U.S. patent application number 16/928061 was filed with the patent office on 2020-10-29 for automated cell processing instruments comprising reagent cartridges.
The applicant listed for this patent is Inscripta, Inc.. Invention is credited to Phillip Belgrader, Jorge Bernate, Bruce Chabansky, Don Masquelier, Brian Van Hatten.
Application Number | 20200338561 16/928061 |
Document ID | / |
Family ID | 1000005147403 |
Filed Date | 2020-10-29 |
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United States Patent
Application |
20200338561 |
Kind Code |
A1 |
Bernate; Jorge ; et
al. |
October 29, 2020 |
AUTOMATED CELL PROCESSING INSTRUMENTS COMPRISING REAGENT
CARTRIDGES
Abstract
The present disclosure provides a reagent cartridge configured
for use in an automated multi-module cell processing environment.
The reagent cartridges may include an electroporation device, as
well as sample receptacles, reagent receptacles, waste receptacles
and the like, and a script for controlling a processor to dispense
samples and reagents contained in the receptacles, and to porate
cells in the electroporation device. Also described are kits
including the cartridges, automated multi-module cell processing
instruments including the reagent cartridges and methods of using
the reagent cartridges.
Inventors: |
Bernate; Jorge; (Boulder,
CO) ; Masquelier; Don; (Boulder, CO) ;
Belgrader; Phillip; (Pleasanton, CA) ; Van Hatten;
Brian; (Boulder, CO) ; Chabansky; Bruce;
(Boulder, CO) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Inscripta, Inc. |
Boulder |
CO |
US |
|
|
Family ID: |
1000005147403 |
Appl. No.: |
16/928061 |
Filed: |
July 14, 2020 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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16827222 |
Mar 23, 2020 |
10737271 |
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16928061 |
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16749757 |
Jan 22, 2020 |
10639637 |
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16827222 |
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16596940 |
Oct 9, 2019 |
10576474 |
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16749757 |
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16525152 |
Jul 29, 2019 |
10478822 |
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16596940 |
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16451602 |
Jun 25, 2019 |
10406525 |
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16525152 |
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16380828 |
Apr 10, 2019 |
10376889 |
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16451602 |
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62657651 |
Apr 13, 2018 |
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62657654 |
Apr 13, 2018 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
B01L 7/00 20130101; C12M
35/02 20130101; B01L 7/02 20130101; C12M 23/06 20130101; C12M 41/18
20130101; B01L 2300/0829 20130101; B01L 2300/04 20130101; C12M
23/44 20130101; B01L 3/527 20130101; B01L 2300/12 20130101; C12M
41/48 20130101; B01L 2300/042 20130101; C12M 29/04 20130101; B01L
2300/0645 20130101; C12N 13/00 20130101; B01L 2200/028 20130101;
B01L 2200/16 20130101 |
International
Class: |
B01L 3/00 20060101
B01L003/00; C12M 1/36 20060101 C12M001/36; C12M 1/12 20060101
C12M001/12; C12M 3/00 20060101 C12M003/00; C12N 13/00 20060101
C12N013/00; B01L 7/00 20060101 B01L007/00; B01L 7/02 20060101
B01L007/02; C12M 1/02 20060101 C12M001/02; C12M 1/42 20060101
C12M001/42; C12M 1/00 20060101 C12M001/00 |
Claims
1. A method for automated nuclease editing of live cells
comprising: providing a reagent cartridge comprising: a thermal
block with at least three rows of reservoirs defined therein; at
least two strips of co-joined reagent tubes each configured to
reside within one of the reservoirs in the reagent cartridge,
wherein at least one co-joined reagent tube contains cells and at
least one co-joined reagent tube contains nucleic acids; at least
one strip comprising a transformation module configured to reside
within one of the reservoirs in the reagent cartridge; providing a
growth module; providing a cell concentration module; providing an
editing module; and providing a processor, wherein the processor
controls a liquid handling device to transfer the cells from the
reagent cartridge to the growth module, wherein the growth module
grows the cells; transfer the grown cells from the growth module to
the cell concentration module, wherein the cell concentration
module concentrates the grown cells; transfer the concentrated
cells from the cell concentration module to the transformation
module and transfer the nucleic acids from the reagent cartridge to
the transformation module, wherein the transformation module
transforms the concentrated cells with the nucleic acids; and
transfer the transformed cells from the transformation module to
the editing module, wherein the cells are edited.
2. The method of claim 1, wherein the transformation module is a
flow-through electroporation device.
3. The method of claim 2, wherein the flow-through electroporation
device comprises: an inlet and inlet channel for introduction of
the concentrated cells to the flow-through electroporation device;
an outlet and outlet channel for exit of the concentrated cells
from the flow-through electroporation device; a flow channel
intersecting and positioned between the inlet channel and outlet
channel; and two or more electrodes, wherein at least one of the
electrodes is positioned in the flow channel between the
intersection of the flow channel with the first inlet channel and
the constriction in the flow channel and at least one of the
electrodes is positioned between the constriction in the flow
channel and the intersection of the flow channel with the outlet
channel, and wherein the two or more electrodes are in fluid
communication with the concentrated cells in the flow channel but
are not in the flow path of the concentrated cells in the flow
channel nor do the two or more electrodes define the constriction
of the flow channel, and wherein the two or more electrodes are
configured to apply an electric pulse or electric pulses to the
concentrated cells.
4. The method of claim 1, wherein the reagent cartridge comprises a
script to control the processor.
5. The method of claim 4, wherein the script comprises information
on the contents of the co-joined tubes.
6. The method of claim 1, wherein the thermal block is fabricated
from cyclic olefin copolymer (COC).
7. The method of claim 1, wherein the at least two strips of
co-joined reagent tubes and the strip comprising the flow-through
electroporation device are packaged in a kit.
8. The method of claim 1, wherein the reagent cartridge is
configured to accommodate four or more inserts and further
comprising at least one large reservoir strip.
9. The method of claim 8, wherein the large reservoir strip
comprises the cells.
10. The method of claim 8, wherein the at least two strips of
co-joined reagent tubes, one large reservoir strip and the strip
comprising the flow-through electroporation device are packaged in
a kit.
11. The method of claim 1, wherein the reagent cartridge can be
cooled to 0.degree. C. and heated to 40.degree. C.
12. The method of claim 1, wherein the cell growth module comprises
a rotating growth vial.
13. The method of claim of claim 1, wherein the cell concentration
module utilizes tangential flow filtration.
14. The method of claim 1, further comprising the step of providing
a storage module, wherein the processor controls the liquid
handling device to transfer the edited cells to the storage
module.
15. The method of claim 1, further comprising the step of providing
a recovery module, wherein the processor controls a liquid handling
device to transfer the edited cells to the recovery module.
16. A method for automated nuclease editing of live cells
comprising: providing a reagent cartridge comprising: a thermal
block with at least three rows of reservoirs defined therein; at
least one strip of co-joined reagent tubes each configured to
reside within one of the reservoirs in the reagent cartridge,
wherein at least one co-joined reagent tube contains cells and at
least one co-joined reagent tube contains nucleic acids; at least
one strip comprising a transformation module configured to reside
within one of the reservoirs in the reagent cartridge; providing a
nucleic acid assembly module; providing a growth module; providing
a cell concentration module; providing an editing module; and
providing a processor, wherein the processor controls a liquid
handling device to transfer the cells from the reagent cartridge to
the growth module, wherein the growth module grows the cells;
transfer the grown cells from the growth module to the cell
concentration module, wherein the cell concentration module
concentrates the grown cells; transfer the concentrated cells from
the cell concentration module to the transformation module;
transfer the nucleic acids to the nucleic acid assembly module,
wherein the nucleic acid assembly module assembles the nucleic
acids; transfer the assembled nucleic acids from the nucleic acid
assembly module to the transformation module, wherein the
transformation module transforms the concentrated cells with the
assembled nucleic acids; and transfer the transformed cells from
the transformation module to the editing module, wherein the cells
are edited.
17. The method of claim 16, further comprising the step of
providing a recovery module, wherein the processor controls a
liquid handling device to transfer the edited cells to the recovery
module.
18. The method of claim 16, wherein the reagent cartridge is
configured to accommodate at least one large reservoir strip.
19. The method of claim 18, wherein the at least one large
reservoir strip contains the cells.
20. The method of claim 16, wherein the reagent cartridge comprises
a script to control the processor.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. Utility
application Ser. No. 16/827,222, entitled "Automated Cell
Processing Instruments Comprising Reagent Cartridges", filed 23
Mar. 2020, now U.S. Pat. No. 10,738,271; which is a continuation of
U.S. Utility application Ser. No. 16/749,757, entitled "Automated
Cell Processing Instruments Comprising Reagent Cartridges", filed
22 Jan. 2020, now U.S. Pat. No. 10,639,637; which is a continuation
of U.S. Utility application Ser. No. 16/596,940, entitled
"Automated Cell Processing Instruments Comprising Reagent
Cartridges", filed 9 Oct. 2019, now U.S. Pat. No. 10,576,474; which
is a continuation of U.S. Utility application Ser. No. 16/525,152,
entitled "Automated Cell Processing Instruments Comprising Reagent
Cartridges", filed 29 Jul. 2019, now U.S. Pat. No. 10,478,822;
which is a continuation of U.S. Utility application Ser. No.
16/451,601, entitled "Automated Cell Processing Instruments
Comprising Reagent Cartridges", filed Jun. 25, 2019, now U.S. Pat.
No. 10,406,525; which is a continuation of U.S. Utility application
Ser. No. 16/380,828, entitled "Automated Cell Processing
Instruments Comprising Reagent Cartridges", filed Apr. 10, 2019,
now U.S. Pat. No. 10,376,889; all of which claim priority to U.S.
Provisional Patent Application Ser. No. 62/657,651, entitled
"Combination Reagent Cartridge and Electroporation Device," filed
Apr. 13, 2018; and U.S. Provisional Patent Application Ser. No.
62/657,654, entitled "Automated Cell Processing Systems Comprising
Cartridges," filed Apr. 13, 2018; all of which are hereby
incorporated by reference in their entireties for all purposes.
FIELD OF THE INVENTION
[0002] The present disclosure relates to automated multi-module
instruments comprising reagent cartridges, particularly automated
multi-module instruments configured to facilitate nucleic
acid-directed nuclease editing of live cells.
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] Genome editing with engineered nucleases is a method in
which changes to nucleic acids are made to the genome of a living
organism. Certain nucleases create site-specific double-strand
breaks at target regions in the genome, which can be repaired by
homologous recombination, resulting in targeted edits. These
methods, however, have not been compatible with automation due to
low efficiencies and challenges with performing liquid handling,
cell transformation, growth measurement, cell selection, cell
editing and linking modules that perform these functions without
human intervention. Traditional benchtop devices do not necessarily
scale and integrate well into an automated, modular system. Methods
and instruments to create edited cell populations thus remain
cumbersome, and the challenges of introducing multiple rounds of
edits using recursive techniques where the process may take several
days or even weeks has limited the nature and complexity of cell
populations that can be created.
[0005] There is thus a need for automated instruments, modules and
methods for introducing nucleic acids and other biological
molecules into living cells in an automated fashion where the
edited cells may be further processed in the automated instrument
and/or used for further experimentation outside of the automated
instrument. The present disclosure addresses this need.
SUMMARY OF THE INVENTION
[0006] This Summary is provided to introduce a selection of
concepts in a simplified form that are further described below in
the Detailed Description. This Summary is not intended to identify
key or essential features of the claimed subject matter, nor is it
intended to be used to limit the scope of the claimed subject
matter. Other features, details, utilities, and advantages of the
claimed subject matter will be apparent from the following written
Detailed Description including those aspects illustrated in the
accompanying drawings and defined in the appended claims.
[0007] The present disclosure provides a reagent cartridge
configured for use in an automated multi-module cell processing
environment. The reagent cartridges may include an electroporation
device, as well as sample receptacles, reagent receptacles, waste
receptacles and the like, and a script for controlling a processor
to dispense samples and reagents contained in the receptacles, and
to porate cells in the electroporation device. Also described are
kits including the cartridges, automated multi-module cell
processing instruments including the reagent cartridges and methods
of using the reagent cartridges.
[0008] Thus, there is presented herein a reagent cartridge
comprising: a thermal block with block reservoirs defined therein;
a cover disposed upon the thermal block with cover reservoirs
defined therein, wherein the cover reservoirs and block reservoirs
are coincident and together define reservoirs in the reagent
cartridge; a script readable by a processor; and at least two
inserts configured to reside within the reservoirs in the reagent
cartridge. In some embodiments, one or more of the inserts
comprises tubes, a reservoir coincident with the reservoirs in the
reagent cartridge, and/or a flow-through electroporation
device.
[0009] In some aspects when the reagent cartridge comprises and
insert with a flow-through electroporation device, the flow-through
electroporation device comprises: an inlet and inlet channel for
introduction of a cell sample to the flow-through electroporation
device; an outlet and outlet channel for exit of the cell sample
from the flow-through electroporation device; a flow channel
intersecting and positioned between the inlet channel and outlet
channel with a constriction in the flow channel positioned between
the inlet channel and outlet channel; and two or more electrodes,
wherein at least one of the electrodes is positioned in the flow
channel between the intersection of the flow channel with the first
inlet channel and the constriction in the flow channel and at least
one of the electrodes is positioned between the constriction in the
flow channel and the intersection of the flow channel with the
outlet channel, and wherein the two or more electrodes are in fluid
communication with the cell sample in the flow channel but are not
in the flow path of the cell sample in the flow channel nor do the
two or more electrodes define the constriction of the flow channel,
and wherein the two or more electrodes are configured to apply an
electric pulse or electric pulses to a cell sample.
[0010] In some aspects, one, two or all three of the cover, thermal
block, and at least one of the at least two inserts of the reagent
cartridge are fabricated from stainless steel, aluminum, polyvinyl
chloride, cyclic olefin copolymer (COC), polyamide, polyethylene,
polypropylene, acrylonitrile butadiene, polycarbonate,
polyetheretheketone (PEEK), poly(methyl methylacrylate) (PMMA),
polysulfone, or polyurethane, and in some aspects, one, two or all
three of the cover, thermal block, and at least one of the at least
two inserts are fabricated from cyclic olefin copolymer (COC).
[0011] In some aspects, the reagent cartridge is configured to
accommodate three, four, five, six or more inserts. Also, in some
aspects the reagent cartridge can be cooled to 0.degree. C. or less
and heated to 40.degree. C. or more.
[0012] In some aspects, the script of the reagent cartridge
comprises information on the contents of the inserts.
[0013] Other embodiments present an automated multi-module cell
processing instrument comprising the reagent cartridge, wherein the
automated multi-module cell processing instrument comprises a
processor, and the script readable by a processor comprises
commands for performing four or more processes in the automated
multi-module cell processing instrument. In some aspects, the
script readable by a processor comprises commands for performing
all processes in the automated multi-module cell processing
instrument. In some aspects, the script readable by a processor
commands the processor to perform tasks according to encoded
instructions or the script commands the processor to modify tasks
according to encoded scripts.
[0014] In some aspects, the automated multi-module cell processing
instrument further comprises one or more of a cell growth module,
with, e.g., a rotating growth vial; a cell concentration module,
where the cell concentration module comprises a tangential flow
filtration device; a recovery module, where the recovery module
optionally comprises a cell growth module comprising a rotating
growth vial; a nucleic acid assembly module; an editing module;
and/or a singulation module. In some aspects, in lieu of a
singulation module, the automated multi-module cell processing
instrument comprises a bulk liquid editing module.
[0015] In some embodiments the script comprises commands for
retrieving reagents in the reagent cartridge and commands for
electroporating cells in the electroporation device, and in some
embodiments the script readable by a processor comprises commands
for performing one or more additional processes in the automated
multi-module cell processing instrument, and in yet other aspects,
the automated multi-module cell processing the script readable by a
processor comprises commands for performing all processes in the
automated multi-module cell processing instrument.
[0016] Also provided herein is a kit comprising the reagent
cartridge, and further comprising reagents dispensed in one or more
of the reagent reservoirs. In some aspects, one or more of the
reagent reservoirs therein is sealed. In some aspects, the kit
comprises a top cover for the reagent cartridge disposed over the
reagent cartridge and over the reagent cartridge seals, if present.
In some aspects, one or more of the reagent reservoirs comprises
cells, and in some aspects, the kit further comprises a
flow-through electroporation device.
[0017] Also in one embodiment there is provided an automated
multi-module cell processing instrument comprising: a reagent
cartridge, wherein the reagent cartridge comprises a thermal block
with block reservoirs defined therein; a cover disposed upon the
thermal block with cover reservoirs defined therein, wherein the
cover reservoirs and block reservoirs are coincident and together
define reservoirs in the reagent cartridge; a script readable by a
processor; and inserts configured to reside within the reservoirs
in the reagent cartridge, wherein one insert comprises a
flow-through electroporation device, and wherein the flow-through
electroporation device comprises: an inlet and inlet channel for
introduction of a cell sample to the flow-through electroporation
device; an outlet and outlet channel for exit of the cell sample
from the flow-through electroporation device; a flow channel
intersecting and positioned between the inlet channel and outlet
channel with a constriction in the flow channel positioned between
the inlet channel and outlet channel; and two or more electrodes,
wherein at least one of the electrodes is positioned in the flow
channel between the intersection of the flow channel with the first
inlet channel and the constriction in the flow channel and at least
one of the electrodes is positioned between the constriction in the
flow channel and the intersection of the flow channel with the
outlet channel, and wherein the two or more electrodes are in fluid
communication with the cell sample in the flow channel but are not
in the flow path of the cell sample in the flow channel nor define
the flow channel, and wherein the two or more electrodes are
configured to apply an electric pulse or electric pulses to a cell
sample; the processor; a cell growth module; a cell concentration
module; a recovery module; and a storage module.
[0018] These aspects and other features and advantages of the
invention are described below in more detail.
BRIEF DESCRIPTION OF THE DRAWINGS
[0019] The foregoing and other features and advantages of the
present disclosure will be more fully understood from the following
detailed description of illustrative embodiments taken in
conjunction with the accompanying drawings in which:
[0020] FIGS. 1A-1D depict the structure and components of one
embodiment of a reagent cartridge.
[0021] FIGS. 2A-2E depict the structure and components of an
alternative embodiment of a reagent cartridge.
[0022] FIGS. 3A-3T depict the structure and components of yet
another alternative embodiment of a reagent cartridge.
[0023] FIGS. 4A-4D depict an automated multi-module instrument and
components thereof with which to generate the edited cells.
[0024] FIG. 5A depicts one embodiment of a rotating growth vial for
use with the cell growth module described herein. FIG. 5B
illustrates a perspective view of one embodiment of a rotating
growth vial in a cell growth module. FIG. 5C depicts a cut-away
view of the cell growth module from FIG. 5B. FIG. 5D illustrates
the cell growth module of FIG. 5B coupled to LED, detector, and
temperature regulating components.
[0025] FIG. 6A is a model of the process of tangential flow
filtration used in the TFF device presented herein. FIG. 6B depicts
a top view of a lower member of one embodiment of an exemplary TFF
device showing the serpentine geometry of the flow channel. FIG. 6C
depicts a top view of upper and lower members and a membrane of an
exemplary TFF device. FIG. 6D depicts a bottom view of upper and
lower members and a membrane of an exemplary TFF device. FIGS.
6E-6I depict various views of an embodiment of a TFF module
comprising a TFF device and having fluidically coupled reservoirs
for retentate, filtrate, and exchange buffer.
[0026] FIGS. 7A and 7B are top perspective and bottom perspective
views, respectively, of flow-through electroporation devices (here,
there are six such devices co-joined). FIG. 7C is a top view of one
embodiment of an exemplary flow-through electroporation device.
FIG. 7D depicts a top view of a cross section of the
electroporation device of FIG. 7C. FIG. 7E is a side view cross
section of a lower portion of the electroporation devices of FIGS.
7C and 7D.
[0027] FIG. 8A depicts a simplified graphic of a workflow for
singulating, editing and normalizing cells after nucleic
acid-guided nuclease genome editing in a solid wall device. FIG. 8B
is a photograph of one embodiment of a solid wall device. FIGS.
8C-8E are photographs of E. coli cells singulated (via Poisson
distribution) and grown into colonies in microwells in a solid wall
device with a permeable bottom at low, medium, and high
magnification, respectively. FIG. 8F is a simplified block diagram
of methods for enriching for live cells that have been edited via
nucleic acid-guided nuclease editing that do not involve
singulation or a singulation device and instead utilize cell growth
in liquid and induction of editing. FIG. 8G depicts a typical
growth curve for cells in culture. FIG. 8H is a graphic depiction
of methods for growing, inducing, editing, enriching, and screening
for edited cells in a population of cells.
[0028] FIG. 9 is a flow chart of an example method for automated
multi-module cell editing to produce the cell libraries as
described herein.
[0029] FIG. 10 is a simplified flow chart of two exemplary methods
(1000a and 1000b) that may be performed by an automated
multi-module cell editing instrument comprising a singulation
device.
[0030] FIG. 11 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.
[0031] FIG. 12 is a simplified block diagram of an alternative
embodiment of an exemplary automated multi-module cell processing
instrument comprising a solid wall
singulation/growth/editing/normalization module, in this case, used
for recursive editing.
[0032] FIG. 13 is a simplified process diagram of yet another
embodiment of an exemplary automated multi-module cell processing
instrument, in this case without a singulation module.
[0033] FIG. 14 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. 15 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. 16 is a graph demonstrating the effectiveness of a
4-paddle rotating growth vial and cell growth device as described
herein for growing an EC83 cell culture vs. a conventional orbital
cell shaker.
[0036] FIG. 17 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. 18 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. 19 is a graph demonstrating real-time monitoring of
growth of S. cerevisiae 288 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. 20A 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. 20B is a graph plotting filtrate conductivity against
filter processing time for a S. cerevisiae yeast culture processed
in the cell concentration device/module described herein.
[0041] FIG. 21A is a bar graph showing the results of
electroporation of E. coli using a flow-through electroporation
device and a comparator (NEPA) electroporation device.
[0042] FIG. 21B 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.
[0043] FIG. 22 is a bar graph showing the results of
electroporation of S. cerevisiae yeast using an FTEP device of the
disclosure and a comparator electroporation method.
[0044] It should be understood that the drawings are not
necessarily to scale, and that like reference numbers refer to like
features.
DETAILED DESCRIPTION
[0045] 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.
[0046] 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.
[0047] 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," and/or "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.
[0048] 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.
[0049] 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.
[0050] 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.
[0051] The term "complementary" as used herein refers to
Watson-Crick base pairing between nucleotides and specifically
refers to nucleotides hydrogen bonded to one another with thymine
or uracil residues linked to adenine residues by two hydrogen bonds
and cytosine and guanine residues linked by three hydrogen bonds.
In general, a nucleic acid includes a nucleotide sequence described
as having a "percent complementarity" or "percent homology" to a
specified second nucleotide sequence. For example, a nucleotide
sequence may have 80%, 90%, or 100% complementarity to a specified
second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10
of 10 nucleotides of a sequence are complementary to the specified
second nucleotide sequence. For instance, the nucleotide sequence
3'-TCGA-5' is 100% complementary to the nucleotide sequence
5'-AGCT-3'; and the nucleotide sequence 3'-TCGA-5' is 100%
complementary to a region of the nucleotide sequence
5'-TTAGCTGG-3'.
[0052] 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.
