RU2773331C1 - Tools, modules and methods for improved detection of edited sequences in live cells - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: group of inventions relates to tools for optimising detection of genome changes in live cells. Disclosed is a unit for a module for solid-wall cell isolation, substantial isolation, growth, editing induction, normalization and selection (SWIIN), intended for cell isolation, cell growth, creation of conditions for editing cells, and normalization or selection of small cell colonies, containing a retaining element, a perforated element, a filter, a seal surrounding the perforated element and the filter; a permeable element, and means for connecting the retaining element, the perforated element, the filter, the seal, and the permeable element. Also disclosed are SWIIN modules.

EFFECT: group of inventions ensures automated highly productive and extremely sensitive screening for identifying edited cells.

20 cl, 78 dwg, 6 tbl, 15 ex

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This international patent application claims the priority of U.S. Provisional Applications Nos. 62/718449, filed August 14, 2018; 62/735365, filed September 24, 2018; 62/781112, filed December 18, 2018; and 62/779119, filed December 13, 2018; as well as U.S. Patent No. 10253316, issued April 09, 2019.

FIELD OF TECHNOLOGY TO WHICH THE INVENTION RELATES

[0002] The present invention relates to improved tools, modules and methods for screening, selecting, and thus optimizing the detection of genome changes in living cells.

BACKGROUND OF THE INVENTION

[0003] In the following discussion, certain articles and methods will be described for reference and introductory purposes. Nothing contained herein should be construed as an "admission" of prior art. Applicant expressly reserves the right to demonstrate, where appropriate, that the methods referred to herein are not prior art under applicable law.

[0004] The ability to make precise and targeted changes to the genome of living cells has been a longstanding goal of biomedical research and development. Recently, various nucleases have been identified that allow the manipulation of gene sequences and hence the function of genes. These nucleases include nucleic acid-directed nucleases that allow researchers to make permanent changes to living cells. Current protocols using nucleic acid-driven nuclease systems typically use constituency-expressed nuclease components to provide highly efficient editing. However, in pooled or multiplex formats, persistent expression of editing components can lead to rapid depletion of edited cell types and selective enrichment of cells that have not been edited. This occurs in most cell types because only a small proportion (<1-5%) of cells survive the introduction of double-stranded DNA (dsDNA) breaks, and thus these cells contribute fewer viable cells to the resulting populations compared to unedited, untreated cells. breakage of double-stranded DNA.

[0005] Thus, in the field of gene editing using nucleic acid-directed nucleases, there is a need for improved tools, modules and methods for editing the genome, as well as for identifying and enriching edited cells. The present invention satisfies this need.

SUMMARY OF THE INVENTION

[0006] This Summary is intended to provide a simplified introduction to the set of concepts that are described in the Detailed Description below. This section is not intended to identify all key or essential features of the claimed subject matter, nor is it intended to limit the scope of the claimed subject matter. Other features, details and advantages of the claimed subject matter will become apparent from the following detailed description, including the aspects illustrated in the accompanying drawings and defined in the appended claims.

[0007] The present invention provides tools, modules, and methods for tightly regulated expression of components of nucleic acid-directed nuclease editing systems that allow separation of transformation and genome editing processes. These compositions and methods use inducible guide RNA (gRNA) constructs resulting in increased observed transformation efficiency and easy to automate control over the time and duration of the editing process. In addition, these tools, modules and methods provide automated, high throughput and extremely sensitive screening for the identification of edited cells. These tools, modules and methods take advantage of singulation or essential singulation, where the term "singulation" in this context refers to the process of separating cells and growing them into clonally isolated formats. The term "substantial singulation" refers to the process of dividing cells in a population of cells into "groups" of 2-100, or 2-50, and preferably 2-10 cells. Singulation, followed by an initial period of growth, editing induction, and growth normalization, results in enrichment of the edited cells. In addition, the tools, modules, and methods described herein facilitate "selection" of edited cell colonies by providing direct selection of edited cells. Singulation or substantial singulation helps overcome the growth bias towards unedited cells that occurs with competitive growth regimes such as bulk liquid culture. Indeed, removing growth rate bias through singulation or significant singulation, induction, and normalization has been found to improve observed editing efficiency up to 4-fold (e.g., from 10% to 40% absolute population-scale efficiency) or more compared to conventional methods, and in addition, colony selection using the methods described herein increases the observed editing efficiency up to 8-fold (eg, from 10% to 80% absolute population-scale efficiency) compared to conventional methods. Thus, the combination of singulation or significant singulation, growth, editing induction, and normalization or selection improves the observed editing efficiency by up to 8 times compared to traditional methods in which singulation or significant singulation, growth, editing induction and normalization or selection are not used.

[0008] One particularly simple module or device for singulation or significant singulation is a solid wall device where cells are essentially isolated, grown in clonal format, editing is induced, and either normalization or selection is used. Solid wall devices or modules and their use are described in detail in this document. These tools, modules, and methods, in some embodiments, normalize edited and unedited cell colonies. Normalization refers to the growth of colonies of cells - edited or unedited - to a final size; that is, to grow the cells until the cells in the colonies enter the senescence stage due to, for example, nutrient depletion or limited space for further growth. Normalization of cell colonies enriches the edited cells, as the edited cells get an equal standing with the unedited cells. In addition, these tools, modules, and methods facilitate the "selection" of colonies. The selection provides a direct selection of the edited cells by exploiting the edit-induced growth inhibition of the edited colonies. Colony selection using the tools, modules and methods described herein can more than double the observed editing efficiency resulting from singulation or significant singulation.

[0009] Some embodiments of these tools, modules, and methods provide for the enrichment of edited cells during nucleic acid-directed nuclease editing, wherein the methods comprise transforming cells with one or more vectors comprising a promoter that directs nuclease expression, a promoter that directs transcription of the target nucleic acid, and a donor DNA sequence, wherein one or both of the promoters driving the transcription of the nuclease or the target nucleic acid are an inducible promoter; diluting the transformed cells to a cell concentration sufficient to substantially singulate the transformed cells onto the substrate; growing substantially singulated cells on a substrate; initiating editing by inducing an inducible promoter(s); and either 1) growing colonies of cells derived from induced cells to final size colonies (eg, normalizing cell colonies) and harvesting normalized cell colonies; or 2) monitoring the growth of cell colonies on the substrate, followed by the selection of slowly growing colonies.

[0010] Thus, in some embodiments, a solid wall singulation assembly for singulation or significant singulation, growth, editing induction, and normalization or selection ("solid wall isolation/induction/normalization module" or "SWIIN") is provided, comprising: a holding element comprising an upper surface and a lower surface, which contains at least one distribution channel of the retained substance, which crosses the holding element from its upper surface to its lower surface for most of the length of the holding element; in which the lower surface of the retaining element contains retaining ribs, between which there are guides for the flow of the retained substance; wherein the retaining member further comprises one or more retaining member openings configured to supply cells and fluid to the retaining member and remove cells and fluid from the retaining member; and in which the openings of the retaining element are hydraulically connected with the retained substance distribution channel and the retained substance flow guides; a perforated element with a top surface and a bottom surface, in which the upper surface of the perforated element is located below and adjacent to the bottom surface of the retaining element, and in which the perforated element contains at least 25,000 holes; a filter with a top surface and a bottom surface, in which the top surface of the filter is located below and adjacent to the bottom surface of the perforated element, and in which the bottom surface of the filter is located above and adjacent to the upper surface of the perforated element; a seal surrounding the perforated element and the filter; and a permeable element comprising an upper surface and a lower surface, which contains at least one permeate distribution channel that intersects the permeable element from its lower surface to its upper surface for most of the length of the permeate element; in which the upper surface of the permeable element contains permeable ribs, between which are permeate flow guides; in which the permeable element further comprises one or more openings of the permeable element, configured to supply liquid to the permeable element and remove liquid from the permeable element; and wherein the orifices of the permeate member are in fluid communication with the permeate distribution channel and the permeate flow guides; as well as a means for connecting the retaining element, the perforated element, the filter, the seal and the permeable element.

[0011] In some aspects of an embodiment of the singulation assembly, the means for connecting the retaining element, the perforated element, the filter, the seal, and the permeable element comprises ultrasonic welding, and in other aspects, the means comprises a pressure-sensitive adhesive, a solvent bond, mating fittings, or a combination of adhesives, welding, solvent bonding and mating fittings; and other such fasteners and connections. In some aspects of the singulation node, the perforated element contains at least 50,000; 100000; 150000; 200,000, 250,000 holes or more, and in some aspects, the SWIIN is a composite SWIIN, and each part of the composite SWIIN contains a perforated element having at least 50,000; 100000; 150000; 200,000, 250,000 holes. In some aspects, the retaining and permeable elements are made from polycarbonate, a cyclic olefin copolymer, or poly(methyl methacrylate); and in some aspects, the retaining and permeable members are 75 mm to 350 mm long, 50 mm to 250 mm wide, and 2 mm to 15 mm thick. In some aspects of the singulation assembly, the retaining and permeable elements are 150 mm to 250 mm long, 100 mm to 150 mm wide, and 4 mm to 8 mm thick. In some aspects of the singulation assembly, there are two permeate distribution channels and/or two retention channels, and in some aspects the retention channels and/or permeate distribution channels are approximately 150 mm long and 1 mm wide. In some aspects, the retaining and/or permeate fins are about 0.5 mm high and 80 mm long, and in some aspects the retainer and/or permeate flow guides are about 5 mm wide. In some aspects, the volume of the singulation node is from 15 ml to 100 ml.

[0012] Some embodiments of the present invention provide a SWIIN module comprising a singulation assembly and further comprising: a reservoir assembly comprising at least two reservoirs, wherein the first reservoir is 1) in fluid communication with at least one reservoir opening through which liquids and /or the cells enter from outside the SWIIN module into a first reservoir 2) in fluid communication with a reservoir/channel opening through which fluids and/or cells flow into one or more of the retaining member's openings; and 3) pneumatically connected to a pressure source; the second reservoir is 1) in fluid communication with at least one reservoir opening through which fluids enter from outside the SWIIN module into the second reservoir, 2) in fluid communication with a reservoir/channel opening through which fluids flow into one or more permeable element apertures; and 3) pneumatically connected to a pressure source; and a SWIIN cap. In some aspects of an embodiment of the SWIIN module, the SWIIN module further comprises two additional reservoirs, wherein the first and third reservoirs are 1) fluidly connected to at least two reservoir openings through which fluids and/or cells enter from outside the SWIIN module into the first and third reservoirs, 2) fluidly connected to the opening of the reservoir/channel through which fluids and/or cells flow into at least two openings of the retaining element; and 3) pneumatically connected to a pressure source; and the second and fourth reservoirs are 1) in fluid communication with at least two reservoir openings through which fluids enter the outside of the SWIIN module into the second and fourth reservoirs, 2) in fluid communication with the reservoir/channel opening through which fluids flow into at least two openings of the permeable element; and 3) pneumatically connected to a pressure source.

[0013] In some aspects of an embodiment of a SWIIN module, the SWIIN cover comprises a tank cover portion of the SWIIN cover, wherein the tank cover portion comprises 1) at least two tank access openings that provide access to the tank openings, and 2) at least two openings pneumatic access, which provide access to at least these two tanks and provide a pressure below and above atmospheric pressure in these at least two tanks. In some aspects, the SWIIN module is configured to follow the growth of a cell colony after editing is induced, and further comprises a means for selecting slow growing cell colonies. In some aspects of the SWIIN module, editing is caused by an inducible promoter, which is a temperature inducible promoter, and the temperature to induce transcription of the nuclease and/or target nucleic acid is provided in the SWIIN module using a Peltier device.

[0014] Other embodiments of the present invention provide an automated multi-module cell editing tool, comprising: a SWIIN module; a housing configured to receive all or some of the modules; a receiver configured to receive cells; one or more receivers configured to obtain nucleic acids; cultivation module; a transformation module configured to introduce nucleic acids into cells; and a processor configured to control the automated multi-module cell editing tool based on user input and/or selection of a pre-programmed script.

[0015] In some aspects of the automated multi-module cell editing tool, the transformation module comprises an electroporation flow device; and in some aspects, the automated multi-module cell editing tool further comprises a cell concentration module. In some aspects, the cell concentration module is a tangential flow filtration module. In some aspects, the fluid handling system moves fluids between modules. In some aspects, the multi-module automated cell processing system performs the processes of growing cells, concentrating and making cells electrocompetent, transforming cells with nucleic acid-directed nuclease editing components, singulating transformed cells, inducing editing in singulated cells, and growing and enriching cells, all without intervention. person.

[0016] In other embodiments, an automated multi-module cell editing tool is provided, comprising: a SWIIN module; a housing configured to receive all or some of the modules; a receiver configured to receive cells; one or more receivers configured to obtain nucleic acids; a transformation module configured to introduce nucleic acids into cells; cell concentration module; and a processor configured to control the automated multi-module cell editing tool based on user input and/or selection of a pre-programmed script. In some aspects of the automated multi-module cell processing tool, the fluid handling system moves reagents into and between modules as well as the SWIIN module. In some aspects, a stand-alone, integrated, multi-module automated cell processing system includes a growth module containing a rotating growth vial, a nucleic acid assembly module, or a reagent cartridge. In some aspects, the multi-module automated cell processing system performs the processes of growing cells, concentrating and making cells electrocompetent, transforming cells with nucleic acid-directed nuclease editing components, singulating transformed cells, inducing editing in singulated cells, and growing and enriching cells, all without intervention. person. Like other embodiments, this embodiment of an automated multi-module cell editing tool includes a fluid handling system to move fluids between modules.

[0017] In another further embodiment, a method is provided for enriching edited cells during nucleic acid-guided nuclease editing, comprising: transforming cells with one or more vectors containing an inducible promoter driving nuclease expression, an inducible promoter driving transcription of the target nucleic acid, and the sequence of the donor DNA; diluting the transformed cells to such a concentration as to substantially singulate the transformed cells on the substrate; growing cells on substrate for 2-200 doublings; initiating editing by inducing an inducible promoter(s) directing transcription of the guide nucleic acid and nuclease; growing induced cells in a colony; and selecting the induced cells from the substantially singulated colonies from a substrate in which the induced substantially singulated colonies are enriched in the edited cells.

[0018] In some aspects of this embodiment of the inducible transcription method, the target nucleic acid is the pL promoter and the inducible promoter driving nuclease expression is the pL promoter. In some aspects, the nuclease, guide nucleic acid, and donor DNA sequence are on the same vector. In other aspects, the nuclease is on the first vector and the guide nucleic acid and donor DNA sequence are on the second vector, and two transforming steps are required. In some aspects, the donor DNA sequence further comprises a PAM altering sequence, and in some aspects, each of the one or more vectors further comprises a gene for a selectable marker. In some aspects, the method further comprises adding selective agents to the substrate medium to select selectable marker(s) on one or more vectors. In some aspects, the substantially singulated selected colonies induced are small colonies, and in other aspects, the cells are grown into a colony of final size.

[0019] In one embodiment, the method also provides a method for enriching edited cells during nucleic acid-driven nuclease editing, comprising: transforming cells with one or more vectors containing a promoter driving nuclease expression, inducible promoter-driven transcription of the guide nucleic acid, and the sequence donor DNA; diluting the transformed cells to a concentration such that the transformed cells are substantially singulated on the first substrate; growing cells to form colonies on the first substrate; selecting cells from substantially singulated colonies from the first substrate and placing the selected cells on the second substrate; creating a duplicate of the second substrate, forming a third substrate; growing and inducing cells on the second substrate; growing and inducing cells on a third substrate under conditions that do not allow repair of the genome; comparison of cell growth on the second and third substrates; and selecting cells that grow on the second substrate but do not grow on the third substrate.

[0020] In some aspects of method embodiments, the promoter driving transcription of the guide nucleic acid is the pL promoter, and in some aspects, the promoter driving nuclease expression is an inducible promoter. In some aspects, the inducible promoter driving the expression of each of the target nucleic acid and nuclease is the same inducible promoter, and is the pL promoter. In some aspects, the donor DNA sequence further comprises a PAM altering sequence, and the method further comprises adding selective agents to the first substrate medium to select one or more vectors. In some aspects, the guide nucleic acid is a guide RNA, and in some aspects the cells are bacterial cells, and the motor vector further comprises a recombination system.

[0021] Another embodiment of the method provides a method for enriching edited cells during nucleic acid-guided nuclease editing, comprising: transforming cells with one or more vectors containing a promoter that controls nuclease expression, an inducible promoter that drives transcription of the target nucleic acid, and the sequence donor DNA; diluting the transformed cells to such a concentration as to substantially singulate the transformed cells on the substrate; growing cells on substrate for 2-200 doublings; initiating editing by inducing an inducible promoter and growing cells to form colonies; and selecting small colonies from substantially singulated colonies from a substrate in which the induced substantially singulated colonies are enriched in edited cells.

[0022] Another additional embodiment provides a method for enriching edited cells during nucleic acid-guided nuclease editing, comprising: transforming cells with one or more vectors containing a promoter that directs nuclease expression, an inducible promoter that directs transcription of the target nucleic acid, and the sequence donor DNA; diluting the transformed cells to a concentration such that the transformed cells are substantially singulated on the first substrate; growing cells on the first substrate for 2-200 doublings; initiating editing by inducing an inducible promoter; and growing cells to form colonies of the size limit. Some aspects of this embodiment further comprise a pooling of cut-off colonies, and some aspects of this embodiment further comprise selecting cut-off colonies.

[0023] These aspects and other features and advantages of the present invention are described in more detail below.

BRIEF DESCRIPTION OF THE DRAWINGS

[0024] FIG. 1A is a simplified flowchart of exemplary methods for enriching and selecting edited cells. Fig. 1B is a plot of absorbance versus time showing growth curves for edited cells (dotted line) and unedited cells (solid line). Fig. 1C depicts an exemplary inducible expression system for regulating gRNA and/or nuclease transcription.

[0025] FIG. 2A depicts a prior art, standard protocol for performing nucleic acid-guided nuclease editing of a genome. Fig. 2B-2F depict improved protocols using singulation or substantial singulation, induction, and normalization or selection (eg, selection) to identify edited cells in a population that have undergone nucleic acid-guided nuclease editing of the genome. Fig. 2B depicts a protocol for functional deconvolution of the editing process, either by placing the cells in 96-well plates containing various media or by placing the cells in a culture dish containing various media. Fig. 2C depicts a protocol for collecting colonies from a culture dish, positioning colonies on a 96-well plate, and then performing functional deconvolution. Fig. 2E shows the protocol for selection, and FIG. 2F depicts the protocol used to confirm that selection is extremely efficient for selecting edited cells.

