed to recover
164 in medium without antibiotic, and then at step 165, the cells
are diluted, if necessary, or transferred into medium containing
chloramphenicol and carbenicillin to select for both the engine and
editing vectors.
[0110] After transfer to a larger liquid volume 166b for bulk
liquid editing, the cells are outgrown; that is, the cells are
grown to saturation (see FIG. 1B). Like the curing step, it has
been determined that editing in a bulk liquid culture is optimized
when the cells have been grown to late log phase or saturation
before inducing editing. Again, the cells are grown through at
least 60% of log phase, or at least 75% of log phase, 80% of log
phase, 85% of log phase, 90% of log phase, 95% of log phase, or are
in a stationary phase of growth before inducing curing. Thus, the
cells are outgrown, then arabinose is added to the medium, inducing
the lambda Red recombination system encoded by, e.g., the engine
vector (see, e.g., FIGS. 1C and 1D). Once the lambda Red
recombination system is induced for, e.g., 30 minutes to an hour,
the temperature is increased 42.degree. C. for 2 hours, thereby
inducing the pL promoters driving transcription of the nuclease on
the engine vector and the editing cassette on the editing vector
and thus editing. After 2 hours at 42.degree. C., the cells are
grown at 30.degree. C. for at least 9 more hours.
[0111] At step 173, the cells are washed to, e.g., remove the
carbenicillin, and resuspended in medium containing chloramphenicol
(continuing to select for the engine vector) and the cells may be
grown for another length of time to assure that the cells are in
the stationary phase of growth for curing. The editing vectors in
the cells are then cured 168 by inducing transcription of the
editing gRNA that cuts the pUC origin of replication on the editing
vector. Induction of the anti-pUC gRNA is accomplished by first
raising the temperature of the culture to 42.degree. C. for 2
hours, thereby inducing the pL promoter driving transcription of
the nuclease, and second by the addition of 2,4
diacetylphloroglucinol (DAPG) to induce the pPhIF promoter driving
transcription of the anti-pUC gRNA (see, e.g., the vector
architecture of the engine and editing vectors in FIGS. 1C and 1D).
After 2 hours at 42.degree. C., the temperature of the cell culture
is lowered to 30.degree. C., and the cells are allowed to recover
and grow for 6 additional hours. The co-expression of the nuclease
and the anti-pUC gRNA permits targeting of the pUC origin of
expression on the editing vector. At step 169, the cells are washed
with medium containing chloramphenicol (again to select for cells
comprising the engine vector), and the cells are allowed to
recover. At this point, the edited cells can be grown to a proper
OD (e.g., OD=0.5), made electrocompetent once more 170, and
subjected to a second round of editing 171.
[0112] FIG. 1H depicts the exemplary recursive method using the
bulk liquid editing protocol of FIG. 1G. FIG. 1H depicts an
exemplary protocol 1000 for performing nucleic acid-guided nuclease
genome editing and curing. FIG. 1H depicts the protocol 160b shown
in FIG. 1G for editing cells. First, a library or collection of
editing vectors 1002 (editing vectors each comprising an editing
cassette) is introduced 1003 (e.g., electroporated) into cultured
cells 1004 that comprise a coding sequence for a nuclease (e.g.,
MAD7) under the control of an inducible promoter, contained on an
engine vector (along with a selectable marker) that has already
been transformed into the cells or already integrated into the
genome of the cells being transformed. The editing vectors 1002
comprise a donor DNA comprising a PAM or spacer-altering sequence,
a coding sequence for an editing gRNA under the control of an
inducible promoter, and a selectable marker. Depending on whether
the system is an engine curing system or a self-curing system, a
coding sequence for a curing gRNA under the control of an inducible
promoter is located on the engine vector or editing vector,
respectively.
[0113] At step 1005, cells are grown until they reach stationary
phase, or nearly so. Once the cells reach the stationary phase
1006, editing is induced 1007 where transcription of the nuclease
and gRNA is induced and the cells in the culture 1008 are edited
and then allowed to recover from editing. Induction of editing in
some embodiments comprises raising the temperature of the bulk
liquid culture to 42.degree. C. to activate the pL promoter driving
transcription of the nuclease, editing gRNA, and donor DNA. Once
recovered, the cells are washed, and resuspended in medium 1009 and
again outgrown so that the cells are in the stationary phase of
growth or nearly so. The editing vectors in the cells are then
cured 1010 by inducing transcription of the curing gRNA that cuts
the pUC origin of replication on the editing vector. Induction of
the anti-pUC gRNA is accomplished by first raising the temperature
of the culture to 42.degree. C. for 2 hours, thereby inducing the
pL promoter driving transcription of the nuclease, and second by
the addition of 2,4 diacetylphloroglucinol (DAPG) to induce the
pPhIF promoter driving transcription of the anti-pUC gRNA (see the
vector architectures of the exemplary engine and editing vectors in
FIGS. 1C and 1D).
[0114] After 2 hours at 42.degree. C., the temperature of the cell
culture is lowered to 30.degree. C., and the cells are allowed to
recover and grow for 6 additional hours. The co-expression of the
nuclease and the anti-pUC gRNA permits targeting of the pUC origin
of expression on the editing vector. Growing the cells further
performs "passive editing" as described above. At step 1011, the
cells are washed with medium containing chloramphenicol (again to
select for cells comprising the engine vector), and the cells are
allowed to recover. At this point, the edited cells can be made
electrocompetent once more 1012 and be subjected to a second round
of editing.
[0115] FIG. 1 depicts an exemplary recursive method 160c using a
solid wall isolation device. The recursive method 160c--as with the
recursive methods 160a and 160b--begins with competent cells 162,
for example, E. coli cells that have been previously transformed
with an engine vector expressing a nucleic acid-guided nuclease
such as MAD7 and a selective marker, such as a chloramphenicol
resistance gene. At step 163, the competent cells are transformed
with a library of editing vectors, where each editing vector in the
library of editing vectors comprises an editing cassette with a
sequence coding for an editing gRNA and a donor DNA, and each
editing vector also comprises an antibiotic resistance gene such as
a gene conferring resistance to carbenicillin or other antibiotic
gene different from the antibiotic gene on the engine vector.
Following transformation, the cells are allowed to recover 164 in
medium without antibiotic, and then at step 165, the cells are
diluted, if necessary, and loaded onto a solid wall singulation,
induction, isolation and normalization device (a SWIIN, described
in detail below in relation to FIGS. 6B-6E) 166c where the editing
process takes place. The cells are loaded into the SWIIN in a
Poisson or substantial Poisson distribution (described in detail
below) and are grown for at 30.degree. C. for approximately 8-9
hours. After the initial growth phase, medium exchange is
performed, adding arabinose to the culture medium to induce the
lambda Red recombination system encoded by, e.g., the engine vector
(see, e.g., the exemplary vector architectures of FIGS. 1C and
1D).
[0116] Once the lambda Red recombination system is induced for,
e.g., 30 minutes to an hour, the temperature of the SWIIN is
increased 42.degree. C. for 2 hours, thereby inducing the pL
promoters driving transcription of the nuclease on the engine
vector and the editing cassette (editing gRNA and donor DNA) on the
editing vector and thus inducing editing. After 2 hours at
42.degree. C., the cells are grown at 30.degree. C. for at least 9
more hours.
[0117] At step 175, the cells are recovered from the SWIIN and
washed to, e.g., remove the carbenicillin. The cells are then
resuspended in medium containing chloramphenicol (continuing to
select for the engine vector) and out-grown so that the cells are
in late log phase or stationary phase. The editing vectors in the
cells are then cured 168 by inducing transcription of the curing
gRNA that cuts the pUC origin of replication on the editing vector.
Induction of the anti-pUC gRNA is accomplished by first raising the
temperature of the culture to 42.degree. C. for 2 hours, thereby
inducing the pL promoter driving transcription of the nuclease, and
second by performing media exchange to medium with of 2,4
diacetylphloroglucinol (DAPG) added to induce the pPhIF promoter
driving transcription of the anti-pUC gRNA (see FIGS. 1C and
1D).
[0118] After 2 hours at 42.degree. C., the temperature of the cell
culture is lowered to 30.degree. C., and the cells are allowed to
recover and grow for 6 hours. The co-expression of the nuclease and
the anti-pUC gRNA permits targeting of the pUC origin of expression
on the editing vector. At step 169, the cells are washed with
medium containing chloramphenicol (again to select for cells
comprising the engine vector), and the cells are allowed to recover
and are grown to be made electrocompetent. Growing the cells also
performs "passive" curing, where the editing vector is "diluted
out" of the growing cell population, as removing from the medium
the antibiotic that selects for the editing vector removes pressure
on cells to retain the editing vector. At this point, the edited
cells can be made electrocompetent once more 170 and be subjected
to a second round of editing 171. FIG. 6A depicts and the
description of FIG. 6A presents this method in additional
detail.
Automated Cell Editing Instruments and Modules to Perform Nucleic
Acid-Guided Nuclease Editing including Curing
Automated Cell Editing Instruments
[0119] FIG. 2A depicts an exemplary automated multi-module cell
processing instrument 200 to, e.g., perform one of the exemplary
recursive workflows for targeted gene editing of live yeast cells.
The instrument 200, for example, may be and preferably is designed
as a stand-alone desktop instrument for use within a laboratory
environment. The instrument 200 may incorporate a mixture of
reusable and disposable components for performing the various
integrated processes in conducting automated genome cleavage and/or
editing in cells without human intervention. Illustrated is a
gantry 202, providing an automated mechanical motion system
(actuator) (not shown) that supplies XYZ axis motion control to,
e.g., an automated (i.e., robotic) liquid handling system 258
including, e.g., an air displacement pipettor 232 which allows for
cell processing among multiple modules without human intervention.
In some automated multi-module cell processing instruments, the air
displacement pipettor 232 is moved by gantry 202 and the various
modules and reagent cartridges remain stationary; however, in other
embodiments, the liquid handling system 258 may stay stationary
while the various modules and reagent cartridges are moved.
[0120] Also included in the automated multi-module cell processing
instrument 200 are reagent cartridges 210 comprising reservoirs 212
and transformation module 230 (e.g., a flow-through electroporation
device as described in detail in relation to FIGS. 5C-5G and an
exemplary reagent cartridge is described in relation to FIGS. 5A
and 5B), as well as wash reservoirs 206, cell input reservoir 251
and cell output reservoir 253. The wash reservoirs 206 may be
configured to accommodate large tubes, for example, wash solutions,
or solutions that are used often throughout an iterative process.
Although two of the reagent cartridges 210 comprise a wash
reservoir 206 in FIG. 2A, the wash reservoirs instead could be
included in a wash cartridge where the reagent and wash cartridges
are separate cartridges. In such a case, the reagent cartridge 210
and wash reservoir 206 may be identical except for the consumables
(reagents or other components contained within the various inserts)
inserted therein.