[0053] As used herein the term "donor DNA" or "donor nucleic acid"
refers to nucleic acid that is designed to introduce a DNA sequence
modification (insertion, deletion, substitution) into a locus by
homologous recombination using nucleic acid-guided nucleases. For
homology-directed repair, the donor DNA must have sufficient
homology to the regions flanking the "cut site" or site to be
edited in the genomic target sequence. The length of the homology
arm(s) will depend on, e.g., the type and size of the modification
being made. In many instances and preferably, the donor DNA will
have two regions of sequence homology (e.g., two homology arms) to
the genomic target locus. Preferably, an "insert" region or "DNA
sequence modification" region--the nucleic acid modification that
one desires to be introduced into a genome target locus in a
cell--will be located between two regions of homology. The DNA
sequence modification may change one or more bases of the target
genomic DNA sequence at one specific site or multiple specific
sites. A change may include changing 1, 2, 3, 4, 5, 10, 15, 20, 25,
30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more base
pairs of the target sequence. A deletion or insertion may be a
deletion or insertion of 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, 50,
75, 100, 150, 200, 300, 400, or 500 or more base pairs of the
target sequence.
[0054] As used herein, "enrichment" refers to enriching for edited
cells by singulation, optionally inducing editing, and growth of
singulated or substantially singulated cells into terminal-sized
colonies (e.g., saturation or normalization of colony growth).
Alternatively, "enrichment" may be performed on a bulk liquid
culture, by inducing editing when the cells are at the end of the
logarithmic stage of growth or just after the cells enter growth
senescence. Inducing editing entails inducing transcription of the
nuclease, gRNA or both.
[0055] 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.
[0056] "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.
[0057] "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.
[0058] A "promoter" or "promoter sequence" is a DNA regulatory
region capable of binding RNA polymerase and initiating
transcription of a polynucleotide or polypeptide coding sequence
such as messenger RNA, ribosomal RNA, small nuclear or nucleolar
RNA, guide RNA, or any kind of RNA transcribed by any class of any
RNA polymerase I, II or III. Promoters may be constitutive or
inducible, and in some embodiments--particularly many embodiments
in which enrichment is employed--the transcription of at least one
component of the nucleic acid-guided nuclease editing system is
under the control of an inducible promoter.
[0059] As used herein the term "selectable marker" refers to a gene
introduced into a cell, which confers a trait suitable for
artificial selection. General use selectable markers are well-known
to those of ordinary skill in the art. Drug selectable markers such
as ampicillin/carbenicillin, kanamycin, chloramphenicol,
erythromycin, tetracycline, gentamicin, bleomycin, streptomycin,
rifampicin, puromycin, hygromycin, blasticidin, and G418 may be
employed. In other embodiments, selectable markers include, but are
not limited to sugars such as rhamnose, human nerve growth factor
receptor (detected with a MAb, such as described in U.S. Pat. No.
6,365,373); truncated human growth factor receptor (detected with
MAb); mutant human dihydrofolate reductase (DHFR; fluorescent MTX
substrate available); secreted alkaline phosphatase (SEAP;
fluorescent substrate available); human thymidylate synthase (TS;
confers resistance to anti-cancer agent fluorodeoxyuridine); human
glutathione S-transferase alpha (GSTA1; conjugates glutathione to
the stem cell selective alkylator busulfan; chemoprotective
selectable marker in CD34+cells); CD24 cell surface antigen in
hematopoietic stem cells; human CAD gene to confer resistance to
N-phosphonacetyl-L-aspartate (PALA); human multi-drug resistance-1
(MDR-1; P-glycoprotein surface protein selectable by increased drug
resistance or enriched by FACS); human CD25 (IL-2.alpha.;
detectable by Mab-FITC); Methylguanine-DNA methyltransferase (MGMT;
selectable by carmustine); and Cytidine deaminase (CD; selectable
by Ara-C). "Selective medium" as used herein refers to cell growth
medium to which has been added a chemical compound or biological
moiety that selects for or against selectable markers.
[0060] The terms "target genomic DNA sequence", "target sequence",
or "genomic target locus" refer to any locus in vitro or in vivo,
in a nucleic acid (e.g., genome) of a cell or population of cells,
in which a change of at least one nucleotide is desired using a
nucleic acid-guided nuclease editing system. The target sequence
can be a genomic locus or extrachromosomal locus.
[0061] 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, YACs,
BACs, synthetic chromosomes, and the like. As used herein, the
phrase "engine vector" comprises a coding sequence for a nuclease
to be used in the nucleic acid-guided nuclease systems and methods
of the present disclosure. The engine vector may also comprise, in
a bacterial system, the .lamda. Red recombineering system or an
equivalent thereto. Engine vectors also typically comprise a
selectable marker. As used herein the phrase "editing vector"
comprises a donor nucleic acid, optionally including an alteration
to the target sequence that prevents nuclease binding at a PAM or
spacer in the target sequence after editing has taken place, and a
coding sequence for a gRNA. The editing vector may also comprise a
selectable marker and/or a barcode. In some embodiments, the engine
vector and editing vector may be combined; that is, all editing and
selection components may be found on a single vector. Further, the
engine and editing vectors comprise control sequences operably
linked to, e.g., the nuclease coding sequence, recombineering
system coding sequences (if present), donor nucleic acid, guide
nucleic acid, and selectable marker(s).
Exemplary Reagent Cartridges for Use in Automated Multi-Module Cell
Processing Instruments
[0062] The present disclosure relates to reagent cartridges for
providing reagents in automated multi-module cell processing
instruments. The reagent cartridges include sample receptacles,
reagent receptacles, and/or waste receptacles, etc.; additionally,
in certain embodiments, the reagent cartridge will comprise a
script readable by a processor for dispensing the reagents and
controlling the electroporation device contained within the reagent
cartridge and may, in some embodiments, further comprise an
electroporation device. Further, the reagent cartridge is a part of
an automated multi-module cell processing instrument and the script
may comprise instructions for performing some, many, or all
processes in the automated multi-module cell processing instrument.
Also described are kits including the reagent cartridges comprising
instructions for functioning of various modules of the automated
multi-module cell processing instrument. The automated multi-module
cell processing instrument can be used to process many different
types of cells in a controlled, contained, and reproducible manner,
including bacterial cells, mammalian cells, non-mammalian
eukaryotic cells, yeast cells, fungi, archaea, and the like.
[0063] FIG. 1A depicts an exemplary combination reagent cartridge
and electroporation device 100 ("cartridge" or "reagent cartridge")
that may be used in an automated multi-module cell processing
instrument. Cartridge 100 comprises a body 102, and reagent
receptacles or reservoirs 104. Additionally, cartridge 100
comprises an electroporation device 106 (an exemplary embodiment of
which is described in detail in relation to FIGS. 7A-7E), which is
preferably a flow-through electroporation device. Cartridge 100 may
be disposable or may be configured to be reused. Preferably,
cartridge 100 is disposable. Cartridge 100 may be made from any
suitable material, including stainless steel, aluminum, paper or
other fiber, 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. If the cartridge is
disposable, preferably it is made of plastic or paper. Preferably
the material used to fabricate the cartridge is
thermally-conductive, as in certain embodiments the cartridge 100
contacts a thermal device (not shown) that heats or cools reagents
in the reagent receptacles or reservoirs 104. In some embodiments,
the thermal device is a Peltier device or thermoelectric cooler.
Reagent receptacles or reservoirs 104 may be receptacles into which
individual tubes of reagents are inserted as shown in FIG. 1A,
receptacles into which one or more multiple co-joined tubes are
inserted (e.g., a row of four tubes that are co-joined are inserted
into the reagent receptacles; see, e.g., FIGS. 2A and 3A), or the
reagent receptacles may hold the reagents without inserted tubes
with the reagents dispensed directly into the receptacles or
reservoirs. Additionally, the receptacles 104 in a reagent
cartridge 100 may be configured for any combination of tubes,
co-joined tubes, and direct-fill of reagents.
[0064] In one embodiment, the reagent receptacles or reservoirs 104
of reagent cartridge 100 are configured to hold various size tubes,
including, e.g., 250 ml tubes, 25 ml tubes, 10 ml tubes, 5 ml
tubes, and Eppendorf (e.g., microcentrifuge) tubes. In yet another
embodiment, all receptacles may be configured to hold the same size
tube, e.g., 5 ml tubes, and reservoir inserts may be used to
accommodate smaller tubes in the reagent reservoir (seen, e.g., in
FIG. 1B). In yet another embodiment--particularly in an embodiment
where the reagent cartridge 100 is disposable--the reagent
reservoirs 104 hold reagents without inserted tubes. In this
disposable embodiment, the reagent cartridge may be part of a kit,
where the reagent cartridge is pre-filled with reagents and the
receptacles or reservoirs sealed with, e.g., foil, film, heat seal
acrylic or the like and presented to a consumer where the reagent
cartridge can then be used in an automated multi-module cell
processing instrument. The reagents contained in the reagent
cartridge 100 will vary depending on work flow; that is, the
reagents will vary depending on the processes to which the cells
are subjected in the automated multi-module cell processing
instrument.
[0065] FIG. 1B also depicts an exemplary combination reagent
cartridge and electroporation device 100 ("cartridge" or "reagent
cartridge") that may be used in an automated multi-module cell
processing instrument. The reagent cartridge and electroporation
device 100 of FIG. 1B is very similar to the reagent cartridge and
electroporation device 100 of FIG. 1A but illustrates additional
detail. Like the reagent cartridge and electroporation device 100
of FIG. 1A, reagent cartridge 100 in FIG. 1B comprises a body 102,
reagent receptacles or reservoirs 104, and an electroporation
device 106. Again, reagent cartridge 100 may be disposable, or may
be configured to be reused. Preferably, reagent cartridge 100 is
disposable. The combination reagent cartridge and electroporation
device 100 of FIG. 1B further comprises a cover 152 for the reagent
cartridge 100, and a cavity or recess 150 into which the
electroporation device 106 is placed.
[0066] Further, FIG. 1B shows additional detail for an embodiment
of a reagent cartridge and electroporation device 100 where reagent
receptacles or reservoirs 104 are configured to accept tubes 160 or
thermal spacers 162. In addition, within thermal spacer 162 is a
small tube 154 positioned in adaptor 156, such as an Eppendorf or
microcentrifuge tube, useful when only small reagent volumes are
required. Thermal spacer 162 is thermally conductive assuring heat
or cooling is transferred to reagents contained in small tubes,
e.g., Eppendorf (e.g., microfuge) tubes 154. As discussed above, in
some embodiments, the body of the reagent cartridge 100 itself is
thermally conductive and is in contact with a thermal device to
warm or cool the reagents contained therein as desired by a user.
Also seen in FIG. 1B are foil or film seals 158 used to seal tubes
160 and 154. Alternatively, if the reagent cartridge is reusable,
the reagent cartridge may comprise a thermal device to heat and
cool reagents contained within, as opposed to contacting a thermal
device (not shown). In most embodiments, the thermal device will
cool the reagents.
[0067] FIG. 1C depicts an exemplary matrix configuration 140 for
the reagents contained in the reagent cartridges of FIGS. 1A and
1B; where this matrix embodiment is a 4.times.4 reagent matrix.
Through a matrix configuration, a user (or programmed processor)
can locate the proper reagent for a given process. That is,
reagents such as cell samples, enzymes, buffers, nucleic acid
vectors, expression cassettes, reaction components (such as, e.g.,
MgCl.sub.2, dNTPs, isothermal nucleic acid assembly reagents, Gap
Repair reagents, and the like), wash solutions, ethanol, and
magnetic beads for nucleic acid purification and isolation, etc.,
are positioned in the matrix 140 at a known position. For example,
reagents are located at positions A1 (110), A2 (111), A3 (112), A4
(113), B1 (114), B2 (115) and so on through, in this embodiment, to
position D4 (125). FIG. 1A is labeled to show where several
reservoirs 104 correspond to matrix 140; see receptacles 110, 113,
121 and 125. Although the reagent cartridges 100 of FIGS. 1A and 1B
and the matrix configuration 140 of FIG. 1C shows a 4.times.4
matrix, matrices of the reagent cartridge and electroporation
devices can be any configuration, such as, e.g., 2.times.2,
2.times.3, 2.times.4, 2.times.5, 2.times.6, 3.times.3, 3.times.5,
4.times.6, 6.times.7, or any other configuration, including
asymmetric configurations, or two or more different matrices
depending on the reagents needed for the intended workflow. Note in
FIG. 4A the matrix configuration is a 5.times.3+1 matrix.
[0068] In preferred embodiments of reagent cartridge 100 shown in
FIGS. 1A and 1B, the reagent cartridge comprises a script (not
shown) readable by a processor (not shown) for dispensing the
reagents via a liquid handling device (ADP head shown at 432) and
controlling the electroporation device contained within reagent
cartridge 100. Also, the reagent cartridge 100 as one component in
an automated multi-module cell processing instrument may comprise a
script specifying two, three, four, five, ten or more processes
performed by the automated multi-module cell processing instrument,
or even specify all processes performed by the automated
multi-module cell processing instrument. In certain embodiments,
the reagent cartridge is disposable and is pre-packaged with
reagents tailored to performing specific cell processing protocols,
e.g., genome editing or protein production. Because the reagent
cartridge contents vary while components of the automated
multi-module cell processing instrument may not, the script
associated with a particular reagent cartridge matches the reagents
used and cell processes performed. Thus, e.g., reagent cartridges
may be pre-packaged with reagents for genome editing and a script
that specifies the process steps (or a script that modifies the
steps of a pre-programmed script based on, e.g., an updated reagent
in the reagent cartridge) for performing genome editing in an
automated multi-module cell processing instrument such as described
in relation to FIGS. 4A-4D. For example, the reagent cartridge 100
of FIGS. 1A and 1B may comprise a script to pipette
electrocompetent cells from reservoir A2 (111), transfer the cells
to the electroporation device 106, pipette a nucleic acid solution
comprising an editing vector from reservoir C3 (120), transfer the
nucleic acid solution to the electroporation device, initiate the
electroporation process for a specified time, then move the porated
cells to a reservoir D4 (125) in the reagent cassette or to another
module such as the rotating growth vial (See, e.g., 418 or 420 of
FIG. 4A) in the automated multi-module cell processing instrument
in FIG. 4A. In another example, the reagent cartridge may comprise
a script to pipette transfer of a nucleic acid solution comprising
a vector from reservoir C3 (120), nucleic acid solution comprising
editing oligonucleotide cassettes in reservoir C4 (121), and
isothermal nucleic acid assembly reaction mix from A1 (110) to the
isothermal nucleic acid assembly/desalting reservoir (See, e.g.,
414 of FIG. 4A). The script may also specify process steps
performed by other modules in the automated multi-module cell
processing instrument. For example, the script may specify that the
isothermal nucleic acid assembly/desalting module be heated to
50.degree. C. for 30 min to generate an assembled isothermal
nucleic acid product; and desalting of the assembled isothermal
nucleic acid product via magnetic bead-based nucleic acid
purification involving a series of pipette transfers and mixing of
magnetic beads in reservoir B2 (115), ethanol wash in reservoir B3
(116), and water in reservoir CI (118) to the isothermal nucleic
acid assembly/desalting reservoir (See, e.g., 414 of FIG. 4A).
[0069] FIG. 1D is an embodiment of a wash or reagent cartridge 130
with a body 132 and reagent receptacles or reservoirs 134 that can
be separate from or combined with reagent cartridge 100 of FIG. 1A
or 1B; that is, reagent cartridges 100 and 130 may be combined into
a single housing/body. Wash or reagent cartridge 130 in the
embodiment shown in FIG. 1D is configured to accommodate large
tubes, for example, wash solutions, or solutions that are used
often throughout an iterative process. For example, wash or reagent
cartridge 130 may be configured to remain in place when two or more
reagent cartridges 100 are sequentially used and replaced. As with
reagent cartridge 100, wash or reagent cartridge 130 may be
disposable, or may be configured to be reused. Preferably reagent
cartridge 130 is disposable.
[0070] Wash or reagent cartridge 130 may be made from any suitable
material as described above for reagent cartridge 100. If cartridge
130 is disposable, preferably it is fabricated from plastic.
Reagent receptacles or reservoirs 134 may be receptacles into which
individual tubes of reagents are inserted as shown in FIG. 1D,
receptacles into which one or more multiple co-joined tubes are
inserted (e.g., a row of tubes or rows and columns of tubes that
are co-joined and can be inserted into the reagent receptacles such
as seen in FIGS. 2A and 3A), or the reagent receptacles may hold
the reagents without inserted tubes. Additionally, the receptacles
in wash or reagent cartridge 130 may be configured for any
combination of tubes, co-joined tubes, and direct-fill of reagents.
In one embodiment--particularly in an embodiment where wash or
reagent cartridge 130 is disposable--the reagent receptacles hold
reagents without inserted tubes. As with reagent cartridge 100,
cartridge 130 may be fabricated from thermally conductive material
such as plastic and be in contact with a thermal device to keep
reagents contained within wash or reagent cartridge 130 at a
temperature specified by a user or script (typically cool at, e.g.,
4.degree. C.). In this disposable embodiment, the wash or reagent
cartridge 130 may be part of a kit with reagent cartridge 100,
where the reagent cartridges are, e.g., pre-filled with reagents
and the receptacles or reservoirs sealed with e.g., foil, film,
heat seal acrylic, or the like and presented to a consumer to be
used in an automated cell processing instrument.
[0071] FIGS. 2A-2E depict the structure and components of an
alternative embodiment of a reagent cartridge. In FIG. 2A, reagent
cartridge 200 comprises a body 202, which has reservoirs 204. One
reservoir 204 is shown empty, and two of the reservoirs have
individual tubes (not shown) inserted therein, with individual tube
covers 205. Additionally shown are rows of reservoirs into which
have been inserted co-joined rows of large tubes 203a, and
co-joined rows of small tubes 203b. The co-joined rows of tubes are
presented in a strip, with outer flanges 207 that mate on the
backside of the outer flange (not shown) with an indentation 209 in
the body 202, so as to secure the co-joined rows of tubes (203a and
203b) to the reagent cartridge 200. Shown also is a base 211 of
reagent cartridge body 202. Note that the reservoirs 204 in body
202 are shaped generally like the tubes in the co-joined tubes that
are inserted into these reservoirs 204 FIG. 2B depicts the reagent
cartridge 200 in FIG. 2A with a row of co-joined large tubes 203a,
a row of co-joined small tubes 203b, and one large tube 260 with a
cover 205 removed from (i.e., depicted above) the reservoirs 204 of
the reagent cartridge 200. Again, the co-joined rows of tubes are
presented in a strip, with individual large tubes 261 shown, and
individual small tubes 255 shown. Again, each strip of co-joined
tubes comprises outer flanges 207 that mate on the backside (not
shown) of the outer flange with an indentation 209 in the body 202,
to secure the co-joined rows of tubes (203a and 203b) to the
reagent cartridge 200. As in FIG. 2A, reagent cartridge body 202
comprises a base 211. Reagent cartridge 200, like reagent cartridge
100 shown in FIGS. 1A-1D, 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 200 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 200 may contact a thermal device (not shown)
that heats or cools reagents in the reagent reservoirs 204,
including reagents in co-joined tubes. In some embodiments, the
thermal device is a Peltier device or thermoelectric cooler.
[0072] FIG. 2C is a perspective view of a cross section 221 of the
reagent cartridge 200 depicted in FIG. 2A. Again, reagent cartridge
200 comprises a base 211 and reservoirs 204. The two reservoirs in
the left-most row of reservoirs are empty. In the next two rows of
reservoirs, co-joined large tubes 203a are inserted, including
large tubes 261. The three right-most rows of reservoirs have
co-joined small tubes 203b inserted therein. In the right-most
reservoirs small tubes 255 can be seen disposed in reservoirs 204.
FIG. 2D is a perspective view and FIG. 2E is a side view of one
strip of co-joined large tubes 203a comprising large tubes 261.
Outer flange 207 can be seen in these figures, as can an inner
flange 213, which engages with indentation 209 on the reagent
cartridge 200 (not shown in these figures but see FIGS. 2A and
2B).
[0073] FIGS. 3A-3M depict the structure and components of yet
another embodiment of a reagent cartridge useful for multi-module
automated processing of cells. FIG. 3A depicts a
partially-assembled reagent cartridge 300, comprising a cover 301,
reservoirs 304, a reservoir cover 305 comprising an outer flange
307 with an inner flange (not shown), that engages with an
indentation (indentations 309 seen with uncovered reservoirs 304).
Outer flange 307 provides a grip for handling the reservoir cover
305 or other inserts for the reservoirs described below. Like
reagent cartridge 200 depicted in FIGS. 2A-2C, reservoirs 304 in
reagent cartridge 300 in FIG. 3A accommodate strips of co-joined
tubes, including as shown here a strip of co-joined large tubes
303a (with individual tubes 361). However, in contrast to the
reservoirs 204 in the embodiment of the reagent cartridge 200 shown
in FIGS. 2A-2C, the reservoirs 304 in the embodiment of the reagent
cartridge 300 shown in FIG. 3A (and in FIGS. 3B-3F) are
"slot"-shaped and are configured to be "universal": that is, the
slot-shaped reservoirs 304 are configured to accommodate many
different types of reservoir inserts, as described below. The strip
of co-joined large tubes 303a comprises an outer flange 307 with an
inner flange (not shown), configured to engage with an indentation
309 in the cover 301 of the reagent cartridge 300, as well as a tab
317 configured to engage with tab engagement member 311 in cover
301. Any or all reservoir inserts may be covered by a protective
foil, film or peel-off strip to maintain sterility of the reservoir
and the contents thereof (e.g., see 371 of FIG. 3N).
[0074] Also shown in FIG. 3A is an insert 308 comprising a
flow-through electroporation device 306. The flow-through
electroporation device 306 is configured to transform or transfect
nucleic acids or other materials into cells and is described in
detail in relation to FIGS. 7A-7E. Note that the embodiments of
reagent cartridges shown in FIGS. 1 and 2 may also be configured to
accommodate a transformation (e.g., flow-through electroporation)
device or module. The flow-through electroporation device insert
308 comprises both a tab 317, and an outer flange 307. As with the
strip of co-joined large tubes 303a, the outer flange 307 comprises
an inner flange (not shown), configured to engage with an
indentation 309 in the cover 301 of the reagent cartridge 300, as
well as a tab 317 configured to engage with tab engagement member
311 in cover 301.
[0075] FIG. 3B depicts the reagent cartridge 300 from FIG. 3A
assembled, with the strip of co-joined large tubes 303a and
flow-through electroporation device insert 308 inserted into the
left-most reservoirs 304. Again, reagent cartridge 300 comprises a
cover 301, reservoirs 304 with one reservoir covered with reservoir
cover 305. Reservoir cover 305, strip of co-joined large tubes
303a, and flow-through electroporation device insert 308 each
comprise an outer flange 307 with an inner flange (not shown) that
engages with an indentation (indentations 309 seen with uncovered
reservoirs 304), as well as a tab 317 (not shown) configured to
engage with tab engagement member 311 in cover 301. The reagent
reservoirs 304 of reagent cartridge 300 are configured to hold
various size tubes, including, e.g., 250 ml tubes, 25 ml tubes, 10
ml tubes, 5 ml tubes, and Eppendorf or microcentrifuge tubes.
[0076] FIG. 3C depicts the assembled reagent cartridge 300 of FIG.
3B with cover 301 detached from thermal body 302. Thermal body 302
comprises reservoirs 304a that mate with reservoirs 304 in cover
301. Again, a strip of co-joined large tubes 303a and flow-through
electroporation device insert 308 are shown inserted into the
left-most reservoirs 304, and one reservoir 304 comprises reservoir
cover 305. Reservoir cover 305, strip of co-joined large tubes
303a, and flow-through electroporation device insert 308 each
comprise an outer flange 307 with an inner flange (not shown) that
engages with an indentation (indentations 309 seen with uncovered
reservoirs 304). The thermal body 302 and cover 301 may be
configured to be reusable. Thermal body 302 is made from any
thermally-conductive material such as stainless steel, aluminum, or
thermally-conductive plastics including aluminum, copper, stainless
steel, high thermal conductivity plastic. Typically, reagents in
the reagent cartridge are kept cool at, e.g., 4.degree. C.