[0026] FIG. 3A depicts a simplified block diagram for singulating, editing, and normalizing cells in a solid wall device. Fig. 3B depicts a simplified variation of the flowchart for substantial singulation, editing, and normalization of cells in a solid wall device. Fig. 3C is a photograph of one embodiment of a solid wall device. Fig. 3D-3F are photographs of E. coli cells heavily singulated (via a substantial Poisson distribution) and colony grown in microwells in a permeable-bottom solid wall device, at low, medium, and high magnification, respectively.

[0027] FIG. 4A-4E are photographs of the perforated element and the microwells therein. Fig. 4F-4R depict components of three exemplary embodiments of a singulation assembly comprising a retaining and permeable element, and a perforated element/filter/seal assembly. Fig. 4S-4Z depict a singulation, growth, edit induction and normalization or selection module assembly (eg, "solid wall isolation/induction/normalization module" or "SWIIN"). Fig. 4AA and 4BB depict SWIIN composite modules containing two (FIG. 4AA) or four (FIG. 4BB) singulation nodes. Fig. 4CC is an exemplary pneumatic architecture diagram for the SWIIN module described with reference to FIG. 4A-4Z.

[0028] FIG. 5A-5D depict a stand-alone, integrated, automated multi-module tool and its components, including a singulation module, with which edited cells can be produced and identified.

[0029] FIG. 6A depicts one embodiment of a rotating growth flask for use with the cell culture module described herein. Fig. 6B illustrates a perspective view of one embodiment of a rotating culture device in a cell culture module housing. Fig. 6C is a sectional view of the cell culture module shown in FIG. 6b. Fig. 6D illustrates the cell culture module shown in FIG. 6B connected to LED, detector and temperature control components.

[0030] FIG. 7A is a model of the tangential flow filtration (TFF) process used in the TFF module presented herein. Fig. 7B is a plan view of the bottom element of one embodiment of an exemplary TFF device/module. Fig. 7C is an exploded view of the top and bottom members and membrane of an exemplary TFF module. Fig. 7D is an upside down exploded view of the top and bottom members and membrane of an exemplary TFF module. Fig. 7E-7H depict various views of one embodiment of a TFF module having fluidly coupled retentate, filtrate, and exchange buffer reservoirs.

[0031] FIG. 8A and 8B are top and bottom perspective views, respectively, of flow-through electroporation devices (here, six such devices connected together). Fig. 8C is a plan view of one embodiment of an exemplary electroporation flow apparatus. Fig. 8D is a top cross-sectional view of the electroporation device shown in FIG. 8C. Fig. 8E is a cross-sectional side view of the bottom of the electroporation devices shown in FIG. 8C and 8D.

[0032] FIG. 9 is a simplified block diagram of one embodiment of an exemplary automated multi-module cell processing tool comprising a singulation or substantial singulation/growing/editing and normalization or selection module ("solid wall isolation/induction/normalization module" or "SWIIN").

[0033] FIG. 10 is a simplified block diagram of one alternative embodiment of an exemplary automated multi-module cell processing instrument comprising a singulation or substantial singulation/growing/editing and normalization or selection module ("solid wall isolation/induction/normalization module" or "SWIIN") .

[0034] FIG. 11A is a map of an exemplary motor vector that may be used in the methods described herein; and Fig. 11B is a map of an exemplary editing vector (with editing cassette) that can be used in the methods described herein.

[0035] FIG. 12 is a bar graph showing the transformation efficiency observed for galK gRNA targeting cassettes under various promoters.

[0036] FIG. 13 is a plot of cell growth versus time demonstrating that heat induction editing does not affect cell growth or viability.

[0037] FIG. 14 depicts results demonstrating that gRNA-repressed cassettes give high cell viability/transformation efficiency for three exemplary nucleases.

[0038] FIG. 15 illustrates heat maps and growth curves showing OD at 6 hours for uninduced and induced cell populations.

[0039] FIG. 16 shows the results of cell colony normalization for E. coli cells under various conditions.

[0040] FIG. 17A is a photograph of a permeable-bottom solid wall device on agar-agar on which yeast cells were essentially singulated and grown into clonal colonies. Fig. 17B presents photographs of yeast colony growth at various time points.

DETAILED DESCRIPTION

[0041] All functionality described in connection with one embodiment is intended to be applicable to additional embodiments described herein, except where explicitly stated or where a feature or function is incompatible with those additional embodiments. For example, when a given feature or function is explicitly described in connection with one embodiment, but not explicitly mentioned in connection with an alternative embodiment, it should be understood that that feature or function may be deployed, used, or implemented in connection with an alternative embodiment, for unless that feature or function is incompatible with an alternate embodiment.

[0042] In practice, the techniques described herein may use, unless otherwise indicated, conventional methods and descriptions of organic chemistry, polymer technology, molecular biology (including recombinant techniques), cell biology, biochemistry, and sequencing technology that are within the skill of those skilled in the art. the field of technology. Such conventional techniques include template polymer synthesis, hybridization and ligation of polynucleotides, and detection of hybridization using a label. Specific illustrations of suitable techniques can be found in the examples provided herein. However, of course, other equivalent conventional procedures can also be used. Such conventional techniques and descriptions can be found in standard laboratory manuals such as Green, et al., eds., Genome Analysis: A Laboratory Manual Series (Vols. I-IV) (1999); Weiner, Gabriel, Stephens, eds., Genetic Variation: A Laboratory Manual (2007); Dieffenbach, Dveksler, eds., PCR Primer: A Laboratory Manual (2003); Bowtell and Sambrook, DNA Microarrays: A Molecular Cloning Manual (2003); Mount, Bioinformatics: Sequence and Genome Analysis (2004); Sambrook and Russell, Condensed Protocols from Molecular Cloning: A Laboratory Manual (2006); ^Stryer , Biochemistry (4th Ed.) W.H. Freeman, New York NY (1995); Gait, " Oligonucleotide Synthesis: A Practical Approach" (1984), IRL Press, London; Nelson and Cox, Lehninger, Principles of Biochemistry ^3rd Ed., WH Freeman Pub., New York, NY (2000); Berg et al., Biochemistry, 5th ^Ed ., WH Freeman Pub., New York, NY (2002); Doyle & Griffiths, eds., Cell and Tissue Culture: Laboratory Procedures in Biotechnology , Doyle & Griffiths, eds., John Wiley & Sons (1998); G. Hadlaczky, ed. Mammalian Chromosome Engineering - Methods and Protocols, Humana Press (2011); and Lanza and Klimanskaya, eds., Essential Stem Cell Methods , Academic Press (2011), all of which are incorporated herein by reference in their entirety for all purposes. CRISPR-specific techniques can be found, for example, in Appasani and Church, Genome Editing and Engineering From TALENs and CRISPRs to Molecular Surgery (2018); and Lindgren and Charpentier, CRISPR: Methods and Protocols (2015); both of which are incorporated herein by reference in their entirety for all purposes.

[0043] It should be noted that as used herein and in the appended claims, the grammatical forms of the singular also include the plural, unless the context clearly indicates otherwise. Thus, for example, reference to "cell" refers to one or more cells, and reference to "system" includes reference to equivalent steps, methods, and devices known to one of skill in the art, and so on. In addition, it should be understood that terms such as "left", "right", "top", "bottom", "front", "back", "side", "height", "length", "width" , "upper", "lower", "interior", "exterior", "internal", and/or "external" as may be used herein merely describe orientations and do not necessarily limit embodiments of the present invention to any particular orientations. or configurations. In addition, terms such as "first", "second", "third", etc. simply identify one of the many parts, components, steps, operations, functions, and/or landmarks disclosed herein, and likewise do not necessarily limit the embodiments of the present invention to any particular orientations or configurations.

[0044] In addition, the terms "about", "approximately", "not significant", and similar terms generally refer to ranges that, in some embodiments, include an identified value within 20%, 10%, or preferably 5%, as well as any value in between.

[0045] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which the present invention belongs. All publications mentioned herein are incorporated herein by reference for the purpose of describing and disclosing devices, methods, and cell populations that may be used in connection with the present invention.

[0046] Where a range of values is specified, it is understood that every intermediate value between the upper and lower limits of this range, as well as any other set or intermediate value in this specified range, is covered by the present invention. The upper and lower limits of these smaller ranges may be independently included in the smaller ranges, and are also covered by the present invention, subject to any specifically excluded limit within that stated range. When a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the present invention.

[0047] In the following description, numerous specific details are set forth in order to provide a more complete understanding of the present invention. However, those skilled in the art will appreciate that the present invention may be practiced without one or more of these specific details. In other cases, features and procedures well known to those skilled in the art are not described so as not to obscure the present invention.

[0048] As used herein, the term "complementary" refers to the Watson-Crick base pairing between nucleotides, and specifically refers to nucleotides that are linked to each other by hydrogen bonds: by thymine or uracil residues and by adenine residues by two hydrogen bonds, and with the help of cytosine and guanine residues by three hydrogen bonds. Typically, a nucleic acid includes a nucleotide sequence described as having "percent complementarity" or "percent homology" with said second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity with said second nucleotide sequence, meaning that 8 out of 10, 9 out of 10, or 10 out of 10 nucleotides of the sequence are complementary with said second nucleotide sequence. For example, the 3'-TCGA-5' nucleotide sequence is 100% complementary to the 5'-AGCT-3' nucleotide sequence; and the 3'-TCGA-5' nucleotide sequence is 100% complementary to the 5'-TTAGCTGG-3' nucleotide sequence region.

[0049] The term “control sequences” of DNA collectively refers 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 replication, transcription and translation of the coding sequence in the recipient cell. Not all of these types of control sequences need to be present, provided that the selected coding sequence is capable of replication, transcribing and, for some components, being translated in the appropriate host cell.

[0050] As used herein, the term "donor DNA" or "donor nucleic acid" refers to a nucleic acid that is designed to introduce a DNA sequence modification (insert, deletion, substitution) into a locus via homologous recombination using nucleic acid-directed nucleases. For homology-directed reparative synthesis, the donor DNA must have sufficient homology to the regions located on the sides of the "cut" or edited region in the genomic target sequence. The length of the homologous arm(s) will depend, for example, on the type and size of the modification being performed. For example, the donor DNA will have at least one homologous sequence region (eg, one homologous arm) for the target genomic locus. In many cases, and preferably, the donor DNA will have two homologous sequence regions (eg, two homologous arms) for the target genomic locus. Preferably, an "insert" region or a "DNA sequence modification" region - a nucleic acid modification that is desired to be introduced into a target genomic locus in a cell - will lie between the two regions of homology. DNA sequence modification can change one or more bases of the target genomic DNA sequence at one particular site or at several specific sites. The change may include a change in 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more nitrogenous base pairs target sequence. The deletion or insertion may be a deletion or insertion 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. The donor DNA optionally further includes a change in the target sequence, such as a PAM mutation, which prevents the nuclease from binding to the PAM or spacer in the target sequence after the edit has taken place.

[0051] As used herein, the term "enrichment" refers to the enrichment of edited cells by singulating or substantially singulating cells, initially growing cells in a cell colony, inducing editing, and growing cell colonies in a colony size limit (e.g., saturating or normalizing colony growth) . As used herein, the term "selection" or "selection of edited cells" refers to the process of using a combination of singulation or significant singulation, initially growing cells in a colony, inducing editing, and then using cell growth - as measured by colony size, concentration of metabolites or waste products, or other characteristics that correlate with cell growth rate - to select cells that have been edited based on growth retardation caused by editing.

[0052] The terms "targeting nucleic acid" or "guide RNA" or "gRNA" refer to a polynucleotide containing 1) a targeting sequence capable of hybridizing to a target genomic locus, and 2) a backbone sequence capable of interacting with or complexing with the targeting nucleic acid nuclease.

[0053] The terms "homology" or "identity" or "similarity" refers to the sequence similarity between two peptides or, more commonly in the context of the present invention, between two nucleic acid molecules. The term "homologous region" or "homologous arm" refers to a region on the donor DNA that has a certain degree of homology with the target genomic DNA sequence. Homology can be determined by comparing the position in each sequence, which can be aligned for comparison purposes. When a position in the compared sequence is occupied by the same base or amino acid, then the molecules are homologous at that position. The degree of homology between sequences is a function of the number of corresponding or homologous positions shared by those sequences.

[0054] The term "operably linked" refers to such an arrangement of elements in which the described components are configured to perform their normal function. Thus, control sequences operably linked to a coding sequence are capable of effecting transcription, and in some cases translation, of the coding sequence. Control sequences need not be adjacent to a coding sequence if they are capable of directing expression of the coding sequence. Thus, for example, intermediate untranslated but transcribed sequences may be present between the promoter sequence and the coding sequence, and the promoter sequence may still be considered "operably linked" to the coding sequence. In fact, such sequences do not have to be on the same contiguous DNA molecule (ie, chromosome) and may still have interactions leading to altered regulation.

[0055] A "promoter" or "promoter sequence" is a DNA regulatory region capable of binding an 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 RNA transcribed by any class of any RNA polymerase I, II or III. Promoters may be constitutive or inducible. In the methods described herein, the promoters that direct the transcription of gRNA are inducible.

[0056] As used herein, the term "selectable marker" refers to a gene introduced into a cell that confers a trait suitable for artificial selection. General purpose selectable markers are well known to those skilled in the art. Selectable drug markers such as ampicillin/carbenicillin, kanamycin, chloramphenicol, erythromycin, tetracycline, gentamicin, bleomycin, streptomycin, puromycin, hygromycin, blasticidin and G418 can be used. In other embodiments, selectable markers include, but are not limited to, human nerve growth factor receptor (detectable by MAb, for example, as described in US Pat. No. 6,365,373); truncated human growth factor receptor (detected by MAb); mutant human dihydrofolate reductase (DHFR; MTX fluorescent substrate available); secreted alkaline phosphatase (SEAP; fluorescent substrate available); human thymidylate synthase (TS; confers resistance to the anticancer agent fluorodeoxyuridine); human glutathione S-transferase alpha (GSTA1; conjugates glutathione to the selective stem cell alkylating agent busulfan; chemoprotective selectable marker in CD34+ cells); surface antigen CD24 in hematopoietic stem cells; rhamnose; the human CAD gene conferring resistance to N-phosphonacetyl-L-aspartate (PALA); human multi-resistance-1 (MDR-1; P-glycoprotein surface protein selected for increased drug resistance or enriched in FACS); human CD25 (IL-2α; detectable by MAb-FITC); methylguanine DNA methyltransferase (MGMT; selected by carmustine); and cytidine deaminase (CD; selected by Ara-C). As used herein, the term "selective medium" refers to a cell growth medium to which a chemical compound or biological moiety has been added that selects for or against selectable markers.

[0057] As used herein, the terms "singulate" or "singulate" mean the separation of individual cells so that each cell (as well as the colonies formed from each cell) is separate from other cells; for example, a single cell in a single microwell, or 100 single cells, each in its own microwell. "Singulation" or "singulated cells" in one embodiment results from a Poisson distribution in ordered cells. The terms "substantially singulated", "substantially singulated" and "substantially singulated" mean that the cells are largely separated from each other in small groups or batches. Namely, when 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 are placed in the microwell , 24, 25, 26, 27, 28, 29, 30 or up to 50, but preferably 10 or less cells. "Substantially singulated" or "substantially singulated" cells in one embodiment result from a "substantial Poisson distribution" in ordered cells. With more complex libraries of edits - or with libraries that may contain lethal edits or edits with widely varying fitness effects - it is preferable that the cells singulate through a Poisson distribution.

[0058] The terms "target genomic DNA sequence", "target sequence", or "genomic target locus" refers to any locus in vitro or in vivo , or in the nucleic acid (e.g., genome) of a cell or population of cells, in which a change is desired at least one nucleotide using a nucleic acid-directed nuclease editing system. The target sequence may be a genomic locus or an extrachromosomal locus.

[0059] A "vector" is any of a variety of nucleic acids that contain the desired sequence or sequences to be delivered and/or expressed in a cell. Vectors usually consist of DNA, although RNA vectors are also available. Vectors include, but are not limited to, plasmids, fosmids, phagemids, viral genomes, YACs, BACs, mammalian synthetic chromosomes, and the like. As used herein, the phrase "engine vector" contains a coding sequence for a nuclease (optionally driven by an inducible promoter) for use in systems and methods based on the nucleic acid-directed nuclease of the present invention. The motor vector may also contain, in the bacterial system, the λ Red recombination system or its equivalent, as well as a selectable marker. As used herein, an "editing vector" contains a donor nucleic acid comprising a change in the target sequence that prevents the nuclease from binding to a PAM or spacer in the target sequence after editing, as well as a coding sequence for gRNA under the control of an inducible promoter. The editing vector may also contain a selectable marker and/or a barcode. In some embodiments, the motor vector and the edit vector may be combined; that is, the content of the engine vector can be found on the edit vector.

Inducible editing in genomic systems based on nucleic acid-directed nuclease in general

[0060] The present invention provides nucleic acid-directed nuclease-based genome editing tools, modules, and methods that provide 1) improved observed editing efficiency of nucleic acid-directed nuclease-based editing methods, and 2) improved screening and detection of cells whose genomes have been properly edited to include high throughput screening methods. In current protocols using nuclease systems, constitutively expressed nuclease components are commonly used to perform highly efficient editing. However, in pooled or multiplexed formats, constitutive expression of editing components can result in selective enrichment of cells that are not being edited due to the absence of DNA double strand breaks that occur during editing. In addition, constitutively expressed components of the nucleic acid-targeted nuclease (eg, nuclease and target nucleic acid) immediately edit freshly transformed physiologically fragile cells, resulting in reduced cell viability. This document provides tools for tightly regulated expression of components of a nucleic acid-guided nuclease editing system (including tight regulation of the transcription of the guide nucleic acid and optionally and preferably a nuclease (or a nuclease and optionally and preferably a guide nucleic acid)), allowing separation transformation and editing processes. In addition, the methods described herein take advantage of singulation (separating cells and growing them into clonal colonies) and either normalizing cell colonies or selecting slow growing colonies. Singulation or substantial singulation, initial growth followed by edit induction and normalization overcomes growth bias of unedited cells, and replacement selection for normalization allows direct selection of edited cells. These tools, modules, and methods can be applied to all cell types, including protozoan, prokaryotic, and eukaryotic (eg, yeast, fungi, plant, and animal) cells.