[0121] In some implementations, the reagent cartridges 210 are
disposable kits comprising reagents and cells for use in the
automated multi-module cell processing/editing instrument 200. For
example, a user may open and position each of the reagent
cartridges 210 comprising various desired inserts and reagents
within the chassis of the automated multi-module cell editing
instrument 200 prior to activating cell processing. Further, each
of the reagent cartridges 210 may be inserted into receptacles in
the chassis having different temperature zones appropriate for the
reagents contained therein.
[0122] Also illustrated in FIG. 2A is the robotic liquid handling
system 258 including the gantry 202 and air displacement pipettor
232. In some examples, the robotic handling system 258 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 215
may be provided in a pipette transfer tip supply 214 for use with
the air displacement pipettor 232.
[0123] Inserts or components of the reagent cartridges 210, in some
implementations, are marked with machine-readable indicia (not
shown), such as bar codes, for recognition by the robotic handling
system 258. For example, the robotic liquid handling system 258 may
scan one or more inserts within each of the reagent cartridges 210
to confirm contents. In other implementations, machine-readable
indicia may be marked upon each reagent cartridge 210, and a
processing system (not shown, but see element 237 of FIG. 2B) of
the automated multi-module cell editing instrument 200 may identify
a stored materials map based upon the machine-readable indicia. In
the embodiment illustrated in FIG. 2A, a cell growth module
comprises a cell growth vial 218 (described in greater detail below
in relation to FIGS. 3A-3D). Additionally seen is the TFF module
222 (described above in detail in relation to FIGS. 4A-4E). Also
illustrated as part of the automated multi-module cell processing
instrument 200 of FIG. 2A is a singulation module 240 (e.g., a
solid wall isolation, incubation and normalization device (SWIIN
device) is shown here) described herein in relation to FIGS. 6B-6E,
served by, e.g., robotic liquid handing system 258 and air
displacement pipettor 232. Additionally seen is a selection module
220. Also note the placement of three heatsinks 255.
[0124] FIG. 2B is a simplified representation of the contents of
the exemplary multi-module cell processing instrument 200 depicted
in FIG. 2A. Cartridge-based source materials (such as in reagent
cartridges 210), for example, may be positioned in designated areas
on a deck of the instrument 200 for access by an air displacement
pipettor 232 moved by gantry 202 with pipette tips supplied by
pipette transfer tip supply 214. The deck of the multi-module cell
processing instrument 200 may include a protection sink such that
contaminants spilling, dripping, or overflowing from any of the
modules of the instrument 200 are contained within a lip of the
protection sink. Also seen are reagent cartridges 210, which are
shown disposed with thermal assemblies 211 which can create
temperature zones appropriate for different regions. Note that one
of the reagent cartridges also comprises a flow-through
electroporation device 230 (FTEP), served by FTEP interface (e.g.,
manifold arm) and actuator 231. Also seen is TFF module 222 with
adjacent thermal assembly 225, where the TFF module is served by
TFF interface (e.g., manifold arm) and actuator 233. Thermal
assemblies 225, 235, and 245 encompass thermal electric devices
such as Peltier devices, as well as heatsinks, fans and coolers.
The rotating growth vial 218 is within a growth module 234, where
the growth module is served by two thermal assemblies 235.
Selection module is seen at 220. Also seen is the SWIIN module 240,
comprising a SWIIN cartridge (not shown), where the SWIIN module
also comprises a thermal assembly 245, illumination 243 (in this
embodiment, backlighting), evaporation and condensation control
249, and where the SWIIN module is served by SWIIN interface (e.g.,
manifold arm) and actuator 247. Also seen in this view is touch
screen display 201, display actuator 203, illumination 205 (one on
either side of multi-module cell processing instrument 200),
cooling grate 264, and cameras 239 (one illumination device on
either side of multi-module cell processing instrument 200).
Finally, element 237 comprises electronics, such as circuit control
boards, high-voltage amplifiers, power supplies, and power entry;
as well as pneumatics, such as pumps, valves and sensors.
[0125] FIG. 2C illustrates a front perspective view of multi-module
cell processing instrument 200 for use in as a desktop version of
the automated multi-module cell editing instrument 200. For
example, a chassis 290 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 290 may be and preferably is designed to hold all modules
and disposable supplies used in automated cell processing and to
perform all processes required without human intervention; that is,
chassis 290 is configured to provide an integrated, stand-alone
automated multi-module cell processing instrument. As illustrated
in FIG. 2C, chassis 290 includes touch screen display 201, cooling
grate 264, which allows for air flow via an internal fan (not
shown). The touch screen display provides information to a user
regarding the processing status of the automated multi-module cell
editing instrument 200 and accepts inputs from the user for
conducting the cell processing. In this embodiment, the chassis 290
is lifted by adjustable feet 270a, 270b, 270c and 270d (feet
270a-270c are shown in this FIG. 2C). Adjustable feet 270a-270d,
for example, allow for additional air flow beneath the chassis
290.
[0126] Inside the chassis 290, in some implementations, will be
most or all of the components described in relation to FIGS. 2A and
2B, including the robotic liquid handling system disposed along a
gantry, reagent cartridges 210 including a flow-through
electroporation device (not shown in this FIG. 2C), a rotating
growth vial 218 in a cell growth module 234 (not shown in this FIG.
2C), a tangential flow filtration module 222 (not shown in this
FIG. 2C), a SWIIN module 240 as well as interfaces and actuators
for the various modules (not shown in this FIG. 2C). In addition,
chassis 290 houses control circuitry, liquid handling tubes, air
pump controls, valves, sensors, thermal assemblies (e.g., heating
and cooling units) and other control mechanisms (not shown in this
FIG. 2C). For examples of multi-module cell editing instruments,
see U.S. Pat. Nos. 10,253,316; 10,329,559; 10,323,242; 10,421,959;
10,465,185; 10,519,437; 10,584,333; and 10,584,334 and U.S. Ser.
No. 16/750,369, filed 23 Jan. 2020; Ser. No. 16/822,249, filed 18
Mar. 2020; and Ser. No. 16/837,985, filed 1 Apr. 2020, all of which
are herein incorporated by reference in their entirety.
The Rotating Cell Growth Module
[0127] FIG. 3A shows one embodiment of a rotating growth vial 300
for use with the cell growth device and in the automated
multi-module cell processing instruments described herein. The
rotating growth vial 300 is an optically-transparent container
having an open end 304 for receiving liquid media and cells, a
central vial region 306 that defines the primary container for
growing cells, a tapered-to-constricted region 318 defining at
least one light path 310, a closed end 316, and a drive engagement
mechanism 312. The rotating growth vial 300 has a central
longitudinal axis 320 around which the vial rotates, and the light
path 310 is generally perpendicular to the longitudinal axis of the
vial. The first light path 310 is positioned in the lower
constricted portion of the tapered-to-constricted region 318.
Optionally, some embodiments of the rotating growth vial 300 have a
second light path 308 in the tapered region of the
tapered-to-constricted region 318. 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 are not affected by the rotational speed of the growth
vial. The first light path 310 is shorter than the second light
path 308 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 308 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).
[0128] The drive engagement mechanism 312 engages with a motor (not
shown) to rotate the vial. In some embodiments, the motor drives
the drive engagement mechanism 312 such that the rotating growth
vial 300 is rotated in one direction only, and in other
embodiments, the rotating growth vial 300 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 300 (and the cell culture contents) are
subjected to an oscillating motion. Further, the choice of whether
the culture is subjected to oscillation and the periodicity
therefor may be selected by the user. 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 400 may be oscillated at a
first periodicity (e.g., every 60 seconds), and then a later stage
of cell growth the rotating growth vial 300 may be oscillated at a
second periodicity (e.g., every one second) different from the
first periodicity.
[0129] The rotating growth vial 300 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 304 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
system. 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 304 may optionally
include an extended lip 302 to overlap and engage with the cell
growth device. In automated systems, the rotating growth vial 300
may be tagged with a barcode or other identifying means that can be
read by a scanner or camera (not shown) that is part of the
automated system.
[0130] The volume of the rotating growth vial 300 and the volume of
the cell culture (including growth medium) may vary greatly, but
the volume of the rotating growth vial 300 must be large enough to
generate a specified total number of cells. In practice, the volume
of the rotating growth vial 300 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 and mixing in the rotating growth vial 300.
Proper aeration promotes uniform cellular respiration within the
growth media. Thus, the volume of the cell culture should be
approximately 5-85% of the volume of the growth vial or from 20-60%
of the volume of the growth vial. For example, for a 30 mL growth
vial, the volume of the cell culture would be from about 1.5 mL to
about 26 mL, or from 6 mL to about 18 mL.
[0131] The rotating growth vial 300 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
cyclic olefin copolymer (COC), glass, polyvinyl chloride,
polyethylene, polyamide, 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.
[0132] FIG. 3B is a perspective view of one embodiment of a cell
growth device 330. FIG. 3C depicts a cut-away view of the cell
growth device 330 from FIG. 3B. In both figures, the rotating
growth vial 300 is seen positioned inside a main housing 336 with
the extended lip 302 of the rotating growth vial 300 extending
above the main housing 336. Additionally, end housings 352, a lower
housing 332 and flanges 334 are indicated in both figures. Flanges
334 are used to attach the cell growth device 330 to
heating/cooling means or other structure (not shown). FIG. 3C
depicts additional detail. In FIG. 3C, upper bearing 342 and lower
bearing 340 are shown positioned within main housing 336. Upper
bearing 342 and lower bearing 340 support the vertical load of
rotating growth vial 300. Lower housing 332 contains the drive
motor 338. The cell growth device 330 of FIG. 3C comprises two
light paths: a primary light path 344, and a secondary light path
350. Light path 344 corresponds to light path 310 positioned in the
constricted portion of the tapered-to-constricted portion of the
rotating growth vial 300, and light path 350 corresponds to light
path 308 in the tapered portion of the tapered-to-constricted
portion of the rotating growth via 316. Light paths 310 and 308 are
not shown in FIG. 3C but may be seen in FIG. 3A. In addition to
light paths 344 and 340, there is an emission board 348 to
illuminate the light path(s), and detector board 346 to detect the
light after the light travels through the cell culture liquid in
the rotating growth vial 300.
[0133] The motor 338 engages with drive mechanism 312 and is used
to rotate the rotating growth vial 300. In some embodiments, motor
338 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 338 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.
[0134] Main housing 336, end housings 352 and lower housing 332 of
the cell growth device 330 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 300 is envisioned in some embodiments to be reusable, but
preferably is consumable, the other components of the cell growth
device 330 are preferably reusable and function as a stand-alone
benchtop device or as a module in a multi-module cell processing
system.
[0135] The processor (not shown) of the cell growth device 330 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 (not shown) of the cell growth device
330--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 device 330, where the
second spectrophotometer is used to read a blank at designated
intervals.