[0077] Cover 301 is configured to cover the exterior of thermal
body 302 and to retain any condensation or "sweating" that may
occur on thermal body 302. Additionally, cover 301 in this
embodiment is shown as having reservoirs 304 that are "universal"
in configuration, which match the shape of the reservoirs 304a in
the thermal body 302; however, the configuration of reservoirs 304
in cover 301 may instead have holes or other-shaped recesses that
accommodate individual tubes (such as the body 202 of reagent
cartridge 200 shown in FIGS. 2A-2C), large reservoir inserts (such
as shown in FIG. 3H), a flow-through electroporation device insert
308, or other configuration of insert. Reagent cartridge cover 301,
like reagent cartridges 200 (FIGS. 2A-2C) and 100 (FIGS. 1A-1D) may
be made from any suitable material, preferably a
thermally-insulating material, including stainless steel, aluminum,
paper, 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. Also seen in FIG. 3D
is an optional hole or port 319 that traverses thermal body 302
through reservoirs 304 to remove condensation and inadvertent
liquid spills from the reservoirs, where the fluid collected is
shunted to a controlled waste collection point.
[0078] The inserts may be made of any appropriate material;
however, the inserts are in most embodiments disposable, so
typically are fabricated from biocompatible 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. Reagent cartridges 300 are versatile, where different
workflows are supported by the different contents of the inserts as
described below in relation to FIGS. 3Q-3T.
[0079] FIG. 3D is a cross section of reagent cartridge 300 taken at
line A-A of FIG. 3C, where the strip of co-joined large tubes 303a
is seen comprising four tubes 361. In this cross section, cover 301
of reagent cartridge 300 is seen, disposed on top of thermal body
302. As a part of the strip of co-joined large tubes 303a, tab 317
can be seen engaged with tab engagement member 311, and inner
flange 313 is engaged with indentation 309. Further, outer flange
307 can be seen. Note again that the reservoirs 304a in thermal
body 302 are "universal" in nature, able to accommodate many
different types of inserts (See, e.g., FIG. 3C). FIG. 3E is a side
perspective view of reagent cartridge 300 with cover 301 comprising
from left to right, a flow-through electroporation device insert
308 comprising a flow-through electroporation device 306, a strip
of co-joined large tubes 303a, a reservoir with a reservoir cover,
a large reservoir insert 315 (uncovered in this figure, but
typically covered with, e.g., a pierceable foil or film cover), a
reservoir 304 with no insert, and a reservoir into which has been
inserted a large tube insert 321. Large tube insert 321 has two
large tubes 323 with large tube covers 325, and lips 327 that
engage with a large tube support (not shown) that is a part of
large tube insert 321. Each of the reservoir inserts comprises an
outer flange 307 with an inner flange (not shown here but see FIG.
3D) that engages with an indentation (indentation 309 seen with
reservoir 304). The large reservoir insert 315 may contain a
reagent that is used in large amounts in the cell processing
protocol, such as wash buffers or cell growth medium, or the large
reservoir insert 315 may be used as a waste receptacle for liquid
or solid waste. Likewise, the large tubes 323 in the large tube
insert 321 may hold reagents that are, e.g., required in larger
quantities, supplied by the user (e.g., a custom reagent that is
not part of a kit) or kept at a different temperature than other
reagents in the reagent cartridge.
[0080] FIG. 3F is a top view of the reagent cartridge 300 depicted
in FIG. 3E, again showing from left to right, a flow-through
electroporation device insert 308 comprising a flow-through
electroporation device 306, a strip of co-joined large tubes 303a,
a reservoir with a reservoir cover 305, a large reservoir insert
315, a reservoir 304 with no insert, and a reservoir into which has
been inserted a large tube insert 321. Large tube insert 321 has
two large tubes (not shown) with large tube covers 325, and lips
327. Each of the reservoir inserts (flow-through electroporation
device insert 308, strip of co-joined large tubes 303a, reservoir
cover 305, large reservoir insert 315, and large tube insert 321)
comprises an outer flange 307 with an inner flange (not shown here
but see FIG. 3D) that engages with an indentation (indentation 309
seen with uncovered reservoir 304).
[0081] FIG. 3G is a side perspective view of a strip of co-joined
large tubes 303a, comprising four large tubes 361, tab 317 and
outer flange 307. FIG. 3H is a side perspective view of large
reservoir insert 315 also comprising tab 317 and outer flange 307.
FIGS. 31 and 3J provide perspective side views of large tube insert
321 comprising large tube support 329, with two large tubes 323
both with covers 325 and lips 327, where the lips comprise a member
on the lower or bottom surface of the lip (not shown) that engages
with slot 331 in the large tube support 329. Large tube support 329
has a tab 317 that engages with tab engagement member 311 of the
reagent cartridge cover 301 (not shown but see FIGS. 3A and 3B) and
outer flange 307, which has an inner flange (not shown) that
engages with an indentation 309 in the reagent cartridge cover
301.
[0082] FIGS. 3K-3M depict three side perspective views of a
flow-through electroporation device insert 308 comprising
flow-through electroporation device 306. Flow-through
electroporation device 306 is configured to transform or transfect
nucleic acids or other materials into cells and is described in
detail in relation to FIGS. 7A-7E. In the embodiment of reagent
cartridge 300 depicted in FIGS. 3A-3M, the flow-through
electroporation device 306 (e.g., transformation module) is located
in the reagent cartridge 300 (also see reagent cartridge 410 with
flow-through electroporation device 430 as one component of an
automated multi-module cell processing instrument 400 in FIG. 4A);
although in alternative embodiments, the transformation module may
be separate from the reagent cartridge. The flow-through
electroporation device insert 308 comprises both a tab 317, and an
outer flange 307. FIG. 3M depicts the flow-through electroporation
device insert 308 with a cover 305 for, e.g., shipping and to keep
the flow-through electroporation device 306 sterile until use.
[0083] FIGS. 3N-3P are various views of an FTEP insert 308. FIG. 3N
is a cross section of the FTEP insert 308, housing FTEP 306
(described in more detail in FIGS. 7A-7E infra) where two
electrodes 370 are shown. FTEP insert 308 comprises an outer flange
307, an FTEP cover 371, and tab 317, which engages with tab
engagement member 311 (not shown) in reagent cartridge cover 301
(also not shown but see FIG. 3A) when inserted into a reagent
cartridge. Also shown is FTEP cover 371, which in this embodiment
is a tear-off foil, film or other type seal that is used to
maintain the sterility of the FTEP until ready for use. FIG. 3O is
a top view of the FTEP insert 308 shown in FIG. 3N. Seen are FTEP
insert cover or seal 371, which protects and keeps sterile the FTEP
device before use and is removable by a user, data 373, and
machine-readable indicia 375. Data 373 may include information such
as a lot number, a serial number, a product number, an expiration
date, or other data pertinent to FTEP insert 308. Machine-readable
indicia 375 may be a barcode, QR code, a Data Matrix code (error
correction-type barcode), RFID or other type of machine-readable
indicia, detected by one or more imaging sensors (e.g., barcode
scanners, cameras, etc.) (not shown) located in an automated
multi-module cell processing instrument to, e.g., confirm the
contents of and optionally to control the operation of FTEP insert
308. Though in FIG. 3O an FTEP insert is shown, any type of
insert--e.g., co-joined large tubes (e.g., 5 mL, 10 mL, 15, mL, 20
mL, 35 mL, or 50 mL tubes), co-joined small tubes (e.g., 0.1 mL,
0.2 mL, 0.5 mL tubes), large reservoir inserts (e.g., 50 mL, 75,
mL, 100, mL, 125 mL, 150 ml, 200 mL, 250 mL reservoirs), and large
tube inserts (e.g., 50 mL, 75, mL, 100, mL tubes)--may and
preferably are also labeled with data 373 and machine-readable
indicia 375. FIG. 3P is a top view of FTEP insert 308 with FTEP
insert cover 371 (seen in FIG. 3O) removed. Again, data 373,
machine-readable indicia 375, and FTEP 306 can be seen. Also,
electrodes 370 of FTEP 306 are seen.
[0084] FIGS. 3Q and 3R show two exemplary reagent cartridges 300.
FIG. 3Q is a top view of an exemplary room temperature reagent
block 300a, comprising from left to right a strip of co-joined
small tubes 303b (e.g., 0.5 mL tubes), a strip co-joined large
tubes or receptables 303a (e.g., 20 mL tubes), a second strip of
co-joined large tubes or receptacles 303a, an open reservoir 304,
and an FTEP insert 308 comprising FTEP 306. In this exemplary room
temperature reagent cartridge 300a, there are 6 reservoirs
available to accept various inserts, although it should be
recognized by the skilled artisan given the teachings herein that
there may be more or less reservoirs in alternative reagent
cartridge configurations. The reagent cartridges 300 may be any
appropriate size as long as the reagent cartridge can accommodate
inserts for the appropriate reagents (or components, such as an
FTEP or waste receptacle) in the proper volumes required for cell
processing. In one example, the room temperature reagent cartridge
300a may be from 40 to 250 mm wide (in this FIG. 3Q, width is from
left to right), or from 50 to 200 mm wide, or from 75 to 175 mm
wide, or from 90 to 150 mm wide; from 40 to 200 mm deep (in this
FIG. 3Q, depth is from bottom to top), or from 50 to 175 mm deep,
or from 70 to 150 mm wide, or from 80 to 125 mm deep; from 40 to
150 mm tall, or from 50 to 130 mm tall, or from 60 to 120 mm tall.
In one embodiment, the room temperature reagent cartridge 300a is
approximately 130 mm wide and 90 mm deep, and can accommodate six
inserts, each of which are approximately 20 mm wide and 80 mm
deep.
[0085] FIG. 3R is a top view of an exemplary cooled (e.g., at
4.degree. C.) reagent cartridge 300b, comprising from left to right
two large tubes 350 and 351, a strip of co-joined small tubes 303b
(with tubes 355), and four open reservoirs 304. Cooled reagent
cartridge 300b may be the same size, smaller, or larger than room
temperature reagent block 300a. In some embodiments, the same
reagent cartridge configuration is used for both room temperature
reagent cartridge 300a and cooled reagent cartridge 300b; that is,
because of the modular nature of reagent cartridge 300 (and 300a
and 300b) and the varying configurations of the inserts, different
reagents, reagent volumes, and components can be placed in reagent
cartridge 300 as needed. Additionally, because a thermal block 302
is used in the reagent cartridge embodiment shown in FIGS. 3A-3F, a
reagent cartridge 300 can be used at room temperature (300a), at
4.degree. C. (300b), or at any other desired temperature.
[0086] FIGS. 3S and 3T are top views of two sets of exemplary
reagent cartridges for, e.g., editing E. coli cells (FIG. 3S) and
S. cerevisiae cells (FIG. 3T). Each set of two reagent cartridges
300 has a room temperature reagent cartridge 300a and a cooled
reagent cartridge 300b. In FIG. 3S, room temperature cartridge 300a
comprises from left to right a strip of co-joined small tubes 303b
(two of which have contents and four of which do not), a strip of
co-joined large tubes or receptacles 303a (all of which have
contents), a second strip of co-joined large tubes or receptacles
303a (two of which have contents, and two of which do not), a large
reservoir inset 315, an open reservoir 304, and an FTEP insert 308
comprising FTEP 306. For editing in E. coli cells, the contents of
the tubes and receptacles may include an editing vector or separate
editing cassettes and a plasmid backbone disposed in small tubes
355 (with tubes 355 having a volume of, e.g., 0.5 mL). Whether the
reagents comprise an editing vector or separate editing cassettes
and plasmid backbone will depend on whether nucleic acid assembly
is performed in the automated multi-module cell processing
instrument.
[0087] The left-most strip of co-joined large tubes 303a (each with
an approximate volume of 20 mL) may comprise different growth
media. For example, a first tube may contain luria broth (LB)
growth medium without a selection component (e.g., antibiotic), a
second tube may contain luria broth (LB) growth medium with a first
selection component (e.g., ampicillin/carbenicillin), a third tube
may contain luria broth (LB) growth medium with a second selection
component (e.g., kanamycin), and a fourth tube may contain luria
broth (LB) growth medium with a third selection component (e.g.,
chloramphenicol) where different selection is performed in various
stages of cell editing. The second strip of co-joined large tubes
303a (each with an approximate volume of, e.g., 20 mL) may comprise
different reagents needed for cell washes or to conduct buffer
exchange to make the E. coli cells electrocompetent (e.g., buffer
with glycerol) or reagents to facilitate loading the cells in the
solid wall singulation device (e.g., phospho-buffered
saline+TWEEN). Likewise, the large reservoir inset 315 (with an
approximate volume of, e.g., 100 mL) may comprise medium, cell wash
reagents or any other reagent needed in a large volume.
Alternatively, large reservoir inset 315 may be used as a waste
receptacle for, e.g., discarded cell wash supernatant or for solid
waste such as pipette tips. Open reservoir 304 may be left empty or
may, like large reservoir inset 315, be used as a waste receptacle.
Finally, in this embodiment FTEP insert 308 comprising FTEP 306 is
located in room temperature reagent cartridge 300a.
[0088] The second reagent cartridge 300 in FIG. 3S is a cooled
reagent cartridge 300b comprising from left to right two large
tubes 350 and 351 (each with an approximate volume of, e.g., 5 mL),
a strip of co-joined small tubes 303b (with tubes 355), and four
open reservoirs 304. In some embodiments large tube 350 may contain
the E. coli cells to be processed in the automated multi-module
cell processing instrument, and large tube 351 may serve as the
reservoir for the E. coli cells that have been edited; that is,
large tube 351 may serve as a cell storage unit (such as storage
unit 1112 seen in FIG. 11, storage unit 1212 seen in FIG. 12, and
storage unit 1312 seen in FIG. 13). As with the open reservoir 304
in room temperature reagent cartridge 300a, the four open
reservoirs 304 in cooled reagent cartridge 300b may be used for,
e.g., discarded cell wash or for solid waste such as pipette
tips.
[0089] The reagent cartridges 300 in FIG. 3T are identical to the
reagent cartridges 300 in FIG. 3S. Both FIGS. 3S and 3T comprise a
set of two reagent cartridges 300, one room temperature reagent
cartridge 300a and one cooled reagent cartridge 300b. The reagents
that are held in the various tubes and reservoirs differ, however,
as the reagent cartridges in FIG. 3T are adapted to edit yeast (S.
cerevisiae) cells. For example, instead of glycerol being used to
render the cells electrocompetent for transformation, sorbitol is
used; instead of luria broth growth medium, YPAD (yeast extract
peptone dextrose medium with adenine sulfate added) growth medium
is used; instead of PBS, a lithium acetate buffer is used; etc. As
with the reagent cartridges in FIG. 3S, the large reservoirs may
contain buffers or wash solutions needed in large quantities for
cell processing or may be used for liquid or solid waste. In some
embodiments, each pair of reagent cartridges 300a and 300b hold all
reagents needed for one round of cell editing (see FIGS. 9, 11 and
13). In embodiments where more than one round of cells editing is
desired (see FIG. 9 (step 924 "additional editing?") and FIG. 12),
reagent cartridges 300a and 300b may be replaced with new, filled
reagent cartridges 300a and 300b for another round of editing.
Alternatively, reagent cartridges 300a and 300b may hold reagents
needed for two or more rounds of editing.
Nucleic Acid-Directed Nuclease Genome Editing Generally
[0090] A recent discovery for editing live cells involves nucleic
acid-guided nuclease (e.g., RNA-guided nuclease) editing. A nucleic
acid-guided nuclease complexed with an appropriate synthetic guide
nucleic acid in a cell can cut the genome of the cell at a desired
location. The guide nucleic acid helps the nucleic acid-guided
nuclease recognize and cut the DNA at a specific target sequence.
By manipulating the nucleotide sequence of the guide nucleic acid,
the nucleic acid-guided nuclease may be programmed to target any
DNA sequence for cleavage as long as an appropriate protospacer
adjacent motif (PAM) is nearby. In certain aspects, the nucleic
acid-guided nuclease editing system may use two separate guide
nucleic acid molecules that combine to function as a guide nucleic
acid, e.g., a CRISPR RNA (crRNA) and trans-activating CRISPR RNA
(tracrRNA). In other aspects, the guide nucleic acid may be a
single guide nucleic acid that includes both the crRNA and tracrRNA
sequences.
[0091] In general, a guide nucleic acid (e.g., gRNA) complexes with
a compatible nucleic acid-guided nuclease and can then hybridize
with a target sequence, thereby directing the nuclease to the
target sequence. A guide nucleic acid can be DNA or RNA;
alternatively, a guide nucleic acid may comprise both DNA and RNA.
In some embodiments, a guide nucleic acid may comprise modified or
non-naturally occurring nucleotides. In cases where the guide
nucleic acid comprises RNA, the gRNA may be encoded by a DNA
sequence on a polynucleotide molecule such as a plasmid, linear
construct, or the coding sequence may reside within an editing
cassette. The sequence for the gRNA may be under the control of a
constitutive promoter, or, in some embodiments and preferably, an
inducible promoter as described below.
[0092] 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.
[0093] In the present methods and compositions, the guide nucleic
acid is provided as a sequence to be expressed from a plasmid or
vector and comprises both the guide sequence and the scaffold
sequence as a single transcript under the control of a promoter,
and in some embodiments, an inducible promoter. The guide nucleic
acid can be engineered to target a desired target sequence by
altering the guide sequence so that the guide sequence is
complementary to a desired target sequence, thereby allowing
hybridization between the guide sequence and the target sequence.
In general, to generate an edit in the target sequence, the
gRNA/nuclease complex binds to a target sequence as determined by
the guide RNA, and the nuclease recognizes a 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. The 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).
[0094] The guide nucleic acid may be part of an editing cassette
that encodes the donor nucleic acid. Alternatively, the guide
nucleic acid may not be part of the editing cassette and instead
may be encoded on the engine or editing vector backbone. For
example, a sequence coding for a guide nucleic acid can be
assembled or inserted into a vector backbone first, followed by
insertion of the donor nucleic acid in, e.g., the editing cassette.
In other cases, the donor nucleic acid in, e.g., an editing
cassette can be inserted or assembled into a vector backbone first,
followed by insertion of the sequence coding for the guide nucleic
acid. In yet other cases, the sequence encoding the guide nucleic
acid and the donor nucleic acid (inserted, for example, in an
editing cassette) are simultaneously but separately inserted or
assembled into a vector. In yet other embodiments, the sequence
encoding the guide nucleic acid and the sequence encoding the donor
nucleic acid are both included in the editing cassette.
[0095] The 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. In
certain embodiments, the genome editing of a target sequence both
introduces a desired DNA change to a target sequence, e.g., the
genomic DNA of a cell, and removes, mutates, or renders inactive a
proto-spacer mutation (PAM) region in the target sequence.
Rendering the PAM at the target sequence inactive precludes
additional editing of the cell genome at that target sequence,
e.g., upon subsequent exposure to a nucleic acid-guided nuclease
complexed with a synthetic guide nucleic acid in later rounds of
editing. Thus, cells having the desired target sequence edit and an
altered PAM can be selected using a nucleic acid-guided nuclease
complexed with a synthetic guide nucleic acid complementary to the
target sequence. The genome of the cells that did not undergo the
first editing event will be cut rendering a double-stranded DNA
break, and thus these cells will not continue to be viable. The
genome of the cells containing the desired target sequence edit and
PAM alteration will not be cut, as these edited cells no longer
contain the necessary PAM site and will thus continue to grow and
propagate.
[0096] The range of target sequences that nucleic acid-guided
nucleases can recognize is constrained by the need for a specific
PAM to be located near the desired target sequence. As a result, it
often can be difficult to target edits with the precision that is
necessary for genome editing. It has been found that nucleases can
recognize some PAMs very well (e.g., canonical PAMs), and other
PAMs less well or poorly (e.g., non-canonical PAMs). Because
certain of the methods disclosed herein allow for identification of
edited cells in a background of unedited cells (see, e.g., FIGS.
8A-8H and the descriptions thereof), 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.
[0097] 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/Cpfl, MAD2, or MAD7 or other MADzymes.
As with the guide nucleic acid, the nuclease may be encoded by a
DNA sequence on a vector (e.g., the engine vector) and be under the
control of a constitutive or inducible promoter. In some
embodiments, the sequence encoding the nuclease is under the
control of an inducible promoter, and the inducible promoter may be
separate from but the same as the inducible promoter controlling
transcription of the guide nucleic acid; that is, a separate
inducible promoter drives the transcription of the nuclease and
guide nucleic acid sequences but the two inducible promoters may be
the same type of inducible promoter (e.g., both are pL promoters).
Alternatively, the inducible promoter controlling expression of the
nuclease may be different from the inducible promoter controlling
transcription of the guide nucleic acid; that is, e.g., the
nuclease may be under the control of the pBAD inducible promoter,
and the guide nucleic acid may be under the control of the pL
inducible promoter.
[0098] Another component of the nucleic acid-guided nuclease system
is the donor nucleic acid. In some embodiments, the donor nucleic
acid is on the same polynucleotide (e.g., editing vector or editing
cassette) as the guide nucleic acid and may be (but not
necessarily) under the control of the same promoter as the guide
nucleic acid (e.g., a single promoter driving the transcription of
both the guide nucleic acid and the donor nucleic acid). The donor
nucleic acid is designed to serve as a template for homologous
recombination with a target sequence nicked or cleaved by the
nucleic acid-guided nuclease as a part of the gRNA/nuclease
complex. A donor nucleic acid polynucleotide may be of any suitable
length, such as about or more than about 20, 25, 50, 75, 100, 150,
200, 500, or 1000 nucleotides in length. In certain preferred
aspects, the donor nucleic acid can be provided as an
oligonucleotide of between 20-300 nucleotides, more preferably
between 50-250 nucleotides. The donor nucleic acid comprises a
region that is complementary to a portion of the target sequence
(e.g., a homology arm). When optimally aligned, the donor nucleic
acid overlaps with (is complementary to) the target sequence by,
e.g., about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more
nucleotides. In many embodiments, the donor nucleic acid comprises
two homology arms (regions complementary to the target sequence)
flanking the mutation or difference between the donor nucleic acid
and the target template. The donor nucleic acid comprises at least
one mutation or alteration compared to the target sequence, such as
an insertion, deletion, modification, or any combination thereof
compared to the target sequence.
[0099] Often the donor nucleic acid is provided as an editing
cassette, which is inserted into a vector backbone where the vector
backbone may comprise a promoter driving transcription of the gRNA
and the coding sequence of the gRNA, or the vector backbone may
comprise a promoter driving the transcription of the gRNA but not
the gRNA itself. Moreover, there may be more than one, e.g., two,
three, four, or more guide nucleic acid/donor nucleic acid
cassettes inserted into an editing vector, where each guide nucleic
acid is under the control of separate different promoters, separate
like promoters, or where all guide nucleic acid/donor nucleic acid
pairs are under the control of a single promoter. In some
embodiments, the promoter driving transcription of the gRNA and the
donor nucleic acid (or driving more than one gRNA/donor nucleic
acid pair) is an inducible promoter and the promoter driving
transcription of the nuclease is an inducible promoter as well. For
additional information regarding editing cassettes, see U.S. Pat.