[0061] The tools, modules, and methods described herein use editing cassettes containing a guide RNA (gRNA) sequence covalently linked to a donor DNA sequence, where the gRNA is under the control of an inducible promoter (e.g., the editing cassettes are CREATE cassettes; see U.S. Patent Nos. 9/982278 issued May 29, 2019 and 10/240167 issued March 26, 2019; 10/266849 issued April 23, 2019; and U.S. Patent Applications Nos. 15/948785 filed April 09, 2018; 16/275439 filed February 14, 2019; and 16/275465 filed February 14, 2019, all of which are incorporated herein by reference in their entirety). These editing cassettes provide increased transformation efficiency and control over the timing and duration of the editing process. The disclosed methods allow cells to be transformed, essentially singulated and grown for several doublings, after which editing is induced in the cells. This combination of process effectively negates the impact of unedited cells on the cell population. The combination of significant cell singulation, then initial growth followed by induction of gRNA (and/or nuclease) transcription and cell colony normalization or cell selection results in 2-250x, 10-225x, 25-200x, 40-175x, 50-150x, 60- 100x, or 50-100x increased identification of edited cells compared to prior art methods and allows the generation of grouped or pooled edited cells containing genome-edited cell libraries. In addition, these methods can be used to create iterative editing systems to create combinatorial libraries of cells with two or more changes in each cell genome. Inducible gRNA constructs (and/or inducible nuclease constructs) and methods for using “pulsed” action on cells of active editing components 1) allow cells to be grouped (for example, heavily singulated) before editing is initiated, 2) reduce off-target activity, 3) provide identify rare cell changes, and 4) enrich edited cells or allow high-throughput screening applications to identify edit activity using cell growth as an edit indicator, such as by measuring optical density, colony size or metabolic by-products or other characteristics, thereby enriching the edited population cells.

[0062] The tools, compositions, and methods described herein enhance CRISPR editing systems in which nucleic acid-targeted nucleases (eg, RNA-targeted nucleases) are used to edit specific target regions in an organism's genome. A nucleic acid-targeted nuclease complexed with an appropriate synthetic target nucleic acid in a cell can cut the cell's genome at the desired position. The targeting nucleic acid helps the nucleic acid-targeted nuclease to recognize and cut DNA at a specific target sequence. By manipulating the nucleotide sequence of the targeting nucleic acid, the targeting nucleic acid nuclease can be programmed to target any DNA sequence for cleavage, as long as the appropriate neighboring protospacer motif (PAM) is nearby. In some aspects, a nucleic acid-targeted nuclease editing system may use two separate target nucleic acid molecules that combine to function as a target nucleic acid, such as CRISPR RNA (crRNA) and CRISPR transactivating RNA (tracrRNA). In other aspects, the guide nucleic acid may be a single guide nucleic acid that includes both crRNA and tracrRNA sequences, or a single guide nucleic acid that does not require tracrRNA. In the compositions and methods described herein, if two separate RNA molecules are combined to function as a guide nucleic acid, at least one of the components of the guide nucleic acid is under the control of an inducible promoter. If a single guide nucleic acid is used, that single guide nucleic acid is under the control of an inducible promoter.

[0063] Typically, a target nucleic acid (eg, gRNA) is complexed with a compatible target nucleic acid nuclease and can then hybridize to a target sequence, thereby directing the nuclease to the target sequence. The target nucleic acid may be DNA or RNA; alternatively, the target nucleic acid may comprise both DNA and RNA. In some embodiments, the guide nucleic acid may contain modified or non-naturally occurring nucleotides. Where the target nucleic acid contains RNA, the gRNA is encoded by a DNA sequence on a polynucleotide molecule, such as a plasmid, a linear construct, or is within an editing cassette and is preferably under the control of an inducible promoter.

[0064] The targeting nucleic acid comprises a targeting sequence, which is a polynucleotide sequence having sufficient complementarity with the target sequence for hybridization with the target sequence and direct sequential binding of the complexed nuclease directed by the nucleic acid to the target sequence. The degree of complementarity between the guide sequence and the corresponding target sequence, when optimally aligned using a suitable alignment algorithm, is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more. The optimal alignment can be determined using any suitable sequence alignment algorithm. In some embodiments, the targeting sequence (the portion of the targeting nucleic acid that hybridizes to the target sequence) is about or greater 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, the 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 in length, or 15, 16, 17, 18, 19 or 20 nucleotides in length.

[0065] In the present methods and compositions, the guide nucleic acid is provided as a sequence expressed from a plasmid or vector and contains both the guide sequence and the backbone sequence as a single transcript under the control of an inducible promoter. Alternatively, guide nucleic acids can be transcribed from two separate sequences, at least one of which is under the control of an inducible promoter. A targeting nucleic acid can be designed to target a desired target DNA sequence by changing the targeting sequence so that the targeting sequence is complementary to the target DNA sequence, thereby allowing hybridization between the targeting sequence and the target DNA sequence. In most cases, in order to make an edit in a target DNA sequence, the gRNA/nuclease complex binds to the 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 (DNA or RNA), endogenous or exogenous to a prokaryotic or eukaryotic cell, or in vitro . For example, the target sequence may be a polynucleotide found in the nucleus of a eukaryotic cell. The target sequence can be a gene product coding sequence (eg a protein) and/or a non-coding sequence (eg a regulatory polynucleotide, intron, PAM or junk DNA).

[0066] The target nucleic acid may be part of an editing cassette that encodes a donor nucleic acid; that is, the editing cassette may be a CREATE cassette (see, for example, U.S. Patent Nos. 9/982278, issued May 29, 2019, and 10/240167, issued March 26, 2019; 10/266849, issued April 23, 2019 15/948785, filed April 09, 2018; 16/275439, filed February 14, 2019; and 16/275465, filed February 14, 2019, all of which are incorporated herein. document by reference in their entirety). The target nucleic acid and the donor nucleic acid can be, and usually are, under the control of a single (in this case preferably an inducible) promoter. Again, it should be noted that either the target nucleic acid or the nuclease must be under the control of an inducible promoter, and in some embodiments and preferably both the target nucleic acid and the nuclease are under the control of an inducible promoter. In fact, it is important that the inducible promoter(s) driving the transcription of the target nucleic acid and/or (most preferably) the nuclease be tightly controlled. In particular, it is important that nuclease activity be controlled so as to avoid significant bias and loss of efficiency prior to singulation and growth of singulated cells. Thus, the extent to which the editing apparatus is able to reach the off status is important regardless of the process of singulation or significant singulation. Alternatively, the targeting nucleic acid may not be part of the editing cassette, and may instead be encoded on the motor or editing vector strand. For example, the sequence encoding for a guide nucleic acid may be first assembled or inserted into a vector strand, followed by insertion of a donor nucleic acid. In other cases, the donor nucleic acid may be first inserted or assembled into the vector strand, followed by insertion of the coding sequence for the target nucleic acid. In other cases, the encoding of the guide nucleic acid sequence and the donor nucleic acid (inserted, for example, in an editing cassette) simultaneously, but separately, are inserted or assembled into a vector. In other embodiments (and preferably), encoding the guide nucleic acid sequence and encoding the donor nucleic acid sequence are both included in the editing cassette.

[0067] The target sequence binds to PAM, which is a short nucleotide sequence recognized by the gRNA/nuclease complex. The exact PAM sequence and length requirements for different nucleic acid-targeted nucleases vary; however, PAMs are typically 2-7 base pair sequences adjacent or close to the target sequence and, depending on the nuclease, may be 5' or 3' to the target sequence. The development of a PAM-interacting domain of a nucleic acid-targeted nuclease may allow the specificity of PAM to be modified, improve target site recognition accuracy, decrease target site recognition accuracy, and increase the versatility of the nucleic acid-targeted nuclease. In some embodiments, editing the genome of a target sequence both introduces a desired DNA change into a target sequence, such as the genomic DNA of a cell, and removes, alters, or renders inactive a proto-spacer (PAM) region in the target sequence; that is, the donor DNA often includes a change in the target sequence that prevents the nuclease from binding to the PAM in the target sequence after the edit has taken place. Deactivation of a PAM at a target sequence prevents further editing of the cellular genome at that target sequence, for example, by subsequent exposure to a nucleic acid-targeted nuclease complexed with a synthetic target nucleic acid in later editing rounds. Thus, cells having the desired target sequence change and PAM alteration can be selected using a nucleic acid-targeted nuclease complexed with a synthetic target nucleic acid complementary to the target sequence. Cells that have not undergone the first edit will be cut, resulting in double-stranded DNA breaks, and thus no longer viable. Cells containing the desired target sequence change and the modified PAM will not be cut as these edited cells no longer contain the required PAM region and will continue to grow and spread.

[0068] The range of target sequences that can recognize nucleic acid-directed nucleases is limited by the need to locate a particular PAM near the desired target sequence. As a result, it can often be difficult to target changes with the precision required for genome editing. It has been found that nucleases can recognize some PAMs very well (eg canonical PAMs) and other PAMs less well or poorly (eg non-canonical PAMs). Since the methods disclosed herein allow the identification of edited cells against a large background of unedited cells, these methods allow the identification of edited cells in cases where PAM is suboptimal; that is, the methods for identifying edited cells herein allow the identification of edited cells even if the editing efficiency is very low. In addition, the present methods expand the scope of target sequences that can be edited as changes are more easily identified, including cells in which genome changes are associated with less functional PAMs.

[0069] With regard to the nuclease component of a nucleic acid-directed nuclease editing system, a polynucleotide sequence encoding a nucleic acid-directed nuclease can be codon optimized for expression in specific cells, such as protozoan, prokaryotic, or eukaryotic cells. Eukaryotic cells can be yeast, fungal, algae, plant, animal, or human cells. Eukaryotic cells can be cells or derived from a particular organism, such as a mammal, including, but not limited to, a human, a mouse, a rat, a rabbit, a dog, or a non-human mammal, including non-human primates. The selection of the nucleic acid-targeted nuclease to use depends on many factors such as the type of editing to be done on the target sequence and whether a suitable PAM is close to the desired target sequence. Nucleases used in the methods described herein include, but are not limited to, Cas 9, Cas 12/CpfI, MAD2 or MAD7, or other MADs. As with a guide nucleic acid, the nuclease can be encoded by a DNA sequence on a vector (eg, motor vector) and be driven by a constitutive or, preferably, an inducible promoter. Again, at least one, and preferably both, of the nuclease and the guide nucleic acid are under the control of an inducible promoter. In some embodiments, the nuclease coding sequence is under the control of an inducible promoter, and the inducible promoter may be separate from but the same as the inducible promoter driving transcription of the guide nucleic acid; that is, a single inducible promoter directs transcription of the nuclease and guide nucleic acid sequences, but the two inducible promoters may be of the same type (eg, both may be pL promoters). Alternatively, the inducible promoter driving nuclease expression may be different from the inducible promoter driving transcription of the guide nucleic acid; that is, for example, the nuclease may be driven by the inducible pBAD promoter and the target nucleic acid may be driven by the inducible pL promoter. Again, it should be noted that it is important that the inducible promoter(s) driving the transcription of the guide nucleic acid and nuclease be tightly controlled. In particular, it is important that nuclease activity be controlled so as to avoid significant bias and loss of efficiency prior to singulation and the process of growing singulated cells. Thus, the extent to which the editing apparatus is able to reach the off status is important regardless of the process of singulation or significant singulation.

[0070] Another component of the nucleic acid-directed nuclease system is a donor nucleic acid. In some embodiments, the donor nucleic acid is on the same polynucleotide (eg, vector or editing cassette (CREATE)) as the target nucleic acid. The donor nucleic acid is intended to be used as a template for homologous recombination with a target sequence cut or cleaved by a nucleic acid directed nuclease as part of a gRNA/nuclease complex. The donor nucleic acid polynucleotide may be any suitable length, such as about or greater than about 30, 35, 40, 45, 50, 75, 100, 150, 200, 500, 1000, 2500, 5000 nucleotides or more. In some preferred aspects, the donor nucleic acid may be provided as an oligonucleotide of 40-300 nucleotides, more preferably 50-250 nucleotides. The donor nucleic acid contains a region that is complementary to some portion of the target sequence (eg, a homologous arm). In optimal alignment, the donor nucleic acid overlaps with (becomes complementary to) the target sequence, for example, by about 10, 15, 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more nucleotides. In many embodiments, the donor nucleic acid contains two homologous arms (regions complementary to the target sequence) flanked by a mutation or difference between the donor nucleic acid and the target template. The donor nucleic acid contains at least one mutation or change from the target sequence, such as an insertion, deletion, modification, or any combination thereof, from the target sequence.

[0071] Often, the donor nucleic acid is provided as an editing cassette that is inserted into a vector strand, which may contain a promoter that directs the transcription of the gRNA and a coding sequence for the gRNA, or the vector strand may contain a promoter that directs the transcription of the gRNA, but not the gRNA itself. In addition, there may be more than one, for example, two, three, four or more target nucleic acid/donor nucleic acid cassettes inserted into the motor vector, where the target nucleic acids are under the control of separate different promoters, separate identical promoters, or where all guide nucleic acid/donor nucleic acid pairs are under the control of a single promoter. (See, for example, US Patent Document No. 16/275465, filed February 14, 2019, on multiple CREATE cassettes). A promoter driving the transcription of a gRNA and a donor nucleic acid (or driving more than one gRNA/donor nucleic acid pair) is preferably an inducible promoter, and a promoter driving the transcription of a nuclease is optionally also an inducible promoter. Thus, at least one - and preferably both - of the guide nucleic acid (gRNA) and the nuclease is under the control of an inducible promoter.

[0072] Inducible editing is advantageous in that significantly or heavily singulated cells can be grown in a few or many cell doublings before editing is initiated, which increases the likelihood that cells with changes will survive as double helix cuts caused by active editing, are largely toxic to cells. This toxicity results in both necrosis of cells in the edited colonies and growth retardation of the edited cells, which survive but need to be repaired and regenerated after editing. However, once the edited cells have a chance to recover, the colony size of the edited cells will eventually equal the colony size of the unedited cells (e.g., the process of "normalizing" or growing colonies to "final size"; see e.g. Fig. 1B described below) .

[0073] In addition to the donor nucleic acid, the editing cassette may contain one or more primer regions. Primer regions can be used to enhance the editing cassette by using oligonucleotide primers; for example, if the primer sites are located on the sides of one or more other components of the editing cassette.

[0074] In addition, as described above, the donor nucleic acid may contain - in addition to at least one mutation relative to the target sequence - one or more PAM sequence changes that alter, delete, or render inactive a PAM site in the target sequence. Changing the PAM sequence in the target sequence renders the PAM region "resistant" to the nucleic acid-directed nuclease, and prevents the target sequence from being further edited in subsequent editing cycles if the same nuclease is used.

[0075] The editing cassette may also contain a bar code. The barcode is a unique DNA sequence that matches that of the donor DNA, so that the barcode can identify a change made to the corresponding target sequence. A barcode may contain more than four nucleotides. In some embodiments, the editing cassettes comprise a set of donor nucleic acids representing, for example, gene or genomic libraries of donor nucleic acids. The editing cassette library is cloned into a vector strand where, for example, each different donor nucleic acid design is associated with a different barcode, or alternatively, each different cassette molecule is associated with a different barcode.

[0076] In addition, in some embodiments, the expression vector or coding components of a nucleic acid-targeted nuclease system cassette further encode a nucleic acid-targeted nuclease comprising one or more nuclear localization sequences (NLS), e.g., about or greater than 1, 2 , 3, 4, 5, 6, 7, 8, 9, 10 or more NLS. In some embodiments, the engineered nuclease contains NLS at or near the amino terminus, NLS at or near the carboxy terminus, or a combination.

Sample Workflows for Editing, Enriching, and Selecting Edited Cells

[0077] The methods described herein provide improved observed editing performance of nucleic acid-directed nuclease-based editing methods as a result of a combination of singulation or substantial singulation, initial cell growth, editing induction and normalization of resulting cell colonies, or selection of slow growing cell colonies. The combination of singulation or substantial singulation processes, initial cell growth, editing induction and normalization overcomes the growth bias in favor of unedited cells, as well as editing suitability effects (including different editing speeds), thus giving all cells an equal opportunity. The combination of singulation or essential singulation, primary cell growth, editing induction and selection provides direct selection of edited cell colonies. The result of the methods described herein is that even in nucleic acid-directed nuclease-based systems where editing is not optimal (eg, in systems where non-canonical PAMs are targeted), there is an increase in observed editing efficiency; that is, edited cells can be identified even against a large background of unedited cells. The observed editing efficiency can be improved up to 80% or more. Singulation, initial growth, editing induction and cell colony normalization or cell colony selection results in 2-250x, 10-225x, 25-200x, 40-175x, 50-150x, 60-100x, or 5-100x enhancement in the identification of edited cells compared to with prior art methods and allows the generation of clustered or pooled edited cells containing genome libraries. In addition, these tools, modules, and methods can be used to create iterative editing systems to create combinatorial libraries, identify rare cell changes, and enable high-throughput enrichment applications to identify editing activity.

[0078] FIG. 1A shows simplified flowcharts for two alternative exemplary cell singulation methods 100a and 100b for enrichment (100a) and selection (100b). On FIG. 1A, method 100 begins by transforming cells 110 with the components needed to perform editing using a nucleic acid-directed nuclease. For example, cells can be transformed simultaneously with a separate engine vector and edit vector; the cells may have already been transformed with a nuclease-expressing motor vector (e.g., the cells may have already been transformed with a motor 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 cells; or the cells may be transformed with a single vector containing all components required to perform genome editing using a nucleic acid-directed nuclease.

[0079] Various delivery systems can be used to introduce (eg, transform or transfect) components of a nucleic acid-directed nuclease editing system into host cell 110. 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 exosomes. Alternatively, molecular Trojan liposomes can be used to deliver nucleic acid-targeted nuclease components across the blood-brain barrier. Of interest, especially in the context of an automated multi-module tool for cell editing, is the use of electroporation, in particular flow electroporation (as a stand-alone tool or as a module in an automated multi-module system), described, for example, in US patent documents No. 16/147120, filed September 28, 2018 G.; No. 16/147353 filed September 28, 2018; No. 16/147865 filed September 30, 2018; and No. 16/147871, filed September 30, 2018. If the solid wall singulation or essential singulation/growth/induction editing and normalization module is one module in the automated multi-module cell editing tool described herein below, the cells can be transformed into automated cell transformation module.

[0080] After cells are transformed with the components necessary to perform nucleic acid-directed nuclease editing, the cells are substantially or substantially singulated 120; that is, the cells are diluted (if necessary) in the liquid culture medium so that the cells, upon delivery to the singulation substrate, are substantially separated from each other and can form colonies substantially separated from each other. For example, if a solid wall device (described below with reference to Figures 3A-3E and 4A-4CC) is used, the cells are diluted such that, upon delivery to the solid wall device, they fill the microwells of the solid wall device according to the Poisson or essentially a Poisson distribution. In one example (illustrated in Fig. 3A), singulation is achieved when, on average, half of the cells are delivered to each microwell; that is, when some microwells contain one cell and other microwells do not contain cells. Alternatively, a substantially Poisson cell distribution occurs when two or more (2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30 or up to 50, but preferably 10 or less, or preferably 5 or less) cells are delivered per microwell (illustrated in Fig. 3B).