[0136] FIG. 3D illustrates a cell growth device 330 as part of an
assembly comprising the cell growth device 330 of FIG. 3B coupled
to light source 390, detector 392, and thermal components 394. The
rotating growth vial 300 is inserted into the cell growth device.
Components of the light source 390 and detector 392 (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 332 that
houses the motor that rotates the rotating growth vial 300 is
illustrated, as is one of the flanges 334 that secures the cell
growth device 330 to the assembly. Also, the thermal components 394
illustrated are a Peltier device or thermoelectric cooler. In this
embodiment, thermal control is accomplished by attachment and
electrical integration of the cell growth device 330 to the thermal
components 394 via the flange 334 on the base of the lower housing
332. 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 300 is controlled to approximately
+/-0.5.degree. C.
[0137] 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 300 by piercing
though the foil seal or film. The programmed software of the cell
growth device 330 sets the control temperature for growth,
typically 30.degree. C., then slowly starts the rotation of the
rotating growth vial 300. The cell/growth media mixture slowly
moves vertically up the wall due to centrifugal force allowing the
rotating growth vial 300 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.
[0138] One application for the cell growth device 330 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 minutes. While
the cell growth device 330 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. As with optional measure of cell growth in relation to
the solid wall device or module described supra, spectroscopy using
visible, UV, or near infrared (NIR) light allows monitoring the
concentration of nutrients and/or wastes in the cell culture and
other spectroscopic measurements may be made; that is, other
spectral properties can be measured via, e.g., dielectric impedance
spectroscopy, visible fluorescence, fluorescence polarization, or
luminescence. Additionally, the cell growth device 330 may include
additional sensors for measuring, e.g., dissolved oxygen, carbon
dioxide, pH, conductivity, and the like. For additional details
regarding rotating growth vials and cell growth devices see U.S.
Pat. Nos. 10,435,662; 10,443,031; 10,590,375 and U.S. Ser. No.
16/80,640, filed 3 Feb. 2020.
The Cell Concentration Module
[0139] As described above in relation to the rotating growth vial
and cell growth module, in order to obtain an adequate number of
cells for transformation or transfection, cells typically are grown
to a specific optical density in medium appropriate for the growth
of the cells of interest; however, for effective transformation or
transfection, it is desirable to decrease the volume of the cells
as well as render the cells competent via buffer or medium
exchange. Thus, one sub-component or module that is desired in cell
processing systems for the processes listed above is a module or
component that can grow, perform buffer exchange, and/or
concentrate cells and render them competent so that they may be
transformed or transfected with the nucleic acids needed for
engineering or editing the cell's genome.
[0140] FIG. 4A shows a retentate member 422 (top), permeate member
420 (middle) and a tangential flow assembly 410 (bottom) comprising
the retentate member 422, membrane 424 (not seen in FIG. 4A), and
permeate member 420 (also not seen). In FIG. 4A, retentate member
422 comprises a tangential flow channel 402, which has a serpentine
configuration that initiates at one lower corner of retentate
member 422--specifically at retentate port 428--traverses across
and up then down and across retentate member 422, ending in the
other lower corner of retentate member 422 at a second retentate
port 428. Also seen on retentate member 422 are energy directors
491, which circumscribe the region where a membrane or filter (not
seen in this FIG. 4A) is seated, as well as interdigitate between
areas of channel 402. Energy directors 491 in this embodiment mate
with and serve to facilitate ultrasonic welding or bonding of
retentate member 422 with permeate/filtrate member 420 via the
energy director component 491 on permeate/filtrate member 420 (at
right). Additionally, countersinks 423 can be seen, two on the
bottom one at the top middle of retentate member 422. Countersinks
423 are used to couple and tangential flow assembly 410 to a
reservoir assembly (not seen in this FIG. 4A but see FIG. 4B).
[0141] Permeate/filtrate member 420 is seen in the middle of FIG.
4A and comprises, in addition to energy director 491, through-holes
for retentate ports 428 at each bottom corner (which mate with the
through-holes for retentate ports 428 at the bottom corners of
retentate member 422), as well as a tangential flow channel 402 and
two permeate/filtrate ports 426 positioned at the top and center of
permeate member 420. The tangential flow channel 402 structure in
this embodiment has a serpentine configuration and an undulating
geometry, although other geometries may be used. Permeate member
420 also comprises countersinks 423, coincident with the
countersinks 423 on retentate member 420.
[0142] At bottom of FIG. 4A is a tangential flow assembly 410
comprising the retentate member 422 and permeate member 420 seen in
this FIG. 4A. In this view, retentate member 422 is "on top" of the
view, a membrane (not seen in this view of the assembly) would be
adjacent and under retentate member 422 and permeate member 420
(also not seen in this view of the assembly) is adjacent to and
beneath the membrane. Again countersinks 423 are seen, where the
countersinks in the retentate member 422 and the permeate member
420 are coincident and configured to mate with threads or mating
elements for the countersinks disposed on a reservoir assembly (not
seen in FIG. 4A but see FIG. 4B).
[0143] A membrane or filter is disposed between the retentate and
permeate members, where fluids can flow through the membrane but
cells cannot and are thus retained in the flow channel disposed in
the retentate member. 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 20 .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 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 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.
[0144] The length of the channel structure 402 may vary depending
on the type and volume of the cell culture to be grown and the
optical density of the cell culture to be concentrated. The length
of the channel structure typically is from 60 mm to 300 mm, or from
70 mm to 200 mm, or from 80 mm to 100 mm. The cross-section
configuration of the flow channel 402 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 402 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.
Moreover, the volume of the channel in the retentate 422 and
permeate 420 members may be different depending on the depth of the
channel in each member.
[0145] FIG. 4B shows front perspective (top) and rear perspective
(bottom) views of a reservoir assembly 450 configured to be used
with the tangential flow assembly 410 seen in FIG. 4A. Seen in the
front perspective view (e.g., "front" being the side of reservoir
assembly 450 that is coupled to the tangential flow assembly 410
(seen in FIG. 4A) are retentate reservoirs 452 on either side of
permeate reservoir 454. Also seen are permeate ports 426, retentate
ports 428, and three threads or mating elements 425 for
countersinks 423 (countersinks 423 not seen in this FIG. 4B).
Threads or mating elements 425 for countersinks 423 are configured
to mate or couple the tangential flow assembly 410 (seen in FIG.
4A) to reservoir assembly 450. Alternatively or in addition,
fasteners, sonic welding or heat stakes may be used to mate or
couple the tangential flow assembly 410 to reservoir assembly 450.
In addition is seen gasket 445 covering the top of reservoir
assembly 450, with pipette tip 405 shown inserted into the
left-most retentate reservoir. Gasket 445 is described in detail in
relation to FIG. 4E. At left in FIG. 4B is a rear perspective view
of reservoir assembly 450, where "rear" is the side of reservoir
assembly 450 that is not coupled to the tangential flow assembly.
Seen are retentate reservoirs 452, permeate reservoir 454, gasket
445, with pipette tip 405 shown inserted into the right-most
retentate reservoir.
[0146] The TFF device 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.
[0147] FIG. 4C depicts a top-down view of the reservoir assemblies
450 shown in FIG. 4B. FIG. 4D depicts a cover 444 for reservoir
assembly 450 shown in FIGS. 4B and 4E depicts a gasket 445 that in
operation is disposed on cover 444 of reservoir assemblies 450
shown in FIG. 4B. FIG. 4C is a top-down view of reservoir assembly
450, showing the tops of the two retentate reservoirs 452, one on
either side of permeate reservoir 454. Also seen are grooves 432
that will mate with a pneumatic port (not shown), and fluid
channels 434 that reside at the bottom of retentate reservoirs 452,
which fluidically couple the retentate reservoirs 452 with the
retentate ports 428 (not shown), via the through-holes for the
retentate ports in permeate member 420 and membrane 424 (also not
shown). FIG. 4D depicts a cover 444 that is configured to be
disposed upon the top of reservoir assembly 450. Cover 444 has
round cut-outs at the top of retentate reservoirs 452 and
permeate/filtrate reservoir 454. Again at the bottom of retentate
reservoirs 452 fluid channels 434 can be seen, where fluid channels
434 fluidically couple retentate reservoirs 452 with the retentate
ports 428 (not shown). Also shown are three pneumatic ports 430 for
each retentate reservoir 452 and permeate/filtrate reservoir 454.
FIG. 4E depicts a gasket 445 that is configured to be disposed upon
the cover 444 of reservoir assembly 450. Seen are three fluid
transfer ports 442 for each retentate reservoir 452 and for
permeate/filtrate reservoir 454. Again, three pneumatic ports 430,
for each retentate reservoir 452 and for permeate/filtrate
reservoir 454, are shown.
[0148] The overall work flow for cell growth comprises loading a
cell culture to be grown into a first retentate reservoir,
optionally bubbling air or an appropriate gas through the cell
culture, passing or flowing the cell culture through the first
retentate port then tangentially through the TFF channel structure
while collecting medium or buffer through one or both of the
permeate ports 426, collecting the cell culture through a second
retentate port 428 into a second retentate reservoir, optionally
adding additional or different medium to the cell culture and
optionally bubbling air or gas through the cell culture, then
repeating the process, all while measuring, e.g., the optical
density of the cell culture in the retentate reservoirs
continuously or at desired intervals. Measurements of optical
densities (OD) at programmed time intervals are accomplished using
a 600 nm Light Emitting Diode (LED) that has been columnated
through an optic into the retentate reservoir(s) containing the
growing cells. The light continues through a collection optic to
the detection system which consists of a (digital) gain-controlled
silicone photodiode. Generally, optical density is 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 TFF
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.
[0149] In the channel structure, the membrane bifurcating the flow
channels retains the cells on one side of the membrane (the
retentate side 422) and allows unwanted medium or buffer to flow
across the membrane into a filtrate or permeate side (e.g.,
permeate member 420) of the device. Bubbling air or other
appropriate gas through the cell culture both aerates and mixes the
culture to enhance cell growth. During the process, medium that is
removed during the flow through the channel structure is removed
through the permeate/filtrate ports 406. Alternatively, cells can
be grown in one reservoir with bubbling or agitation without
passing the cells through the TFF channel from one reservoir to the
other.
[0150] 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. As with the cell growth
process, 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 permeate/filtrate side
(e.g., permeate member 420) 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 ports
428, and the medium/buffer that has passed through the membrane is
collected through one or both of the permeate/filtrate ports 426.
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.
[0151] The medium or buffer used to suspend the cells in the cell
concentration device/module may be any suitable medium or buffer
for the type of cells being transformed or transfected, such as LB,
SOC, TPD, YPG, YPAD, MEM, DMEM, IMDM, RPMI, Hanks', PBS and
Ringer's solution, where the media may be provided in a reagent
cartridge as part of a kit.