Nos. 9,982,278 and 10,240,167, and U.S. Ser. Nos. 15/116,616;
15/948,785; 16/056,310; 16/275,439; and 16/275,465.
[0100] 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
(e.g., growth of edited cell colonies and unedited cell colonies
become "normalized"). Further, a guide nucleic acid may be
efficacious directing the edit of more than one donor nucleic acid
in an editing cassette; e.g., if the desired edits are close to one
another in a target sequence.
[0101] In addition to the donor nucleic acid, an editing cassette
may comprise one or more primer sites. The primer sites can be used
to amplify the editing cassette by using oligonucleotide primers;
for example, if the primer sites flank one or more of the other
components of the editing cassette.
[0102] Also, as described above, the donor nucleic acid may
optionally comprise--in addition to the at least one mutation
relative to a target sequence--one or more PAM sequence alterations
that mutate, delete or render inactive the PAM site in the target
sequence. The PAM sequence alteration in the target sequence
renders the PAM site "immune" to the nucleic acid-guided nuclease
and protects the target sequence from further editing in subsequent
rounds of editing if the same nuclease is used.
[0103] In addition, the editing cassette may comprise a barcode. A
barcode is a unique DNA sequence that corresponds to the donor DNA
sequence such that the barcode can identify the edit made to the
corresponding target sequence. The barcode typically comprises four
or more nucleotides. In some embodiments, the editing cassettes
comprise a collection of donor nucleic acids representing, e.g.,
gene-wide or genome-wide libraries of donor nucleic acids. The
library of editing cassettes is cloned into vector backbones where,
e.g., each different donor nucleic acid is associated with a
different barcode.
[0104] 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 thereof.
[0105] The engine and editing vectors comprise control sequences
operably linked to the component sequences to be transcribed. As
stated above, the promoters driving transcription of one or more
components of the nucleic acid-guided nuclease editing system may
be inducible such as one or both of the gRNA and the nuclease. A
number of gene regulation control systems have been developed for
the controlled expression of genes in plant, microbe, and animal
cells, including mammalian cells, including the pL promoter
(induced by heat inactivation of the CI857 repressor), the pBAD
promoter (induced by the addition of arabinose to the cell growth
medium), and the rhamnose inducible promoter (induced by the
addition of rhamnose to the cell growth medium). Other systems
include the tetracycline-controlled transcriptional activation
system (Tet-On/Tet-Off, Clontech, Inc. (Palo Alto, Calif.); Bujard
and Gossen, PNAS, 89(12):5547-5551 (1992)), the Lac Switch
Inducible system (Wyborski et al., Environ Mol Mutagen,
28(4):447-58 (1996); DuCoeur et al., Strategies 5(3):70-72 (1992);
U.S. Pat. No. 4,833,080), the ecdysone-inducible gene expression
system (No et al., PNAS, 93(8):3346-3351 (1996)), the cumate
gene-switch system (Mullick et al., BMC Biotechnology, 6:43
(2006)), and the tamoxifen-inducible gene expression (Zhang et al.,
Nucleic Acids Research, 24:543-548 (1996)) as well as others. In
the present methods used in the modules and instruments described
herein, it is preferred that at least one of the nucleic
acid-guided nuclease editing components (e.g., the nuclease and/or
the gRNA) is under the control of a promoter that is activated by a
rise in temperature, as such a promoter allows for the promoter to
be activated by an increase in temperature, and de-activated by a
decrease in temperature, thereby "turning off" the editing process.
Thus, in the scenario of a promoter that is de-activated by a
decrease in temperature, editing in the cell can be turned off
without having to change media; to remove, e.g., an inducible
biochemical in the medium that is used to induce editing.
Automated Cell Editing Instruments and Modules
Automated Cell Editing Instruments
[0106] FIG. 4A depicts an exemplary automated multi-module cell
processing instrument 400 to, e.g., perform one of the exemplary
workflows described infra, comprising one or more reagent
cartridges as described herein. The instrument 400, for example,
may be and preferably is designed as a desktop instrument for use
within a laboratory environment. The instrument 400 may incorporate
a mixture of reusable and disposable components for performing
various staged processes in conducting automated genome cleavage
and/or editing in cells. Illustrated is a gantry 402, providing an
automated mechanical motion system (actuator) (not shown) that
supplies XYZ axis motion control to, e.g., an automated liquid
handling system 458 including, e.g., an air displacement pipettor
which allows for cell processing among multiple modules without
human intervention. In some automated multi-module cell processing
instruments, the air displacement pipettor 432 is moved by gantry
402 and the various modules and reagent cartridges remain
stationary; however, in other embodiments, the liquid handling
system may stay stationary while the various modules and reagent
cartridges are moved. Also included in the automated multi-module
cell processing instrument 400 is reagent cartridge 410 comprising
reservoirs 412 and transformation module 430 (e.g., a flow-through
electroporation device as described in relation to FIGS. 3K-3P and
7A-7E), as well as a wash cartridge 404 comprising reservoirs 406.
The wash cartridge 404 may be configured to accommodate large
tubes, for example, wash solutions, or solutions that are used
often throughout an iterative process. In one example, wash
cartridge 404 may be configured to remain in place when two or more
reagent cartridges 410 are sequentially used and replaced. Although
reagent cartridge 410 and wash cartridge 404 are shown in FIG. 4A
as separate cartridges, the contents of wash cartridge 404 may be
incorporated into reagent cartridge 410. The reagent cartridge and
wash cartridge may be identical except for the consumables
(reagents or other components contained within the various inserts)
inserted therein. Note in this embodiment, transformation module
430 is contained within reagent cartridge 410; however, in
alternative embodiments, transformation module 430 is contained
within its own module or may be part of another module, such as a
growth module.
[0107] The wash and reagent cartridges 404 and 410, in some
implementations, comprise disposable kits (one or more of the
various inserts and reagents) provided for use in the automated
multi-module cell editing instrument 400. For example, a user may
open and position each of the reagent cartridge 410 and the reagent
and wash cartridges 404 comprising various desired inserts and
reagents within a chassis of the automated multi-module cell
editing instrument 400 prior to activating cell processing.
[0108] Also illustrated is the automated liquid handling system 458
including the gantry 402 and air displacement pipettor 432. In some
examples, the robotic liquid handling system 458 may include an
automated liquid handling system such as those manufactured by
Tecan Group Ltd. of Mannedorf, Switzerland, Hamilton Company of
Reno, Nev. (see, e.g., WO2018015544A1), or Beckman Coulter, Inc. of
Fort Collins, Colo. (see, e.g., US20160018427A1). Pipette tips may
be provided in a pipette transfer tip supply (not shown) for use
with the air displacement pipettor 432.
[0109] Inserts or components of the wash and reagent cartridges
404, 410, in some implementations, are marked with machine-readable
indicia (not shown), such as bar codes, for recognition by the
robotic handling system 458. For example, the robotic liquid
handling system 458 may scan one or more inserts within each of the
wash and reagent cartridges 404, 410 to confirm contents. In other
implementations, machine-readable indicia may be marked upon each
cartridge 404, 410, and the processing system 426 (shown in FIG.
4D) of the automated multi-module cell editing instrument 400 may
identify a stored materials map based upon the machine-readable
indicia. The exemplary automated multi-module cell processing
instrument 400 of FIG. 4A further comprises a cell growth module
434. (Note, all modules recited briefly here are described in
detail below.) In the embodiment illustrated in FIG. 4A, the cell
growth module 434 comprises two cell growth vials 418, 420
(described in greater detail below in relation to FIGS. 5A-5D) as
well as a cell concentration module 422 (described in detail in
relation to FIGS. 6A-6F). In alternative embodiments, the cell
concentration module 422 may be separate from cell growth module
434, e.g., in a separate, dedicated module. Also illustrated as
part of the automated multi-module cell processing instrument 400
of FIG. 4A is an optional enrichment module 440, served by, e.g.,
robotic liquid handling system 458 and air displacement pipettor
432. Also seen are an optional nucleic acid assembly/desalting
module 414 comprising a reaction chamber or tube receptacle (not
shown) and a magnet 416 to allow for purification of nucleic acids
using, e.g., magnetic solid phase reversible immobilization (SPRI)
beads (Applied Biological Materials Inc., Richmond, BC), as well as
a cell enrichment module (described in relation to FIGS. 8A-8H
below). The cell growth module, cell concentration module,
transformation module, enrichment module, reagent cartridge, and
nucleic acid assembly module are described in greater detail
below.
[0110] FIG. 4B is a plan view of the front of the exemplary
multi-module cell processing instrument 400 depicted in FIG. 4A.
Cartridge-based source materials (such as in reagent cartridge
410), for example, may be positioned in designated areas on a deck
402 of the cell processing instrument 400 for access by a robotic
handling instrument (not shown in this figure). As illustrated in
FIG. 4B, the deck 402 may include a protection sink such that
contaminants spilling, dripping, or overflowing from any of the
modules of the instrument 400 are contained within a lip of the
protection sink. In addition to reagent cartridge 410, also seen in
FIG. 4B is wash cartridge 404, optional enrichment module 440, and
a portion of growth module 434. Also seen in this view is touch
screen display 450, transformation module controls 438, electronics
rack 436, and processing system 426.
[0111] FIGS. 4C through 4D illustrate side and front views of
multi-module cell processing instruments 480 comprising chassis 490
for use in desktop versions of the cell processing instrument 480.
For example, the chassis 490 may have a width of about 24-48
inches, a height of about 24-48 inches and a depth of about 24-48
inches. Chassis 490 may be and preferably is designed to hold
multiple modules and disposable supplies used in automated cell
processing. Further, chassis 490 may mount a robotic liquid
handling system 458 for moving materials between modules. As
illustrated, the chassis 490 includes a cover 452 having a handle
454 and hinges 456a-456c for lifting the cover 452 and accessing
the interior of the chassis 490. A cooling grate 464 shown in FIG.
4C allows for air flow via an internal fan (not shown). Further,
the chassis 490 is lifted by adjustable feet 470 (feet 470a-470c
are shown). The feet 470a-470c, for example, may provide additional
air flow beneath the chassis 490. A control button 466, in some
embodiments, allows for single-button automated start and/or stop
of cell processing within the chassis 490.
[0112] Inside the chassis 490, in some implementations, a robotic
liquid handling system 458 is disposed along a gantry 402 above
reagent cartridges 404 and 410 (reagent cartridge 410 is not seen
in these figures). Control circuitry, liquid handling tubes, air
pump controls, valves, thermal units (e.g., heating and cooling
units) and other control mechanisms, in some embodiments, are
disposed below a deck of the chassis 490, in a control box region
468. Also seen in FIG. 4D is enrichment module 440 and nucleic acid
assembly module 414 comprising a magnet 416
[0113] Although not illustrated, in some embodiments a display
screen may be positioned on the front face of the chassis 490, for
example, covering a portion of the cover (e.g., see touch screen
display 450 FIG. 4B). The display screen may provide information to
the user regarding the processing status of the automated
multi-module cell editing instrument. In another example, the
display screen may accept inputs from the user for conducting the
cell processing.
Rotating Cell Growth Module
[0114] FIG. 5A shows one embodiment of a rotating growth vial 500
for use with the cell growth device described herein. The rotating
growth vial is an optically-transparent container having an open
end 504 for receiving liquid media and cells, a central vial region
506 that defines the primary container for growing cells, a
tapered-to-constricted region 518 defining at least one light path
510, a closed end 516, and a drive engagement mechanism 512. The
rotating growth vial has a central longitudinal axis 520 around
which the vial rotates, and the light path 510 is generally
perpendicular to the longitudinal axis of the vial. The first light
path 510 is positioned in the lower constricted portion of the
tapered-to-constricted region 518. Optionally, some embodiments of
the rotating growth vial 500 have a second light path 508 in the
tapered region of the tapered-to-constricted region 518. Both light
paths in this embodiment are positioned in a region of the rotating
growth vial that is constantly filled with the cell culture
(cells+growth media) and is not affected by the rotational speed of
the growth vial. The first light path 510 is shorter than the
second light path 508 allowing for sensitive measurement of OD
values when the OD values of the cell culture in the vial are at a
high level (e.g., later in the cell growth process), whereas the
second light path 508 allows for sensitive measurement of OD values
when the OD values of the cell culture in the vial are at a lower
level (e.g., earlier in the cell growth process). Also shown is lip
502, which allows the rotating growth vial to be seated in a growth
module (not shown) and further allows for easy handling by the
user.
[0115] In some configurations of the rotating growth vial, the
rotating growth vial has two or more "paddles" or interior features
disposed within the rotating growth vial, extending from the inner
wall of the rotating growth vial toward the center of the central
vial region 506. In some aspects, the width of the paddles or
features varies with the size or volume of the rotating growth
vial, and may range from 1/20 to just over 1/3 the diameter of the
rotating growth vial, or from 1/15 to 1/4 the diameter of the
rotating growth vial, or from 1/10 to 1/5 the diameter of the
rotating growth vial. In some aspects, the length of the paddles
varies with the size or volume of the rotating growth vial, and may
range from 4/5 to 1/4 the length of the main body of the rotating
growth vial 500, or from 3/4 to 1/3 the length of the central body
region 506 of the rotating growth vial, or from 1/2 to 1/3 the
length of the central body region 506 of the rotating growth vial
500. In other aspects, there may be concentric rows of raised
features disposed on the inner surface of the main body of the
rotating growth vial arranged horizontally or vertically; and in
other aspects, there may be a spiral configuration of raised
features disposed on the inner surface of the main body of the
rotating growth vial. In alternative aspects, the concentric rows
of raised features or spiral configuration may be disposed upon a
post or center structure of the rotating growth vial. Though
described above as having two paddles, the rotating growth vial 500
may comprise 3, 4, 5, 6 or more paddles, and up to 20 paddles. The
number of paddles will depend upon, e.g., the size or volume of the
rotating growth vial 500. The paddles may be arranged symmetrically
as single paddles extending from the inner wall of the vial into
the interior of the vial, or the paddles may be symmetrically
arranged in groups of 2, 3, 4 or more paddles in a group (for
example, a pair of paddles opposite another pair of paddles)
extending from the inner wall of the vial into the interior of the
vial. In another embodiment, the paddles may extend from the middle
of the rotating growth vial out toward the wall of the rotating
growth vial, from, e.g., a post or other support structure in the
interior of the rotating growth vial.
[0116] The drive engagement mechanism 512 engages with a motor (not
shown) to rotate the vial. In some embodiments, the motor drives
the drive engagement mechanism 512 such that the rotating growth
vial is rotated in one direction only, and in other embodiments,
the rotating growth vial is rotated in a first direction for a
first amount of time or periodicity, rotated in a second direction
(i.e., the opposite direction) for a second amount of time or
periodicity, and this process may be repeated so that the rotating
growth vial (and the cell culture contents) are subjected to an
oscillating motion. Further, the choice of whether the culture is
subject 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 may be oscillated at a first periodicity
(e.g., every 60 seconds), and then at a later stage of cell growth,
the rotating growth vial may be oscillated at a second periodicity
(e.g., every one second) different from the first periodicity.
[0117] The rotating growth vial 500 may be reusable or, preferably,
the rotating growth vial is consumable. In some embodiments, the
rotating growth vial is consumable and is presented to the user
pre-filled with growth medium, where the vial is hermetically
sealed at the open end 504 with a foil or film seal. A
medium-filled rotating growth vial packaged in such a manner may be
part of a kit for use with a stand-alone cell growth device or with
a cell growth module that is part of an automated multi-module cell
processing instrument. To introduce cells into the vial, a user
need only pipette up a desired volume of cells and use the pipette
tip to punch through the foil or film seal of the vial. Open end
504 may optionally include an extended lip 502 to overlap and
engage with the cell growth device (not shown). In automated
systems, the rotating growth vial 500 may be tagged with a barcode
or other identifying means that can be read by a scanner or camera
that is part of the automated instrument (not shown).
[0118] The volume of the rotating growth vial 500 and the volume of
the cell culture (including growth medium) may vary greatly, but
the volume of the rotating growth vial 500 must be large enough for
the cell culture in the growth vial to get proper aeration while
the vial is rotating and to generate an adequate number of cells.
In practice, the volume of the rotating growth vial 500 may range
from 1-250 ml, 2-100 ml, from 5-80 ml, 10-50 ml, or from 12-35 ml.
Likewise, the volume of the cell culture (cells+growth media)
should be appropriate to allow proper aeration in the rotating
growth vial. Thus, the volume of the cell culture should be
approximately 5-85% of the volume of the growth vial or from 20-60%
of the volume of the growth vial. For example, for a 35 ml growth
vial, the volume of the cell culture would be from about 1.8 ml to
about 27 ml, or from 5 ml to about 21 ml.
[0119] The rotating growth vial 500 preferably is fabricated from a
bio-compatible optically transparent material--or at least the
portion of the vial comprising the light path(s) is transparent.
Additionally, material from which the rotating growth vial is
fabricated should be able to be cooled to about 4.degree. C. or
lower and heated to about 55.degree. C. or higher to accommodate
both temperature-based cell assays and long-term storage at low
temperatures. Further, the material that is used to fabricate the
vial must be able to withstand temperatures up to 55.degree. C.
without deformation while spinning. Suitable materials include
glass, cyclic olefin copolymer (COC), 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.
[0120] FIGS. 5B-5D show an embodiment of a cell growth module 550
comprising a rotating growth vial 500. FIG. 5B is a perspective
view of one embodiment of a cell growth module 550. FIG. 5C depicts
a cut-away view of the cell growth module 550 from FIG. 5B. In both
figures, the rotating growth vial 500 is seen positioned inside a
main housing 526 with the extended lip 502 of the rotating growth
vial 500 extending above the main housing 526. Additionally, end
housings 522, a lower housing 532, and flanges 524 are indicated in
both figures. Flanges 524 are used to attach the cell growth
device/module to heating/cooling means or to another structure (not
shown). FIG. 5C depicts additional detail. In FIG. 5C, upper
bearing 542 and lower bearing 530 are shown positioned in main
housing 526. Upper bearing 542 and lower bearing 530 support the
vertical load of rotating growth vial 500. Lower housing 532
contains the drive motor 536. The cell growth device 550 of FIG. 5C
comprises two light paths: a primary light path 534, and a
secondary light path 530. Light path 534 corresponds to light path
510 positioned in the constricted portion of the
tapered-to-constricted portion of the rotating growth vial, and
light path 530 corresponds to light path 508 in the tapered portion
of the tapered-to-constricted portion of the rotating growth vial.
Light paths 510 and 508 are not shown in FIG. 5C but may be seen
in, e.g., FIG. 5A. In addition to light paths 534 and 530, there is
an emission board 528 to illuminate the light path(s), and detector
board 546 to detect the light after the light travels through the
cell culture liquid in the rotating growth vial 500.
[0121] The drive motor 536 used to rotate the rotating growth vial
500 in some embodiments is a brushless DC type drive motor with
built-in drive controls that can be set to hold a constant
revolution per minute (RPM) between 0 and about 3000 RPM.
Alternatively, other motor types such as a stepper, servo, brushed
DC, and the like can be used. Optionally, the drive motor 506 may
also have direction control to allow reversing of the rotational
direction, and a tachometer to sense and report actual RPM. The
motor is controlled by a processor (not shown) according to, e.g.,
standard protocols programmed into the processor and/or user input,
and the motor may be configured to vary RPM to cause axial
precession of the cell culture thereby enhancing mixing, e.g., to
prevent cell aggregation, increase aeration, and optimize cellular
respiration.
[0122] Main housing 526, end housings 522 and lower housing 532 of
the cell growth device/module 550 may be fabricated from any
suitable, robust material including aluminum, stainless steel, and
other thermally conductive materials, including plastics. These
structures or portions thereof can be created through various
techniques, e.g., metal fabrication, injection molding, creation of
structural layers that are fused, etc. Whereas the rotating growth
vial 500 is envisioned in some embodiments to be reusable but
preferably is consumable, the other components of the cell growth
device 550 are preferably reusable and can function as a
stand-alone benchtop device or, as here, as a module in a
multi-module cell processing instrument.
[0123] The processor (not shown) of the cell growth system may be
programmed with information to be used as a "blank" or control for
the growing cell culture. A "blank" or control is a vessel
containing cell growth medium only, which yields 100% transmittance
and 0 OD, while the cell sample will deflect light rays and will
have a lower percent transmittance and higher OD. As the cells grow
in the media and become denser, transmittance will decrease and OD
will increase. The processor of the cell growth system may be
programmed to use wavelength values for blanks commensurate with
the growth media typically used in cell culture (whether, e.g.,
mammalian cells, bacterial cells, animal cells, yeast cells, etc.).
Alternatively, a second spectrophotometer and vessel may be
included in the cell growth system, where the second
spectrophotometer is used to read a blank at designated
intervals.
[0124] FIG. 5D illustrates a cell growth device/module 550 as part
of an assembly comprising the cell growth device 550 of FIG. 5B
coupled to light source 590, detector 592, and thermoelectric
components 594. The rotating growth vial 500 is inserted into the
cell growth device 550. Components of the light source 590 and
detector 592 (e.g., such as a photodiode with gain control to cover
5-log) are coupled to the main housing of the cell growth device
550. The lower housing 532 that houses the motor that rotates the
rotating growth vial is illustrated, as is one of the flanges 524
that secures the cell growth device to the assembly. Also
illustrated is a Peltier device or thermoelectric component 594. In
this embodiment, thermal control is accomplished by attachment and
electrical integration of the cell growth device 500 to the
thermoelectric component 594 via the flange 504 on the base of the
lower housing 532. Thermoelectric coolers/devices 594 are capable
of "pumping" heat to either side of a junction, either cooling a
surface or heating a surface depending on the direction of current
flow. In one embodiment, a thermistor is used to measure the
temperature of the main housing and then, through a standard
electronic proportional-integral-derivative (PID) controller loop,
the rotating growth vial 500 is controlled to approximately
+/-0.5.degree. C.
[0125] In certain embodiments, a rear-mounted power entry module
contains the safety fuses and the on-off switch, which when
switched on powers the internal AC and DC power supplies (not
shown) activating the processor. Measurements of optical densities
(OD) at programmed time intervals are accomplished using a 600 nm
Light Emitting Diode (LED) (not shown) that has been columnated
through an optic into the lower constricted portion of the rotating
growth vial which contains the cells of interest. The light
continues through a collection optic to the detection system which
consists of a (digital) gain-controlled silicone photodiode.
Generally, optical density is normally shown as the absolute value
of the logarithm with base 10 of the power transmission factors of
an optical attenuator: OD=-log 10 (Power out/Power in). Since OD is
the measure of optical attenuation--that is, the sum of absorption,
scattering, and reflection--the cell growth device OD measurement
records the overall power transmission, so as the cells grow and
become denser in population, the OD (the loss of signal) increases.
The OD system is pre-calibrated against OD standards with these
values stored in an on-board memory accessible by the measurement
program.
[0126] 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 500 by piercing
though the foil or film seal. The programmed software of the cell
growth device 550 sets the control temperature for growth,
typically 30.degree. C., then slowly starts the rotation of the
rotating growth vial. The cell/growth media mixture slowly moves
vertically up the wall due to centrifugal force allowing the
rotating growth vial to expose a large surface area of the mixture
to a normal oxygen environment. The growth monitoring system takes
either continuous readings of the OD or OD measurements at pre-set
or pre-programmed time intervals. These measurements are stored in
internal memory and if requested the software plots the
measurements versus time to display a growth curve. If enhanced
mixing is required, e.g., to optimize growth conditions, the speed
of the vial rotation can be varied to cause an axial precession of
the liquid, and/or a complete directional change can be performed
at programmed intervals. The growth monitoring can be programmed to
automatically terminate the growth stage at a pre-determined OD,
and then quickly cool the mixture to a lower temperature to inhibit
further growth.