[0081] After the cells are substantially or substantially singulated 120, they are allowed to grow, for example, up to 2-130, or up to 5-120, or up to 10-100 doublings, forming clonal colonies 140. Thereafter once colonies have formed, editing is induced, eg, when one or more inducible promoters drive transcription of one or both of the gRNA and nuclease (preferably both), as well as, for example, components of the λ red recombinant system in bacterial systems. For an exemplary inducible system, see FIG. 1C, detailed below. After editing is complete in enrichment method 100a, cells are grown into a final colony size of 150; that is, colonies arising from substantially or substantially singulated cells are grown in colonies until cell growth peaks and normalizes or saturates for both edited and unedited cells. Normalization occurs when the nutrients in the environment around the growing cell colony are depleted and/or the growing cells fill the microwells and further growth is physically restricted. Colonies of final size are pooled 160 by, for example, scraping colonies from a plate containing a solid medium or other substrate, or by washing clonal cell colonies from microwells in a solid wall device or module in order to pool cells from normalized cell colonies. Again, since singulation or significant singulation overcomes the growth shift in favor of unedited cells or cells showing fitness effects as a result of the changes made, singulation or significant singulation, initial growth, editing induction, and normalization alone enriches the overall cell population with edited cells; that is, singulation or significant singulation combined with primary cell growth, editing induction, and normalization (eg, growing colonies to size limit) provides for high-throughput enrichment of edited cells.

[0082] The method 100b shown in FIG. 1A is similar to method 100a in that the cells of interest are transformed 110 with the components needed to perform editing based on nucleic acid-directed nuclease. As described above, the cells may be transformed simultaneously with a motor vector and an editing vector, the cells may already express a nuclease (e.g., the cells may have already been transformed with a motor vector, or the coding sequence for a nuclease may be stably integrated into the cellular genome) so that only the editing vector needs to be transformed into cells; or the cells may be transformed with a single vector containing all components required to perform genome editing using a nucleic acid-directed nuclease. In addition, if the module of singulation or significant singulation of cells, primary cell growth, induction of editing and normalization or selection ("solid wall isolation/induction/normalization module" or "SWIIN") is one module in an automated multi-module cell editing tool, the transformation cells can be performed in an automated transformation module as described with reference to FIG. 8A-8E below.

[0083] After 110 cells are transformed with the components needed to perform nucleic acid-directed nuclease editing, the cells are diluted (if necessary) in a liquid medium so that the cells, upon delivery to the singulation device or module, are separated from each other and could form colonies that separate from each other. For example, if a solid wall device (described with reference to Figs. 3A-3E and 4A-4CC) is used, the cells are diluted such that, upon delivery to the solid wall device, they fill the microwells of the solid wall device according to the Poisson or essentially a Poisson distribution.

[0084] After the cells are substantially or substantially singulated 120, they grow to, for example, 2-150, or 5-120, or 10-100 doublings, forming clonal colonies 130. After colony formation, editing is induced 140 by, for example, activating inducible promoters that direct the transcription of at least one of the gRNA and nuclease (as described above, preferably both) and, in the case of bacteria, the recombination system. When editing is induced 140, many of the edited cells in the clonal colonies die due to DNA double-strand breaks that occur during the editing process; however, in the percentage of edited cells, the genome is edited and the double-stranded DNA break is properly repaired. When given a chance to recover, these edited cells begin to grow and regenerate cell populations within each isolated partition or colony; however, the growth of edited colonies tends to lag behind the growth of those clonal colonies where editing has not taken place (eg, runaway cells). If the growth of these colonies is monitored 170 , small or slow growing colonies (edited cells) can be identified and then selected or selected 180 based on observed differences in colony size.

[0085] FIG. 1B is a plot of absorbance versus time for unedited cells (solid line) and edited cells (dashed line). Fig. 1B shows how edited and unedited colonies are normalized. It should be noted that the OD (e.g., growth) of edited cells initially lags behind unedited cells, but eventually catches up, for example, due to unedited cells depleting nutrients in the medium, becoming physically confined within a microwell or other restricted area. growth (such as a droplet), or otherwise stop growing in a logarithmic phase. The cell colonies are allowed to grow long enough for the growth of the edited colonies to catch up (approximately in size, eg cell count) to the unedited colonies.

[0086] FIG. 1C depicts an exemplary inducible expression system—in this example, a pL-inducible system—for regulating gRNA activity. At the top of Fig. 1C shows a portion of an exemplary engine vector 111 containing an origin of replication 112, a promoter 114 driving the expression of the c1857 repressor gene 116, and a first promoter 118 pL driving the expression of nuclease 121. At the top of FIG. 1C also shows a portion of an exemplary editing vector 131 containing an origin of replication 132 and a second pL promoter 134 directing the transcription of an editing cassette 136 (e.g., the CREATE cassette), which includes coding sequences for both gRNA and donor DNA. The middle part of Fig. 1C depicts the c1857 repressor gene 116 gene product 124 on motor vector 111 actively repressing the first 118 pL promoter driving the transcription of nuclease 121 and the second 134 pL promoter driving the transcription of the editing cassette 136 on the editing vector 131. Finally, the bottom of FIG. 1C shows protein product 126 of c1857 repressor gene 116 on motor vector 111 unfolding/degrading at elevated temperatures. The unfolded or disrupted protein product 126 cannot bind to the 118 pL first promoter or the 134 pL second promoter; thus, the 118 pL promoter is active and drives the transcription of nuclease 121 on the engine vector 111, and the 134 pL promoter is active and drives the transcription of the editing cassette 136 on the editing vector 131.

[0087] In FIG. 1C, transcription of both nucleases and gRNA is under the control of the pL promoter; however, in other embodiments, different inducible promoters may be used to drive transcription of the nuclease and gRNA, or in some embodiments, only one of the nuclease and gRNA is under the control of an inducible promoter. For example, the pL and pBAD promoters are shown in association with an exemplary motor and edit vector in FIG. 11A and 11B, and a number of gene regulation control systems have been developed for controlled gene expression in plant, microbial, and animal cells, including mammalian cells. These systems include the tetracycline-driven transcription activation system (Tet-On/Tet-Off, Clontech, Inc. (Palo Alto, CA); Bujard and Gossen, PNAS, 89(12):5547-5551 (1992)), the inducible Lac Switch system (Wyborski et al., Environ Mol Mutagen, 28(4):447-58 (1996); DuCoeur et al., Strategies 5(3):70-72 (1992); and US Pat. No. 4,833,080) , the ecdysone-induced 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 tamoxifen inducible gene expression (Zhang et al., Nucleic Acids Research, 24:543-548 (1996)), etc. However, the pL promoter is a particularly useful inducible promoter because it is activated by increasing the temperature to, for example, 42°C , and is deactivated by returning the temperature to, for example, 30°C. In the case of other inducible systems that are activated by the presence or absence of a particular molecular compound, activation of the inducible promoter requires the addition or removal of the molecular compound from the culture medium, which requires fluid handling, medium replacement, washing steps, and the like.

[0088] FIG. 2A depicts a standard, conventional prior art protocol 200 for performing nucleic acid-directed nuclease-based genome editing, where constitutively expressed nuclease components are typically used to drive high-throughput editing. On FIG. 2A, a library or collection of editing vectors 202 is introduced 203 (eg, electroporated) into cultured cells 204 that contain a coding sequence for a nuclease driven by a constitutive or inducible promoter. In some embodiments, the coding sequence for the nuclease is contained on a "motor vector", although in other embodiments, the coding sequence for the nuclease may be integrated into the cell's genome. In yet another alternative, the motor vector and edit vector components may be combined. Editing vectors 202 contain an editing sequence containing a sequence for the desired editing in a nucleic acid sequence endogenous to the cell, as well as an optional PAM alteration sequence (most often a sequence that turns off PAM in a target sequence in the genome) encoding a sequence for gRNA under the control of a constitutive promoter and a selectable marker. After the cells have been transformed with the editing vectors, the cells are placed 205 on a selection medium 206 to select cells that have both the motor vector and the editing vector, and the cells are grown to form colonies. Cells are then selected 207 from the colonies and grown, for example, in 96-well plates 208 in order to prepare them for genome or plasmid sequencing. Cells that grow on selective medium plate 206 must contain both a motor vector and an edit vector; however, it is likely that nucleic acid-directed editing components may be non-functional in some cells. In pooled or multiplex formats, selective enrichment of cells with a non-functional editing system is likely because unedited cells are not subject to DNA double-strand breaks that occur during editing. Rigid selection of cells with non-functional nucleic acid-directed nuclease editing systems results in a disproportionate representation of unedited cells in the final cell population and compromises the integrity of libraries generated by multiplexed nuclease editing systems.

[0089] FIG. 2B-2F depict improved protocols for performing genome editing based on a nucleic acid-directed nuclease. Fig. 2B depicts a first embodiment 201 of an improved protocol for performing nucleic acid-directed nuclease-based genome editing using an inducible promoter to drive gRNA expression. On FIG. 2B, a library or collection of editing vectors 202 is introduced 203 (eg, electroporated) into cultured cells 204 that contain a coding sequence for a nuclease driven by a constitutive or inducible promoter; The most tightly controlled nucleic acid-directed nuclease system is achieved by using an inducible promoter to drive nuclease expression, so it is preferred that an inducible promoter be used to drive nuclease transcription. In some embodiments, the coding sequence for the nuclease is contained on a "motor plasmid" (most often together with, for example, a selectable marker) that has already been transformed into cells, although in other embodiments, the coding sequence for the nuclease may be integrated into the genome of the cells. In other embodiments, the coding sequence for the nuclease may be located on an edit vector (ie, a combined motor vector and edit vector). Editing vectors 202 contain an editing sequence that optionally includes a PAM altering sequence (most commonly a sequence that turns off PAM at a target site in the genome), an inducible promoter-driven gRNA coding sequence, and a selectable marker.

[0090] After the cells 204 have been transformed with editing vectors, the cells are placed 205 on a selection medium to select cells that have both the motor vector and the editing vector, and the cells are grown to form colonies 206. The cells are then selected 207 from the colonies and grown overnight, for example, in the first 96-well plate 208 in a medium that selects both a motor vector and an edit vector. In the next step 209, cells from the first 96-well plate 208 are replicated into the second 96-well plate 210 in a medium containing an additional selective component, such as, for example, arabinose, to stimulate a strong induction of the λ red recombination system (homologous repair mechanism). The inducible pBAD promoter (induced by the presence of arabinose in the growth medium) drives the λ red recombination system and is described with reference to FIG. 11A below. Again, although the recombination system is illustrated here in a bacterial editing system, recombination systems are generally not needed in eukaryotic editing systems. After initial cell growth at 30° C., cutting and editing of the cell genome is induced by increasing the temperature to 42° C. in the second 96-well plate 210 for, for example, 0.5 to about 2 hours (depending on cell type) to activate pL-inducible promoters that control the transcription of nuclease and gRNA. After induction of cutting and editing (for example, within about 2 hours), the temperature returns to 30°C to allow the cells to recover.

[0091] In addition, at step 211, cells from the first 96-well plate 208 replicate into the third 96-well plate 214 in a medium that does not contain a selective component that induces a homologous repair mechanism (in this case, arabinose, so that recombination the λ Red system is not activated). However, the third 96-well plate 214 - except that it does not contain arabinose in the growth medium - is subjected to the same conditions as the second 96-well plate; that is, initially growing cells at 30°C, increasing the temperature to 42°C for, for example, 2 hours to activate the pL-inducible promoter driving nuclease and gRNA expression, and then decreasing the temperature to 30°C to give the cells opportunity to recover. As an alternative to the 96-well plates 210 and 214, two agar plates 212 and 216 are shown on which the cells from plate 206 are placed. well plate 214 containing growth medium without arabinose. It should be noted that, as an alternative to performing the method depicted in FIG. 2B, with +/- arabinose, the method can be performed with +/- temperature induction of the cut. For example, in a +/- temperature induction experiment, plate 210 would be a non-inducible culture plate (no temperature increase) and plate 214 would be temperature-induced. The same general interpretation applies; however, the cut activity is isolated to avoid the need to deconvolve the cut from the paste.

[0092] Looking at the second and third 96-well plates (210 and 214, respectively) and plates 212 and 216, cell colonies can be seen. The second 96-well plate 210 has two types of colonies: 226 (white wells) and 228 (hatched wells). The third 96-well plate 214 has two types of colonies: 220 (black wells) and 226 (white wells). Replication of seeding cells from the first 96-well plate to the second and third 96-well plates with different media provides functional deconvolution of the resulting cell populations. In the third 96-well plate, 214 cells were cultured in medium without arabinose. Without arabinose, the λ Red recombination system is inactive, and cells that have active expression of gRNA and nuclease (induced by increasing the temperature of cultured cells to 42°C) are not viable due to cutting of the active gRNA and nuclease double helix without repair by the λ Red recombination system. These 222 cell colonies are shown as white wells. Cell colonies 220, shown as black wells, represent cells having inactive gRNA, so that the genomes of the cells in these colonies are not cut by the nuclease-gRNA complex. Inactive gRNAs are generated at a frequency of approximately 2-15% in a typical cell editing experiment, and are usually the result of errors in the guide sequence portion (homologous portion) of the gRNA.

[0093] In a second 96-well plate 210, cells were cultured in arabinose medium, thereby activating the λ Red recombination system. Cells with active expression of gRNA and nuclease (induced by increasing the temperature of cultured cells to 42°C) are cut, edited, and the λ Red recombination system repairs DNA double strand breaks, and thus these cells are viable. However, cells with inactive gRNAs (or, less frequently, inactive nucleases) are also viable. All viable colonies in plate 210 are shown as shaded wells 228. Finally, cells that have active gRNAs but are not edited appropriately (due to, for example, an inactive recombination system) are indicated as white wells 226. Comparing the second 96-well plate with the third 96-well plate plate, it is possible to determine the function of the system based on the directed nucleic acid nuclease in each colony. For example, if colonies of cells grow without arabinose (plate 214, black wells 220) and with arabinose (plate 210, shaded wells 228), these cells should not have active gRNA. If these cells had active gRNA, they would not be viable in an arabinose-free medium (eg, without an active λ Red recombination system). If cell colonies do not grow in the medium without arabinose (plate 214, white wells 226) and grow when arabinose is added to the culture medium (plate 210, shaded wells 228), the cells in these wells contain an active system based on nucleic acid-directed nuclease - with active gRNA and nuclease components - and are likely to be properly edited. Finally, if cell colonies do not grow without arabinose (plate 214, white wells 226) or with arabinose (plate 210, white wells 226), the gRNA is active, but the cells cannot repair cuts for some reason. Thus, the method shown in Fig. 2B allows the identification of edited cell colonies by functional deconvolution of the various components of the editing "mechanism" in a single experiment.

[0094] In plates 212 and 216, the phenotypic readout is the same as in 96-well plates 210 and 214. Like the 96-well plate 210, the medium in the culture dish 212 contains arabinose, and like plate 214, the medium in the culture dish 216 does not contain arabinose. Thus, in culture dish 216, cells with inactive gRNAs grow because there are no changes (double-strand breaks) to repair. However, in cells with active gRNA, editing takes place, but there is no active mechanism to repair the changes, and the cells are non-viable and do not form colonies. In a culture dish 212 that contains arabinose, cells having inactive gRNAs form colonies, as do cells that have active gRNAs but are properly edited. Cells that are not viable for any reason do not form colonies. As with 96-well plates 210 and 214, comparison of culture dishes 212 and 214 allows deconvolution of the various components of the editing mechanism. If cells are growing on both 212 and 214 culture dishes, the gRNA is most likely inactive. If cells grow on culture dish 212 but not on culture dish 214, the gRNA is most likely active and proper editing has taken place. If the cells do not grow on either the culture dish 212 or the culture dish 214, the cells probably have active gRNA but the changes are not properly repaired.

[0095] Thus, the method 201 depicted in FIG. 2B allows the identification of cells with non-functional gRNAs that, due to the absence of double-stranded DNA breaks, have a growth advantage. Most importantly, the method 201 depicted in FIG. 2B allows identification of cells that have been properly edited. An aliquot of colonies 228 from the second 96-well plate 210 or from the agar plate 212 - colonies confirmed to have active gRNA - can be selected and sequenced to confirm the edit. The 96-well plate 210 or agar plate 212 can be stored so that when the correct edit is confirmed, for example by sequencing, one can return to the plate 210 or 212 and retrieve the properly edited cells. Plates 210 and 212 may be referred to as "cage hotels" or "cage storage", for example. It should be noted that the method 201 shown in FIG. 2B first substantially or substantially singulates the cells by seeding them on a selective medium at an appropriate dilution so that single cells form single colonies. Again, the singulation or significant singulation overcomes the bias growth that is characteristic of unedited cells. This method then allows functional deconvolution of different colonies by seeding replicate colonies in 96-well microtiter plates with different media in different sections to ensure positive identification of the edited colonies. The specific protocol for this exemplary method is described below in Example 6.

[0096] FIG. 2C depicts a second exemplary embodiment of an improved protocol 240 for performing nucleic acid-directed nuclease-based genome editing using an inducible promoter to drive expression of gRNA, and preferably also nuclease. On FIG. 2C, as in FIG. 2B, a library or collection of editing vectors 202 is introduced 203 (eg, electroporated) into cultured cells 204 that contain a coding sequence for a nuclease driven by a constitutive or inducible promoter; the tightest regulation of the nucleic acid-directed nuclease system is achieved by using an inducible promoter to drive nuclease expression and is therefore preferred. Same as in Fig. 2B, in some embodiments, the coding sequence for the nuclease is contained on a "motor plasmid" (most often together with, for example, a selectable marker) that has already been transformed into cells, although in other embodiments, the coding sequence for the nuclease may be integrated into the genome. cells. In other embodiments, the coding sequence for the nuclease may be located on an edit vector (ie, a combined motor vector and edit vector). Editing vectors 202 contain an editing sequence with the desired edit to the endogenous nucleic acid sequence in the cell, together with a PAM altering sequence (most commonly a sequence that turns off PAM at a target site in the genome), a coding sequence for gRNA, preferably under the control of an inducible promoter, and selectable marker.