[0152] In both the cell growth and concentration processes, passing
the cell sample through the TFF device and collecting the cells in
one of the retentate ports 404 while collecting the medium in one
of the permeate/filtrate ports 406 is considered "one pass" of the
cell sample. The transfer between retentate reservoirs "flips" the
culture. The retentate and permeate ports collecting the cells and
medium, respectively, for a given pass reside on the same end of
TFF device/module with fluidic connections arranged so that there
are two distinct flow layers for the retentate and
permeate/filtrate sides, but if the retentate port 404 resides on
the retentate member of device/module (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 permeate/filtrate port 406 will reside on the permeate member
of device/module 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).
Due to the high pressures used to transfer the cell culture and
fluids through the flow channel of the TFF device, the effect of
gravity is negligible.
[0153] At the conclusion of a "pass" in either of the growth and
concentration processes, the cell sample is collected by passing
through the retentate port 428 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 port 428 and into retentate reservoir
(not shown) on the opposite end of the device/module from the
retentate port 428 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 permeate port 426 on
the opposite end of the device/module from the permeate port 426
that was used to collect the filtrate during the first pass, or
through both ports. This alternating process of passing the
retentate (the concentrated cell sample) through the device/module
is repeated until the cells have been grown to a desired optical
density, and/or concentrated to a desired volume, and both permeate
ports (i.e., if there are more than one) 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 growth may (and
typically do) take place simultaneously, and buffer exchange and
cell concentration may (and typically do) take place
simultaneously. For further information and alternative embodiments
on TFFs see, e.g., U.S. Ser. No. 16/98,302, filed 22 Feb. 2020.
Nucleic Acid Assembly Module
[0154] 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.
[0155] 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.
[0156] Regulatory elements are 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.
[0157] 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.
[0158] 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 US 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. Nos. 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).
[0159] 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.
[0160] 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
[0161] FIGS. 5A and 5B depict the structure and components of an
embodiment of an exemplary reagent cartridge useful in the
automated multi-module instrument described therein. In FIG. 5A,
reagent cartridge 500 comprises a body 502, which has reservoirs
504. One reservoir 504 is shown empty, and two of the reservoirs
have individual tubes (not shown) inserted therein, with individual
tube covers 505. Additionally shown are rows of reservoirs into
which have been inserted co-joined rows of large tubes 503a, and
co-joined rows of small tubes 503b. The co-joined rows of tubes are
presented in a strip, with outer flanges 507 that mate on the
backside of the outer flange (not shown) with an indentation 509 in
the body 502, so as to secure the co-joined rows of tubes (503a and
503b) to the reagent cartridge 500. Shown also is a base 511 of
reagent cartridge body 502. Note that the reservoirs 504 in body
502 are shaped generally like the tubes in the co-joined tubes that
are inserted into these reservoirs 504.
[0162] FIG. 5B depicts the reagent cartridge 500 in FIG. 5A with a
row of co-joined large tubes 503a, a row of co-joined small tubes
503b, and one large tube 560 with a cover 505 removed from (i.e.,
depicted above) the reservoirs 504 of the reagent cartridge 500.
Again, the co-joined rows of tubes are presented in a strip, with
individual large tubes 561 shown, and individual small tubes 555
shown. Again, each strip of co-joined tubes comprises outer flanges
507 that mate on the backside (not shown) of the outer flange with
an indentation 509 in the body 502, to secure the co-joined rows of
tubes (503a and 503b) to the reagent cartridge 500. As in FIG. 5A,
reagent cartridge body 502 comprises a base 511. Reagent cartridge
500 may be made from any suitable material, including stainless
steel, aluminum, or plastics including polyvinyl chloride, cyclic
olefin copolymer (COC), polyethylene, polyamide, polypropylene,
acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK),
poly(methyl methylacrylate) (PMMA), polysulfone, and polyurethane,
and co-polymers of these and other polymers. Again, if reagent
cartridge 500 is disposable, it preferably is made of plastic. In
addition, in many embodiments the material used to fabricate the
cartridge is thermally-conductive, as reagent cartridge 500 may
contact a thermal device (not shown) that heats or cools reagents
in the reagent reservoirs 504, including reagents in co-joined
tubes. In some embodiments, the thermal device is a Peltier device
or thermoelectric cooler.
[0163] FIGS. 5C and 5D are top perspective and bottom perspective
views, respectively, of an exemplary FTEP device 550 that may be
part of (e.g., a component in) reagent cartridge 500 in FIGS. 5A
and 5B or may be a stand-alone module; that is, not a part of a
reagent cartridge or other module. FIG. 5C depicts an FTEP device
550. The FTEP device 550 has wells that define cell sample inlets
552 and cell sample outlets 554. FIG. 5D is a bottom perspective
view of the FTEP device 550 of FIG. 5C. An inlet well 552 and an
outlet well 554 can be seen in this view. Also seen in FIG. 5D are
the bottom of an inlet 562 corresponding to well 552, the bottom of
an outlet 564 corresponding to the outlet well 554, the bottom of a
defined flow channel 566 and the bottom of two electrodes 568 on
either side of flow channel 566. The FTEP devices may comprise
push-pull pneumatic means to allow multi-pass electroporation
procedures; that is, cells to electroporated may be "pulled" from
the inlet toward the outlet for one pass of electroporation, then
be "pushed" from the outlet end of the FTEP device toward the inlet
end to pass between the electrodes again for another pass of
electroporation. Further, this process may be repeated one to many
times. For additional information regarding FTEP devices, see,
e.g., U.S. Pat. Nos. 10,435,713; 10,443,074; 10,323,258; and
10,508,288. Further, other embodiments of the reagent cartridge may
provide or accommodate electroporation devices that are not
configured as FTEP devices, such as those described in U.S. Ser.
No. 16/109,156, filed 22 Aug. 2018. For reagent cartridges useful
in the present automated multi-module cell processing instruments,
see, e.g., U.S. Pat. Nos. 10,376,889; 10,406,525; 10,576,474; and
U.S. Ser. No. 16/749,757, filed 22 Jan. 2020; and Ser. No.
16/827,222, filed 23 Mar. 2020.
[0164] Additional details of the FTEP devices are illustrated in
FIGS. 5E-5G. Note that in the FTEP devices in FIGS. 5E-5G the
electrodes are placed such that a first electrode is placed between
an inlet and a narrowed region of the flow channel, and the second
electrode is placed between the narrowed region of the flow channel
and an outlet. FIG. 5E shows a top planar view of an FTEP device
550 having an inlet 552 for introducing a fluid containing cells
and exogenous material into FTEP device 550 and an outlet 554 for
removing the transformed cells from the FTEP following
electroporation. The electrodes 568 are introduced through channels
(not shown) in the device.
[0165] FIG. 5F shows a cutaway view from the top of the FTEP device
550, with the inlet 552, outlet 554, and electrodes 568 positioned
with respect to a flow channel 566. FIG. 5G shows a side cutaway
view of FTEP device 550 with the inlet 552 and inlet channel 572,
and outlet 554 and outlet channel 574. The electrodes 568 are
positioned in electrode channels 576 so that they are in fluid
communication with the flow channel 566, but not directly in the
path of the cells traveling through the flow channel 566. Note that
the first electrode is placed between the inlet and the narrowed
region of the flow channel, and the second electrode is placed
between the narrowed region of the flow channel and the outlet. The
electrodes 568 in this aspect of the device are positioned in the
electrode channels 576 which are generally perpendicular to the
flow channel 566 such that the fluid containing the cells and
exogenous material flows from the inlet channel 572 through the
flow channel 566 to the outlet channel 574, and in the process
fluid flows into the electrode channels 576 to be in contact with
the electrodes 568. In this aspect, the inlet channel, outlet
channel and electrode channels all originate from the same planar
side of the device. In certain aspects, however, the electrodes may
be introduced from a different planar side of the FTEP device than
the inlet and outlet channels.
[0166] In the FTEP devices of the disclosure, the toxicity level of
the transformation results in greater than 30% viable cells after
electroporation, preferably greater than 35%, 40%, 45%, 50%, 55%,
60%, 70%, 75%, 80%, 85%, 90%, 95% or even 99% viable cells
following transformation, depending on the cell type and the
nucleic acids being introduced into the cells.
[0167] The housing of the FTEP device can be made from many
materials depending on whether the FTEP device is to be reused,
autoclaved, or is disposable, including stainless steel, silicon,
glass, resin, polyvinyl chloride, polyethylene, polyamide,
polystyrene, polyethylene, polypropylene, acrylonitrile butadiene,
polycarbonate, polyetheretheketone (PEEK), polysulfone and
polyurethane, co-polymers of these and other polymers. Similarly,
the walls of the channels in the device can be made of any suitable
material including silicone, resin, glass, glass fiber, polyvinyl
chloride, polyethylene, polyamide, polyethylene, polypropylene,
acrylonitrile butadiene, polycarbonate, polyetheretheketone (PEEK),
polysulfone and polyurethane, co-polymers of these and other
polymers. Preferred materials include crystal styrene, cyclo-olefin
polymer (COP) and cyclic olephin co-polymers (COC), which allow the
device to be formed entirely by injection molding in one piece with
the exception of the electrodes and, e.g., a bottom sealing film if
present.
[0168] The FTEP devices described herein (or portions of the FTEP
devices) can be created or fabricated via various techniques, e.g.,
as entire devices or by creation of structural layers that are
fused or otherwise coupled. For example, for metal FTEP devices,
fabrication may include precision mechanical machining or laser
machining; for silicon FTEP devices, fabrication may include dry or
wet etching; for glass FTEP devices, fabrication may include dry or
wet etching, powderblasting, sandblasting, or photostructuring; and
for plastic FTEP devices fabrication may include thermoforming,
injection molding, hot embossing, or laser machining.
[0169] The components of the FTEP devices may be manufactured
separately and then assembled, or certain components of the FTEP
devices (or even the entire FTEP device except for the electrodes)
may be manufactured (e.g., using 3D printing) or molded (e.g.,
using injection molding) as a single entity, with other components
added after molding. For example, housing and channels may be
manufactured or molded as a single entity, with the electrodes
later added to form the FTEP unit. Alternatively, the FTEP device
may also be formed in two or more parallel layers, e.g., a layer
with the horizontal channel and filter, a layer with the vertical
channels, and a layer with the inlet and outlet ports, which are
manufactured and/or molded individually and assembled following
manufacture.
[0170] In specific aspects, the FTEP device can be manufactured
using a circuit board as a base, with the electrodes, filter and/or
the flow channel formed in the desired configuration on the circuit
board, and the remaining housing of the device containing, e.g.,
the one or more inlet and outlet channels and/or the flow channel
formed as a separate layer that is then sealed onto the circuit
board. The sealing of the top of the housing onto the circuit board
provides the desired configuration of the different elements of the
FTEP devices of the disclosure. Also, two to many FTEP devices may
be manufactured on a single substrate, then separated from one
another thereafter or used in parallel. In certain embodiments, the
FTEP devices are reusable and, in some embodiments, the FTEP
devices are disposable. In additional embodiments, the FTEP devices
may be autoclavable.