[0127] One application for the cell growth device 550 is to
constantly measure the optical density of a growing cell culture.
One advantage of the described cell growth device is that optical
density can be measured continuously (kinetic monitoring) or at
specific time intervals; e.g., every 5, 10, 15, 20, 30 45, or 60
seconds, or every 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes. While
the cell growth device has been described in the context of
measuring the optical density (OD) of a growing cell culture, it
should, however, be understood by a skilled artisan given the
teachings of the present specification that other cell growth
parameters can be measured in addition to or instead of cell
culture OD. For example, spectroscopy using visible, UV, or near
infrared (NIR) light allows monitoring the concentration of
nutrients and/or wastes in the cell culture. Additionally,
spectroscopic measurements may be used to quantify multiple
chemical species simultaneously. Nonsymmetric chemical species may
be quantified by identification of characteristic absorbance
features in the NIR. Conversely, symmetric chemical species can be
readily quantified using Raman spectroscopy. Many critical
metabolites, such as glucose, glutamine, ammonia, and lactate have
distinct spectral features in the IR, such that they may be easily
quantified. The amount and frequencies of light absorbed by the
sample can be correlated to the type and concentration of chemical
species present in the sample. Each of these measurement types
provides specific advantages. FT-NIR provides the greatest light
penetration depth and can be used for thicker samples. FT-mid-IR
(MIR) provides information that is more easily discernible as being
specific for certain analytes as these wavelengths are closer to
the fundamental IR absorptions. FT-Raman is advantageous when
interference due to water is to be minimized. Other spectral
properties can be measured via, e.g., dielectric impedence
spectroscopy, visible fluorescence, fluorescence polarization, or
luminescence. Additionally, the cell growth device may include
additional sensors for measuring, e.g., dissolved oxygen, carbon
dioxide, pH, conductivity, and the like.
Cell Concentration Module
[0128] FIGS. 6A-6I depict variations on one embodiment of a cell
concentration/buffer exchange cassette and module that utilizes
tangential flow filtration. Once cells are grown to a desired OD,
it is advantageous--particularly in the context of automated cell
processing where small volumes are preferred--to decrease the
volume of the cell culture as well as to render the cells
electrocompetent through, e.g., buffer exchange. One embodiment of
a cell concentration device contemplated herein operates using
tangential flow filtration (TFF), also known as crossflow
filtration, in which the majority of the feed flows tangentially
over the surface of the filter thereby reducing cake (retentate)
formation as compared to dead-end filtration, in which the feed
flows into the filter. Secondary flows relative to the main feed
are also exploited to generate shear forces that prevent filter
cake formation and membrane fouling thus maximizing particle
recovery, as described below.
[0129] The TFF device described herein was designed to take into
account two primary design considerations. First, the geometry of
the TFF device leads to filtering of the cell culture over a large
surface area so as to minimize processing time. Second, the design
of the TFF device is configured to minimize filter fouling. FIG. 6A
is a general model 690 of tangential flow filtration. The TFF
device operates using tangential flow filtration, also known as
cross-flow filtration. FIG. 6A shows cells flowing over a membrane
694, where the feed flow of the cells 692 in medium or buffer is
parallel to the membrane 694. TFF is different from dead-end
filtration where both the feed flow and the pressure drop are
perpendicular to a membrane or filter.
[0130] FIG. 6B depicts a top view of the lower member of one
embodiment of a TFF device/module providing tangential flow
filtration. As can be seen in the embodiment of the TFF device of
FIG. 6B, the lower member 620 the TFF device/module comprises a
channel structure 616 comprising a flow channel 602b through which
a cell culture is flowed. The channel structure 616 comprises a
single flow channel 602b that is horizontally bifurcated by a
membrane (not shown) through which buffer or medium may flow, but
cells cannot. This particular embodiment comprises an undulating
serpentine geometry 614 (i.e., the small "wiggles" in the flow
channel 602) and a serpentine "zig-zag" pattern where the flow
channel 602 crisscrosses the device from one end at the left of the
device to the other end at the right of the device. The serpentine
pattern allows for filtration over a high surface area relative to
the device size and total channel volume, while the undulating
contribution creates a secondary inertial flow to enable effective
membrane regeneration preventing membrane fouling. Although an
undulating geometry and serpentine pattern are exemplified here,
other channel configurations may be used as long as the channel can
be bifurcated by a membrane, and as long as the channel
configuration provides for flow through the TFF module in
alternating directions. In addition to the flow channel 602b,
portals 604 and 606 as part of the channel structure 616 can be
seen, as well as recesses 608. Portals 604 collect cells passing
through the channel on one side of a membrane (not shown) (the
"retentate"), and portals 606 collect the medium ("filtrate" or
"permeate") passing through the channel on the opposite side of the
membrane (not shown). The culture is driven through the device by a
pressure source, and valves are used to direct, collect, and remove
fluids. In this embodiment, recesses 608 accommodate screws or
other fasteners (not shown) that allow the components of the TFF
device to be secured to one another.
[0131] The length 610 and width 612 of the channel structure 616
may vary depending on the volume of the cell culture. The length
610 of the channel structure 616 typically is from 1 mm to 300 mm,
or from 50 mm to 250 mm, or from 60 mm to 200 mm, or from 70 mm to
150 mm, or from 80 mm to 100 mm. The width 612 of the channel
structure 616 typically is from 1 mm to 120 mm, or from 20 mm to
100 mm, or from 30 mm to 80 mm, or from 40 mm to 70 mm, or from 50
mm to 60 mm. The cross-section configuration of the flow channel
602 may be round, elliptical, oval, square, rectangular,
trapezoidal, or irregular. If square, rectangular, or another shape
with generally straight sides, the cross section may be from about
10 .mu.m to 1000 .mu.m wide, or from 200 .mu.m to 800 .mu.m wide,
or from 300 .mu.m to 700 .mu.m wide, or from 400 .mu.m to 600 .mu.m
wide; and from about 10 .mu.m to 1000 .mu.m high, or from 200 .mu.m
to 800 .mu.m high, or from 300 .mu.m to 700 .mu.m high, or from 400
.mu.m to 600 .mu.m high. If the cross section of the flow channel
602 is generally round, oval or elliptical, the radius of the
channel may be from about 50 .mu.m to 1000 .mu.m in hydraulic
radius, or from 5 .mu.m to 800 .mu.m in hydraulic radius, or from
200 .mu.m to 700 .mu.m in hydraulic radius, or from 300 .mu.m to
600 .mu.m wide in hydraulic radius, or from about 200 to 500 .mu.m
in hydraulic radius.
[0132] When looking at the top view of the lower member 620 of TFF
device/module of FIG. 6B, note that there are two retentate portals
604 and two filtrate portals 606, where there is one of each type
portal at both ends (e.g., the narrow edge) of the TFF device. In
other embodiments, retentate and filtrate portals can reside on the
same surface of the same member (e.g., upper or lower member), or
they can be arranged on the side surfaces of the assembly. Unlike
other TFF devices that operate continuously, the TFF device/module
described herein uses an alternating method for concentrating
cells. The overall work flow for cell concentration using the TFF
device/module involves flowing a cell culture or cell sample
tangentially through the channel structure. The membrane
bifurcating the flow channels retains the cells on one side of the
membrane and allows unwanted medium or buffer to flow across the
membrane into a filtrate side (e.g, lower member 620) of the
device. In this process, a fixed volume of cells in medium or
buffer is driven through the device until the reduced-volume cell
sample is collected into one of the retentate portals 604, and the
medium/buffer that has passed through the membrane is collected
through one or both of the filtrate portals 606. All types of
prokaryotic and eukaryotic cells--both adherent and non-adherent
cells--can be grown in the TFF device. Adherent cells may be grown
on beads (e,g, microcarriers) or other cell scaffolds suspended in
medium that flow through the TFF device.
[0133] In the cell concentration process, passing the cell sample
through the TFF device and collecting the cells in one of the
retentate portals 604 while collecting the medium in one of the
filtrate portals 606 is considered "one pass" of the cell sample.
The transfer between retentate reservoirs "flips" the culture. The
retentate and filtrate portals collecting the cells and medium,
respectively, for a given pass reside on the same end of TFF
device/module 600 with fluidic connections arranged so that there
are two distinct flow layers for the retentate and filtrate sides,
but if the retentate portal 604 resides on the upper member of TFF
device/module 600 (that is, the cells are driven through the
channel above the membrane and the filtrate (medium) passes to the
portion of the channel below the membrane), the filtrate portal 606
will reside on the lower member 620 of TFF device/module 100 and
vice versa (that is, if the cell sample is driven through the
channel below the membrane, the filtrate (medium) passes to the
portion of the channel above the membrane). This configuration can
be seen more clearly in FIGS. 6C-6D, where the retentate flows 660
from the retentate portals 604 and the filtrate flows 670 from the
filtrate portals 606. Alternatively, the TFF device may be
positioned on its side; that is, the membrane or filter may divide
the flow channel 602 from left to right, with upper member 622
being on, e.g., the right side of the device, and lower member 620
being on, e.g., the left side of the device. Because the fluids are
driven through the device by pressure, the effect of gravity on the
cell culture is negligible.
[0134] At the conclusion of a "pass" in the growth concentration
process, the cell sample is collected by passing through the
retentate portal 604 and into the retentate reservoir (not shown).
To initiate another "pass", the cell sample is passed again through
the TFF device, this time in a flow direction that is reversed from
the first pass. The cell sample is collected by passing through the
retentate portal 604 and into retentate reservoir (not shown) on
the opposite end of the device/module from the retentate portal 604
that was used to collect cells during the first pass. Likewise, the
medium/buffer that passes through the membrane 624 on the second
pass is collected through the filtrate portal 606 on the opposite
end of the device/module from the filtrate portal 606 that was used
to collect the filtrate during the first pass, or through both
portals. This alternating process of passing the retentate (the
concentrated cell sample) through the device/module is repeated
until the cells have been concentrated to a desired volume, and
both filtrate portals can be open during the passes to reduce
operating time. In addition, buffer exchange may be effected by
adding a desired buffer (or fresh medium) to the cell sample in the
retentate reservoir, before initiating another "pass", and
repeating this process until the old medium or buffer is diluted
and filtered out and the cells reside in fresh medium or buffer.
Note that buffer exchange and cell concentration may (and typically
do) take place simultaneously.
[0135] FIG. 6C depicts a top view of upper (622) and lower (620)
members of an exemplary TFF module. Again, portals 604 and 606 are
seen. As noted above, recesses--such as the recesses 608 seen in
FIG. 6B--provide a means to secure the components (upper member
622, lower member 620, and membrane 624) of the TFF device/membrane
to one another during operation via, e.g., screws or other like
fasteners. However, in alternative embodiments an adhesive, such as
a pressure sensitive adhesive, ultrasonic welding, or solvent
bonding may be used to couple the upper member 622, lower member
620, and membrane 624 together. Indeed, one of ordinary skill in
the art given the guidance of the present disclosure can find yet
other configurations for coupling the components of the TFF device,
such as e.g., clamps; mated fittings disposed on the upper and
lower members; combination of adhesives, welding, solvent bonding,
and mated fittings; and other such fasteners and couplings.
[0136] Note that there is one retentate portal 604 and one filtrate
portal 606 on each "end" (e.g., the narrow edges) of the TFF
device/module 600. The retentate and filtrate portals on the left
side of the TFF device/module 600 will collect cells (flow path at
660) and medium (flow path at 670), respectively, for the same
pass. Likewise, the retentate and filtrate portals on the right
side of the device/module will collect cells (flow path at 660) and
medium (flow path at 670), respectively, for the same pass. In this
embodiment, the retentate is collected from portals 604 on the top
surface of the TFF device, and filtrate is collected from portals
606 on the bottom surface of the device. The cells are maintained
in the TFF flow channel 602a above the membrane 624, while the
filtrate (medium) flows through membrane 624 and then through
filtrate portals 606; thus, the top/retentate portals 604 and
bottom/filtrate portals 606 configuration is practical. It should
be recognized, however, that other configurations of retentate 604
and filtrate 606 portals may be implemented such as positioning
both the retentate 604 and filtrate 606 portals on the side (as
opposed to the top and bottom surfaces) of the TFF device 600, for
example, if the TFF device is positioned on its "side" with the
lower and upper members (620, 622) being positioned as "left" or
"right" members. In FIG. 6C, the flow channel 602b can be seen on
the lower member 620 of the TFF device 600. However, in other
embodiments, retentate 604 and filtrate 606 portals can reside on
the same surface of the TFF device.
[0137] Also seen in FIG. 6C is membrane or filter 624. Filters or
membranes appropriate for use in the TFF device/module are those
that are solvent resistant, are contamination free during
filtration, and are able to retain the types and sizes of cells of
interest. For example, in order to retain small cell types such as
bacterial cells, pore sizes can be as low as 0.2 .mu.m, however for
other cell types, the pore sizes can be as high as 5 .mu.m. Indeed,
the pore sizes useful in the TFF device/module include filters 624
with sizes from 0.20 .mu.m, 0.21 .mu.m, 0.22 .mu.m, 0.23 .mu.m,
0.24 .mu.m, 0.25 .mu.m, 0.26 .mu.m, 0.27 .mu.m, 0.28 .mu.m, 0.29
.mu.m, 0.30 .mu.m, 0.31 .mu.m, 0.32 .mu.m, 0.33 .mu.m, 0.34 .mu.m,
0.35 .mu.m, 0.36 .mu.m, 0.37 .mu.m, 0.38 .mu.m, 0.39 .mu.m, 0.40
.mu.m, 0.41 .mu.m, 0.42 .mu.m, 0.43 .mu.m, 0.44 .mu.m, 0.45 .mu.m,
0.46 .mu.m, 0.47 .mu.m, 0.48 .mu.m, 0.49 .mu.m, 0.50 .mu.m and
larger. The filters 624 may be fabricated from any suitable
non-reactive material including cellulose mixed ester (cellulose
nitrate and acetate) (CME), polycarbonate (PC), polyvinylidene
fluoride (PVDF), polyethersulfone (PES), polytetrafluoroethylene
(PTFE), nylon, glass fiber, or metal substrates as in the case of
laser or electrochemical etching. The TFF device 600 shown in FIGS.
6C and 6D does not show a seat in the upper 622 and lower 620
members where the filter 624 can be seated or secured (for example,
a seat half the thickness of the filter in each of upper 622 and
lower 620 members); however, such a seat is contemplated in some
embodiments.
[0138] FIG. 6D depicts a bottom view of upper and lower components
of the exemplary TFF module shown in FIG. 6C. FIG. 6D depicts a
bottom view of upper (622) and lower (620) members of an exemplary
TFF module 600. Again portals 604 and 606 are seen. Note again that
there is one retentate portal 604 and one filtrate portal 606 on
each end of the TFF device/module 600. On the left side of the TFF
device 600, the retentate portals 604 will collect cells (flow path
at 660) and the filtrate portals 606 will collect medium (flow path
at 670), respectively, for the same pass. Likewise, on the right
side of the TFF device 600, the retentate portals 604 will collect
cells (flow path at 660) and the filtrate portals 606 will collect
medium (flow path at 670), respectively, for the same pass. In FIG.
6D, the flow channel 602a can be seen on the upper member 622 of
the TFF device 600. Thus, looking at FIGS. 6C and 6D, note that
there is a flow channel 602 (602a and 602b) in both the upper
member 622 and lower member 620, with a membrane 624 between the
upper 622 and lower 620 members of the flow channel. The flow
channel 602 of the upper 622 and lower 620 members (602a and 602b,
respectively) mate to create the flow channel 602 with the membrane
624 positioned horizontally between the upper and lower members of
the TFF device/module thereby bifurcating the flow channel 602.
[0139] Buffer exchange during cell concentration and/or rendering
the cells competent is performed on the TFF device/module by adding
fresh medium to growing cells or a desired buffer to the cells
concentrated to a desired volume; for example, after the cells have
been concentrated at least 20-fold, 30-fold, 40-fold, 50-fold,
60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold or
more. A desired exchange medium or exchange buffer is added to the
cells either by addition to the retentate reservoir (not shown) or
through the membrane 624 from the filtrate side (e.g. to cells in
retentate reservoir) and the process of passing the cells through
the TFF device 600 is repeated until the cells have been
concentrated to a desired volume in the exchange medium or buffer.
This process can be repeated any number of desired times so as to
achieve a desired level of exchange of the buffer and a desired
volume of cells. The exchange buffer may comprise, e.g., glycerol
or sorbitol thereby rendering the cells competent for
transformation in addition to decreasing the overall volume of the
cell sample.
[0140] The TFF device 600 may be fabricated from any robust
material in which channels (and channel branches) may be milled
including stainless steel, silicon, glass, aluminum, or plastics
including cyclic-olefin copolymer (COC), cyclo-olefin polymer
(COP), polystyrene, polyvinyl chloride, polyethylene, polyamide,
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 600 is disposable, preferably it
is made of plastic. In some embodiments, the material used to
fabricate the TFF device/module 600 is thermally-conductive so that
the cell culture may be heated or cooled to a desired temperature.
In certain embodiments, the TFF device 600 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 these mass production techniques.
[0141] FIG. 6E depicts an exemplary configuration of an assembled
TFF device 6000, where, like the other configurations, the upper
member 6022 and lower member 6020 in combination form a flow
channel with a membrane 6024 disposed between the upper and lower
members; however, in this configuration, in addition to the
retentate reservoirs, there is an optional buffer or medium
reservoir positioned between the retentate reservoirs, and a lower
filtrate or permeate reservoir. In the TFF device 6000
configuration shown in FIG. 6E, 6044 is the top or cover of the TFF
device 6000, having three ports 6046, where there is a pipette tip
6048 disposed in the right-most port 6046. The top 6044 of the TFF
device 6000 is adjacent to and, in operation, is coupled with a
combined reservoir and upper member structure 6050. Combined
reservoir and upper member structure 6050 comprises a top surface
that is adjacent the top or cover 6044 of the TFF device 6000, a
bottom surface which comprises the upper member 6022 of the TFF
device, where the upper member 6022 of the TFF device defines the
upper portion of the flow channel (not shown) disposed on the
bottom surface of the upper member 6022 of the combined reservoir
and upper member structure 6050. Additionally, combined reservoir
and upper member structure 6050 comprises two retentate reservoirs
6080 and an optional buffer or medium reservoir 6082. The retentate
reservoirs 6080 are fluidically coupled to the upper portion of the
flow channel, and the buffer or medium reservoir 6082 is
fluidically coupled to the retentate reservoirs 6080. Also seen in
this assembled view of TFF device 6000 is membrane 6024, lower
member 6020 which, as described previously, comprises on its top
surface the lower portion of the tangential flow channel (not
shown), where the flow channels of the upper member 6022 and lower
member 6020 (neither shown in this view) mate to form a single flow
channel. Beneath and adjacent to lower member 6020 is a gasket
6040, which is interposed between lower member 6020 and an optional
filtrate (or permeate) reservoir 6042. The filtrate reservoir 6042
is in fluid connection with the lower portion of the flow channel,
as a receptacle for the filtrate or permeate that is removed from
the cell culture. In operation, top 6044, combined reservoir and
upper member structure 6050, membrane 6024, lower member 6020,
gasket 6040, and filtrate reservoir 6042 are coupled and secured
together to be fluid- and air-tight. The assembled TFF device 6000
typically is from 4 to 25 cm in height, or from 5 to 20 cm in
height, or from 7 to 15 cm in height; from 5 to 30 cm in length, or
from 8 to 25 cm in length, or from 10 to 20 cm in length; and is
from 3 to 15 cm in depth, or from 5 to 10 cm in depth. An exemplary
TFF device is 11 cm in height, 12 cm in length, and 8 cm in depth.
The retentate reservoirs, buffer or medium reservoir, and
tangential flow channel-forming structures may be configured to be
cooled to 4.degree. C. for cell maintenance. The dimensions for the
serpentine channel recited above, as well as the specifications and
materials for the filter and the TFF device, apply to the
embodiment of the device shown in FIGS. 6E-6I. In embodiments
including the present embodiment, up to 120 mL of cell culture can
be grown and/or filtered, or up to 100 mL, 90 mL, 80 mL, 70 mL, 60
mL, 50 mL, 40 mL, 30 mL or 20 mL of cell culture can be grown
and/or filtered.
[0142] FIG. 6F depicts an exploded perspective side view of TFF
device 6000. In this configuration, 6044 is the top or cover of the
TFF device 6000, having three ports 6046, where there is a pipette
tip 6048 disposed in the left-most port 6046. The top 6044 of the
TFF device 6000 is, in operation, coupled with a combined reservoir
and upper member structure 6050. Combined reservoir and upper
member structure 6050 comprises a top surface that, in operation,
is adjacent the top or cover 6044 of the TFF device, a bottom
surface which comprises the upper member 6022 of the TFF device,
where the upper member 6022 of the TFF device defines the upper
portion of the tangential flow channel (not shown). Combined
reservoir and upper member structure 6050 comprises two retentate
reservoirs 6080 and an optional buffer or medium reservoir 6082.
The retentate reservoirs 6080 are fluidically coupled to the upper
portion of the flow channel, and the optional buffer or medium
reservoir 6082 is fluidically coupled to the retentate reservoirs
6080. Also seen in this exploded view of TFF device 6000 is lower
member 6020 which, as described previously, comprises on its top
surface the lower portion of the tangential flow channel 6002b
(seen on the top surface of lower member 6020), where the upper and
lower portions of the flow channels of the upper member 6022 and
lower member 6020, respectively, when coupled mate to form a single
flow channel (the membrane that is interposed between the upper
member 6022 and lower member 6020 in operation is not shown).
Beneath lower member 6020 is gasket 6040, which in operation is
interposed between lower member 6020 and filtrate (or permeate)
reservoir 6042. In operation, top 6044, combined reservoir and
upper member structure 6050, membrane (not shown), lower member
6020, gasket 6040, and filtrate reservoir 6042 are coupled and
secured together to be fluid- and air-tight. In FIG. 6F, fasteners
are shown that can be used to couple the various structures (top
6044, combined reservoir and upper member structure 6050, membrane
(not shown), lower member 6020, gasket 6040, and filtrate reservoir
6042) together. However, as an alternative to screws or other like
fasteners, the various structures of TFF device 6000 can be coupled
using an adhesive, such as a pressure sensitive adhesive;
ultrasonic welding; or solvent bonding. Further, a combination of
fasteners, adhesives, and/or welding types may be employed to
couple the various structures of the TFF device. One of ordinary
skill in the art given the guidance of the present disclosure could
find yet other configurations for coupling the components of TFF
device 6000, such as e.g., clamps, mated fittings, and other such
fasteners.
[0143] FIG. 6G depicts combined reservoir and upper member
structure 6050, comprising two retentate reservoirs 6080 and an
optional buffer or medium reservoir 6082, as well as upper member
6020, which is disposed on the bottom of combined reservoir and
upper member structure 6050. Upper member 6022 of the TFF device
defines the upper portion of the tangential flow channel (not
shown) disposed on the bottom surface of the combined reservoir and
upper member structure 6050. FIG. 6H is a top-down view of the
upper surface of combined reservoir and upper member structure
6050, depicting the top of retentate reservoirs 6080 and buffer or
medium reservoir 6082, as well as fluid or vacuum ports (not
shown). The retentate reservoirs 6080 are fluidically coupled to
the upper portion of the flow channel, and the buffer or medium
reservoir 608 is fluidically coupled to the retentate reservoirs.