[0097] After the cells 204 have been transformed with editing vectors, the cells are seeded 235 on a selective medium on a substrate or plate 236 to select cells that have both a motor vector and an editing vector. Cells are diluted prior to seeding so that they are substantially or substantially singulated - separated enough that they and the colonies they form are separated from other cell colonies - and these cells are then grown 237 on a plate or substrate 236 until colonies begin to form 238. Cells are allowed to grow, for example at 30° C., for example up to 2-150, or up to 5-120, or up to 10-100 doublings, forming clonal colonies. This initial cell growth must accumulate enough clonal cells in the colony in order to survive editing induction. Once colonies have formed, cutting and editing of the cellular genome is induced by inducing or activating promoters driving one or both of the gRNA and nuclease and the λ Red recombination system (if present). If the λ Red recombination system is present and under the control of an inducible promoter, preferably this inducible promoter is different from the inducible promoter driving the transcription of gRNA and nuclease and is activated (induced) before the induction of gRNA and nuclease. The λ Red recombination system works as a "patch" or repair system for double-strand breaks in bacteria, and in some bacterial species must be present to repair double-strand breaks that occur during editing. The λ Red recombination system may be driven by, for example, the pBAD promoter. The pBAD promoter, like the pL promoter, is an inducible promoter; however, the pBAD promoter is regulated (induced) by the addition of arabinose to the growth medium. Thus, if arabinose is contained in a substrate selective medium or plate 236, then the λ Red recombination system will be activated when the cells are grown 237. With regard to induction of editing 239, if gRNA and nuclease transcription is driven by the pL promoter, gRNA and nuclease transcription is induced by increasing temperature up to 42°C for, for example, 0.5-2 hours (or more, depending on the cell type), which activates the pL-inducible promoter. After inducing cutting and editing and a two hour incubation at 42°C, the temperature returns to 30°C in order to allow the cells to recover and turn off the pL promoter system.

[0098] After the cells recover and grow at 30°C, they are grown on the substrate or plate 236 in the colony size limit 244; that is, colonies arising from substantially or significantly singulated cells are grown in the colony until cell growth peaks and becomes normalized (eg, saturates) for both edited and unedited cells. As described above, normalization occurs when the nutrients in the medium around the growing cell colony are depleted and/or the growing cells fill microwells or are otherwise restricted; that is, the cells are in the aging stage. Critical size colonies are then pooled 241 by, for example, scraping colonies from the substrate or plates (or, for example, by washing clonal cell colonies from microwells in a solid wall device) in order to pool 280 cells from normalized cell colonies. It should be noted that the method 240 illustrated in FIG. 2C uses singulation or substantial singulation, initial growth, induction, normalization, and pooling of resulting cell colonies. Again, since singulation or significant singulation overcomes the growth shift in favor of unedited cells or cells showing fitness effects as a result of the changes made, the combination of singulation or significant singulation, initial growth, editing induction, and normalization enriches the total cell population with those cells that have been edited. . It should be noted, however, that unlike the methods 201, 250 depicted in FIG. 2B and FIG. 2D, respectively, the method 240 depicted in FIG. 2C does not contain steps for deconvolution of the editing mechanism.

[0099] FIG. 2D depicts a third exemplary embodiment of an improved protocol 250 for performing nucleic acid-directed nuclease-based genome editing using an inducible promoter to drive the expression of gRNA, and in some embodiments and preferably, also the nuclease. Fig. 2D depicts a protocol 250 for activity-based debugging and relocation. On FIG. 2D, as in FIG. 2B and 2C, a library or collection of editing vectors 202 is introduced 203 (e.g., electroporated) into cultured cells 204 that contain the coding sequence for a nuclease driven by a constitutive or inducible promoter (preferably an inducible one) contained on a "motor plasmid" (e.g., together with a selectable marker) that has already been transformed into cells or integrated into the cell genome. Alternatively, the coding sequence for the nuclease may be located on an editing vector. Editing vectors 202 contain an editing sequence that alters a PAM sequence (most commonly a sequence that disables PAM at a target site in the genome), a coding sequence for gRNA, preferably under the control of an inducible promoter, and a selectable marker.

[0100] After cells have been transformed with editing vectors, cells are diluted and plated 205 on selective medium 206 to select cells that have both a motor vector and an editing vector (e.g., chloramphenicol and carbenicillin medium) and grown to colony formation 221, again in a process that substantially or substantially singulates the cells. Cells are then selected 207 from colonies 221 and grown overnight, for example in a first 96-well plate 208 in media that does not contain selective media. In the next step, cells from the first 96-well plate are replicated 211 into the second 96-well plate 214 in arabinose-free medium, resulting in wells with colonies (black wells 220) and wells without cell colonies (white wells 222). This process identifies cells with active gRNAs, since cells with active gRNAs will not survive because, without arabinose, the λ Red recombination system is inactive and cannot repair cuts in the genome made by gRNAs. Thus, wells 222 are highly likely to be active gRNA wells. The colonies that grow on this second 96-well plate 214 are very likely to have inactive gRNA, and thus there are no cuts to repair and the cells remain viable (shown as black wells 220).

[0101] After cells with active gRNAs are identified (wells 222), these cells are then selected 213 from the first 96-well plate and placed in the third 96-well plate 252 for storage as a cell store or cell hotel. All colonies in the third 96-well plate 252 are cells that have been identified as highly likely to have active gRNAs 222. Using this cell storage 252, aliquots of these selected cells (eg, cells with active gRNAs) can then be placed 255 in a fourth 96 -well plate 254 into the medium with arabinose. After a period of initial growth, the cells in this fourth 96-well plate 254 are subjected to cutting and editing conditions, for example by increasing the temperature to 42° C. for two hours in order to induce the pL-inducible promoter driving nuclease and gRNA expression. After cutting and editing processes, colonies containing properly edited cells can be identified by monitoring colony growth and selecting slow growing colonies, or by targeted or whole genome sequencing of cells taken from the 254 plate. Once cells with the desired changes have been identified, they can be extracted from 96-well plate 252 (for example, cell storage or cell hotel). Alternatively, cell colonies with putatively active gRNAs on plate 252 can be pooled 253 in a mixed cell culture 256 and either analyzed or subjected to an additional round of editing.

[0102] FIG. 2E depicts another exemplary embodiment of an improved protocol 270 for performing nucleic acid-directed nuclease-based genome editing using an inducible promoter to direct gRNA and/or nuclease expression. Protocol 270 in FIG. 2D does not contain a functional deconvolution of the cell editing mechanism, but depicts a protocol for high throughput screening using colony morphology to identify edited cells. Again, in edited cells, cell viability declines in the post-editing period. The present method uses colony growth arrest of the edited cells to identify the edited cells. In some embodiments, the colony size of the edited cells is 20% smaller than that of the unedited cells. In some aspects, the colony size of the edited cells is 30%, 40%, 50%, 60%, 70%, 80%, or 90% smaller than that of the unedited cells. In many embodiments, the edited cell colony size is 30-80% smaller than the unedited cell colonies, and in some embodiments, the edited cell colony size is 40-70% smaller than the unedited cell colonies.

[0103] In FIG. 2E, as in FIG. 2B-2D, a library or collection of editing vectors 202 is introduced 203 (e.g., electroporated) into cultured cells 204 that contain the coding sequence for a nuclease driven by a constitutive or inducible promoter (preferably an inducible one) contained 1) on a "motor plasmid" (more often together with the selectable marker) that has already been transformed into cells; 2) integrated into the genome of transformed cells; or 3) the coding sequence for the nuclease may be located on an editing vector. Editing vectors 202 contain an editing sequence and optionally include a PAM altering sequence (eg, a sequence that turns off PAM at a target site in the genome), a coding sequence for gRNA driven by a preferably inducible promoter, and a selectable marker. In many embodiments, an inducible promoter is included in the backbone of an editing vector and the editing cassette is inserted at the 3' position of the inducible promoter, where the editing cassette contains, 5' to 3': a coding sequence for the gRNA and a donor DNA containing the desired change for the target sequence, as well as a PAM-changing sequence (for example, a CREATE cassette). It should be noted that transcription of one or both of the gRNA and nuclease must be under the control of an inducible promoter. Preferably, however, both gRNA and nuclease transcription are under the control of an inducible promoter.

[0104] In step 271, the transformed cells are diluted and plated (e.g., substantially or substantially singulated) onto selective medium 272 that selects both a motor vector and an editing vector (e.g., a medium containing chloramphenicol and carbenicillin), and additionally contains arabinose to activate the λ Red recombination system. After seeding, cells are grown 273 at 30° C. for 6-8 hours so that colonies form, then the temperature is increased to 42° C., for example, 0.5-2 hours to induce nuclease and gRNA expression for cutting and editing. After sufficient time for editing has elapsed, the temperature returns to 30° C. so that the cells grow to re-establish colonies on plate 272. After colonies appear, large 278 and small 276 colonies are observed. Small size 276 colonies indicate active gRNA and are highly likely to have been edited because double-strand cuts caused by active editing are highly toxic to cells, resulting in both cell death in the edited colonies and growth stunting for the edited cells. which survive but must be repaired and restored after editing. Small colonies (edited cells) are selected 277 and lined up on a 96-well plate 282. Cells in a 96-well plate 282 can be cultured and aliquots from this 96-well plate 282 can be sequenced to identify colonies with the desired changes. This 96-well plate can be stored as a cell hotel or cell store, and once cells that have been properly edited are identified, cells with the desired modification can be retrieved from the 96-well cell hotel plate 282.

[0105] Alternatively, small colonies 276 can be selected and pooled 275 for additional edits, since the edited cells were selected in the first edit and are likely to contain changes from the first edit. Again, it should be noted that this method does not provide functional deconvolution of the editing mechanism; however, the method shown in FIG. 2E uses singulation or significant singulation and selection, and thus allows the high throughput diagnostic method to identify, based on colony size morphology, those cells that are highly likely to be edited, and once identified, the population of edited cells can be enriched. Screening out a significant proportion of cells with non-functional gRNAs provides easier identification of edited cells. Eliminating growth rate bias by singulation or significant singulation has been found to improve observed editing efficiency up to 2x, 3x, 4x or more over conventional methods, and furthermore, colony selection using the methods described herein in 1, 5x, 1.75x, 2.0x, 2.5x or more increases the observed efficiency of singulation editing. Thus, the combination of significant singulation and selection improves the observed editing efficiency by 8x compared to conventional methods. Example 9 below describes materials and methods for this embodiment.

[0106] While a method for screening edited cells using cell growth as a feature of editing has been described herein in the context of measuring the size of cell colonies on an agar plate, the optical density (OD) of growing cell colonies can be measured instead, for example, on microtiter plate or in a series of tubes. In addition, other cell growth parameters may be measured in addition to or instead of cell colony size or OD. For example, spectroscopy using visible, UV, or near infrared (NIR) light can monitor the concentration of nutrients and/or waste products in a cell culture. In addition, spectral measurements can be used to quantify multiple chemicals simultaneously. Unsymmetrical chemical compounds can be quantified by identifying characteristic absorption features in the near infrared range. Conversely, symmetrical chemical compounds can easily be quantified using Raman spectroscopy. Many critical metabolites, such as glucose, glutamine, ammonia, and lactate, have distinct IR spectral features so that they can be easily quantified. The amount and frequency of light absorbed by a sample can be correlated with the type and concentration of chemicals present in the sample. Each of these measurement types provides specific benefits. FT-NIR provides the greatest depth of light penetration and therefore can be used on a thicker sample resulting in a higher degree of light scattering. Mid-IR (MIR) FT provides information that is easier to discern as it is specific to some analytes because these wavelengths are closer to fundamental IR absorbance values. Raman FT is beneficial when water interference is to be minimized. Other spectral properties can be measured by, for example, dielectric impedance spectroscopy, visible fluorescence, fluorescence polarization, or luminescence. In addition, sensors for measuring, for example, dissolved oxygen, carbon dioxide, pH value and/or electrical conductivity can be used to evaluate cell growth rate.

[0107] FIG. 2F depicts additional details of the exemplary embodiment shown in FIG. 2E using a specific example based on the experiments performed (see Example 1 below). Fig. 2F shows high throughput screening 290 using colony morphology to identify or select edited cells. As described above, in edited cells, cell viability decreases in the post-editing period. The present method uses colony growth arrest of the edited cells to identify the edited cells. On FIG. 2F transformed cells are diluted and plated on medium containing arabinose 274 and grown for some time, eg at 30°C. Editing is initiated, for example, by raising the temperature to 42° C. for a period of time, then lowering the temperature to 30° C. (eg, the transformed cells essentially or substantially singulate). The colonies are allowed to grow, and small 276 and large 278 colonies appear. Colonies from this plate are selected 291 and lined up on a second plate 292 containing a selective medium, such as a medium to select for successful galK editing resulting in white (as opposed to red) colonies when plated on MacConkey agar containing galactose as the sole carbon source. . Of note, selection of small colonies 293 from the first plate results mainly in edited 296 cells (white colonies shown here as white circles), and at a much lower frequency to some cells in which gRNA is inactive 294 (red colonies shown here). like black circles). Confirmation of colonies in which the gRNA is inactive is shown by selecting 295 large colonies 278 from the first plate and seeding them on the second plate (colonies 298), where these cells lead to red colonies when grown on MacConkey agar containing galactose as the only source. carbon, thus confirming an inactive gRNA or other part of the editing mechanism. Thus, using the morphology of small and large colonies as a feature of edited and unedited cells, respectively, provides a high throughput and simple screening method for edited cells. It should be noted that the methods depicted in FIG. 2E and 2F use both singulation or essential singulation and selection strategies.

[0108] The exemplary workflows described herein use the concept of singulation. Singulation overcomes the growth shift in favor of the unedited cells, thus placing the edited cells on an equal footing with the unedited cells. In addition, these methods take advantage of a highly regulated inducible system to separate edited cells from non-edited cells (both cell types in which the gRNA is non-functional, and cells in which the gRNA is functional but some component of the nucleic acid-directed nuclease system is non-functional). ). Screening can be done using replica plates and identifying runaways or cells in which the gRNA is non-functional, screening can be done using the growth inhibition benefits of edited cells versus unedited cells, or enrichment can be done by using a combination of singulation or essential singulation, initial growth, editing induction and normalization. The result of these methods is that even in nucleic acid-directed nuclease-based systems where editing is not optimal (eg, in systems where non-canonical PAMs are targeted), there is an increase in observed editing efficiency; that is, edited cells can be identified even against a large background of unedited cells.

[0109] It should be noted that the methods depicted in FIG. 2B-2F show cell colony singulation, initial growth, and induction of editing on solid media in culture dishes or 96-well plates. One skilled in the art, given the discussion herein, will appreciate that singulation, initial growth, and edit induction can be performed in other formats, such as in the solid wall devices described with reference to FIGS. 3A-3F and 4A-4CC or as described in US Patent Document No. 62/735365 entitled "Detection of Nuclease-Edited Sequences in Automated Modules and Systems" filed September 24, 2018 and US Patent Document No. 62/781112 , entitled "Improved Detection of Nuclease-Edited Sequences in Automated Modules and Systems", filed December 18, 2018, which includes descriptions of singulation or significant singulation by isolating cells on functionalized islands, singulation or significant singulation inside water droplets carried in a hydrophobic carrier liquid, or gel beads in emulsion (GEM, see e.g. 10X Genomics, Pleasanton, CA), or singulation or substantial singulation within a polymerized alginate scaffold (for this singulation embodiment, see also US Patent Document No. 62/769805 , titled "Improved Detection of Edited Nucleases sequencing in automated modules and tools through mass cell culture”, filed November 20, 2018).

Exemplary Modules for Editing, Enriching, and Selecting Edited Cells

[0110] The tools, methods, and modules described herein provide improved observed editing efficiency of nucleic acid-directed nuclease editing methods as a result of singulation or substantial singulation, initial cell growth, editing induction, and normalization. The combination of singulation or substantial singulation processes, initial growth, editing induction and normalization overcomes the growth bias in favor of unedited cells, as well as editing suitability effects (including different editing speeds), thus providing equal opportunities for all cells. The result of the tools, modules, and methods described herein is that even in nucleic acid-directed nuclease-based systems where editing is not optimal (eg, in systems where non-canonical PAMs are targeted), there is an increase in observed editing efficiency; that is, edited cells can be identified even against a large background of unedited cells. The observed editing efficiency can be improved up to 80% or more.

[0111] FIG. 3A depicts a solid wall device 350 and a workflow for singulating cells in microwells in a solid wall device where one or both—preferably both—of gRNA and nuclease are under the control of an inducible promoter in this workflow. On FIG. (i) shows a solid wall device 350 with microwells 352. A section 354 of substrate 350 is shown in FIG. (ii), microwells 352 are also shown. FIG. (iii) showing a cross section of a solid wall device 350 where microwells 352 are loaded, Poisson or substantially Poisson loading in this embodiment; that is, each microwell has one or no cells, and there is little chance that any one microwell has more than one cell. On FIG. (iv) a workflow 340 is shown where substrate 350 having microwells 352 shows microwells 356 with one cell per microwell, microwells 357 with no cells per microwell, and one microwell 360 with two cells per microwell. In step 351, cells in microwells are allowed to double approximately 2-150 times to form clonal colonies (v), then editing is induced 353 by heating the substrate (eg, for thermally induced editing), or by flowing chemicals under or over the substrate (eg, sugars, antibiotics for chemically induced editing), or by moving the solid wall device to another environment, particularly if the solid wall device is placed on the membrane that forms the bottom of the microwells 352 (membrane not shown).

[0112] After induction of 353 edit, many cells in colonies of cells that have been edited die as a result of double strand cuts caused by active editing, and there is a growth retardation of edited cells that survive but must be repaired and restored after editing (microwells 358), then how unedited cells thrive (microwells 359) (vi). All cells are allowed to continue to grow to form colonies and normalize, with colonies of edited cells in microwells 358 catching up in size and/or number of cells with cells in microwells 359 that are not edited (vii). After the cell colonies are normalized, either pooling 360 of all cells in the microwells can take place, in which case the cells are enriched in edited cells by eliminating bias due to unedited cells and fitness effects from editing; alternatively, colony growth in the microwells is monitored after editing, and slow growing colonies (eg, cells in microwells 358) are identified and selected 361 (eg, "selected"), further enriching the edited cells.

[0113] When growing cells, the medium used will, of course, depend on the type of cells being edited, eg, bacterial, yeast, or mammalian cells. For example, bacteria growth media includes LB, SOC, M9 minimal media, and Magic media; yeast cell growth medium includes TPD, YPG, YPAD and synthetic minimal medium; and the mammalian cell growth medium includes MEM, DMEM, IMDM, RPMI, and Hank's medium. For culturing adherent cells, the cells can be placed on beads or other type of scaffold suspended in the medium. Most normal mammalian tissue-derived cells, with the exception of those derived from the hematopoietic system, are anchorage-dependent and require support on a surface or in cell culture for normal proliferation. The rotating growth flask described herein uses microcarrier technology. Specific microcarriers are typically 100-300 µm in diameter and have a slightly higher density than the culture medium (enabling easy separation of cells and medium, e.g. for medium exchange), but the density should also be low enough to ensure complete suspension of the media. at a minimum stirring speed to avoid hydrodynamic cell damage. Many different types of microcarriers are available, and different microcarriers are optimized for different cell types. Positively charged carriers are available such as Cytodex 1 (dextran-based, GE Healthcare), DE-52 (cellulose-based, Sigma-Aldrich Labware), DE-53 (cellulose-based, Sigma-Aldrich Labware), HLX 11-170 (based on polystyrene); collagen or ECM (extracellular matrix) coated carriers such as Cytodex 3 (dextran based, GE Healthcare) or HyQ-sphere Pro-F 102-4 (polystyrene based, Thermo Scientific); uncharged carriers such as HyQspheres P 102-4 (Thermo Scientific); or macroporous carriers based on gelatin (Cultisphere, Percell Biolytica) or cellulose (Cytopore, GE Healthcare).