[0171] The electrodes 508 can be formed from any suitable metal,
such as copper, stainless steel, titanium, aluminum, brass, silver,
rhodium, gold or platinum, or graphite. One preferred electrode
material is alloy 303 (UNS330300) austenitic stainless steel. An
applied electric field can destroy electrodes made from of metals
like aluminum. If a multiple-use (i.e., non-disposable)
flow-through FTEP device is desired-as opposed to a disposable,
one-use flow-through FTEP device--the electrode plates can be
coated with metals resistant to electrochemical corrosion.
Conductive coatings like noble metals, e.g., gold, can be used to
protect the electrode plates.
[0172] As mentioned, the FTEP devices may comprise push-pull
pneumatic means to allow multi-pass electroporation procedures;
that is, cells to be electroporated may be "pulled" from the inlet
toward the outlet for one pass of electroporation, then be "pushed"
from the outlet end of the flow-through FTEP device toward the
inlet end to pass between the electrodes again for another pass of
electroporation. This process may be repeated one to many
times.
[0173] Depending on the type of cells to be electroporated (e.g.,
bacterial, yeast, mammalian) and the configuration of the
electrodes, the distance between the electrodes in the flow channel
can vary widely. For example, where the flow channel decreases in
width, the flow channel may narrow to between 10 .mu.m and 5 mm, or
between 25 .mu.m and 3 mm, or between 50 .mu.m and 2 mm, or between
75 .mu.m and 1 mm. The distance between the electrodes in the flow
channel may be between 1 mm and 10 mm, or between 2 mm and 8 mm, or
between 3 mm and 7 mm, or between 4 mm and 6 mm. The overall size
of the FTEP device may be from 3 cm to 15 cm in length, or 4 cm to
12 cm in length, or 4.5 cm to 10 cm in length. The overall width of
the FTEP device may be from 0.5 cm to 5 cm, or from 0.75 cm to 3
cm, or from 1 cm to 2.5 cm, or from 1 cm to 1.5 cm.
[0174] The region of the flow channel that is narrowed is wide
enough so that at least two cells can fit in the narrowed portion
side-by-side. For example, a typical bacterial cell is 1 .mu.m in
diameter; thus, the narrowed portion of the flow channel of the
FTEP device used to transform such bacterial cells will be at least
2 .mu.m wide. In another example, if a mammalian cell is
approximately 50 .mu.m in diameter, the narrowed portion of the
flow channel of the FTEP device used to transform such mammalian
cells will be at least 100 .mu.m wide. That is, the narrowed
portion of the FTEP device will not physically contort or "squeeze"
the cells being transformed.
[0175] In embodiments of the FTEP device where reservoirs are used
to introduce cells and exogenous material into the FTEP device, the
reservoirs range in volume from 100 .mu.L to 10 mL, or from 500
.mu.L to 75 mL, or from 1 mL to 5 mL. The flow rate in the FTEP
ranges from 0.1 mL to 5 mL per minute, or from 0.5 mL to 3 mL per
minute, or from 1.0 mL to 2.5 mL per minute. The pressure in the
FTEP device ranges from 1-30 psi, or from 2-10 psi, or from 3-5
psi.
[0176] To avoid different field intensities between the electrodes,
the electrodes should be arranged in parallel. Furthermore, the
surface of the electrodes should be as smooth as possible without
pin holes or peaks. Electrodes having a roughness Rz of 1 to 10
.mu.m are preferred. In another embodiment of the invention, the
flow-through electroporation device comprises at least one
additional electrode which applies a ground potential to the FTEP
device. Flow-through electroporation devices (either as a
stand-alone instrument or as a module in an automated multi-module
system) are described in, e.g., U.S. Pat. Nos. U.S. Pat. Nos.
10,435,713; 10,443,074; 10,323,258; and 10,508,288.
Cell Singulation and Enrichment Device
[0177] FIG. 6A depicts a solid wall device 6050 and a workflow for
singulating cells in microwells in the solid wall device. At the
top left of the figure (i), there is depicted solid wall device
6050 with microwells 6052. A section 6054 of substrate 6050 is
shown at (ii), also depicting microwells 6052. At (iii), a side
cross-section of solid wall device 6050 is shown, and microwells
6052 have been loaded, where, in this embodiment, Poisson or
substantial Poisson loading has taken place; that is, each
microwell has few, one or no cells. At (iv), workflow 6040 is
illustrated where substrate 6050 having microwells 6052 shows
microwells 6056 with one cell per microwell, microwells 6057 with
no cells in the microwells, and one microwell 6060 with two cells
in the microwell. In step 6051, the cells in the microwells are
allowed to double approximately 2-150 times to form clonal colonies
(v), then editing is allowed to occur 6053.
[0178] After editing 6053, 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 a lag in growth for the edited cells
that do survive but must repair and recover following editing
(microwells 6058), where cells that do not undergo editing thrive
(microwells 6059) (vi). All cells are allowed to continue grow to
establish colonies and normalize 6055, where the colonies of edited
cells in microwells 6058 catch up in size and/or cell number with
the cells in microwells 6059 that do not undergo editing (vii).
Once the cell colonies are normalized, either pooling 6060 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 6058) are
identified and selected 6061 (e.g., "cherry picked") resulting in
even greater enrichment of edited cells.
[0179] 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 yeast cell growth includes
LB, SOC, TPD, YPG, YPAD, MEM and DMEM.
[0180] A module useful for performing the methods depicted in FIG.
6A is a solid wall isolation, incubation, and normalization (SWIIN)
module. FIG. 6B depicts an embodiment of a SWIIN module 650 from an
exploded top perspective view. In SWIIN module 650 the retentate
member is formed on the bottom of a top of a SWIIN module component
and the permeate member is formed on the top of the bottom of a
SWIIN module component.
[0181] The SWIIN module 650 in FIG. 6B comprises from the top down,
a reservoir gasket or cover 658, a retentate member 604 (where a
retentate flow channel cannot be seen in this FIG. 6B), a
perforated member 601 swaged with a filter (filter not seen in FIG.
6B), a permeate member 608 comprising integrated reservoirs
(permeate reservoirs 652 and retentate reservoirs 654), and two
reservoir seals 662, which seal the bottom of permeate reservoirs
652 and retentate reservoirs 654. A permeate channel 660a can be
seen disposed on the top of permeate member 608, defined by a
raised portion 676 of serpentine channel 660a, and ultrasonic tabs
664 can be seen disposed on the top of permeate member 608 as well.
The perforations that form the wells on perforated member 601 are
not seen in this FIG. 6B; however, through-holes 666 to accommodate
the ultrasonic tabs 664 are seen. In addition, supports 670 are
disposed at either end of SWIIN module 650 to support SWIIN module
650 and to elevate permeate member 608 and retentate member 604
above reservoirs 652 and 654 to minimize bubbles or air entering
the fluid path from the permeate reservoir to serpentine channel
660a or the fluid path from the retentate reservoir to serpentine
channel 660b (neither fluid path is seen in this FIG. 6B). Also
seen is a gasket 658, which covers the permeate and retentate
reservoir access apertures 632a, 632b, 632c, and 632d, as well as
pneumatic ports 633a, 633b, 633c and 633d.
[0182] In this FIG. 6B, it can be seen that the serpentine channel
660a that is disposed on the top of permeate member 608 traverses
permeate member 608 for most of the length of permeate member 608
except for the portion of permeate member 608 that comprises
permeate reservoirs 652 and retentate reservoirs 654 and for most
of the width of permeate member 608. As used herein with respect to
the distribution channels in the retentate member or permeate
member, "most of the length" means about 95% of the length of the
retentate member or permeate member, or about 90%, 85%, 80%, 75%,
or 70% of the length of the retentate member or permeate member. As
used herein with respect to the distribution channels in the
retentate member or permeate member, "most of the width" means
about 95% of the width of the retentate member or permeate member,
or about 90%, 85%, 80%, 75%, or 70% of the width of the retentate
member or permeate member.
[0183] In this embodiment of a SWIIN module, the perforated member
includes through-holes to accommodate ultrasonic tabs disposed on
the permeate member. Thus, in this embodiment the perforated member
is fabricated from 316 stainless steel, and the perforations form
the walls of microwells while a filter or membrane is used to form
the bottom of the microwells. Typically, the perforations
(microwells) are approximately 150 .mu.m-200 .mu.m in diameter, and
the perforated member is approximately 125 .mu.m deep, resulting in
microwells having a volume of approximately 2.5 nl, with a total of
approximately 200,000 microwells. The distance between the
microwells is approximately 279 .mu.m 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. As for the filter or
membrane, like the filter described previously, filters appropriate
for use are solvent resistant, 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 0.10 sm, however for
other cell types (e.g., such as for mammalian cells), the pore
sizes can be as high as 10.0 .mu.m-20.0 .mu.m or more. Indeed, the
pore sizes useful in the cell concentration device/module include
filters with sizes from 0.10 .mu.m, 0.11 .mu.m, 0.12 .mu.m, 0.13
.mu.m, 0.14 .mu.m, 0.15 .mu.m, 0.16 .mu.m, 0.17 .mu.m, 0.18 .mu.m,
0.19 .mu.m, 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.
[0184] The cross-section configuration of the mated serpentine
channel 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
2 mm to 15 mm wide, or from 3 mm to 12 mm wide, or from 5 mm to 10
mm wide. If the cross section of the mated serpentine channel is
generally round, oval or elliptical, the radius of the channel may
be from about 3 mm to 20 mm in hydraulic radius, or from 5 mm to 15
mm in hydraulic radius, or from 8 mm to 12 mm in hydraulic
radius.
[0185] Serpentine channels 660a and 660b can have approximately the
same volume or a different volume. For example, each "side" or
portion 660a, 660b of the serpentine channel may have a volume of,
e.g., 2 mL, or serpentine channel 660a of permeate member 608 may
have a volume of 2 mL, and the serpentine channel 660b of retentate
member 604 may have a volume of, e.g., 3 mL. The volume of fluid in
the serpentine channel may range from about 2 mL to about 80 mL, or
about 4 mL to 60 mL, or from 5 mL to 40 mL, or from 6 mL to 20 mL
(note these volumes apply to a SWIIN module comprising a, e.g.,
50-500K perforation member). The volume of the reservoirs may range
from 5 mL to 50 mL, or from 7 mL to 40 mL, or from 8 mL to 30 mL or
from 10 mL to 20 mL, and the volumes of all reservoirs may be the
same or the volumes of the reservoirs may differ (e.g., the volume
of the permeate reservoirs is greater than that of the retentate
reservoirs).