FIG. 6I is a bottom-up view of the lower surface of combined
reservoir and upper member structure 6050, showing the upper member
6022 with the upper portion of the tangential flow channel 6002a
disposed on the bottom surface of upper member 6022. The flow
channel 6002a disposed on the bottom surface of upper member 6022,
in operation, is mated to the bottom portion of the tangential flow
channel disposed on the top surface of the lower member (not shown
in this view, but see, e.g., flow channel 6002b in FIG. 6F), where
the upper and lower portions of the flow channel mate to form a
single flow channel.
[0144] As an alternative to the TFF module 6000 described above, a
cell concentration module comprising a hollow filter may be
employed. Examples of filters suitable for use in the present
disclosure include membrane filters, ceramic filters and metal
filters. The filter may be used in any shape; the filter may, for
example, be cylindrical or essentially flat. Preferably, the filter
used is a membrane filter, most preferably a hollow fiber filter.
The term "hollow fiber" is meant to include a tubular membrane. The
internal diameter of the tube is at least 0.1 mm, more preferably
at least 0.5 mm, most preferably at least 0.75 mm and preferably
the internal diameter of the tube is at most 10 mm, more preferably
at most 6 mm, most preferably at most 1 mm. Filter modules
comprising hollow fibers are commercially available from various
companies, including G.E. Life Sciences (Marlborough, Mass.) and
InnovaPrep (Drexel, Mo.). Specific examples of hollow fiber filter
systems that can be used, modified or adapted for use in the
present methods and systems include, but are not limited to, U.S.
Pat. Nos. 9,738,918; 9,593,359; 9,574,977; 9,534,989; 9,446,354;
9,295,824; 8,956,880; 8,758,623; 8,726,744; 8,677,839; 8,677,840;
8,584,536; 8,584,535; and 8,110,112.
Nucleic Acid Assembly Module
[0145] 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,
BACs, YACs, and other synthetic chromosomes.
[0146] 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.
[0147] In some embodiments, a regulatory element is operably linked
to one or more elements of a targetable nuclease system so as to
drive transcription, and for some nucleic acid sequences,
translation and expression of the one or more components of the
targetable nuclease system.
[0148] 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 linked to a polynucleotide
sequence, which, when transcribed, forms a guide RNA.
[0149] The nucleic acid assembly module can be configured to
perform a wide variety of different nucleic acid assembly
techniques in an automated fashion. Nucleic acid assembly
techniques that can be performed in the nucleic acid assembly
module of the disclosed automated multi-module cell editing
instruments include, but are not limited to, those assembly methods
that use restriction endonucleases, including PCR, BioBrick
assembly (U.S. Pat. No. 9,361,427), Type IIS cloning (e.g.,
GoldenGate assembly, European Patent Application Publication EP 2
395 087 A1), and Ligase Cycling Reaction (de Kok, ACS Synth Biol.,
3(2):97-106 (2014); Engler, et al., PLoS One, 3(11):e3647 (2008);
and U.S. Pat. No. 6,143,527). In other embodiments, the nucleic
acid assembly techniques performed by the disclosed automated
multi-module cell editing instruments are based on overlaps between
adjacent parts of the nucleic acids, such as Gibson Assembly.RTM.,
CPEC, SLIC, Ligase Cycling etc. Additional assembly methods include
gap repair in yeast (Bessa, Yeast, 29(10):419-23 (2012)), gateway
cloning (Ohtsuka, Curr Pharm Biotechnol, 10(2):244-51 (2009)); U.S.
Pat. 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).
[0150] 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.
[0151] 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.
Cell Transformation Module
[0152] In addition to the modules for cell growth, cell
concentration, and nucleic acid assembly, FIGS. 7A-7E depict
variations on one embodiment of a cell transformation module (in
this case, a flow-through electroporation device) that may be
included in a cell growth/concentration/transformation instrument.
FIGS. 7A and 7B are top perspective and bottom perspective views,
respectively, of six co-joined flow-through electroporation devices
750. FIG. 7A depicts six flow-through electroporation units 750
arranged on a single substrate 756. Each of the six flow-through
electroporation units 750 have wells 752 that define cell sample
inlets and wells 754 that define cell sample outlets. FIG. 7B is a
bottom perspective view of the six co-joined flow-through
electroporation devices of FIG. 7A also depicting six flow-through
electroporation units 750 arranged on a single substrate 756. Six
inlet wells 752 can be seen, one for each flow-through
electroporation unit 750, and one outlet well 754 can be seen (the
outlet well of the left-most flow-through electroporation unit
750). Additionally seen in FIG. 7B are an inlet 702, outlet 704,
flow channel 706 and two electrodes 708 on either side of a
constriction in flow channel 706 in each flow-through
electroporation unit 750. Once the six flow-through electroporation
units 750 are fabricated, they can be separated from one another
(e.g., "snapped apart") and used one at a time, or alternatively,
in some embodiments two or more flow-through electroporation units
750 may be used in parallel without separation.
[0153] The flow-through electroporation devices achieve high
efficiency cell electroporation with low toxicity. The flow-through
electroporation devices of the disclosure allow for particularly
easy integration with robotic liquid handling instrumentation that
is typically used in automated systems such as air displacement
pipettors. Such automated instrumentation includes, but is not
limited to, off-the-shelf automated liquid handling systems from
Tecan (Mannedorf, Switzerland), Hamilton (Reno, Nev.), Beckman
Coulter (Fort Collins, Colo.), etc.
[0154] Generally speaking, microfluidic electroporation--using cell
suspension volumes of less than approximately 10 ml and as low as 1
.mu.l--allows more precise control over a transfection or
transformation process and permits flexible integration with other
cell processing tools compared to bench-scale electroporation
devices. Microfluidic electroporation thus provides unique
advantages for, e.g., single cell transformation, processing and
analysis; multi-unit electroporation device configurations; and
integrated, automatic, multi-module cell processing and
analysis.
[0155] In specific embodiments of the flow-through electroporation
devices of the disclosure, the toxicity level of the transformation
results in greater than 10% viable cells after electroporation,
preferably greater than 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%,
55%, 60%, 70%, 75%, 80%, 85%, 90%, or even 95% viable cells
following transformation, depending on the cell type and the
nucleic acids being introduced into the cells.
[0156] The flow-through electroporation device 750 described in
relation to FIGS. 7A-7E comprises a housing with an electroporation
chamber, a first electrode and a second electrode configured to
engage with an electric pulse generator, by which electrical
contacts engage with the electrodes of the electroporation device.
In certain embodiments, the electroporation devices are
autoclavable and/or disposable, and may be packaged with reagents
in a reagent cartridge. The electroporation device may be
configured to electroporate cell sample volumes between 1 .mu.l to
2 ml, 10 .mu.l to 1 ml, 25 .mu.l to 750 .mu.l, or 50 .mu.l to 500
.mu.l. The cells that may be electroporated with the disclosed
electroporation devices include mammalian cells (including human
cells), plant cells, yeasts, other eukaryotic cells, bacteria,
archaea, and other cell types.
[0157] In one exemplary embodiment, FIG. 7C depicts a top view of a
flow-through electroporation device 750 having an inlet 702 for
introduction of cells and an exogenous reagent to be electroporated
into the cells ("cell sample") and an outlet 704 for the cell
sample following electroporation. Electrodes 708 are introduced
through electrode channels (not shown) in the device. FIG. 7D shows
a cutaway view from the top of flow-through electroporation device
750, with the inlet 702, outlet 704, and electrodes 708 positioned
with respect to a constriction in flow channel 706. The
constriction at the narrowest is configured to be at least 2.times.
the diameter of the cells being porated, and in some embodiments at
least 10.times. or more the diameter of the cells being porated. A
side cutaway view of the bottom portion of flow-through
electroporation device 750 in FIG. 7E illustrates that electrodes
708 in this embodiment are positioned in electrode channels 710 and
perpendicular to flow channel 706 such that the cell sample flows
from the inlet channel 712 through the flow channel 706 to the
outlet channel 714, and in the process the cell sample flows into
the electrode channels 710 to be in contact with electrodes 708;
however, the electrodes are not directly in the path of the cells
as the cells flow through flow channel 706. In fact, the electrodes
708 are disposed in electrode channels 710 generally perpendicular
to flow channel 706. In this aspect, the inlet channel 712, outlet
channel 714 and electrode channels 710 all originate from the top
planar side of the device 750; however, the flow-through
electroporation architecture depicted in FIGS. 7C-7E is but one
architecture useful with the reagent cartridges described herein.
Additional electrode architectures are described, e.g., in U.S.
Ser. No. 16/147,120, filed 24 Sep. 2018; Ser. No. 16/147,865, filed
30 Sep. 2018; and Ser. No. 16/147,871, filed 30 Sep. 2018.
Cell Enrichment Module
[0158] One optional aspect provides automated modules and
instruments for nucleic acid-guided nuclease genome editing that
implement enrichment techniques for cells whose genomes have been
properly edited. The enrichment module performs methods that use
cell singulation and normalization to reduce growth competition
between edited and unedited cells or utilizes a method that takes
advantage of inducing editing at a specific time during cell
growth. Singulation overcomes growth bias from unedited cells or
cells containing edits conferring growth advantages or
disadvantages. The methods, modules and instruments may be applied
to all cell types including, archaeal, prokaryotic, and eukaryotic
(e.g., yeast, fungal, plant and animal) cells.
[0159] Singulating, optional induction of editing, and
normalization of cell colonies leads to 2-250.times.,
10-225.times., 25-200.times., 40-175.times., 50450.times.,
60-100.times., or 5-100.times. gains in identifying edited cells
over prior art methods and generates arrayed or pooled edited cells
comprising genome libraries. Additionally, the methods, modules,
and instruments may be leveraged to create iterative editing
systems to generate combinatorial libraries, identify rare cell
edits, and enable high-throughput enrichment applications to
identify editing activity.
[0160] The compositions and methods described herein improve
nucleic acid-guided nuclease editing systems in which nucleic
acid-guided nucleases (e.g., RNA-guided nucleases) are used to edit
specific target regions in an organism's genome. FIG. 8A depicts a
solid wall device 850 and a workflow for singulating cells in
microwells in the solid wall device, where in this workflow one or
both of the gRNA and nuclease are under the control of an inducible
promoter. At the top left of the figure (i), there is depicted
solid wall device 850 with microwells 852. A section 854 of solid
wall device 850 is shown at (ii), also depicting microwells 852. At
(iii), a side cross-section of solid wall device 850 is shown, and
microwells 852 have been loaded, where, in this embodiment, Poisson
loading has taken place; that is, each microwell has one (e.g.,
microwells 852, 856) or no cells, and the likelihood that any one
microwell has more than one cell is low. At (iv), workflow 840 is
illustrated where substrate 850 having microwells 852 shows
microwells 856 with one cell per microwell, microwells 857 with no
cells in the microwells, and one microwell 260 with two cells in
the microwell. In step 851, the cells in the microwells are allowed
to double approximately 2-50 times to form clonal colonies (v),
then editing is induced 853 by heating the substrate (e.g., for
temperature-induced editing) or flowing chemicals under or over the
substrate (e.g., sugars, antibiotics for chemical-induced editing)
or by moving the solid wall device to a different medium, which is
particularly facile if the solid wall device is placed on a fluid
permeable membrane which forms the bottom of microwells 852. After
induction of editing 853, many cells in the colonies of cells that
have been edited die as a result of the double-strand cuts caused
by active editing, and there is possibly a lag in growth for the
edited cells that do survive but must repair and recover following
editing (microwells 858), where cells that do not undergo editing
thrive (microwells 859) (vi). All cells are allowed to grow to
continue to establish colonies and normalize, where the colonies of
edited cells in microwells 858 catch up in size and/or cell number
with the cells in microwells 859 that do not undergo editing (vii)
due to cell senescence as the unedited cells reach stationary
phase. Once the cell colonies are normalized, either pooling of all
cells in the microwells can take place, in which case the cells are
enriched for edited cells by eliminating the bias from non-editing
cells and fitness effects from editing; alternatively, colony
growth in the microwells is monitored after editing, and slow
growing colonies (e.g., the cells in microwells 858) are identified
and selected (e.g., "cherry picked") resulting in even greater
enrichment of edited cells.
[0161] In growing the cells, the medium used will depend, of
course, on the type of cells being edited--e.g., bacterial, yeast
or mammalian. For example, medium for bacterial growth includes LB,
SOC, M9 Minimal medium, and Magic medium; medium for yeast cell
growth includes TPD, YPG, YPAD, and synthetic minimal medium; and
medium for mammalian cell growth includes MEM, DMEM, IMDM, RPMI,
and Hanks.
[0162] FIG. 8B is a photograph of one embodiment of a solid wall
device comprising microwells for singulating cells. As can be seen
from the photo, the solid wall device is approximately 2 inches
(.about.47 mm) in diameter. The solid device seen in this
photograph is essentially a perforated disk of 816 stainless steel,
where the perforations form the walls of the microwells, and a
filter or membrane is used to form the bottom of the microwells.
Use of a filter or membrane (such as a 0.22.mu. PVDF Duropore.TM.
woven membrane filter) allows for medium and/or nutrients to enter
the microwells but prevents the cells from flowing down and out of
the microwells. Filter or membrane members that may be used in the
solid wall singulation/growth/editing/normalization devices and
modules are those that are solvent resistant, are contamination
free during filtration, and are able to retain the types and sizes
of cells of interest. For example, in order to retain small cell
types such as bacterial cells, pore sizes can be as low as 0.2
.mu.m, however for other cell types, the pore sizes can be as high
as 0.5 .mu.m. Indeed, the pore sizes useful in the cell
concentration device/module include filters with sizes from 0.20
.mu.m, 0.21 .mu.m, 0.22 .mu.m, 0.23 .mu.m, 0.24 .mu.m, 0.25 .mu.m,
0.26 .mu.m, 0.27 .mu.m, 0.28 .mu.m, 0.29 .mu.m, 0.30 .mu.m, 0.31
.mu.m, 0.32 .mu.m, 0.33 .mu.m, 0.34 .mu.m, 0.35 .mu.m, 0.36 .mu.m,
0.37 .mu.m, 0.38 .mu.m, 0.39 .mu.m, 0.40 .mu.m, 0.41 .mu.m, 0.42
.mu.m, 0.43 .mu.m, 0.44 .mu.m, 0.45 .mu.m, 0.46 .mu.m, 0.47 .mu.m,
0.48 .mu.m, 0.49 .mu.m, 0.50 .mu.m and larger. The filters may be
fabricated from any suitable material including cellulose mixed
ester (cellulose nitrate and acetate) (CME), polycarbonate (PC),
polyvinylidene fluoride (PVDF), polyethersulfone (PES),
polytetrafluoroethylene (PTFE), nylon, or glass fiber.
[0163] In the photograph shown in FIG. 8B, the perforations are
approximately 152 nM in diameter, resulting in the microwells
having a volume of approximately 2.5 nL, with a total of
approximately 30,000 wells. The distance between the microwells is
approximately 279 nM center-to-center. Though here the microwells
have a volume of approximately 2.5 nL, the volume of the microwells
may be from 1 to 25 nL, or preferably from 2 to 10 nL, and even
more preferably from 2 to 4 nL. The preferred size/volume of the
microwells will depend on cell type (e.g., bacterial, yeast,
mammalian). The perforated disk shown here is made of 316 stainless
steel; however other bio-compatible metals and materials may be
used. The solid wall device may be disposable or it may be
reusable. The solid wall device shown in FIG. 8B is round, but can
be of any shape, for example, square, rectangular, oval, etc. Round
solid wall devices are useful if petri dishes are used to supply
the solid wall device with nutrients via solid medium. The filters
used to form the bottom of the wells of the solid wall device
include 0.22.mu. PVDF Duropore.TM. woven membrane filters. Further,
though a 2-inch (.about.47 mm) diameter solid wall device is shown,
the solid wall devices may be smaller or larger as desired and the
configuration of the solid wall device will depend on how nutrients
are supplied to the solid wall device, and how media exchange is
performed. Although a round solid wall device is described here,
the solid wall devices can be of any shape and size, including
rectangular solid wall devices with 100K, 200K or more wells, in
addition to configurations of solid wall devices and cassettes that
are multiplexed, e.g., stacked.
[0164] FIGS. 8C-8E are photographs of E. coli cells at low, medium
and high magnification, respectively, singulated via Poisson
distribution in microwells in a solid wall device with a membrane
bottom. FIG. 8C shows digital growth at low magnification where the
darker microwells are microwells where cells are growing. FIG. 8D
is a top view of microwells in a solid wall device where the darker
microwells are microwells where cells are growing. FIG. 8E is a
photograph of microwells where the membrane (e.g., the permeable
membrane that forms the bottom of the microwells) has been removed,
where unpatterned (smooth) microwells are microwells where cells
are not growing, and microwells with irregular pigment/patterned
are microwells where cells are growing, and, in this photograph,
have filled the microwells in which they are growing. In these
photographs, a 0.2 .mu.m filter (membrane) was pressed against the
perforated metal solid wall device such as the round solid wall
device depicted in FIG. 8B. The perforated metal solid wall device
formed the walls of the microwells, and the 0.2 .mu.m filter formed
the bottom of the microwells. To load the solid wall device, the E.
coli cells were pulled into the microwells using a vacuum. The
solid wall device+filter was then placed on an LB agar plate
membrane-side down, and the cells were grown overnight at
30.degree. C., then two days at room temperature. The membrane was
removed and the bottomless microwells were photographed by light
microscopy. Note the ease with which different selective media can
be used to select for certain cell phenotypes; that is, one need
only transfer the solid wall device+filter to a different plate or
petri dish comprising a desired selective medium or flow a desired
selective medium into a substrate onto which the solid wall device
and coupled membrane are positioned.
[0165] In addition to the solid wall cell singulation device
described in relation to FIGS. 8A-8E, other cell singulation
devices may be employed in the multi-module cell processing
instrument, such as those described in U.S. Ser. No. 62/735,365,
entitled "Detection of Nuclease Edited Sequences in Automated
Modules and Systems", filed 24 Sep. 2018, and U.S. Ser. No.
62/781,112, entitled "Improved Detection of Nuclease Edited
Sequences in Automated Modules and Systems", filed 18 Dec. 2018,
including singulation by plating on agar, singulation by isolating
cells on functionalized islands, singulation within aqueous
droplets carried in a hydrophobic carrier fluid or Gel
Bead-in-Emulsion (GEMs, see, e.g., 10.times. Genomics, Pleasanton,
Calif.), or singulation within a polymerized alginate scaffold (for
this embodiment of singulation, also see U.S. Ser. No. 62/769,805,
entitled "Improved Detection of Nuclease Edited Sequences in
Automated Modules and Instruments via Bulk Cell Culture", filed 20
Nov. 2018.
[0166] As an alternative to singulation, inducing editing via an
inducible promoter driving one or both of the gRNA and the nuclease
at a specific time in the cell growth cycle may be employed. FIG.
8F shows a simplified flow chart for exemplary methods 8000 for
enriching for edited cells. Looking at FIG. 8F, method 8000 begins
by designing and synthesizing editing cassettes 8002. As described
in relation to nucleic acid-guided editing above, each editing
cassette typically comprises a gRNA, a donor DNA, and a PAM or
spacer mutation. Once the individual editing cassettes have been
synthesized, the individual editing cassettes may be "linked" or
"assembled" together and are amplified and assembled into editing
vector backbones 8004. The editing vectors comprising the editing
cassettes are then used to transform cells 8006 thereby creating a
library of transformed cells. In addition to the vectors comprising
the assembled editing cassettes, the cells may be transformed
simultaneously with a separate engine vector comprising a coding
sequence for a nuclease. Alternatively, the cells may already be
expressing the nuclease (e.g., the cells may have already been
transformed with an engine vector or the coding sequence for the
nuclease may be stably integrated into the cellular genome) such
that only the editing vector needs to be transformed into the
cells; or the cells may be transformed with a single vector
comprising all components required to perform nucleic acid-guided
nuclease genome editing (e.g., all of the nuclease and an editing
cassette), which is advantageous when employing curing and
recursive rounds of editing.
[0167] A variety of delivery systems may be used to introduce
(e.g., transform or transfect) nucleic acid-guided nuclease editing
system components into a host cell 8008. These delivery systems
include the use of yeast systems, lipofection systems,
microinjection systems, biolistic systems, virosomes, liposomes,
immunoliposomes, polycations, lipid:nucleic acid conjugates,
virions, artificial virions, viral vectors, electroporation, cell
permeable peptides, nanoparticles, nanowires, and/or exosomes.
Alternatively, molecular trojan horse liposomes may be used to
deliver nucleic acid-guided nuclease components across the blood
brain barrier. Of particular interest is the use of
electroporation, particularly flow-through electroporation (either
as a stand-alone instrument or as a module in an automated
multi-module system) as described above in relation to FIGS. 7A-7E
and in, e.g., U.S. Ser. No. 16/024,831 filed 30 Jun. 2018; Ser. No.
16/024,816 filed 30 Jun. 2018; Ser. No. 16/147,353 filed 28 Sep.
2018; Ser. No. 16/147,865 filed 30 Sep. 2018; and Ser. No.
16/147,871 filed 30 Jun. 2018. If the screening/selection module is
one module in an automated multi-module cell editing system, the
cells are likely transformed in an automated cell transformation
module.
[0168] Once transformed 8006, the cells can then be subjected to
selection using a selectable marker 8008. Selectable markers are
employed to select for cells that have received both the engine and
editing vectors, or for cells that have been transformed with a
single, combined engine and editing vector. Commonly used
selectable markers include drug selectable markers such as
ampicillin/carbenicillin, kanamycin, chloramphenicol, erythromycin,
tetracycline, gentamicin, bleomycin, streptomycin, rhamnose,
puromycin, hygromycin, blasticidin, and G418.
[0169] Once cells that have been properly transformed are selected
8008, the next step in method 8000 is to grow cells in liquid
medium until the cells enter (or are close to entering) the
stationary phase of growth. Once the cells are in stationary phase
8010 (or nearly so), editing is induced 8012 in the cells by
induction of transcription of one or both of the nuclease and gRNA.
Once editing is induced 8012, the cells can be grown, rendered
electrocompetent, and subjected to another round of editing
8014.
[0170] FIG. 8G depicts a typical growth curve 8020 for cells in
culture (optical density versus time). Initially there is a lag
phase 8022, then the cells enter log phase 8024 where they grow
quickly, and finally the cells reach stationary phase 8028 where
the cells are no longer dividing. The present methods employ
inducing transcription of either or both the nuclease and/or gRNA
at timepoint 8026 or later when the cells are in the stationary
phase 8028 of growth or nearly so; that is, the cells are induced
at a timepoint at least 60% into the log phase 8024 of growth, or
at least 65% into the log phase 8024 of growth, or at least 70%
into the log phase 8024 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 8024 of growth,
and at any time during the stationary phase 8028 of growth.
[0171] FIG. 8H depicts an exemplary protocol 8050 for performing
nucleic acid-guided nuclease genome editing. FIG. 8H depicts the
protocols shown in FIG. 8F for editing cells. First, a library or
collection of editing vectors 8052 (editing vectors each comprising
an editing cassette) is introduced 8053 (e.g., electroporated) into
cultured cells 8054 that comprise a coding sequence for a nuclease
under the control of a constitutive or inducible promoter
(preferably an inducible promoter), contained 1) on an "engine
plasmid" (most often along with a selectable marker) that has
already been transformed into the cells; 2) integrated into the
genome of the cells being transformed; or 3) the coding sequence
for the nuclease may be located on the editing vector. The editing
vectors 8052 comprise a donor DNA, a PAM or spacer-altering
sequence (most often a sequence that disables the PAM at the target
site in the genome), a coding sequence for a gRNA under the control
of an inducible promoter, and a selectable marker.