[0114] FIG. 3B depicts a solid wall device 350 and a workflow for substantially singulating cells in microwells in a solid wall device, where in this workflow (as in the workflow depicted in FIG. 3A) one or both—preferably both—of gRNA and nucleases are under the control of an inducible promoter. On FIG. (i) shows a solid wall device 350 with microwells 352. A section 354 of substrate 350 is shown in FIG. (ii), microwells 352 are also shown. FIG. (iii) shows a cross-section of a solid wall device 350 where microwells 352 are loaded, with substantially Poisson loading in this embodiment; that is, some microwells 357 do not contain any cells and some microwells 376, 378 contain few cells. On FIG. 3B cells with active gRNA are shown as black circles and cells with inactive gRNA are shown as white circles. On FIG. (iv) shows a workflow 370 where a substrate 350 having microwells 352 shows three microwells 376 with several cells, all with active gRNAs, a microwell 357 with no cells, and two microwells 378 with some cells having active gRNAs and some cells having inactive gRNA. In step 371, the cells in the microwells are allowed to double approximately 2-150 times to form clonal colonies (v), then editing is induced 373 by heating the substrate (eg, for thermally induced editing), or by flowing chemicals under or over the substrate (eg, sugars, antibiotics for chemically induced editing), or by moving the solid wall device to another environment, particularly if the solid wall device is placed on the membrane that forms the bottom of the microwells 352.

[0115] After induction of edit 373, many cells in the colonies of cells that have been edited die as a result of double strand cuts caused by active editing, and there is a growth retardation of the edited cells that survive, but must be repaired and restored after editing (microwells 376), then how unedited cells thrive (microwells 378) (vi). Thus, in microwells 376, which contain only cells with active gRNA (depicted by black circles), most of the cells die; however, in microwells 378 containing cells with inactive gRNA (shown as white circles), the cells continue to grow and are not subjected to active editing. The cells in each microwell (376 and 378) are allowed to grow to continue colony formation and normalization, where the edited cell colonies in microwells 376 catch up in size and/or cell number to the unedited cells in microwells 378 that are not edited (vii). It should be noted that in this workflow, the 370 cell colonies in the microwells are not clonal; that is, not all cells in a well are descended from a single cell. Instead, the cell colonies in a well may be mixed colonies originating in many wells from two or more different cells. After the cell colonies are normalized, either pooling 390 of all cells in the microwells can take place, in which case the cells are enriched in edited cells by eliminating bias due to unedited cells and fitness effects from editing; alternatively, microwell colony growth is monitored post-editing, and slow growing colonies (eg, cells in microwells 376) are identified and selected 391 (eg, "selected"), further enriching the edited cells.

[0116] FIG. 3C is a photograph of one embodiment of a solid wall substrate containing microwells for cell singulation. As seen in this photograph, the solid wall substrate is a perforated disc of metal approximately 2 inches (~47 mm) in diameter. The perforated disc shown in this photo is made from 316 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. The use of a filter or membrane (such as a 0.22 µm PVDF Duropore ^™ woven membrane filter) allows media and/or nutrients to enter the microwells, but prevents cells from flowing down the microwells. The filter or membrane elements that can be used in solid wall singulation or essential singulation/initial growth/induction editing and normalization or selection modules are solvent-resistant, not contaminated during filtration, and are capable of retaining cell types and sizes of interest. For example, to retain small cells such as bacterial cells, pore sizes may be as small as 0.10 µm, however, for other cell types, pore sizes may be as low as 0.5 µm. In fact, pore sizes useful in a cell concentration device/module include filters with aperture sizes of 0.10 µm, 0.11 µm, 0.12 µm, 0.13 µm, 0.14 µm, 0.15 µm, 0.16 µm, 0.17 µm, 0.18 µm, 0.19 µm, 0.20 µm, 0.21 µm, 0.22 µm, 0.23 µm, 0.24 µm, 0.25 µm, 0.26 µm, 0.27 µm, 0.28 µm, 0.29 µm, 0.30 µm, 0.31 µm, 0.32 µm, 0.33 µm, 0.34 µm, 0.35 µm, 0.36 µm, 0.37 µm, 0.38 µm, 0.39 µm, 0.40 µm, 0.41 µm, 0.42 µm, 0.43 µm, 0.44 µm, 0.45 µm, 0.46 µm, 0.47 µm, 0.48 µm, 0.49 µm, 0.50 µm or more. Filters can be made from any suitable material, including mixed cellulose ether (cellulose nitrate and acetate) (CME), polycarbonate (PC), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, or glass fiber.

[0117] In the photograph shown in FIG. 3C, the perforations are approximately 150 μm - 200 μm in diameter, resulting in microwells having a volume of approximately 2.5 nL, for a total of approximately 30,000 microwells. The distance between the centers of the microwells is approximately 279 nm. Although the microwells here have a volume of approximately 2.5 nl, the volume of the microwells can 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 microwells will depend on the cell type (eg, bacterial, yeast, mammalian cells). The perforated disc shown here is 316 stainless steel; however, other biocompatible metals and materials may be used. The solid wall device can be disposable or reusable. The solid wall device shown in FIG. 3C is round but may be any shape such as square, rectangular, oval, etc. (see, for example, Fig. 4A). Round perforated discs are useful when Petri dishes are used to supply a solid wall module with nutrients via a solid medium, such as in a Petri dish or other cell culture dish. Filters used to bottom the microwells of the solid wall device include 0.22 µm PVDF Duropore ^™ woven membrane filters. Also, although a 2 inch (~47 mm) diameter perforated disc is shown, the perforated discs can be smaller or larger as desired and the configuration of the solid wall module will depend on how nutrients are supplied to the solid wall module and how the environment replacement. For example, see the perforated element in FIG. 4A and solid wall module embodiments shown in FIG. 4F-4BB.

[0118] FIG. 3D-3F are photographs of E. coli cells substantially or substantially singulated by a Poisson or substantially Poisson distribution in microwells or holes in a perforated membrane-bottomed disc at low, medium and high magnification, respectively. Fig. 3D shows growth at low magnification where the darker microwells are microwells with growing cells. Fig. 3E is a top view of microwells in a perforated disc, where the darker microwells are microwells with growing cells. Fig. 3F is a photograph of microwells where a membrane (e.g., a permeable membrane that forms the bottom of microwells) has been removed, where unpatterned (smooth) microwells are microwells in which cells do not grow, and microwells with irregular pigment/pattern are microwells, in which cells grow, and in this photo they filled the microwells in which they grow. In these photographs, a 0.2 µm filter (membrane) has been hot stamped under high pressure to a perforated disc, such as the round perforated disc shown in FIG. 3C. The perforated disk formed the walls of the microwells, and the filter formed the bottom of the microwells. To load the perforated filter disc, E. coli cells were drawn into the microwells using vacuum (see description of methods in Example 6). The perforated filter disc was then placed membrane side down on an LB agar plate and the cells were grown overnight at 30° C. and then two days at room temperature. Then the membrane was removed, and the microwells that opened from below were photographed using an optical microscope. Of note is the ease with which different selective media can be used to select for specific cell phenotypes; that is, it is only necessary to transfer the perforated filter disc to another plate or Petri dish containing the desired selective medium. Typically, the number of cells loaded into the singulation device or assembly ranges from about 0.1X to 2.5X the number of perforations or microwells, or from about 0.3X to 2.0X the number of perforations or microwells, or from about 0.5X to 1, 5X the number of perforations or microwells.

[119] FIG. 4A-4BB depict various components of various embodiments and components of a solid wall singulation or significant singulation, growth, editing induction and normalization or selection module ("solid wall isolation/induction/normalization module" or "SWIIN") suitable for singulation ( or significant singulation) of cells of all types, growing cells for initial, for example, 2-150 cycles of cell division, inducing editing and normalizing or selecting the resulting cell colonies. The presented SWIIN modules can be stand-alone devices, or often a single module in an automated multi-module cell processing tool. Fig. 4A shows a perforated metal sheet or perforated member 401, which is typically rectangular in shape. Holes shown are not to scale. As with the perforated disc described with reference to FIG. 3C-3F, the perforated element 401 is made of 316 stainless steel, where the holes form the walls of the microwells, and a filter or membrane (not shown in Fig. 4A) is used to form the bottom of the microwells.

[0120] In the scanning electron microscope photograph shown in FIG. 4B, the perforations (microwells) are approximately 150 µm to 200 µm in diameter and the perforation depth is approximately 125, resulting in a microwell volume of approximately 2.5 nL for a total of approximately 200,000 microwells. The distance between the centers of the microwells is approximately 279 µm. Although the microwells here have a volume of approximately 2.5 nl, the volume of the microwells can be from 1 to 25 nl, or preferably from 2 to 10 nl, and even more preferably from 2 to 4 nl. The perforated element shown in Fig. 4A is approximately 14 cm long and 10 cm wide (140 mm x 100 mm); however, smaller (such as the one shown in Fig. 3C) or larger perforated elements may be used depending on the density of microwells in the perforated element and the number of microwells or septa required. For example, the greater the plexity (eg, complexity) of a library used to edit a population of cells, the greater the number of microwells or partitions is preferred. If, for example, a 10,000-plex library is being used to edit a population of cells, a perforated element with 200,000 microwells or baffles will be more than enough; however, if a 50,000-plex library is used to edit a population of cells, a perforated element (or two or more elements) with a total of 400,000 or more microwells or septa may be preferred. Typically, the number of cells loaded into the singulation device or assembly ranges from about 0.1X to 2.5X the number of perforations or microwells, or from about 0.3X to 2.0X the number of perforations or microwells, or from about 0.5X to 1, 5X the number of perforations or microwells; thus, the number of cells loaded onto a perforated element containing approximately 200,000 perforations will vary from approximately 20,000 to approximately 500,000, or from approximately 60,000 to approximately 400,000, or from approximately 100,000 to approximately 300,000. The preferred microwell size/volume will depend on the type of microwells being edited. cells (eg, archaea, bacteria, yeast, non-mammalian and/or eukaryotic cells). The perforated element shown here is 316 stainless steel; however, other biocompatible metals and materials such as titanium, cobalt-based alloys, and ceramics can also be used. A SWIIN may be disposable, or it may be reusable. When reused, the SWIIN can be heated to 55°C or higher to sterilize it, or alternatively it can be flushed with a stream of antibiotics.

[0121] FIG. 4B-4E are electron microscope photographs of a portion of the perforated element 401 (FIG. 4B), as well as enlarged views of one microwell 402 without (FIG. 4C) and with (FIGS. 4D and 4E) the filter membrane forming the bottom of the microwell 402 Fig. 4B shows a perforated element 401 and approximately 30 microwells 402. FIG. 4C also shows a perforated element 401 and a single microwell 402 having approximately 172 microns in diameter. 4D shows a perforated member 401 and approximately 8 microwells 402, each having a filter or membrane portion 403 forming the bottom of the microwell 402. FIG. 4E is a higher magnification micrograph of one of the micropits 402 of the perforated member 401 shown in FIG. 4D. As described above for FIG. 3C-3F, the use of a filter or membrane (such as a 0.22 µm PVDF Duropore ^™ woven membrane filter) allows media and/or nutrients to enter the microwells but prevents cells from flowing down the microwells. The filter or membrane elements that can be used to form the bottom of the microwells of the perforated element in the singulation or essential singulation/initial growth/editing and normalization/selection induction module with a solid wall are solvent resistant, not contaminated during filtration, rupture under the pressure required for media exchange and cell loading, and are capable of holding cell types and sizes of interest. For example, to retain small cells such as bacterial cells, pore sizes may be as small as 0.2 µm, however, for other cell types, pore sizes may be 0.5 µm or larger. Filters can be made from any suitable material, including mixed cellulose ether (cellulose nitrate and acetate) (CME), polycarbonate (PC), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, or glass fiber. The perforated element 401 and the filter 403 are hot stamped together; that is, the perforated member 401 and the filter 403 are compressed under high pressure (eg, 20 ksi). In some embodiments, the holes or septa in the perforated element are etched with a slight taper and the filter material is pressed into the hole and then loosened to form an effective crimp that provides an adhesive-free connection. Alternatively, glue may be used.

[0122] FIG. 4F depicts one embodiment of the singulation node 420a in a top perspective view that represents the "retaining side" of the singulation node 420a. As used herein, the "singulation assembly" comprises a retaining element 404 as the top element, a permeable element 408 as the bottom element, with a seal 416 surrounding the perforated element 401 and the filter 403, where the seal 416, the perforated element 401 and the filter 403 are sandwiched between the retaining element 404 and a permeable element 408. The retaining and permeable elements 404 and 408, respectively, in the embodiments illustrated in FIG. 4F - 4Q and 4Y are transparent and are approximately 200 mm long, 130 mm wide and 4 mm thick, although in other embodiments the retained and permeable members may be 75 mm to 350 mm long, or 100 mm to 300 mm, or from 150 mm to 250 mm; width from 50 mm to 250 mm, or from 75 mm to 200 mm, or from 100 mm to 150 mm; and thickness from 2 mm to 15 mm, or from 4 mm to 10 mm, or from 5 mm to 8 mm. In the embodiments depicted in FIG. 4F - 4BB, holding elements are made from PMMA (polymethyl methacrylate); however, other materials may be used, including polycarbonate, cyclic olefin copolymer (COC), glass, polyvinyl chloride, polyethylene, polyamide, polypropylene, polysulfone, polyurethane, as well as copolymers of these and other polymers. Seal 416 is a flat, circular circumferential arrangement surrounding perforated element 401 and filter 403, and is made of rubber, silicone, nitrile rubber, polytetrafluoroethylene, plastic polymer such as polychlorotrifluoroethylene, or other easily compressible material.

[0123] The retainer 404 has a generally smooth upper surface; a single distribution channel 405 (in this case centrally located) that traverses the retaining member 404 from its upper surface to its lower surface and for most of the length of the retaining member 404 (details of which are described in connection with FIG. 4I); and ridges 406a, which in this case are located on the lower surface of the retaining element 404 (for example, next to the perforated element 401), where the ridges 406a cross the bottom of the retaining element 404 from one side to the other (for example, from left to right) (in this embodiment, there is approximately 28 combs 406a). As used herein for distribution channels in a containment or vent, the expression "most of the length" means approximately 95% of the length of the containment or vent, or approximately 90%, 85%, 80%, 75%, or 70% of the length of the containment, or permeable element. Flow guides 406 are formed between the ridges 406a. In addition, the retaining member 404 has a single opening 407 that allows cells to be introduced into the singulation node 420a; and there is also a distribution channel cover 413 that covers a single distribution channel 405 in the retaining member 404. In this embodiment, the distribution channel 405 is approximately 150 mm long and 1 mm wide; the retainer ridges 406a are approximately 0.5 mm high and 80 mm long; and the retainer flow guides 406 are approximately 5 mm wide. The volume of fluid in the singulation node is 5 ml to 100 ml, or 7.5 ml to 60 ml, or 10 ml to 40 ml (for a singulation node with 200,000 perforations).

[0124] In addition to the holding element 404, in FIG. 4F also shows fasteners 412, a central composite layer containing a seal 416 surrounding a perforated element 401 located above the filter or membrane 403 (the individual components are not visible in FIG. 4F). The bottom layer of the singulation node 420a shown in FIG. 4F is formed by the vent 408. The vent 408, like the containment 404, includes one or more vent distribution channels, vent ridges, vent flow guides, and one or more openings (none of which are shown in FIG. 4F but shown in Fig. 4G). Singulation nodes 420 and SWIIN modules 400 containing singulation nodes 420 are made from a material that can withstand temperatures from 4°C to 60°C. Heating and cooling of the SWIIN modules is provided by a Peltier device or a thermoelectric cooler; or a combination of systems may be used, such as in multi-layer SWIIN modules (see, for example, Figs. 4BB and 4CC), such as Rankine reverse compression refrigeration or absorption heat pumps.

[0125] In the modules of singulation or essential singulation, growth, induction of editing and normalization and/or selection with a solid wall (“solid wall isolation/induction/normalization module” or “SWIIN”) described in FIG. 4A-4CC, cells and media (at a dilution suitable for Poisson or substantially Poisson distribution of cells in the microwells of the perforated element) flow into the distribution channel 405 through one or more openings (there is one opening 407 in FIG. 4F) in the retaining element 404, and the cells settle in the microwells with a Poisson or substantially Poisson distribution of cells in the microwells. The cells are held in the microwells as they cannot pass through the filter 403. After the cells are loaded, a suitable medium can be introduced into the singulation node through the retainer; that is, the medium is introduced through the holes 411 (FIG. 4G) and the medium passes into the distribution channels 409 (FIG. 4G) in the permeable element 408 and then distributed by the flow guides 410 (FIG. 4G) in the permeable element 408. The medium in the permeable element 408 can flow upward through the filter 403 to feed the cells loaded into the microwells. During operation, when cells are placed in microwells, they are grown for initial, for example, approximately 2-100 doublings, editing is induced, for example, by raising the SWIIN temperature to 42°C to induce a temperature-inducible promoter, or by removing the growth medium from permeable element (back through holes 411, see Fig. 4G) and replacing the growth medium with medium containing a chemical component that induces an inducible promoter. After editing, the SWIIN temperature can be reduced, or the inducing medium can be removed and replaced with fresh medium containing no chemical component, and thus the inducible promoter is deactivated. The cells are then allowed to continue growing in the SWIIN until the growth of cell colonies in the microwells returns to normal. Once normalization has occurred, the colonies are washed out of the microwells (eg, by applying pressure to the permeable element 408, and thus to the filter 403), and the cells in the colonies are pooled; or alternatively, the growth of cell colonies in the microwells is monitored and slow growing colonies are selected directly, for example by pooling cells from slow growing colonies by pipetting slow growing cells from microwells into a tube or other vessel, or colonies from individual microwells can be selected and pipetted , for example, into individual wells of a 96 or 384 well plate.