[0186] The serpentine channel portions 660a and 660b of the
permeate member 608 and retentate member 604, respectively, are
approximately 200 mm long, 130 mm wide, and 4 mm thick, though in
other embodiments, the retentate and permeate members can be from
75 mm to 400 mm in length, or from 100 mm to 300 mm in length, or
from 150 mm to 250 mm in length; from 50 mm to 250 mm in width, or
from 75 mm to 200 mm in width, or from 100 mm to 150 mm in width;
and from 2 mm to 15 mm in thickness, or from 4 mm to 10 mm in
thickness, or from 5 mm to 8 mm in thickness. Embodiments the
retentate (and permeate) members may be fabricated from PMMA
(poly(methyl methacrylate) or other materials may be used,
including polycarbonate, cyclic olefin co-polymer (COC), glass,
polyvinyl chloride, polyethylene, polyamide, polypropylene,
polysulfone, polyurethane, and co-polymers of these and other
polymers. Preferably at least the retentate member is fabricated
from a transparent material so that the cells can be visualized
(see, e.g., FIG. 6E and the description thereof). For example, a
video camera may be used to monitor cell growth by, e.g., density
change measurements based on an image of an empty well, with phase
contrast, or if, e.g., a chromogenic marker, such as a chromogenic
protein, is used to add a distinguishable color to the cells.
Chromogenic markers such as blitzen blue, dreidel teal, virginia
violet, vixen purple, prancer purple, tinsel purple, maccabee
purple, donner magenta, cupid pink, seraphina pink, scrooge orange,
and leor orange (the Chromogenic Protein Paintbox, all available
from ATUM (Newark, Calif.)) obviate the need to use fluorescence,
although fluorescent cell markers, fluorescent proteins, and
chemiluminescent cell markers may also be used.
[0187] Because the retentate member preferably is transparent,
colony growth in the SWIIN module can be monitored by automated
devices such as those sold by JoVE (ScanLag.TM. system, Cambridge,
Mass.) (also see Levin-Reisman, et al., Nature Methods, 7:737-39
(2010)). Automated colony pickers may be employed, such as those
sold by, e.g., TECAN (Pickolo.TM. system, Mannedorf, Switzerland);
Hudson Inc. (RapidPick.TM., Springfield, N.J.); Molecular Devices
(QPix 400 .TM. system, San Jose, Calif.); and Singer Instruments
(PIXL.TM. system, Somerset, UK).
[0188] Due to the heating and cooling of the SWIIN module,
condensation may accumulate on the retentate member which may
interfere with accurate visualization of the growing cell colonies.
Condensation of the SWIIN module 650 may be controlled by, e.g.,
moving heated air over the top of (e.g., retentate member) of the
SWIIN module 650, or by applying a transparent heated lid over at
least the serpentine channel portion 660b of the retentate member
604. See, e.g., FIG. 6E and the description thereof infra.
[0189] In SWIIN module 650 cells and medium-at a dilution
appropriate for Poisson or substantial Poisson distribution of the
cells in the microwells of the perforated member--are flowed into
serpentine channel 660b from ports in retentate member 604, and the
cells settle in the microwells while the medium passes through the
filter into serpentine channel 660a in permeate member 608. The
cells are retained in the microwells of perforated member 601 as
the cells cannot travel through filter 603. Appropriate medium may
be introduced into permeate member 608 through permeate ports 611.
The medium flows upward through filter 603 to nourish the cells in
the microwells (perforations) of perforated member 601.
Additionally, buffer exchange can be effected by cycling medium
through the retentate and permeate members. In operation, the cells
are deposited into the microwells, are grown for an initial, e.g.,
2-100 doublings, editing may be induced by, e.g., raising the
temperature of the SWIIN to 42.degree. C. to induce a
temperature-inducible promoter or by removing growth medium from
the permeate member and replacing the growth medium with a medium
comprising a chemical component that induces an inducible
promoter.
[0190] Once editing has taken place, the temperature of the SWIIN
may be decreased, or the inducing medium may be removed and
replaced with fresh medium lacking the chemical component thereby
de-activating the inducible promoter. The cells then continue to
grow in the SWIIN module 650 until the growth of the cell colonies
in the microwells is normalized. For the normalization protocol,
once the colonies are normalized, the colonies are flushed from the
microwells by applying fluid or air pressure (or both) to the
permeate member serpentine channel 660a and thus to filter 603 and
pooled. Alternatively, if cherry picking is desired, the growth of
the cell colonies in the microwells is monitored, and slow-growing
colonies are directly selected; or, fast-growing colonies are
eliminated.
[0191] FIG. 6C is a top perspective view of a SWIIN module with the
retentate and perforated members in partial cross section. In this
FIG. 6C, it can be seen that serpentine channel 660a is disposed on
the top of permeate member 608 is defined by raised portions 676
and traverses permeate member 608 for most of the length and width
of permeate member 608 except for the portion of permeate member
608 that comprises the permeate and retentate reservoirs (note only
one retentate reservoir 652 can be seen). Moving from left to
right, reservoir gasket 658 is disposed upon the integrated
reservoir cover 678 (cover not seen in this FIG. 6C) of retentate
member 604. Gasket 658 comprises reservoir access apertures 632a,
632b, 632c, and 632d, as well as pneumatic ports 633a, 633b, 633c
and 633d. Also at the far left end is support 670. Disposed under
permeate reservoir 652 can be seen one of two reservoir seals 662.
In addition to the retentate member being in cross section, the
perforated member 601 and filter 603 (filter 603 is not seen in
this FIG. 6C) are in cross section. Note that there are a number of
ultrasonic tabs 664 disposed at the right end of SWIIN module 650
and on raised portion 676 which defines the channel turns of
serpentine channel 660a, including ultrasonic tabs 664 extending
through through-holes 666 (not seen in this FIG. 6C but see FIG.
6B) of perforated member 601. There is also a support 670 at the
end distal reservoirs 652, 654 of permeate member 608.
[0192] FIG. 6D is a side perspective view of an assembled SWIIIN
module 650, including, from right to left, reservoir gasket 658
disposed upon integrated reservoir cover 678 (not seen) of
retentate member 604. Gasket 658 may be fabricated from rubber,
silicone, nitrile rubber, polytetrafluoroethylene, a plastic
polymer such as polychlorotrifluoroethylene, or other flexible,
compressible material. Gasket 658 comprises reservoir access
apertures 632a, 632b, 632c, and 632d, as well as pneumatic ports
633a, 633b, 633c and 633d. Also at the far-left end is support 670
of permeate member 608. In addition, permeate reservoir 652 can be
seen, as well as one reservoir seal 662. At the far-right end is a
second support 670.
[0193] Imaging of cell colonies growing in the wells of the SWIIN
is desired in most implementations for, e.g., monitoring both cell
growth and device performance and imaging is necessary for
cherry-picking implementations. Real-time monitoring of cell growth
in the SWIIN requires backlighting, retentate plate (top plate)
condensation management and a system-level approach to temperature
control, air flow, and thermal management. In some implementations,
imaging employs a camera or CCD device with sufficient resolution
to be able to image individual wells. For example, in some
configurations a camera with a 9-pixel pitch is used (that is,
there are 9 pixels center-to-center for each well). Processing the
images may, in some implementations, utilize reading the images in
grayscale, rating each pixel from low to high, where wells with no
cells will be brightest (due to full or nearly-full light
transmission from the backlight) and wells with cells will be dim
(due to cells blocking light transmission from the backlight).
After processing the images, thresholding is performed to determine
which pixels will be called "bright" or "dim", spot finding is
performed to find bright pixels and arrange them into blocks, and
then the spots are arranged on a hexagonal grid of pixels that
correspond to the spots. Once arranged, the measure of intensity of
each well is extracted, by, e.g., looking at one or more pixels in
the middle of the spot, looking at several to many pixels at random
or pre-set positions, or averaging X number of pixels in the spot.
In addition, background intensity may be subtracted. Thresholding
is again used to call each well positive (e.g., containing cells)
or negative (e.g., no cells in the well). The imaging information
may be used in several ways, including taking images at time points
for monitoring cell growth. Monitoring cell growth can be used to,
e.g., remove the "muffin tops" of fast-growing cells followed by
removal of all cells or removal of cells in "rounds" as described
above, or recover cells from specific wells (e.g., slow-growing
cell colonies); alternatively, wells containing fast-growing cells
can be identified and areas of UV light covering the fast-growing
cell colonies can be projected (or rastered with shutters) onto the
SWIIN to irradiate or inhibit growth of those cells. Imaging may
also be used to assure proper fluid flow in the serpentine channel
660.
[0194] FIG. 6E depicts the embodiment of the SWIIN module in FIGS.
6B-6D further comprising a heat management system including a
heater and a heated cover. The heated cover facilitates the
condensation management that is required for imaging. Assembly 698
comprises a SWIIN module 650 seen lengthwise in cross section,
where one permeate reservoir 652 is seen. Disposed immediately upon
SWIIN module 650 is cover 694 and disposed immediately below SWIIN
module 650 is backlight 680, which allows for imaging. Beneath and
adjacent to the backlight and SWIIN module is insulation 682, which
is disposed over a heatsink 684. In this FIG. 6E, the fins of the
heatsink would be in-out of the page. In addition there is also
axial fan 686 and heat sink 688, as well as two thermoelectric
coolers 692, and a controller 690 to control the pneumatics,
thermoelectric coolers, fan, solenoid valves, etc. The arrows
denote cool air coming into the unit and hot air being removed from
the unit. It should be noted that control of heating allows for
growth of many different types of cells as well as strains of cells
that are, e.g., temperature sensitive, etc., and allows use of
temperature-sensitive promoters. Temperature control allows for
protocols to be adjusted to account for differences in
transformation efficiency, cell growth and viability. For more
details regarding solid wall isolation incubation and normalization
devices see U.S. Pat. Nos. 10,533,152; 10,550,363; 10,532,324;
10,625,212; 10,633,626; and 10,633,627; and U.S. Ser. Nos.
16/693,630, filed 25 Nov. 2019; Ser. No. 16/823,269, filed 18 Mar.
2020; Ser. No. 16/820,292, filed 16 Mar. 2020; Ser. No. 16/820,324,
filed 16 Mar. 2020; and Ser. No. 16/686,066, filed 15 Nov.
2019.
Use of the Automated Multi-Module Yeast Cell Processing
Instrument
[0195] FIG. 7 illustrates an embodiment of a multi-module cell
processing instrument. This embodiment depicts an exemplary system
that performs recursive gene editing on a cell population. The cell
processing instrument 700 may include a housing 726, a reservoir
for storing cells to be transformed or transfected 702, and a cell
growth module (comprising, e.g., a rotating growth vial) 704. 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
cell concentration module 706 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 708. In addition to the reservoir for
storing cells 702, the multi-module cell processing instrument
includes a reservoir for storing the vector pre-assembled with
editing oligonucleotide cassettes 722. The pre-assembled nucleic
acid vectors are transferred to the electroporation device 708,
which already contains the cell culture grown to a target OD. In
the electroporation device 708, the nucleic acids are
electroporated into the cells. Following electroporation, the cells
are transferred into an optional recovery module 710, where the
cells recover briefly post-transformation.