[0172] At step 8059, cells are grown until they reach stationary
phase, or nearly so. Once the cells reach the stationary phase,
editing is induced 8067 (e.g., where transcription of the nuclease,
gRNA or both is induced) and the cells in the culture 8082 are
edited and then allowed to recover from editing. A particularly
useful inducible promoter is the pL promoter system, which is
activated by a rise in temperature. The pL promoter system is
useful because after a period of time for editing, the temperature
of the liquid bulk culture is decreased and editing is terminated
allowing the cells in the culture to recover. Once recovered, the
cells can be plated 8069, grown and pooled 8084. Alternatively, the
cells from culture 8082 can be plated 8081, and slow-growing
colonies are selected 8086 thereby cherry-picking edited colonies.
In yet another alternative, the cells can be retained in liquid
culture 8083, grown to an appropriate OD, rendered
electrocompetent, and subjected to another round of editing 8088.
This method of enrichment of edited cells is particularly desirable
as it may be performed in a high throughput manner and does not
require plating cells and is automatable. Induction at step 8067
can take place by, e.g., using a pL promoter system where the pL
promoter is induced by raising the temperature of the cells in the
medium to 42.degree. C. for, e.g., one to many hours to induce
expression of the nuclease and gRNA for cutting and editing. Once
editing has been induced, the temperature of the culture 8082 is
returned to 30.degree. C.
[0173] In one method 8081, the cells from the bulk liquid culture
are plated or arrays in a 96- or 384-well plate (or other plate
with partitions) and the slow-growing colonies are selected 8086.
In edited cells, cell viability is compromised in the period after
editing is induced. Method 818 shown in FIG. 8H (e.g., selecting
slow growing colonies 8081) takes advantage of the growth lag in
colonies of edited cells to identify edited cells. In some
embodiments, the colony size of the edited cells is 20% smaller
than colonies of non-edited cells. In some aspects the colony size
of the edited cells is 30%, 40%, 50%, 60%, 70%, 80% or 90% smaller
than the colonies of non-edited cells. In many embodiments, the
colony size of the edited cells is 30-80% smaller than colonies of
non-edited cells, and in some embodiments, the colony size of the
edited cells is 40-70% smaller than colonies of non-edited
cells.
Use of the Cell Growth Device
[0174] FIG. 9 is a flow chart of an example method 900 for using an
automated multi-module cell editing instrument such as the systems
illustrated in FIGS. 4A-4D which include the reagent cartridges
described in relation to FIGS. 1-3. A processing system, for
example, directs the processing stage of the method 900. For
example, a software script may identify settings for each
processing stage and instructions for movement of a robotic
handling system to perform the actions of the method 900. In some
embodiments, a software instruction script may be identified by a
reagent cartridge supplied to the automated multi-module cell
editing instrument. For example, the reagent cartridge may include
machine-readable indicia, such as a bar code or QR code, including
identification of a script stored in a memory of the automated
multi-module cell editing instrument. In another example, the
reagent cartridge may contain a downloadable script embedded in
machine-readable indicia such as a radio frequency (RF) tag. In
other embodiments, the user may identify a script, for example
through downloading the script via a wired or wireless connection
to the processing system of the automated multi-module cell editing
instrument or through selecting a stored script through a user
interface of the automated multi-module cell editing instrument. In
a particular example, the automated multi-module cell editing
instrument may include a touch screen interface for submitting user
settings and activating cell processing. Again, the automated
multi-module cell processing instrument is a stand-alone
instrument, and between the script, reagent reservoirs, and liquid
handling system facilitates live cell editing in an entirely
automated manner without human intervention.
[0175] In some implementations, the method 900 begins with
transferring cells to a cell growth module (902). The growth module
may be any growth module amendable to automation such as, for
example, the cell growth module 550 described in relation to FIGS.
5B-5D. In a particular example, the processing system may direct
the robotic handling system to transfer cells to the growth module.
In another example, the cells may be transferred from a reagent
cartridge to the growth module by the robotic handling system. In
some embodiments, the growth vial may contain growth media and be
supplied, e.g., as part of a kit. In other embodiments, the growth
vial may be filled with medium transferred, e.g., via the liquid
handling device, from a reagent container.
[0176] In some embodiments, prior to transferring the cells (e.g.,
from the reagent cartridge or from a vial added to the instrument),
machine-readable indicia may be scanned upon the vial or other
container situated in a position designated for cells to confirm
that the vial or container is marked as containing cells. Further,
the machine-readable indicia may indicate a type of cells provided
to the instrument. The type of cells, in some embodiments, may
cause the instrument to select a particular processing script
(e.g., series of instructions for the robotic handling system and
settings and activation of the various modules).
[0177] In some implementations, the cells are grown in the growth
module to a desired optical density (904). For example, the
processing system may manage a temperature setting of the growth
module for incubating the cells during the growth cycle. The
processing system may further receive sensor signals from the
growth module indicative of optical density and analyze the sensor
signals to monitor growth of the cells. In some embodiments, a user
may set growth parameters for managing growth of the cells. For
example, temperature, and the degree of agitation of the cells.
Further, in some embodiments, the user may be updated regarding the
growth process. The updates, in some examples, may include a
message presented on a user interface of the automated multi-module
cell editing instrument, a text message to a user's cell phone
number, an email message to an email account, or a message
transmitted to an app executing upon a portable electronic device
(e.g., cell phone, tablet, etc.). Responsive to the messages, in
some embodiments, the user may modify parameters, such as
temperature, to adjust cell growth. For example, the user may
submit updated parameters through a user interface of the automated
multi-module cell editing instrument or through a portable
computing device application in communication with the automated
multi-module cell editing instrument, such as a user interface
(see, e.g., touch screen display 450 of FIG. 4B).
[0178] Although described in relation to optical density, in other
implementations cell growth within the growth module may be
monitored using a different measure of cell density and
physiological state such as, in some examples, pH, dissolved
oxygen, released enzymes, acoustic properties, and electrical
properties.
[0179] In some implementations, upon reaching the desired optical
density (904), the cells are transferred from the growth module to
a filtration module or cell wash and concentration module (906).
The robotic handling system, for example, may transfer the cells
from the growth module to the cell concentration module. The cell
concentration module, for example, may be (and typically is)
designed to render the cells electrocompetent. See FIGS. 6A-6I in
relation to the TFF device, above. The cells are rendered
electrocompetent and eluted in the filtration module or cell wash
and concentration module (908). The cells may be eluted using a
wash solution. For example, the cells may be eluted using reagents
from a reagent supply.
[0180] Once the cells have been rendered electrocompetent and
suspended in an appropriate volume such as 50 .mu.L to 10 mL, or
100 .mu.L to 80 mL, or 150 .mu.L to 8 mL, or 250 .mu.L to 7 mL, or
500 .mu.L to 6 mL, or 750 .mu.L to 5 mL for transformation (906),
the cells are transferred to, e.g., an FTEP module (918). The
robotic handling system, for example, may transfer the cells from
the filtration module to the FTEP. The filtration module may be
physically coupled to the FTEP device, or these modules may be
separate.
[0181] In some implementations, nucleic acids are prepared outside
of the automated multi-module cell editing instrument. For example,
an assembled vector or other nucleic acid assembly may be included
as a reagent by a user prior to running the transformation process
and other processes in the method 900.
[0182] However, in other implementations, nucleic acids are
prepared by the automated multi-module cell editing instrument. A
portion of the following steps 910 through 916, in some
embodiments, are performed in parallel with a portion of steps 902
through 908. At least a portion of the following steps, in some
embodiments, are performed before and/or after steps 902 through
908.
[0183] In some implementations, nucleic acids such as an editing
oligonucleotide and a vector backbone, as well as in some examples,
enzymes and other reaction components are transferred to a nucleic
acid assembly module (910). The nucleic acid assembly module may be
configured to perform one or more of a wide variety of different
nucleic acid assembly techniques in an automated fashion. Nucleic
acid assembly techniques that can be performed in the nucleic acid
assembly module may include, but are not limited to, those assembly
methods that use restriction endonucleases, including PCR, BioBrick
assembly, Type IIS cloning, GoldenGate assembly, and Ligase Cycling
Reaction. In other examples, the nucleic acid assembly module may
perform an assembly technique based on overlaps between adjacent
parts of the nucleic acids, such as Gibson Assembly.RTM., CPEC,
SLIC, Ligase Cycling, etc., as described above. Additional example
assembly methods that may be performed by the nucleic acid assembly
module include gap repair in yeast, gateway cloning and
topoisomerase-mediated cloning. In a particular example, the
processing system may direct the robotic handling system to
transfer nucleic acids to the nucleic acid assembly module. In
another example, the nucleic acids may be transferred from a
reagent cartridge to a nucleic acid assembly module by the robotic
handling system.
[0184] In some embodiments--prior to transferring each of the
nucleic acid samples, the enzymes, and other reaction
components--machine-readable indicia may be scanned upon the vials
or other containers situated in positions in the reagent cartridge
designated for these materials to confirm that the vials or
containers are marked as containing the anticipated material.
Further, the machine-readable indicia may indicate a type of one or
more of the nucleic acid samples, the enzymes, and other reaction
components provided to the instrument. The type(s) of materials, in
some embodiments, may cause the instrument to select a particular
processing script (e.g., series of instructions for the robotic
handling system to identify further materials and/or settings and
activation of the nucleic acid assembly module). Again, the
automated multi-module cell processing instrument is a stand-alone
instrument, and between the script, reagent reservoirs, and liquid
handling system facilitates live cell editing in an entirely
automated manner without human intervention.
[0185] In some embodiments, the nucleic acid assembly module is
temperature controlled depending upon the type of nucleic acid
assembly used. For example, when PCR is utilized in the nucleic
acid assembly module, the module can have a thermocycling
capability allowing the temperatures to cycle between denaturation,
annealing and extension steps. When single temperature assembly
methods (e.g., isothermal assembly) are utilized in the nucleic
acid assembly module, the module can have the ability to reach and
hold at the temperature that optimizes the specific assembly
process being performed.
[0186] Temperature control, in most embodiments, is managed by a
processing system of the automated multi-module cell editing
instrument. These temperatures and the duration of maintaining the
temperatures can be determined by a preprogrammed set of parameters
(e.g., identified within the processing script or in another memory
space accessible by the processing system), or manually controlled
by the user through interfacing with the processing system.
[0187] Once sufficient time has elapsed for the assembly reaction
to take place, in some implementations, the nucleic acid assembly
may be transferred to a purification module (914). Alternatively,
the purification module may be the same module as the nucleic acid
assembly module. The processing system, for example, may monitor
timing of the assembly reaction based upon one or more of the type
of reaction, the type of materials, and user settings provided to
the automated multi-module cell editing instrument. The robotic
handling system, for example, may transfer the nucleic acid
assembly to the purification module through a sipper or pipettor
interface. In another example, the robotic handling system may
transfer a vial containing the nucleic acid assembly from a chamber
of the nucleic acid assembly module to a chamber of the
de-salt/purification module.
[0188] In some implementations, the nucleic acid assembly is
de-salted and eluted at the purification module (916). The
purification module, for example, may remove unwanted components of
the nucleic acid assembly mixture (e.g., salts, minerals, etc.). In
some embodiments, the purification module concentrates the
assembled nucleic acids into a smaller volume than the nucleic acid
assembly volume. Examples of methods for exchanging liquid
following nucleic acid assembly include magnetic beads (e.g., SPRI
or Dynal (Dynabeads) by Invitrogen Corp. of Carlsbad, Calif.),
silica beads, silica spin columns, glass beads, precipitation
(e.g., using ethanol or isopropanol), alkaline lysis, osmotic
purification, extraction with butanol, membrane-based separation
techniques, filtration etc. For example, one or more
micro-concentrators fitted with anisotropic, hydrophilic-generated
cellulose membranes of varying porosities may be used. In another
example, the de-salt/purification module may process a liquid
sample including a nucleic acid and an ionic salt by contacting the
mixture with an ion exchanger including an insoluble phosphate
salt, removing the liquid, and eluting nucleic acid from the ion
exchanger.
[0189] In an illustrative embodiment, the nucleic acid assembly may
be combined with magnetic beads, such as SPRI beads, in a chamber
of a purification module. The nucleic acid assembly may be
incubated at a set temperature for sufficient time for the
assembled nucleic acids to bind to the magnetic beads. After
incubation, a magnet may be engaged proximate to the chamber so
that the nucleic acid assembly can be washed and eluted. Once the
nucleic acid assembly has been eluted, the nucleic acid assembly is
transferred to the transformation module (918). The robotic
handling system, for example, may transfer the assembled nucleic
acids to the transformation module through a sipper or pipettor
interface to the FTEP as described above. For example, the
de-salted assembled nucleic acids, during the transfer, may be
combined with the electrocompetent cells from step 908. In other
embodiments, the transformation module may accept each of the
electrocompetent cells and the nucleic acid assembly separately and
enable the mixing (e.g., open one or more channels to combine the
materials in a shared chamber).
[0190] The cells are transformed in the FTEP module (920). A buffer
or medium may be transferred to the transformation module and added
to the cells so that the cells may be suspended in a buffer or
medium that is favorable for cell survival during electroporation.
Prior to transferring the buffer or medium, machine-readable
indicia may be scanned upon the vial or other container or
reservoir situated in the position designated for the buffer or
medium to confirm the contents of the vial, container, or
reservoir. Further, the machine-readable indicia may indicate a
type of buffer or medium provided to the instrument. The type of
buffer or medium, in some embodiments, may cause the instrument to
select a particular processing script (e.g., settings and
activation of the transformation module appropriate for the
particular buffer or medium). For bacterial cell electroporation,
low conductance mediums, such as water or glycerol solutions, may
be used to reduce the heat production by transient high current.
For yeast cells, a sorbitol solution may be used. For mammalian
cell electroporation, cells may be suspended in a highly conductive
medium or buffer, such as MEM, DMEM, IMDM, RPMI, Hanks', PBS, HBSS,
HeBS and Ringer's solution. In a particular example, the robotic
handling system may transfer a buffer solution to FTEP module from
the reagent cartridge. As described in relation to FIGS. 7A-7E, the
FTEP device may be a disposable FTEP device and/or the FTEP device
may be provided as part of the reagent cartridge. Alternatively,
the FTEP device may a separate module.
[0191] Once transformed, the cells are transferred to a second
growth/recovery/editing module (922) such as the cell growth module
described in relation to FIGS. 5A-5D. The robotic handling system,
for example, may transfer the transformed cells to the second
growth module through a sipper or pipettor interface. In another
example, the robotic handling system may transfer a vial containing
the transformed cells from a chamber of the transformation module
to a chamber of the second growth module.
[0192] The second growth module, in some embodiments, acts as a
recovery module, allowing the cells to recover from the
transformation process. In other embodiments, the cells may be
provided to a separate recovery module prior to being transported
to the second growth module. During recovery, the second growth
module allows the transformed cells to uptake and, in certain
aspects, integrate the introduced nucleic acids into the genome of
the cell. The second growth module may be configured to incubate
the cells at any user-defined temperature optimal for cell growth,
preferably 25.degree., 30.degree., or 37.degree. C.
[0193] In some embodiments, the second growth module behaves as a
selection module, selecting the transformed cells based on an
antibiotic or other reagent. In one example, the RNA-guided
nuclease (RGN) protein system is used for selection to cleave the
genomes of cells that have not received the desired edit. In the
example of an antibiotic selection agent, the antibiotic may be
added to the second growth module to enact selection. Suitable
antibiotic resistance genes include, but are not limited to, genes
such as ampicillin-resistance gene, tetracycline-resistance gene,
kanamycin-resistance gene, neomycin-resistance gene,
canavanine-resistance gene, blasticidin-resistance gene,
hygromycin-resistance gene, puromycin-resistance gene, or
chloramphenicol-resistance gene. The robotic handling system, for
example, may transfer the antibiotic to the second growth module
through a sipper or pipettor interface. In some embodiments,
removing dead cell background is aided by using lytic enhancers
such as detergents, osmotic stress by hyponic wash, temperature,
enzymes, proteases, bacteriophage, reducing agents, or chaotropes.
The processing system, for example, may alter environmental
variables, such as temperature, to induce selection, while the
robotic handling system may deliver additional materials (e.g.,
detergents, enzymes, reducing agents, etc.) to aid in selection. In
other embodiments, cell removal and/or media exchange by filtration
is used to reduce dead cell background.
[0194] In further embodiments, in addition to or as an alternative
to applying selection, the second growth module serves as an
editing module, allowing for genome editing in the transformed
cells. Alternatively, in other embodiments, the cells post-recovery
and selection (if performed) are transferred to a separate editing
module. As an editing module, the second growth module induces
editing of the cells' genomes, e.g., through facilitating
expression of the introduced nucleic acids. Expression of the
nuclease and/or editing cassette nucleic acids may involve one or
more of chemical, light, viral, or temperature induction methods.
The second growth module, for example, may be configured to heat or
cool the cells during a temperature induction process. In a
particular illustration, the cells may be induced by heating at
42.degree. C.-50.degree. C. Further to the illustration, the cells
may then be cooled to 0-10.degree. C. after induction. In the
example of chemical or viral induction, an inducing agent may be
transferred to the second growth module to induce editing. If an
inducible nuclease and/or editing cassette was introduced to the
cells during editing, it can be induced through introduction of an
inducer molecule. The inducing agent or inducer molecule, in some
implementations, is transferred to the second growth module by the
robotic handling system, e.g., through a pipettor or sipper
interface.
[0195] In some implementations, if no additional cell editing is
desired (924), the cells may be transferred from the cell growth
module to a storage unit for later removal from the automated
multi-module cell editing instrument (926). The robotic handling
system, for example, may transfer the cells to a storage unit
through a sipper or pipettor interface. In another example, the
robotic handling system may transfer a vial containing the cells
from a chamber of the second growth module to a vial or tube within
the storage unit.
[0196] In some implementations, if additional cell editing is
desired (924), the cells may be transferred to the same or a
different filtration module and rendered electrocompetent (908).
Further, in some embodiments, a new assembled nucleic acid sample
may be prepared by the nucleic acid assembly module at this time,
or, alternatively, a second fully assembled nucleic acid may be
directly introduced to the cells. Prior to recursive editing, in
some embodiments, the automated multi-module cell editing
instrument may require additional materials be supplied by the
user, e.g., through the introduction of one or more separate
reagents vials or cartridge.
[0197] The steps may be the same or different during the second
round of editing. For example, in some embodiments, upon a
subsequent execution of step 904, a selective growth medium is
transferred to the growth module to enable selection of edited
cells from the first round of editing. The robotic handling system
may transfer the selective growth medium from a vial or container
in a reagent cartridge situated in a position designated for
selective growth medium. Prior to transferring the selective growth
medium, machine-readable indicia may be scanned upon the vial or
other container or reservoir situated in the position designated
for the selective growth medium to confirm the contents of the
vial, container, or reservoir. Further, the machine-readable
indicia may indicate a type of selective growth medium provided to
the instrument. The type of selective growth medium, in some
embodiments, may cause the instrument to select a particular
processing script (e.g., settings and activation of the growth
module appropriate for the particular selective growth medium).
Particular examples of recursive editing workflows are described in
relation to FIG. 12.
[0198] In some implementations, the method 900 can be timed to
introduce materials and/or complete the editing cycle or growth
cycle in coordination with a user's schedule. For example, the
automated multi-module cell editing instrument may provide the user
the ability to schedule completion of one or more cell processing
cycles (e.g., one or more recursive edits) such that the method 900
is enacted with a goal of completion at the user's preferred time.
The time scheduling, for example, may be set through a user
interface. For illustration only, a user may set completion of a
first cycle to 4:00 PM so that the user can supply additional
cartridges of materials to the automated multi-module cell editing
instrument to enable overnight processing of another round of cell
editing. Thus, a user may time the programs so that two or more
cycles may be programmed in a specific time period, e.g., a 24-hour
period.
[0199] In some implementations, throughout the method 900, the
automated multi-module cell editing instrument may alert the user
to its current status. For example, the user interface may present
a graphical indication of the present stage of processing. In a
particular example, a front face of the automated multi-module call
processing instrument may be overlaid with a user interface (e.g.,
touch screen) that presents an animated graphic depicting present
status of the cell processing. The user interface may further
present any user and/or default settings associated with the
current processing stage (e.g., temperature setting, time setting,
etc.). In certain implementations, the status may be communicated
to a user via a wireless communications controller.
[0200] Although illustrated as a particular series of operations,
in other embodiments, more or fewer steps may be included in the
method 900. For example, in some embodiments, prior to engaging in
each round of editing, the contents of reservoirs, reagent
cartridges, and/or vials may be screened to confirm appropriate
materials are available to proceed with processing. For example, in
some embodiments, one or more imaging sensors (e.g., barcode
scanners, cameras, etc.) may confirm contents at various locations
within the housing of the automated multi-module cell editing
instrument. In one example, multiple imaging sensors may be
disposed within the housing of the automated multi-module cell
editing instrument, each imaging sensor configured to detect one or
more materials (e.g., machine-readable indicia such as barcodes or
QR codes, shapes/sizes of materials, etc.). In another example, at
least one imaging sensor may be moved by the robotic handling
system to multiple locations to detect one or more materials. In
further embodiments, one or more weight sensors may detect presence
or absence of disposable or replaceable materials. In an
illustrative example, the transfer tip supply holder may include a
weight sensor to detect whether or not tips have been loaded into
the region. In another illustrative example, an optical sensor may
detect that a level of liquid waste has reached a threshold level,
requiring disposal prior to continuation of cell processing or
addition of liquid if the minimum level has not been reached to
proceed. Requests for additional materials, removal of waste
supplies, or other user interventions (e.g., manual cleaning of one
or more elements, etc.), in some implementations, are presented on
a graphical user interface of the automated multi-module cell
editing instrument. The automated multi-module cell editing
instrument, in some implementations, contacts the user with
requests for new materials or other manual interventions, for
example, through a software app, email, or text message.
[0201] FIG. 10 shows simplified flow charts for two alternative
exemplary methods 1000a and 1000b for singulating cells for
enrichment (1000a) and for selection (aka "cherry picking")
(1000b). Looking at FIG. 10, method 1000a begins by transforming
cells 1010 with the components necessary to perform nucleic
acid-guided nuclease editing. For example, the cells may be
transformed simultaneously with separate engine and editing
vectors; the cells may already be expressing the nuclease (e.g.,
the cells may have already been transformed with an engine vector
or the coding sequence for the nuclease may be stably integrated
into the cellular genome) such that only the editing vector needs
to be transformed into the cells; or the cells may be transformed
with a single vector comprising all components required to perform
nucleic acid-guided nuclease genome editing.
[0202] As described above, a variety of delivery systems can be
used to introduce (e.g., transform or transfect) nucleic
acid-guided nuclease editing system components into a host cell
1010. These delivery systems include the use of yeast systems,
lipofection systems, microinjection systems, biolistic systems,
virosomes, liposomes, immunoliposomes, polycations, lipid:nucleic
acid conjugates, virions, artificial virions, viral vectors,
electroporation, cell permeable peptides, nanoparticles, nanowires,
and/or exosomes. Alternatively, molecular trojan horse liposomes
may be used to deliver nucleic acid-guided nuclease components
across the blood brain barrier. Of interest, particularly in the
context of a multi-module cell editing instrument, is the use of
electroporation, particularly flow-through electroporation (either
as a stand-alone instrument or as a module in an automated
multi-module system) as described in, e.g., U.S. Ser. No.
16/147,120, filed 28 Sep. 2018; Ser. No. 16/147,353, filed 28 Sep.