[0126] Colony growth at the singulation node (and thus in the SWIIN module) can be monitored by automated devices such as those sold by JoVE (ScanLag ^™ System, Cambridge, MA) (see also Levin-Reisman, et al., Nature Methods 7:737-39 (2010)). Cell growth, for example for mammalian cells, can be monitored, for example, using a growth monitor sold by IncuCyte (Ann Arbor, MI) (see also Choudhry, PLos One, 11(2): e0148469 (2016)). In addition, automatic colony pickers such as those sold by, for example, TECAN (Pickolo ^™ system, Mannedorf, Switzerland) may be used; Hudson Inc. ( ^RapidPickTM , Springfield, New Jersey); Molecular Devices (QPix ^400TM system, San Jose, CA); and Singer Instruments ( ^PIXLTM system, Somerset, UK).

[0127] FIG. 4G depicts an embodiment of the singulation node 420a shown in FIG. 4F, in a bottom perspective view that represents the bottom of the permeable member 408 of the singulation assembly 420a. The permeable member 408 includes a generally smooth bottom surface (which faces upwards in FIG. 4G); two distribution channels (not shown), covered by a distribution channel cover 414, are located on both sides (left/right) of the bottom of the permeable element 408. Ridges 410a are also visible, which are located here on the upper surface of the permeable element 408 (which in Fig. 4G is facing down ) and cross the top of the permeable member 408 from right to left (in this embodiment, there are approximately 28 ridges 410a), with the flow guides 410 formed by the ridges 410a. In addition, the permeable member 408 has two openings 411 that supply and withdraw liquids to and from the distribution channels (the channels are not visible here because they are covered by the distribution channel covers 414).

[0128] In addition to the permeable element 404, in FIG. 4F also shows fasteners 412, a central composite layer containing a seal 416 surrounding a perforated element 401 crimped by a filter or membrane 403 (individual components not visible in FIG. 4G). The bottommost layer of the singulation node 420a in FIG. 4G is the holding element 404. It should be noted that in the embodiments shown in FIG. 4F-4CC showing fasteners 412; however, other means may be used to secure retainer 404, seal 416, perforated member 401, filter 403, and permeable member 408, such as adhesives such as pressure-sensitive adhesive, ultrasonic welding or bonding, solvent bonding, mating fittings, or combination of adhesives, welding, solvent bonding and mating fittings; and other such fasteners and connections.

[0129] FIG. 4H depicts an embodiment of the singulation node 420a shown in FIG. 4F and 4G, exploded side view in perspective. Seen from above in FIG. 4H is a retaining member 404 comprising a single distribution channel 405 that traverses the retaining member 404 from its top surface to its lower surface for most of the length of the retaining member 404 (details of which are described in connection with Figure 4I); ridges 406a that are located on the bottom surface of the retaining member 404 and cross the bottom surface of the retaining member 404 from left to right (there are approximately 26 ridges 406a in this embodiment), and flow guides 406 that are formed between the ridges 406a. In addition, the retaining member 404 has a single opening 407 that allows cells and other fluids to enter and be removed from the retaining member 404, and thus be introduced or removed from the singulation node 420a. There is also a cover 413 of the distribution channel, made with the ability to insert and close the single channel 405 of the distribution.

[0130] Seal 416, perforated element 401, and filter 403 can be seen more clearly in this exploded view of singulation assembly 420a, where perforated element 401 and filter 403 have very similar, if not identical, dimensions, and seal 416 is configured to surround the perforated element 401 and filter 403 and provide a positive seal between retainer 404 and permeable member 408. FIG. 4H, the breather 408 is shown in a plan view, where the breather 408 includes two distribution channels 409 along both sides (left and right) of the breather 408, ridges 410a, which here are located on the upper surface of the breather 408 and intersect the top of the breather element 408 from one side to the other (left to right) (in this embodiment, there are approximately 28 ridges 410a), and flow guides 410, which are formed between the ridges 410a in the permeable element 408. In addition, the permeable element 408 has two holes 411 (only one of which is visible in Fig. 4H), which supply liquids to the distribution channels 409, take them from there. Also visible are the distribution channel covers 414 that are not in place and do not cover the distribution channels 409 . Also in FIG. 4H, fasteners 412 are visible, although, again, other means for securing the components of the singulation assembly 420a can be and are preferably used for manufacturing singulation assemblies.

[0131] FIG. 4I is an enlarged perspective view of the top surface of the retainer 404, including opening 407 (here luer-locked from the fluid source to opening 407 in the retainer 404), a distribution channel 405 that traverses the retainer 404 from its upper surface to its lower surface. over most of the length of the retaining member 404 and is configured to supply (and remove fluid) to the flow guides 406 on the bottom surface of the retaining member 408. The distribution channel 405 includes flow guide openings 415 that correspond to the flow guides 406 because the flow guides 415 are located in channel 405 distribution between the ridges 406a of the retaining element. The cover 413 of the distribution channel is located on the upper surface of the retaining element 404, closing (and forming the upper surface) of the distribution channel 405.

[0132] FIG. 4J is an enlarged cross-sectional view of the singulation node 420a. On FIG. 4J, retention port 407 is seen (with a luer connection from fluid source to port 407) as well as seal 416, perforated member 401 and filter 403 hot stamped together and positioned between retainer member 404 and permeable member 408. Also visible ridges 406a, which are located on the lower surface of the retaining member 404; and flow guides 406, which are formed between the ridges 406a. In addition, the ridges 410a, the permeable member 408, as well as the flow guides 410 that are formed between the ridges 410a are visible. It should be noted that the ridges 406a on the retaining member 404 and the ridges 410a on the permeable member 408 meet and match each other, and are only separated by the perforated member 401 and the filter 403, so that the guides 406 and the flow guides 410 also match each other. Also visible are the flow guide holes 415 which correspond to the flow guides 406 as they are positioned between the ridges 406a of the retaining member. The ridges 406a on the retainer 404 and the ridges 410a on the vent 408 not only form the flow guides 406 and 410 in the retainer 404 and the vent 408, respectively, but also provide support to the singulation assembly 420 by distributing pressure throughout the singulation assembly 420 and preventing ruptures. filter or membrane 403.

[0133] FIG. 4K depicts another embodiment of the singulation node 420b in a top perspective view that represents the "retaining side" of the singulation node 420b. The embodiment of singulation node 420b shown in FIG. 4K is different from the singulation node 420a shown in FIG. 4F-4H in that the holding member 404 in FIG. 4K-4M has two distribution channels 405 located along both sides (left and right) of the holding element 404 instead of a single distribution channel 405 located in the middle of the holding element 404. As in FIG. 4F-4H holding element 404 shown in FIG. 4J-4M has a generally smooth top surface; two distribution channels 405 that traverse the retaining element 404 from its upper surface to its lower surface for a greater part of the length of the retaining element 404; and ridges 406a that are located on the bottom surface of the retainer 404 and traverse the bottom of the retainer 404 from one side to the other, from left to right (there are approximately 28 retainer ridges 406a in this embodiment). Flow guides 406 are formed between ridges 406a on retainer 404. In addition, retainer 404 has two openings 407 that allow cells and media to enter and exit singulation node 420b. There are also distribution channel covers 413 that cover the two distribution channels 405 and effectively provide the top surface of the distribution channels 405 .

[0134] In addition to the holding element 404, in FIG. 4 also K shows fasteners 412, a central composite layer containing a seal 416 surrounding a perforated element 401 located above the filter or membrane 403 and crimped with it (individual components not visible in Fig. 4K). The bottom layer of the singulation node 420a shown in FIG. 4F is formed by the permeable element 408. The permeable element 408, like the retaining element 404, includes one or more (two in FIG. 4L) distribution channels of the permeable element, a plurality of ridges and flow guides, and one or more holes (which are not shown in Fig. 4L). Fig. 4K but visible in Fig. 4L).

[0135] As previously described with reference to FIG. 4F-4H, in the solid wall singulation or substantial singulation, growth, edit induction and normalization or selection modules ("solid wall isolation/induction/normalization module" or "SWIIN") described in FIG. 4F-4CC, cells and media (at a dilution suitable for Poisson or substantially Poisson distribution of cells in the microwells of the perforated element) flow into the distribution channels 405 from the two holes 407 in the retention element 404 and the cells settle into the Poisson or substantially Poisson microwells. distribution of cells in microwells. The cells are retained in the microwells of the perforated element 401 because they cannot pass through the filter 403. A suitable medium is introduced into the singulation node 420b through the permeable element 408. This medium flows upward through the filter 403 to feed the cells. Thus, during operation, cells are placed in microwells, grown for initial, for example, approximately 2-100 doublings, editing is induced, for example, by raising the SWIIN temperature to 42°C to induce a temperature-inducible promoter, or by removing the growth medium from a permeable element and replacing the growth medium with a medium containing a chemical moiety that induces an inducible promoter. After editing, the SWIIN temperature can be reduced, or the induction medium can be removed and replaced with fresh medium containing no chemical component, thereby activating the inducible promoter. The cells are then allowed to continue growing in the SWIIN until the growth of cell colonies in the microwells returns to normal. As soon as the colonies are normalized, they are washed out of the microwells (by applying pressure to the channels of the permeable element and the flow guides, and thus to the filter) and pooled; alternatively, the growth of cell colonies in the microwells is monitored and slow growing colonies are selected directly, for example by pooling cells from slow growing colonies.

[0136] FIG. 4L depicts an embodiment of the singulation node 420b shown in FIG. 4K, in a bottom perspective view which represents the bottom of the permeable member 408 of the singulation assembly 420b. The permeable member 408 includes a generally smooth bottom surface (which faces upwards in FIG. 4L); two distribution channels (not shown) covered by distribution channel covers 414 (located on both sides (left and right) of the permeable member 408), ridges 410a that are located on the upper surface of the permeable member 408 (which is facing down in FIG. 4L) and intersect the top of the permeable member 408 from one side to the other (in this embodiment, there are approximately 28 ridges 410a), as well as the flow guides 410 that are formed between the ridges 410a. In addition, the permeable member 408 has two openings 411 which supply and withdraw liquids into and out of the distribution channels (the channels are not visible here because they are covered by the distribution channel covers 414) and then directed to the guides 410.

[0137] In addition to the permeable element 404, in FIG. Also visible in FIG. 4L are the fasteners 412, a central composite layer containing a seal 416 surrounding a perforated element 401 located above and crimped with the filter or membrane 403 (individual components not visible in FIG. 4L), and the lowest layer of the singulation assembly 420b in FIG. . 4L is the retaining member 404. Again, it should be noted that in the embodiments depicted in FIG. 4F-4CC showing fasteners 412; however, other means may be used to secure the retainer 408, the seal 416, the perforated member 401, the filter 403, and the permeable member 408, such as adhesives, ultrasonic welding or bonding, solvent bonding, mating fittings, or a combination of adhesives, welding, bonding with solvent and associated fittings; and other such fasteners and connections.

[0138] FIG. 4M depicts an embodiment of the singulation node 420b shown in FIG. 4K and 4L, exploded side view in perspective. From above in Fig. 4M shows a retaining element 404 comprising two distribution channels 405 located on both sides (left and right) of the retaining element 404, both of which intersect the retaining element 404 from its upper surface to its lower surface for most of the length of the retaining element 404; ridges 406a that are located on the bottom surface of the retaining member 404 and traverse the bottom surface of the retaining member 404 from one side to the other (there are approximately 28 ridges 406a in this embodiment), as well as flow guides 406 that are formed between the ridges 406a. In addition, the retaining member 404 has two openings 407 that allow cells and other fluids to enter and exit the singulation node 420a through the distribution channels 405 . There are also two distribution channel covers 413 that are designed to be inserted into and cover the two distribution channels 405 .

[0139] Seal 416, perforation 401, and filter 403 can be seen more clearly in this exploded view of singulation assembly 420b, where perforation 401 and filter 403 are very similar in size, and seal 416 is configured to surround perforation 401 and filter 403 and provide a positive seal between retaining member 404 and permeable member 408. FIG. 4M, the vent 408 is shown in a top view, where the vent 408 includes two distribution channels 409 along both sides (left and right) of the vent 408, both of which intersect the vent 408 from its lower surface to its upper surface for a large part of the length of the permeable member 408, ridges 410a, which here are located on the upper surface of the permeable member 408 and cross the top of the permeable member 408 from one side to the other, and flow guides 410, which are formed between the ridges 410a. In addition, the permeable member 408 has two openings 411 shown in FIG. 4M as openings on the retaining member 404 which, when the singulation assembly 420b is assembled, are in fluid communication with the distribution channels 409 on the permeable member 408, which are connected to the flow distributors 410, and thus to the filter 403 located on the permeable member 408. Also seen distribution channel covers 414, which are shown separately from the singulation assembly 420b in this exploded view and are not inserted into the permeable member 408 to cover the distribution channels 409. Also in FIG. 4M, holes for fasteners 412 are visible (fasteners not shown), although, again, means other than fasteners may be used to secure the components of the singulation assembly 420b.

[0140] FIG. 4N depicts another embodiment of the singulation node 420c in a top perspective view that represents the "retaining side" of the singulation node 420c. The embodiment of singulation node 420c shown in FIG. 4N is different from the singulation node 420a shown in FIG. 4F-4H in that the holding member 404 in FIG. 4N-4P has two distribution channels 405 located along both sides (left and right) of the retaining member 404 instead of a single distribution channel 405 located in the middle of the retaining member 404. The embodiment of the singulation assembly 420c shown in FIG. 4N is different from the singulation node 420b shown in FIG. 4K-4M, in that the configuration of the distribution channels 405 in the retaining member 404 (and the distribution channels 409 in the permeable member 408) in FIG. 4N-4P is different from the configuration in FIG. 4K-4M. Distribution channels 405 (and distribution channels 409) in FIG. 4N-4P are branched; that is, instead of opening 407 delivering liquids directly into distribution channel 405 at the point where opening 407 intersects with distribution channel 405, in the embodiment of the singulation assembly shown in FIG. 4N-4P, liquids flow into openings 407, then into branched distribution channels 405 on both sides (left and right) of containment member 407. The branched distribution channels have a first conduit that ends approximately halfway down the length of containment member 404 where the first conduit then branches into two secondary conduits, these two secondary conduits branch into four tertiary conduits, and these tertiary conduits supply liquids to the final conduit, which runs the entire length of the retaining element, evenly distributing the incoming liquids to the flow guides 406.

[0141] It should be noted that any configuration of distribution channels (e.g., distribution channels 405 on retainer 404 and distribution channels 409 on vent 408) can be used on the retention element 404 or vent 408, provided that the distribution channels properly distribute fluid to the guides 406 or 410 flow. As seen in FIG. 4F-4H, the distribution channels 405 on the retaining member 404 and the distribution channels 409 on the permeable member 408 may differ, or as seen in FIG. 4K-4M and FIG. 4N-4P, the distribution channels 405 on the retaining member 404 and the distribution channels 409 on the permeable member 408 may be the same. As shown in FIG. 4K-4M holding member 404 shown in FIG. 4N-4P has a generally smooth upper surface; two distribution channels 405, left and right of the retaining member 404, which traverse the retaining member 404 from its upper surface to its lower surface and branch, thus distributing liquid along the length of the retaining member 404.

[0142] The retainer 404 also includes ridges 406a that are located on the lower surface of the retainer 404 and cross the bottom of the retainer 404 from one side to the other. In addition, similarly to the previous embodiments, there are flow guides 406 that are formed between the ridges 406a on the holding member 404; and two openings 407 that introduce and dispense cells and media into (and remove cells and media from) retainer 404 of singulation node 420c. Also visible are the distribution channel covers 413 which cover the two branched distribution channels 405 on the holding member 404 and actually provide the upper surface of the branched distribution channels 405. It should be noted that the branching for distribution channels 405 is part of the holding element 404 in this embodiment; however, the retaining member 404 may not include ramifications, and the ramification may be present on the dispensing channel covers 413 that mate with the retaining member 404.

[0143] In addition to the holding member 404 in FIG. 4N, fasteners 412 are also visible, a central composite layer containing a seal 416 surrounding a perforated element 401 located above and crimped together with a filter or membrane 403. The bottom layer of the singulation node 420c shown in FIG. 4F is formed by the permeable member 408. The permeable member 408, like the containment member 404, includes one or more (two in FIG. shown in Fig. 4N but visible in Fig. 4O).

[0144] FIG. 4O depicts an embodiment of the singulation node 420c shown in FIG. 4N, in a bottom perspective view that represents the bottom of the permeable member 408 of the singulation assembly 420c. The permeable member 408 comprises a generally smooth bottom surface (which faces upwards in FIG. 4O); two branched distribution channels 409 closed by distribution channel covers 414, ridges 410a located on the upper surface of the permeable element 408 (which in Fig. 4O faces downward) and crossing the upper surface of the permeable element 408 from one side to the other, as well as guides 410 flow that are formed between the ridges 410a. In addition to this, the permeable element 408 has two holes 411, which supply liquids to the branched distribution channels 409 and to the flow guides 410. As is the case with the retaining member 404 and the distribution channel covers 413, the ramification for the distribution channels 409 in the permeable member 408 is part of the permeable member 408; however, the vent 408 may not contain ramifications, and the ramification may instead be present on the dispensing channel covers 414 that mate with the vent 408.

[0145] In addition to the permeable element 404, in FIG. Also visible in FIG. 4O are the fasteners 412, the central composite layer containing the seal 416 surrounding the perforated element 401 located above the filter or membrane 403 (individual components not visible in FIG. 4M), and the lowest layer of the singulation assembly 420a in FIG. 4O is the holding element 404.

[0146] FIG. 4P depicts an embodiment of the singulation node 420c shown in FIG. 4N and 4O, exploded side view in perspective. From above in Fig. 4P, the retainer 404 is visible, comprising two branched distribution channels 405 located on both sides (left and right) of the retainer 404, both of which intersect the retainer 404 from its upper surface to its lower surface, where the branches intersect most of the length of the retainer 404; ridges 406a that are located on the lower surface of the retaining member 404 and traverse the lower surface of the retaining member 404 from one side to the other; and flow guides 406 that are formed between the ridges 406a. In addition, the retaining member 404 has two openings 407 that are hydraulically connected to the branched distribution channels 405 and flow guides 406 and are configured to allow cells and other fluids to enter the singulation unit 420a, and to exit cells and other fluids from the singulation unit 420a . There are also distribution channel covers 413 which are designed to fit into and close the two branched distribution channels 405 .

[0147] The seal 416, the perforated element 401 and the filter 403 can be seen more clearly in this exploded view of the singulation assembly 420c, where the perforated element 401 and the filter 403 have very similar dimensions, and the seal 416 is configured to surround the perforated element 401 and the filter 403 and provide a positive seal between retaining member 404 and permeable member 408. FIG. 4P, the vent 408 is shown in plan view, where the vent 408 includes two branched distribution channels 409 along both sides (left and right) of the vent 408, both of which traverse the vent 408 from its lower surface to its upper surface and provide branched conduits for most of the length of the permeable element 408. The permeable element 408 further comprises ridges 410a, which in this case are located on the upper surface of the permeable element 408 and cross the top of the permeable element 408 from one side to the other, and flow guides 410, which are formed between the ridges 410a of the permeable element. In addition, the permeable element 408 has two holes 411 (only one hole 411 is visible in Fig. 4P). Also visible are the distribution channel covers 414, which are shown separately from the singulation node 420c in this exploded view and do not cover the branched distribution channels 409. Also in FIG. 4N, fasteners 412 are visible, although again, means other than fasteners may be used to secure the components of singulation assembly 420c.