[0196] After recovery, the cells may be transferred to a storage
module 712, where the cells can be stored at, e.g., 4C for later
processing 714, or the cells may be diluted and transferred to a
selection/singulation/growth/induction/editing/normalization
(SWIIN) module 720. In the SWIIN 720, 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 the cells are treated to conditions that cure the editing
vector from this round. Once cured, the cells can be flushed out of
the microwells and pooled, then transferred to the storage (or
recovery) unit 712 or can be transferred back to the growth module
704 for another round of editing. In between pooling and transfer
to a growth module, there typically is one or more additional
steps, such as cell recovery, medium exchange (rendering the cells
electrocompetent), cell concentration (typically concurrently with
medium exchange by, e.g., filtration. Note that the
selection/singulation/growth/induction/editing/normalization and
editing modules may be the same module, where all processes are
performed in, e.g., a 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/editing module
(solid wall device). Similarly, the cells may be pooled after
normalization, transferred to a separate vessel, and cured in the
separate vessel. 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.
1G and 1H 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
708.
[0197] In electroporation device 708, 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. 7 is controlled by a processor 724
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 724 may
control the timing, duration, and temperature of various processes,
the dispensing of reagents, and other operations of the various
modules of the instrument 700. 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.
[0198] 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. 7, 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.
[0199] 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 the process in which
one or more vectors used in the prior round of editing is
eliminated from the transformed cells as described in detail above
in relation to FIGS. 1A-11. 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 vector.
[0200] FIG. 8 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.
1G and 1H. The cell processing instrument 800 may include a housing
826, a reservoir of cells to be transformed or transfected 802, and
a growth module (a cell growth device) 804. 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 cell
concentration module 830 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 808 (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.
[0201] In addition to the reservoir for storing the cells, the
system 800 may include a reservoir for storing editing cassettes
816 and a reservoir for storing an expression vector backbone 818.
Both the editing oligonucleotide cassettes and the expression
vector backbone are transferred from the reagent cartridge to a
nucleic acid assembly module 828, where the editing oligonucleotide
cassettes are inserted into the expression vector backbone. The
assembled nucleic acids may be transferred into an optional
purification module 822 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 816 or 818. Once the processes carried out by the
purification module 822 are complete, the assembled nucleic acids
are transferred to, e.g., an electroporation device 808, which
already contains the cell culture grown to a target OD and rendered
electrocompetent via an optional filtration module (not shown). In
electroporation device 808, the assembled nucleic acids are
introduced into the cells. Following electroporation, the cells are
transferred into a combined recovery/selection module 830. For
examples of multi-module cell editing instruments, see U.S. Pat.
Nos. 10,253,316; 10,329,559; 10,323,242; 10,421,959; 10,465,185;
10,519,437; 10,584,333; 10,584,334; and U.S. Ser. Nos. 16/412,195,
filed 14 May 2019; Ser. No. 16/750,369, 23 Jan. 2020; Ser. No.
16/822,249, filed 18 Mar. 2020; and Ser. No. 16/837,985, filed 1
Apr. 2020, both of which are herein incorporated by reference in
their entirety.
[0202] Following recovery, and, optionally, selection, the cells
are transferred to a growth, induction, and editing module (bulk
liquid culture) 840. 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.
[0203] The recovery, selection, growth, induction, editing and
storage modules may all be separate, may be arranged and combined
as shown in FIG. 8, 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.
[0204] Once the cells are edited and re-grown (e.g., recovered from
editing), the cells may be stored, e.g., in a storage module 812,
where the cells can be kept at, e.g., 4C until the cells are used
in another round of editing or retrieved 14. The multi-module cell
processing instrument is controlled by a processor 824 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 824 may control the timing, duration, temperature, and
operations of the various modules of the system 800 and the
dispensing of reagents. For example, the processor 824 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
[0205] 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 I: Growth in the Cell Growth Module
[0206] 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 in relation to FIGS. 3A-3D.
[0207] 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. OD.sub.600 was
measured at intervals using a NanoDrop.TM. spectrophotometer
(Thermo Fisher Scientific). The results are shown in FIG. 9. 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. 10. Again, the rotating growth
vial/cell growth device performed better than the cell shaker in
growing the cells to OD.sub.600 1.9.
[0208] 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. 11, 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. OD.sub.600 was measured at
intervals using a NanoDrop.TM. spectrophotometer (Thermo Fisher
Scientific). The results are shown in FIG. 12, 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.
[0209] In yet another experiment, the rotating growth vial/cell
growth device was used to measure OD.sub.600 in real time. FIG. 13
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.
[0210] 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. 14 is a graph showing the results. Note that OD.sub.600 6.0
was reached in 14 hours.
Example II: Cell Concentration
[0211] The TFF module as described above in relation to FIGS. 4A-4E
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:
[0212] 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.
[0213] Filtrate conductivity and filter processing time was
measured for E. coli with the results shown in FIG. 15A. 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 (OD.sub.600) 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 (-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.
[0214] 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. 15B
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 III: Production and Transformation of Electrocompetent E.
coli and S. Cerevisiae
[0215] 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 OD.sub.600
every 1 to 2 hr. When the OD.sub.600 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 were
aliquoted in 1:100 dilutions of cell suspension and ice-cold
glycerol.
[0216] 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.
[0217] 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. 16A. In
FIG. 16A, 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. 16A
the recovery ratio is approximately 0.0001.
[0218] Additionally, a comparison of the NEPAGENE.TM. ELEPO21 and
the FTEP device was made for efficiencies of transformation
(uptake), cutting, and editing. In FIG. 16B, 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. 16B for the
FTEP is greater than 0.001.
[0219] 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 OD.sub.600 of 1.5+/-0.1.
[0220] 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.
[0221] 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.
[0222] 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. 17. 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 IV: Bulk Liquid Protocol: Induction and Outgrowth
[0223] 250 mL baffled shake flasks were prepared with 50 mL of
SOB+100 pg/mL carbenicillin and 25 pg/mL chloramphenicol. For a
full, deconvolution experiment, 3 shake flasks were prepared per
transformation. 500 .mu.L of undiluted culture from each
transformation reaction was transferred into the prepared 250 mL
shake flasks. The following temperature settings were set up on an
incubator 30.degree. C. for 9 hours-42.degree. C. for 2
hours-30.degree. C. for 9 hours. This temperature regime was used
to allow for additional recovery of the cells from transformation
during the first eight hours. The lambda red system was induced one
hour prior to induction of the nuclease, where lambda induction was
triggered by the addition of arabinose (2.5 mL of 20% arabinose) to
the culture, and the nuclease induction was triggered by increasing
the temperature of the cultures to 42.degree. C. For full
deconvolution experiments, arabinose was not added to the UPTAKE
and CUT flasks as those should not express lambda red; further, the
UPTAKE flasks were not shifted to 42.degree. C.
[0224] After the temperature cycling is complete (.about.21 hours),
the shake flasks were removed. For NGS-SinglePlex: serial dilutions
of 10.sup.-5 to 10.sup.-7 of each culture were prepared with 0.8%
NaCl (50 .mu.L of culture into 450 .mu.L of sterile, 0.8% NaCl).
Following dilution, 300 .mu.L of each dilution was plated onto 150
mm LB agar plates with standard concentrations of chloramphenicol
and carbenicillin. The plates were then placed in a 30.degree. C.
incubator for overnight growth and were picked for singleplex NGS
the following day. For NGS-Amplicon: 250 .mu.L of culture from each
shake flask was removed and used as the input for a plasmid
extraction protocol. The OD of this culture was measured to select
a volume based on the desired number of cells to go into the
plasmid purification. Optionally, an undiluted volume from each
shake flask may be plated to see enrichment/depletion of cassettes
and the plates were scraped the following day and processed.
[0225] FIG. 18 is a bar graph showing the various types of edits
observed using constitutive editing in a liquid culture
(approximately 20% editing observed), standard plating procedure
(approximately 76% editing observed), two replica experiments of
induced editing in liquid bulk (approximately 70% and 76% editing
observed), and two replica experiments of induced editing using the
standard plating procedure (approximately 60% and 76% editing
observed). Editing clonality was also measured. The editing
clonality of the standard plating procedure showed mixed clonality
for the 96 wells, with some colonies achieving 100% clonality, most
colonies achieving greater than 50% clonality, and an average
clonality of 70% and 60% for two replicates (data not shown). The
editing clonality of the liquid bulk procotol shows that the
majority of the cells were either 100% edited, or 0% edited (e.g.,
wildtype), with a small number (approximately 8%) between 100% or
0%. The average editing efficiency was similar for these
protocols.
Example V: Singulation, Growth and Editing of E. coli in 200K
SWIIN
[0226] Singleplex automated genomic editing using MAD7 nuclease, a
library with 94 different edits in a single gene (yagP) and
employing a 200K singulation device such as those exemplified in
FIGS. 6B-6E was successfully performed. The engine vector used
comprised MAD7 under the control of the pL inducible promoter, and
the editing vector used comprised the editing cassette being under
the control of the pL inducible promoter, and the .lamda. Red
recombineering system under control of the pBAD inducible promoter
pBAD--with the exception that the editing cassette comprises the 94
yagP gene edits (donor DNAs) and the appropriate corresponding
gRNAs. Two SWIIN workflows were compared, and further were
benchmarked against the standard plating protocol. The SWIIN
protocols differ from one another that in one set of replicates LB
medium containing arabinose was used to distribute the cells in the
SWIIN (arabinose was used to induce the .lamda. Red recombineering
system (which allows for repair of double-strand breaks in E. coli
that are created during editing), and in the other set of
replicates SOB medium without arabinose was used to distribute the
cells in the SWIIN and for initial growth, with medium exchange
performed to replace the SOB medium without arabinose with SOB
medium with arabinose. Approximately 70K cells were loaded into the
200K SWIIN.
[0227] In all protocols (standard plating, LB-SWIIN, and
SOB-SWIIN), the cells were allowed to grow at 30.degree. C. for 9
hours and editing was induced by raising the temperature to
42.degree. C. for 2.5 hours, then the temperature was returned to
30.degree. C. and the cells were grown overnight. The results of
this experiment are shown in FIG. 19 and in Table 1 below. Note
that similar editing performance was observed with the four
replicates of the two SWIIN workflows, indicating that the
performance of SWIIN plating with and without arabinose in the
initial medium is similar. Editing percentage in the standard
plating protocol was approximately 77%, in bulk liquid was
approximately 67%, and for the SWIIN replicates ranged from
approximately 63% to 71%. Note that the percentage of unique edit
cassettes divided by the total number of edit cassettes was similar
for each protocol.