2018; Ser. No. 16/147,865, filed 30 Sep. 2018; and Ser. No.
16/147,871, filed 30 Sep. 2018. If the solid wall
singulation/growth/editing/normalization module is one module in an
automated multi-module cell editing instrument, the cells are
likely transformed in an automated cell transformation module.
[0203] After the cells are transformed with the components
necessary to perform nucleic acid-guided nuclease editing, the
cells are singulated in microwells in a, e.g., solid wall device
1020; that is, the cells are diluted (if necessary) in a liquid
culture medium (in some embodiments, including Tween, at a
concentration of 0.1% or less to effect a good distribution) so
that the cells, when delivered to the solid wall device, fill the
microwells of the solid wall device in a Poisson or substantial
Poisson distribution. Singulation is accomplished when an average
of 1/2 cell is delivered to each microwell; that is, where some
microwells contain one cell and other microwells contain no
cells.
[0204] Once the cells in this embodiment have been singulated in
1000a, the cells are actively editing, as the editing "machinery"
is under the control of a constitutive promoter. As the cells are
editing, they are grown into colonies of terminal size 1030; that
is, the colonies arising from the singulated cells are grown into
colonies to a point where cell growth has peaked and is normalized
or saturated for both edited and unedited cells. Normalization
occurs as the nutrients in the medium around a growing cell colony
are depleted and/or cell growth fills the microwells and further
growth is constrained. Again, in the embodiment 1000a shown in FIG.
10, the editing components are under the control of a constitutive
promoter; thus, editing begins immediately (or almost immediately)
upon transformation. However, in other embodiments such as the
embodiment shown in 1000b described below, one or both of the
nuclease and the guide nucleic acid (as well as, e.g., the .lamda.
red recombination system components in bacterial systems) may be
under the control of an inducible promoter, in which case editing
may be induced after, e.g., a desired number of cell doublings.
Turning back to method 1000a, the terminal-size colonies are pooled
1040 by flushing the clonal cell colonies from the microwells to
mix the cells from the normalized cell colonies. Again, because
singulation overcomes growth bias from unedited cells or cells
exhibiting fitness effects as the result of edits made,
singulation/normalization alone enriches the total population of
cells with cells that have been edited; that is, singulation
combined with normalization (e.g., growing colonies to terminal
size) allows for high-throughput enrichment of edited cells.
[0205] The method 1000b shown in FIG. 10 is similar to the method
1000a in that cells of interest are transformed 1010 with the
components necessary to perform nucleic acid-guided nuclease
editing. As described above, the cells may be transformed
simultaneously with both the engine and editing vectors, the cells
may already be expressing the nuclease (e.g., the cells may have
already been transformed with an engine vector or the coding
sequence for the nuclease may be stably integrated into the
cellular genome) such that only the editing vector needs to be
transformed into the cells, or the cells may be transformed with a
single vector comprising all components required to perform nucleic
acid-guided nuclease genome editing. Further, if the
singulation/growth/editing/normalization solid wall module is one
module in an automated multi-module cell editing instrument, cell
transformation may be performed in an automated transformation
module as described above.
[0206] After the cells are transformed with the components
necessary to perform nucleic acid-guided nuclease editing, the
cells are diluted (if necessary) in liquid medium so that the
cells, when delivered to the solid wall device, fill the microwells
of the solid wall device in a Poisson or substantial Poisson
distribution.
[0207] Once the cells have been singulated in the microwells of the
solid wall device 1020, the cells are allowed to grow to, e.g.,
between 2 and 150, or between 5 and 120, or between 10 and 100
doublings, establishing clonal colonies 1050. After colonies are
established, in this embodiment 1000b, editing is induced 1060 by,
e.g., activating inducible promoters that control transcription of
one or more of the components needed for nucleic acid-guided
nuclease editing, such as, e.g., transcription of the gRNA,
nuclease, or, in the case of bacteria, a recombineering system.
Once editing is induced 1060, many of the edited cells in the
clonal colonies die due to the double-strand DNA breaks that occur
during the editing process; however, in a percentage of edited
cells, the genome is edited and the double strand break is properly
repaired. These edited cells then start growing and re-establish
colonies; however, the growth of edited colonies tends to lag
behind the growth of clonal colonies where an edit has not taken
place. The small or slow-growing colonies (edited cells) are cherry
picked 1070. Alternatively, after induction, the colonies are grown
into colonies of terminal size (normalization) (like step 1030),
and the colonies are then pooled (like step 1040) and used in
research or subjected to an additional round of editing.
[0208] FIG. 11 is a simplified block diagram of an embodiment of an
exemplary automated multi-module cell processing instrument 1100
comprising a singulation/growth/editing/normalization module 1140
for enrichment for edited cells. The cell processing instrument
1100 may include a housing 1144, a reservoir of cells to be
transformed or transfected 1102, and a growth module (a cell growth
device) 1104. The cells to be transformed are transferred from a
reservoir 1102 to the growth module 1104 to be cultured until the
cells hit a target OD. Once the cells hit the target OD, the growth
module 1104 may cool or freeze the cells for later processing, or
the cells may be transferred to a cell concentration module 1130
where the cells are rendered electrocompetent and concentrated to a
volume optimal for cell transformation. Once concentrated, the
cells are then transferred to the electroporation module 1105
(e.g., transformation/transfection module). Exemplary
electroporation devices for use in the automated multi-module cell
processing instruments 1100 include flow-through electroporation
devices such as those described in U.S. Ser. No. 16/147,120, filed
28 Sep. 2018; Ser. No. 16/147,353, filed 28 Sep. 2018; Ser. No.
16/147,865, filed 30 Sep. 2018; and Ser. No. 16/147,871, filed 30
Sep. 2018, all of which are herein incorporated by reference in
their entirety.
[0209] In addition to the reservoir 1102 for storing the cells, the
automated multi-module cell processing instrument 1100 may include
a reservoir for storing editing oligonucleotide cassettes 1116 and
a reservoir for storing an expression vector backbone 1118. Both
the editing oligonucleotide cassettes and the expression vector
backbone are transferred from the reagent cartridge to a nucleic
acid assembly module 1120, where the editing oligonucleotide
cassettes are inserted into the expression vector backbone. The
assembled nucleic acids may be transferred into an optional
purification module 1122 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 1116 or 1118. Once the processes carried out by the
purification module 1122 are complete, the assembled nucleic acids
are transferred to, e.g., an electroporation device 1105, which
already contains the cell culture grown to a target OD and rendered
electrocompetent via cell concentration module 1130. In
electroporation device 1105, the assembled nucleic acids are
introduced into the cells. Following electroporation, the cells are
transferred into a combined recovery/dilution/selection module
1110.
[0210] Following recovery, and, optionally, selection, the cells
are transferred to a singulation, induction, editing, and growth
module 1140, where the cells are diluted and compartmentalized such
that there is an average of one cell per compartment. Once
singulated, the cells are allowed to grow for a pre-determined
number of doublings. Once these initial colonies are established,
editing is induced and the edited cells are allowed to establish
colonies, which are grown to terminal size (e.g., the colonies are
normalized). In some embodiments, editing is induced by one or more
of the editing components being under the control of an inducible
promoter. In some embodiments, the inducible promoter is activated
by a rise in temperature and "deactivated" by lowering the
temperature. Alternatively, in embodiments where the singulation
device is a solid wall device comprising a filter forming the
bottom of the microwell, the solid wall device can be transferred
to a plate (e.g., agar plate or even to liquid medium) comprising a
medium with a component that activates induced editing, then
transferred to a medium that deactivates editing. Once the colonies
are grown to terminal size, the colonies are pooled. Again,
singulation overcomes growth bias from unedited cells and growth
bias resulting from fitness effects of different edits.
[0211] The recovery, selection, singulation, induction, editing and
growth modules may all be separate, may be arranged and combined as
shown in FIG. 11, or may be arranged or combined in other
configurations. In certain embodiments, all of recovery, selection,
singulation, growth, editing, and normalization are performed in a
solid wall device. Alternatively, recovery, selection, and
dilution, if necessary, are performed in liquid medium in a
separate vessel (module), then transferred to the solid wall
singulation/growth/induction/editing/normalization module.
[0212] Once the normalized cell colonies are pooled, the cells may
be stored, e.g., in a storage module 1112, where the cells can be
kept at, e.g., 4.degree. C. until the cells are retrieved 1114 for
further study. Alternatively, the cells may be used in another
round of editing. The multi-module cell processing instrument 1100
is controlled by a processor 1142 configured to operate the
instrument 1100 based on user input, as directed by one or more
scripts, or as a combination of user input or a script. The
processor 1142 may control the timing, duration, temperature, and
operations of the various modules of the instrument 1100 and the
dispensing of reagents. For example, the processor 1142 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 1142 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 cell processing instrument
1100.
[0213] The automated multi-module cell processing instrument 1100
is a nuclease-directed genome editing system and can be used in
single editing systems (e.g., introducing one or more edits to a
cellular genome in a single editing process). The system of FIG.
12, described below, is configured to perform sequential editing,
e.g., using different nuclease-directed systems sequentially to
provide two or more genome edits in a cell; and/or recursive
editing, e.g. utilizing a single nuclease-directed system to
introduce sequentially two or more genome edits in a cell.
[0214] FIG. 12 illustrates another embodiment of a multi-module
cell processing instrument 1200. This embodiment depicts an
exemplary system that performs recursive gene editing on a cell
population. As with the embodiment shown in FIG. 12, the cell
processing instrument 1200 may include a housing 1244, a reservoir
for storing cells to be transformed or transfected 1202, and a cell
growth module (comprising, e.g., a rotating growth vial) 1204. The
cells to be transformed are transferred from a reservoir to the
cell growth module 1204 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 1260 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 or module 1208. In addition to the reservoir
for storing cells, the multi-module cell processing instrument 1200
includes a reservoir for storing the vector pre-assembled with
editing oligonucleotide cassettes 1252. The pre-assembled nucleic
acid vectors are transferred to the electroporation device 1208,
which already contains the cell culture grown to a target OD. In
the electroporation device 1208, the nucleic acids are
electroporated into the cells. Following electroporation, the cells
are transferred into an optional recovery (and optionally,
dilution) module 1256, where the cells are allowed to recover
briefly post-transformation.
[0215] After recovery, the cells may be transferred to a storage
module 1212, where the cells can be stored at, e.g., 4.degree. C.
for later processing, or the cells may be diluted and transferred
to a selection/singulation/growth/induction/editing/normalization
module/device 1258. In the
selection/singulation/growth/induction/editing/normalization module
1258, the cells are arrayed such that there is an average of one
cell per microwell. The arrayed cells may be in selection medium to
select for cells that have been transformed or transfected with the
editing vector(s). Once singulated, the cells grow through 2-50
doublings and establish colonies. Once colonies are established,
editing is induced by providing conditions (e.g., temperature,
addition of an inducing or repressing chemical) to induce editing.
Once editing is initiated and allowed to proceed, the cells are
allowed to grow to terminal size (e.g., normalization of the
colonies) in the microwells and then can be flushed out of the
microwells and pooled, then transferred to the storage unit 1212
(or cell retrieval 1214) or can be transferred to a growth module
1204 for another round of editing. In between pooling and transfer
to a growth module 1204, there may be one or more additional steps,
such as cell recovery, medium exchange, cells concentration, etc.,
by, e.g., filtration. Note that the
selection/singulation/growth/induction/editing and normalization
modules may be the same module, where all processes are performed
in the solid wall device, or selection and/or dilution may take
place in a separate vessel before the cells are transferred to the
solid wall singulation/growth/induction/editing/normalization
module (solid wall device). As an alternative to singulation in,
e.g., a solid wall device, the transformed cells may be grown
in--and editing can be induced in--bulk liquid as described above
in relation to FIGS. 8F-8H above. Once the putatively-edited cells
are pooled, they may be subjected to another round of editing,
beginning with growth, cell concentration and treatment to render
electrocompetent, and transformation by yet another donor nucleic
acid in another editing cassette via the electroporation
device/module 1208.
[0216] In electroporation device 1208, 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 1200 exemplified in FIG. 12 is controlled by a processor
1242 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 1242 may
control the timing, duration, and temperature of various processes,
the dispensing of reagents, and other operations of the various
modules of the instrument 1200. 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.
[0217] 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. 12, then the resulting
edited culture may go through another (or several or many) rounds
of additional editing (e.g., recursive editing) with different
editing vectors. For example, the cells from round 1 of editing may
be diluted and an aliquot of the edited cells edited by editing
vector A may be combined with editing vector B, an aliquot of the
edited cells edited by editing vector A may be combined with
editing vector C, an aliquot of the edited cells edited by editing
vector A may be combined with editing vector D, and so on for a
second round of editing. After round two, an aliquot of each of the
double-edited cells may be subjected to a third round of editing,
where, e.g., aliquots of each of the AB-, AC-, AD-edited cells are
combined with additional editing vectors, such as editing vectors
X, Y, and Z. That is that double-edited cells AB may be combined
with and edited by vectors X, Y, and Z to produce triple-edited
edited cells ABX, ABY, and ABZ; double-edited cells AC may be
combined with and edited by vectors X, Y, and Z to produce
triple-edited cells ACX, ACY, and ACZ; and double-edited cells AD
may be combined with and edited by vectors X, Y, and Z to produce
triple-edited cells ADX, ADY, and ADZ, and so on. In this process,
many permutations and combinations of edits can be executed,
leading to very diverse cell populations and cell libraries. In any
recursive process, it is advantageous to "cure" the previous engine
and editing vectors (or single engine+editing vector in a single
vector system). "Curing" is a process in which one or more vectors
used in the prior round of editing is eliminated from the
transformed cells. Curing can be accomplished by, e.g., cleaving
the vector(s) using a curing plasmid thereby rendering the editing
and/or engine vector (or single, combined vector) nonfunctional;
diluting the vector(s) in the cell population via cell growth (that
is, the more growth cycles the cells go through, the fewer daughter
cells will retain the editing or engine vector(s)), or by, e.g.,
utilizing a heat-sensitive origin of replication on the editing or
engine vector (or combined engine+editing vector). The conditions
for curing will depend on the mechanism used for curing; that is,
in this example, how the curing plasmid cleaves the editing and/or
engine plasmid.
[0218] FIG. 13 is a simplified block diagram of an embodiment of an
exemplary automated multi-module cell processing instrument 1300
comprising, e.g., a bulk liquid growth module for induced editing
and enrichment for edited cells as described above in relation to
FIGS. 8H-8F. The cell processing instrument 1300 may include a
housing 1344, a reservoir of cells to be transformed or transfected
1302, and a growth module (a cell growth device) 1304. The cells to
be transformed are transferred from a reservoir 1302 to the growth
module 1304 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 1330 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 module 1308 (e.g.,
transformation/transfection module). Exemplary electroporation
devices for use in the automated multi-module cell processing
instruments 1300 include flow-through electroporation devices such
as those described in U.S. Ser. No. 16/147,120, filed 28 Sep. 2018;
Ser. No. 16/147,353, filed 28 Sep. 2018; Ser. No. 16/147,865, filed
30 Sep. 2018; and Ser. No. 16/147,871, filed 30 Sep. 2018, all of
which are herein incorporated by reference in their entirety.
[0219] In addition to the reservoir 1302 for storing the cells, the
instrument 1300 may include a reservoir for storing editing
cassettes 1316 and a reservoir for storing an expression vector
backbone 1318. Both the editing oligonucleotide cassettes and the
expression vector backbone are transferred from the reagent
cartridge to a nucleic acid assembly module 1320, where the editing
oligonucleotide cassettes are inserted into the expression vector
backbone. The assembled nucleic acids may be transferred into an
optional purification module 1322 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 1316 or 1318. Once the processes carried out by
the purification module 1322 are complete, the assembled nucleic
acids are transferred to, e.g., an electroporation device or module
1308, which already contains the cell culture grown to a target OD
and rendered electrocompetent via cell concentration module 1330.
In electroporation device 1308, the assembled nucleic acids are
introduced into the cells. Following electroporation, the cells are
transferred into a combined recovery/selection module 1310. For
examples of multi-module cell editing instruments, see U.S. Ser.
Nos. 16/024,816 and 16/024,831, filed 30 Jun. 2018, both of which
are herein incorporated by reference in their entirety.
[0220] Following recovery, and, optionally, selection, the cells
are transferred to a growth, induction, and editing module (bulk
liquid culture) 1340. 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.
[0221] The recovery, selection, growth, induction, editing and
storage modules may all be separate, may be arranged and combined
as shown in FIG. 13, 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.
[0222] Once the cells are edited and re-grown (e.g., recovered from
editing), the cells may be stored, e.g., in a storage module 1312,
where the cells can be kept at, e.g., 4.degree. C. until the cells
are retrieved for further study (e.g., cell retrieval 1314).
Alternatively, the cells may be used in another round of editing.
The multi-module cell processing instrument 1300 is controlled by a
processor 1342 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 1342 may control the timing,
duration, temperature, and operations of the various modules of the
instrument 1300 and the dispensing of reagents. For example, the
processor 1342 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
[0223] 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
[0224] 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.
[0225] In a first example, 20 ml EC23 cells (E. coli cells) in LB
were grown in a 35 ml rotating growth vial 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. OD was measured in the cell
growth device. In parallel, 5 ml EC23 cells in LB were grown in an
orbital shaker in a 5 ml tube at 30.degree. C. and were shaken at
750 rpm; the OD.sub.600 was measured at intervals using a
NanoDrop.TM. spectrophotometer (Thermo Fisher Scientific). The
results are shown in FIGS. 14 and 15. The rotating growth vial/cell
growth device performed better than the cell shaker in growing the
cells to OD.sub.600 2.6 in slightly over 4 hours.
[0226] Two additional experiments were performed, this time
comparing the rotating growth vial/cell growth device with paddles
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. 16, 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. 17, 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.
[0227] In yet another experiment, the rotating growth vial/cell
growth device was used to measure OD.sub.600 in real time. FIG. 18
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.
[0228] 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. 19 is a graph showing the results. Note that OD.sub.600 6.0
was reached in 14 hours.
Example II: Cell Concentration
[0229] The TFF module as described above in relation to FIGS. 6A-6I
has been used successfully to process and perform buffer exchange
on both E. coli and yeast cultures. In concentrating an E. coli
culture, the following steps were performed:
[0230] 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.
[0231] Filtrate conductivity and filter processing time was
measured for E. coli with the results shown in FIG. 20A. 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 (.about.16 .mu.S/cm) was achieved in approximately 30
minutes utilizing three 50 ml 10% glycerol washes and three passes
of the cells through the device for each wash. The volume of the
cells was reduced from 20 ml to 400 .mu.l, and recovery of
approximately 90% of the cells has been achieved.
[0232] 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. 20B
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
[0233] For testing transformation of the FTEP device,
electrocompetent E. coli cells were created. To create a starter
culture, 6 ml volumes of LB chlor-25 (LB with 25 .mu.g/ml
chloramphenicol) were transferred to 14 ml culture tubes. A 25
.mu.l aliquot of E. coli was used to inoculate the LB chlor-25
tubes. Following inoculation, the tubes were placed at a 45.degree.
angle in the shaking incubator set to 250 RPM and 30.degree. C. for
overnight growth, between 12-16 hrs. The OD600 value should be
between 2.0 and 4.0. A 1:100 inoculum volume of the 250 ml LB
chlor-25 tubes were transferred to four sterile 500 ml baffled
shake flasks, i.e., 2.5 ml per 250 ml volume shake flask. The
flasks were placed in a shaking incubator set to 250 RPM and
30.degree. C. The growth was monitored by measuring OD600 every 1
to 2 hr. When the OD600 of the culture was between 0.5-0.6 (approx.
3-4 hrs), the flasks were removed from the incubator. The cells
were centrifuged at 4300 RPM, 10 min, 4.degree. C. The supernatant
was removed, and 100 ml of ice-cold 10% glycerol was transferred to
each sample. The cells were gently resuspended, and the wash
procedure performed three times, each time with the cells
resuspended in 10% glycerol. After the fourth centrifugation, the
cell resuspension was transferred to a 50 ml conical Falcon tube
and additional ice-cold 10% glycerol added to bring the volume up
to 30 ml. The cells were again centrifuged at 4300 RPM, 10 min,
4.degree. C., the supernatant removed, and the cell pellet
resuspended in 10 ml ice-cold glycerol. The cells are aliquoted in
1:100 dilutions of cell suspension and ice-cold glycerol.
[0234] 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.
[0235] The flow-through electroporation experiments were
benchmarked against 2 mm electroporation cuvettes (Bulldog 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. 21A. In
FIG. 21A, 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.
21A, the recovery ratio is approximately 0.0001.
[0236] Additionally, a comparison of the NEPAGENE.TM. ELEPO21 and
the FTEP device was made for efficiencies of transformation
(uptake), cutting, and editing. In FIG. 21B, 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. 21B for the
FTEP is greater-than 0.001.
[0237] For testing transformation of the FTEP device in yeast, S.
Cerevisiae cells were created using the methods as generally set
forth in Bergkessel and Guthrie, Methods Enzymol., 529:311-20
(2013). Briefly, YFAP media was inoculated for overnight growth,
with 3 ml inoculate to produce 100 ml of cells. Every 100 ml of
culture processed resulted in approximately 1 ml of competent
cells. Cells were incubated at 30.degree. C. in a shaking incubator
until they reached an OD600 of 1.5+/-0.1.
[0238] 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.
[0239] 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.
[0240] The flow-through electroporation experiments were
benchmarked against 2 mm electroporation cuvettes (Bulldog 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. 22. 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: Fully-Automated Singleplex RGN-Directed Editing Run
[0241] 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. No.
16/024,831 filed 30 Jun. 2018; Ser. No. 16/024,816 filed 30 Jun.
2018; Ser. No. 16/147,353 filed 28 Sep. 2018; Ser. No. 16/147,865
filed 30 Sep. 2018; and Ser. No. 16/147,871 filed 30 Jun. 2018.
[0242] 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.
[0243] 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.
[0244] 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 V: Fully-Automated Recursive Editing Run
[0245] Recursive editing was successfully achieved using the
automated multi-module cell processing instrument. An ampR plasmid
backbone and a lacZ_V10* editing cassette were assembled via Gibson
Assembly.RTM. into an "editing vector" in an isothermal nucleic
acid assembly module included in the automated system. Similar to
the lacZ_F172 edit, the lacZ_V10 edit functionally knocks out the
lacZ gene. "lacZ_V10" indicates that the edit happens at amino acid
position 10 in the lacZ amino acid sequence. Following assembly,
the product was de-salted in the isothermal nucleic acid assembly
module using AMPure beads, washed with 80% ethanol, and eluted in
buffer. The first assembled editing vector and the
recombineering-ready electrocompetent E. Coli cells were
transferred into a transformation module for electroporation. The
cells and nucleic acids were combined and allowed to mix for 1
minute, and electroporation was performed for 30 seconds. The
parameters for the poring pulse were: voltage, 2400 V; length, 5
ms; interval, 50 ms; number of pulses, 1; polarity, +. The
parameters for the transfer pulses were: Voltage, 150 V; length, 50
ms; interval, 50 ms; number of pulses, 20; polarity, +/-. Following
electroporation, the cells were transferred to a recovery module
(another growth module) allowed to recover in SOC medium containing
chloramphenicol. Carbenicillin was added to the medium after 1
hour, and the cells were grown for another 2 hours. The cells were
then transferred to a centrifuge module and a media exchange was
then performed. Cells were resuspended in TB containing
chloramphenicol and carbenicillin where the cells were grown to
OD600 of 2.7, then concentrated and rendered electrocompetent.
[0246] 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
instrument.
[0247] 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.
[0248] 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,
6.
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