TABLE-US-00001 TABLE 1 SWIIN SWIIN SWIIN SWIIN SOB then SOB then
Standard LB/Ara LB/Ara SOB/Ara SOB/Ara Plating Rep. A Rep. B Rep. A
Rep. B 40006 edit calls/ 0.777 0.633 0.719 0.663 0.695 identified
wells Unique edit 0.49 0.49 0.43 0.50 0.51 cassettes/total edit
cassettes
Example VI: Curing
[0228] Standard Plating Protocol: Three rounds of recursive editing
and curing were performed. Intended edits introduced a stop codon
in three sugar genes (XylA-Y94*, LacZ-F593*, and GalK-E249*). These
mutations cause a loss of function in the target gene. This loss of
function phenotype was observed by growing cells on MacConkey
medium. The editing rate was determined by calculating the ratio of
the number of cells with a loss of function mutation to the number
of total cells. E. coli 181 cells comprising the engine vector
depicted in FIG. 1C on left were made competent and transformed in
a 100 .mu.L volume with the editing vector depicted in FIG. 1C on
right. The cells were allowed to recover for 3 hours in 3.0 mL
total volume SOB medium. The recovered cells were then diluted
1:100 in 20 mL SOB medium containing chloramphenicol and the
appropriate antibiotic for the editing vector. The cells were then
plated on solid SOB medium containing chloramphenicol, the editing
vector antibiotic (e.g., carbenicillin, kanamycin, bleomycin,
streptomycin or nourseothricin N-acetyl transferase, and arabinose.
The arabinose induces the pBAD promoter driving transcription of
the .lamda. Red recombinase system.
[0229] The cells were grown for 9 hours at 30.degree. C., grown for
2 hours at 42.degree. C. to induce the pL promoter driving
transcription of the nuclease and editing gRNA, and then grown for
another 9 hours at 30.degree. C. The cells were scraped from the
plate and diluted in SOB medium with chloramphenicol only. The
cells were then washed 3.times. and 8 mL of cells were suspended in
12 mL SOB+chlor medium and grown to OD=3.0 to assure the cells were
in stationary phase. The temperature of the culture was increased
to 42.degree. C. for two hours to induce the nuclease and 20 .mu.L
DAPG was added to a final concentration of 25 .mu.M to induce
transcription of the curing gRNA (e.g., the anti-pUC gRNA). The
cells were grown at 30.degree. C. for 6 hours. Following curing,
the cells were washed in LB+chlor medium, resuspended in LB+chlor
medium and grown again at 30.degree. C. to OD=0.5. The cells were
washed three times with 10% glycerol to render them
electrocompetent and subjected to another round of editing. The
engine vector was maintained throughout the rounds of editing. The
succession of editing vectors comprised the same editing vector
architecture as shown in at right in FIG. 1C; however, the editing
gRNA/donor DNA and the selectable marker changed with each round.
The editing gRNAs and editing vector antibiotic resistance genes
used for each round of editing and curing are listed in Table
2.
TABLE-US-00002 TABLE 2 SPP experimental description Round 1 2 3
gRNA type editing editing editing Editing locus LacZ GalK XylA
Editing vector Abx Carb Nat Kan
[0230] The results for both editing and curing rates are shown in
FIG. 20A. For editing efficiency, note that after a first round of
editing, 99% of cells had one edit; after a second round of editing
a small percentage of cells had zero edits, approximately 95% of
cells had one edit, approximately 95% of cells had two edits; and
after a third round of editing, a small percentage of cells had
zero edits, approximately 90% of cells had one edit, approximately
80% of cells had two edits, and approximately 38% of cells had all
three edits. Note that these numbers are the fraction of cells that
have at least 1, 2, or 3 edits; thus, if a cell has three edits it
counts for each category, that is, it is the cumulative editing
rate. Curing efficiency was calculated using the following
equation:
1 - CFUcassette + engine CFUengine ##EQU00001##
[0231] As seen in FIG. 20A, curing efficiency was well over 95% for
each round.
[0232] Bulk Liquid Protocol: Four rounds of recursive editing and
curing were performed using sugar editing gRNAs. Intended edits
introduced a stop codon in three sugar genes (XylA-Y194*,
LacZ-F593*, and GalK-E249*). These mutations cause a loss of
function in the target gene. This loss of function phenotype can be
observed by growing cells on MacConkey medium. The editing rate was
determined by calculating the ratio of the number of cells with a
loss of function mutation to the number of total cells. The
fourth-round edit was YiaW_C183 and rate was determined by sampling
colonies and Sanger sequencing 96 colonies at the edit loci. E.
coli 181 strain cells comprising the engine vector depicted in FIG.
1C were made competent and transformed in a 100 .mu.L volume with
the editing vector depicted in FIG. 1C. The cells were allowed to
recover for 3 hours in 3.0 mL total volume SOB medium. The
recovered cells were then diluted 1:100 in 20 mL SOB medium
containing chloramphenicol and the appropriate antibiotic for the
editing vector (e.g., carbenicillin, kanamycin, nourseothricin
N-acetyl transferase). The cells were then plated outgrown for 8
hours in SOB medium containing chloramphenicol and the antibiotic
appropriate for the editing vector. Arabinose was added to the
medium for a final concentration of 1%.
[0233] The cells were then grown for another hour at 30.degree. C.,
grown for 2 hours at 42.degree. C. to induce the pL promoter
driving transcription of the nuclease and editing gRNA, then grown
for another 9 hours at 30.degree. C. The cells were then pelleted,
washed 3.times., and resuspended in 20 mL SOB medium with
chloramphenicol only. 12 mL of additional medium was added to 8 mL
of the cells in suspension. Curing was induced (e.g., transcription
of the nuclease and curing gRNA) by raising the temperature of the
cell culture to 42.degree. C. for 2 hours and by adding 20 .mu.L
DAPG to a final concentration of 25 .mu.M. The cells were then
grown at 30.degree. C. for 6 hours to OD=2.5. Following curing, the
cells were washed in LB+chlor medium, resuspended in LB+chlor
medium and grown again at 30.degree. C. to OD=0.5, and washed with
10% glycerol for another round of editing. The editing gRNAs and
editing vector antibiotic resistance genes are listed in Table 3,
and the results are shown in FIG. 20B. The succession of editing
vectors comprised the same editing vector architecture as shown in
at right in FIG. 1C; however, the editing gRNA/donor DNA and the
selectable marker changed with each round.
[0234] For editing efficiency, note that after a first round of
editing, 100% of cells had zero edits, which was expected since in
the first round the editing vector did not comprise an editing gRNA
or cellular target sequence; after a second round of editing
approximately 30% of cells had zero edits, and approximately 70% of
cells had one edit; and after a third round of editing,
approximately 60% of cells had zero edits, approximately 40% of
cells had one edit, approximately 30% of cells had two edits; and
after a fourth round of editing, approximately 80% of cells had
zero edits, and approximately 5% of cells had each of one, two or
three edits. Note that these numbers are the fraction of cells that
have at least 1, 2, or 3 edits; thus, if a cell has three edits it
counts for each category, that is, it is the cumulative editing
rate. The percentage curing achieved was over 95% after each round
and was over 99% for the first two rounds.
TABLE-US-00003 TABLE 3 Bulk Liquid experimental description Round 1
2 3 4 gRNA type Non-editing editing editing editing Editing locus
None XylA GalK LacZ Editing vector Abx Carb Kan Nat Carb
[0235] SWIIN Protocol: Four rounds of recursive editing and curing
were performed using sugar editing gRNAs. Intended edits introduced
a stop codon in three sugar genes (XylA-Y194*, LacZ-F593*, and
GalK-E249*). These mutations caused a loss of function in the
target gene. This loss of function phenotype can be observed by
growing cells on MacConkey medium. The editing rate was determined
by calculating the ratio of the number of cells with a loss of
function mutation to the number of total cells. The fourth-round
edit was YiaW_C183 and rate was determined by sampling colonies and
Sanger sequencing 96 colonies at the edit loci. E. coli 181 strain
cells comprising the engine vector depicted in FIG. 1C were made
competent and transformed in a 100 .mu.L volume with the editing
vector depicted in FIG. 1C. The cells were allowed to recover for 3
hours in 2.7 mL SOB medium. The recovered cells were suspended in
10 mL and then loaded into a 200K SWIIN and grown for 8 hours in
SOB medium containing chloramphenicol and the antibiotic
appropriate for the editing vector. Medium exchange was performed
with arabinose being added to the medium for a final concentration
of 1%. The cells were then grown for another hour at 30.degree. C.,
grown for 2.5 hours at 42.degree. C. to induce the pL promoter
driving transcription of the nuclease and editing gRNA, then grown
for another 9 hours at 30.degree. C. The cells were then recovered
from the SWIIN, washed 3.times., and resuspended in 20 mL SOB
medium with chloramphenicol only. 12 mL of additional medium was
added to 8 mL of the cells in suspension. The cells were grown to
OD=3.0 and curing was induced (e.g., transcription of the curing
gRNA and nuclease) by adding 20 .mu.L DAPG to a final concentration
of 25 .mu.M and the temperature of the culture was increased to
42.degree. C. for two hours.
[0236] Following curing, the cells were washed in LB+chlor medium,
resuspended in LB+chlor medium and grown again at 30.degree. C. to
OD=0.5 for another round of editing. The editing gRNAs and editing
vector antibiotic resistance genes are listed in Table 4, and the
results for curing and editing efficiency are shown in FIG. 20C.
The succession of editing vectors comprised the same editing vector
architecture as shown in at right in FIG. 1C; however, the editing
gRNA/donor DNA and the selectable marker changed with each round.
Editing on the SWIIN for the first round of editing resulted in
approximately 20% of the cells having zero edits and 80% of the
cells having one edit; for the second round of editing,
approximately 20% of cells had zero edits, 20% had one edit, and
80% had two edits; for the third round of editing approximately 6%
of the cells had zero edits, 4% had one edit, 40% had two edits,
and 60% had three edits; finally after the fourth round of editing,
approximately 80% of the cells had zero edits, 8% had one edit, 6%
had two edits, and 2% had three edits. Note that these numbers are
the fraction of cells that have at least 1, 2, or 3 edits; thus, if
a cell has three edits it counts for each category, that is, it is
the cumulative editing rate. All rounds of editing had a percentage
of curing above 85%, and after rounds one and three, editing
percentage was above 95%.
TABLE-US-00004 TABLE 4 SWIIN experimental description Round 1 2 3 4
gRNA type editing editing editing editing Editing locus LacZ GalK
XylA yiaW Editing vector Abx Carb Nat Kan Carb
Example VII: Fully-Automated Singleplex RGN-directed Editing
Run
[0237] 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 U.S. Ser. Nos.
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.
[0238] 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.
[0239] 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.
[0240] 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 VIII: Fully-Automated Recursive Editing Run
[0241] 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
OD.sub.600 of 2.7, then concentrated and rendered
electrocompetent.
[0242] 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.
[0243] 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.
[0244] While this invention is satisfied by embodiments in many
different forms, as described in detail in connection with
preferred 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,
.dagger-dbl.6.
Curing for Recursive Nucleic Acid-Guided Cell Editing
[0245] The present disclosure provides automated multi-module
instrumentation and automated methods for performing recursive
editing of live cells with curing of editing vectors from prior
rounds of editing.
* * * * *