[0148] FIG. 4Q is an enlarged plan view of the retaining member 404 shown in FIG. 4N and 4P. Visible are the ridges 406a that are located on the bottom surface of the retaining member 404 and traverse the bottom surface of the retaining member 404 from one side to the other, and the flow guides 406 that are formed between the ridges 406a. Additionally, the distribution channel 405 is visible, containing the flow guide holes configured to distribute liquid into the flow guides 406, where the distribution channel 405 is closed by the cover 413 of the distribution channel.

[0149] FIG. 4R is an enlarged cross-sectional view of the retainer 404, with ridges 406a and flow guides 406 that are formed between the ridges 406a. Also visible is the permeable member 408 with ridges 410a and flow guides 410 that are formed between the ridges 410a of the permeable member. Between the retaining member 404 and the permeable member 408 are the seal 416, the perforated member 401, and the filter 403. It should be noted that the ridges 406a on the retaining member 404 and the ridges 410a are congruent with each other, and are separated only by the perforated member 401 and the filter 403. As was previously described, ridges 406a and ridges 410a provide support to perforated element 401 and filter 403 and reduce the chance of filter 403 rupturing during, for example, cell loading or media change.

[0150] FIG. 4S-4W depict various views of one embodiment of a singulation or essential singulation, growth, edit induction, and normalization or solid wall selection (SWIIN) module 400. Fig. 4S shows a perspective view of the SWIIN module 400. SWIIN module 400 includes, for example, one of the exemplary singulation nodes shown in FIG. 4F-4H, 4K-4M and 4N-4P, which are placed in the SWIIN cover 440 and are one part of the SWIIN module. The various components of the SWIIN lid 440 include the reservoir lid 442, grip 441, windows 444 (six windows shown), feet 443, and bar code (or other identifying information) 445. It should be noted that in the embodiments depicted in FIGS. 4S-4Z, windows are round; however, one of ordinary skill in the art will appreciate that the windows may be of any shape. As a general rule, it is desirable that 30% or more of the retainer be viewable in order to obtain acceptable cell loading statistics. Also visible is the tank assembly cover 430a, which in this embodiment is not molded together with the SWIIN cover 440, but is inside the tank cover portion 442 of the SWIIN cover 440. The reservoir assembly cover 430a includes four reservoir access ports (432a, 432b, 432c, and 432d) as well as four pneumatic access ports (433a, 433b, 433c, and 433d). Pneumatic access openings 433a, 433b, 433c, and 433d include filters to prevent contamination in most embodiments.

[0151] Windows 444 can be used to track cell loading and/or cell growth using a camera (eg, video camera). For example, a video camera can be used to monitor cell growth, for example by measuring density change based on an image of an empty well using phase contrast, or if, for example, a chromogenic marker such as a chromogenic protein is used to add a distinct color to the cells. Chromogenic markers such as Blitzen Blue, Dreidel Turquoise, Virginia Violet, Vixen Purple, Prantzer Purple, Brilliant Purple, Maccabi Purple, Donner Magenta, Cupid Pink, Seraphina Pink, Rough Orange, and Leor Orange (ATUM Chromogenic Protein Dyes Set ( Newark, Calif.) eliminate the need for the use of fluorescence, although fluorescent cell markers, fluorescent proteins, and chemiluminescent cell markers can also be used.

[0152] FIG. 4T is a plan view of the SWIIN module 400 showing the SWIIN cover 440 and its various components, including the tank cover 442, gripper 441, windows 444 (six windows shown), legs 443, and bar code (or other identifying information) 445. The cover is also visible. 430a of the tank assembly located within the tank cover portion 442 of the SWIIN cover 440, where the tank assembly cover 430a includes four tank access holes (432a, 432b, 432c, and 432d) and four pneumatic access holes (433a, 433b, 433c, and 433d).

[0153] FIG. 4U is a side view of the SWIIN module 400 showing the SWIIN cover 440, reservoir cover 442, grip 441, and legs 443. FIG. 4V is a view from the barcode end of the 400 SWIIN module to the reservoir end of the 400 SWIIN module. The SWIIN cover 400, reservoir cover 442, gripper 441, and legs 443 are visible. FIG. 4W is a bottom perspective view of the SWIIN module 400 showing the SWIIN cover 440, the reservoir cover 442, the legs 443, and the distribution channel covers 414 located at the bottom of the permeable member 408.

[0154] FIG. 4X-4Z show the reservoir assembly 430. FIG. 4X the reservoir node 430 is located on the node 420 singulation containing the retaining element 404; covers 413 of the distribution channel; a seal, perforated element, and filter assembly (not shown in detail), and a permeable element (also not shown in detail). The tank assembly 430 includes a tank assembly cover 430a having four tank access holes (432a, 432b, 432c and 432d) and four pneumatic access holes (433a, 433b, 433c and 433d).

[0155] FIG. 4Y is a top, cross-sectional perspective view of the reservoir assembly 430 at the "reservoir" end of the SWIIN cover 440. The legs 443 of the 440 SWIIN cover are visible. Reservoirs 431a, 431b, 431c, and 431d are visible, as are reservoir/channel openings 434a, 434b, 434c, and 434d. Reservoirs 431b and 431c are fluidly connected to conduits that are fluidly coupled to distribution channels on the containment member or distribution channels on the permeable member, and reservoirs 431a and 431d are fluidly coupled to conduits that are fluidly coupled to distribution channels on the containment member or distribution channels on the permeable member. element; that is, if reservoirs 431b and 431c are in fluid communication with conduits that are in communication with distribution channels on the containment member, then reservoirs 431a and 431d are in fluid communication with conduits that are in communication with distribution channels on the permeable member. However, any reservoir may be configured to supply fluids and remove fluids to the retaining member 404 or permeable member 408. Reservoirs 431a, 431b, 431c, and 431d typically have a volume of 5.0 to 100 ml, or 7.5 to 60 ml, or 10 to 40 ml (for a singulation node with 200,000 perforations). It should be noted that reservoirs 431a, 431b, 431c, and 431d are funnel-shaped in the portion of the reservoir leading to reservoir/channel openings 434a, 434b, 434c, and 434d, and assist in the supply of liquids from reservoirs 431a, 431b, 431c, and 431d.

[0156] FIG. 4Z shows three different views of four connected tanks 435. The top drawing is a top view of the connected tanks 435, the middle drawing is a side view of the connected tanks 435, and the bottom drawing is a bottom view of the connected tanks 435. The tanks 431a, 431b, 431c and 431d are visible. in all drawings, and the tank/channel openings 434a, 434b, 434c and 434d are visible in the top and bottom drawings.

[0157] FIG. 4AA is an exemplary pneumatic block diagram suitable for the SWIIN module shown in FIG. 4S-4W and, for example, using the singulation nodes described with reference to FIG. 4F-4R. It should be noted that there are two solenoid valves (SV) for each tank - two retention tanks (RR1 and RR2) and two permeate tanks (PR1 and PR2) - where one of the valves is for pneumatics and one of the valves is for blocking the line . It should be noted that there is a rheometer for each pair of solenoid valves. In addition, in this embodiment, there is a single pipeline serving all the tanks; however, other embodiments may use two pipelines, where PR1 and RR1 are served by one pipeline, and PR2 and RR2 are served by another pipeline, or each tank (PR1, RR1, PR2, RR2) can be served by a separate pipeline.

Table 1. Valve states and pressure

Stage Description Stage Manifold/shoulder pump SV1-PR1 SV2-PR2 SV3-RR1 SV4-RR2 SV5-PR3 SV6-PR4 SV7-PR5 SV8-RR4 SV9-Pump "P" SV10-Pump "V" psi Loading the SWIIN module into the instrument one open 0 0 0 0 0 0 0 0 0 0 0 0 Loading 7.5 ml of cell suspension into RR1 and RR3 2 open 0 0 0 0 0 0 0 0 0 0 0 0 Filling the retention layer with cell suspension 3 closed one 0 0 one 0 0 0 one 0 0 0 0.5 Sedimentation of cells to a perforated sheet and into microwells four closed 0 0 0 0 0 0 0 0 0 0 0 0 The introduction of the medium into the layer of the retained substance through the membrane 5 closed one one one 0 0 one one 0 0 one one -5 Pipeline opening 6 open 0 0 0 0 0 0 0 0 0 0 0 0 Removing Excess Media from the PR Using a Pipettor 7 open 0 0 0 0 0 0 0 0 0 0 0 0 Loading 7.5 ml growth media into PR1 and PR3 eight open 0 0 0 0 0 0 0 0 0 0 0 0 Pipeline closure 9 closed 0 0 0 0 0 0 0 0 0 0 0 0 Flushing the permeate layer with growth medium from PR1 to PR2 and PR3 ten closed 0 0 0 0 0 0 0 0 0 0 0 0 Pipeline opening eleven open 0 0 0 0 0 0 0 0 0 0 0 0 Removing Excess Media from the PR Using a Pipettor 12 open 0 0 0 0 0 0 0 one 0 0 0 0.5 Loading 7.5 ml growth media into PR1 and PR3 13 open 0 0 0 0 0 0 0 0 0 0 0 0 Pipeline closure fourteen closed 0 0 0 0 0 0 one 0 0 one one -5 Flushing of the permeate layer with growth medium from PR1 to PR2 and from PR3 to PR4 by gravity fifteen closed 0 0 0 0 0 0 0 0 0 0 0 0 Pipeline opening 16 open 0 0 0 0 0 0 0 0 0 0 0 0 Removing Excess Media from the PR Using a Pipettor 17 open 0 0 0 0 0 0 0 0 0 0 0 0 Pipeline closure eighteen closed 0 0 0 0 0 0 0 0 0 0 0 0 Incubation at 30°C for 4.5 hours 19 closed one 0 0 one 0 0 0 one 0 0 0 0.25 Pipeline opening twenty open 0 0 0 0 0 0 0 0 0 0 0 0 Loading 7.5 ml growth media into PR1 and PR3 21 open 0 0 0 0 0 0 0 0 0 0 0 0 Pipeline closure 22 closed 0 0 0 0 0 0 0 0 0 0 0 0 Incubation at 30°C for 4.5 hours 23 closed one 0 0 one 0 0 0 one 0 0 0 0.25 Induction at 42°C for 2 hours 24 closed one 0 0 one 0 0 0 one 0 0 0 0.25 Incubation at 30°C for 9 hours 25 closed one 0 0 one 0 0 0 one 0 0 0 0.25 Cell aspiration in RR1 and RR2 26 closed one 0 0 one 0 0 one 0 0 one one -5 Pipeline opening 27 open one 0 0 0 0 0 0 0 0 0 0 0 Extraction of cells from RR1 and RR3 using a pipettor 28 open one 0 0 0 0 0 0 0 0 0 0 0 Removal of remaining medium from PR1, PR2, PR3 and PR4 29 open one 0 0 0 0 0 0 0 0 0 0 0

Table 2. Tank volumes

Stage description Stage RR1 and RR3 start/end RR2 and RR4 start/end PR1 and PR3 start/end PR2 and PR4 start/end Temperature (°C) Loading the SWIIN module into the instrument one 0/0 0/0 0/0 0/0 25 Loading 7.5 ml of cell suspension into RR1 and RR3 2 0/7.5 TBD/TBD 0/0 0/0 25 Filling the retention layer with cell suspension 3 7.5/0 TBD/TBD 0/0 0/0 25 Sedimentation of cells to a perforated sheet and into microwells four 0/0 TBD/TBD 0/0 0/0 four The introduction of the medium into the layer of the retained substance through the membrane 5 0/0 TBD/TBD 0/1 0/1 thirty Pipeline opening 6 0/0 TBD/TBD 1/1 1/1 thirty Removal of excess medium from the PR using a pipettor 7 0/0 TBD/TBD 1/0 1/0 thirty Loading 7.5 ml growth media into PR1 and PR3 eight 0/0 TBD/TBD 0/7.5 0/0 thirty Pipeline closure 9 0/0 TBD/TBD 7.5/7.5 0/0 thirty Flushing the permeate layer with growth medium from PR1 to PR2 and PR3 ten 0/0 TBD/TBD 7.5/3.75 0/3.75 thirty Pipeline opening eleven 0/0 TBD/TBD 3.75/3.75 3.75/3.75 thirty Removal of excess medium from the PR using a pipettor 12 0/0 TBD/TBD 3.75/0 3.75/0 thirty Loading 7.5 ml growth media into PR1 and PR3 13 0/0 TBD/TBD 0/7.5 0/0 thirty Pipeline closure fourteen 0/0 TBD/TBD 7.5/7.5 0/0 thirty Flushing of the permeate layer with growth medium from PR1 to PR2 and from PR3 to PR4 by gravity fifteen 0/0 TBD/TBD 7.5/3.75 0/3.75 thirty Pipeline opening 16 0/0 TBD/TBD 3.75/3.75 3.75/3.75 thirty Removal of excess medium from the PR using a pipettor 17 0/0 TBD/TBD 3.75/0 3.75/0 thirty Pipeline closure eighteen 0/0 TBD/TBD 0/0 0/0 thirty Incubation at 30°C for 4.5 hours 19 0/0 TBD/TBD 0/0 0/0 thirty Pipeline opening twenty 0/0 TBD/TBD 0/0 0/0 thirty Loading 7.5 ml growth media into PR1 and PR3 21 0/0 TBD/TBD 0/7.5 0/0 thirty Pipeline closure 22 0/0 TBD/TBD 7.5/3.75 0/3.75 thirty Incubation at 30°C for 4.5 hours 23 0/0 TBD/TBD 3.75/3.75 3.75/3.75 thirty Induction at 42°C for 2 hours 24 0/0 TBD/TBD 3.75/3.75 3.75/3.75 42 Incubation at 30°C for 9 hours 25 0/0 TBD/TBD 3.75/3.75 3.75/3.75 thirty Cell aspiration in RR1 and RR2 26 0/5 TBD/TBD 3.75/1.25 3.75/1.25 room Pipeline opening 27 5/5 TBD/TBD 1.25/1.25 1.25/1.25 room Extraction of cells from RR1 and RR3 using a pipettor 28 5/0 TBD/TBD 1.25/1.25 1.25/1.25 room Removal of remaining medium from PR1, PR2, PR3 and PR4 29 0/0 TBD/TBD 1.25/0 1.25/0 room

[0158] FIG. 4BB shows a two-layer SWIIN module 460, with a housing that contains two singulation nodes (the singulation nodes are not visible in FIG. 4AA). Fig. 4CC graphically depicts a four-layer SWIIN module 465 containing four singulation nodes 420. Fig. 4BB graphically depicts a Peltier device 466 and forced airflow fan 467 468 that can be used to maintain the desired temperature of the four singulation nodes 420; however, it should be noted that multilayer SWIINs may use a combination of systems such as Rankine reverse compression refrigeration or absorption heat pumps.

[0159] FIG. 4DD is a diagram of an exemplary pneumatic architecture for a two-layer SWIIN, showing, along with Tables 1 and 2, one embodiment of the pneumatics used to singulate or substantially singulate, grow, initiate editing, and normalize cells in the SWIIN module described with reference to FIG. 4BB. On FIG. 4DD shows four permeate reservoirs (PR1, PR2, PR3 and PR4) and four retention reservoirs (RR1, RR2, RR3 and RR4). There are four rheometers (FM1, FM2, FM3, and FM4), ten three-way solenoid valves ("3SV"), a pressure transducer, a pressure control reference valve, and a pump capable of delivering -5 to 5 psi. NC means "normally closed", NO means "normally open" and C means "closed". Table 1 for each step of the cell concentration process shows the status of each valve shown in FIG. 4CC and the pressure sensed by pressure sensors 1 and 2. In Table 1 for the pump, 1=on and 0=off. For solenoid valves 1=on and 0=off. Table 2 for each stage of the cell concentration process shows the volume in ml of fluid in each reservoir (ie, four retention reservoirs and four permeate reservoirs). Perforated element 401 is shown as well as temperature zone 499.

[0160] In addition to the Solid Walled Cell Singulation Device (SWIIN) described with reference to FIG. 3A-3E and 4A-4BB, other cell singulation (or essential singulation) devices may be used in a multi-module cell processing tool such as those described in US Pat. ”, filed September 24, 2018, and in US patent document No. 62/781112, entitled “Improved detection of nuclease-edited sequences in automated modules and systems”, filed December 18, 2018, which include singulation or significant singulation by plating on agar-agar, singulation or substantial singulation by isolating cells on functionalized islands, singulation or substantial singulation within aqueous droplets carried in a hydrophobic carrier fluid, or gel beads in emulsion (GEM, see e.g. 10X Genomics, Pleasanton, California), or singulation or significant singulation in Utripolymerized alginate scaffold (for this singulation embodiment, see also US Patent Document No. 62/769805, entitled "Improved Detection of Nuclease-Edited Sequences in Automated Modules and Tools by Bulk Cell Culture", filed November 20, 2018).

Automated Cell Editing Tools and Modules

Automated Cell Editing Tools

[0161] FIG. 5A depicts an exemplary automated multi-module cell processing tool 500 for, for example, performing one of the exemplary workflows described below, comprising one or more of the reagent cartridges described herein. Instrument 500, for example, can be and is preferably designed as a stand-alone benchtop instrument for use in a laboratory environment. Tool 500 may include a mixture of reusable and disposable components to perform various integrated processes when performing automatic genome digestion and/or editing in cells. Portal 502 is illustrated providing an automated mechanical movement system (actuator) (not shown) that provides XYZ movement control for, for example, an automated (i.e., robotic) fluid handling system 558, including, for example, an air displacement pipettor 532, which allows cells to be processed in multiple modules without human intervention. In some automated multi-module cell processing instruments, the air displacement pipettor 532 is moved by portal 502 while the various modules and reagent cartridges remain stationary; however, in other embodiments, the fluid handling system 558 may remain stationary while the various modules and reagent cartridges move. Also included in the automated multi-module cell processing tool 500 is a reagent cartridge 510 containing reservoirs 512 and a transformation module 530 (e.g., the electroporation flow device detailed with reference to FIGS. 8A-8E), as well as a wash cartridge 504 containing reservoirs 506. The wash cartridge 504 can be configured to accommodate large vials, such as wash solutions or solutions that are often used in an iterative process. In one example, flush cartridge 504 may be configured to remain in place when two or more reagent cartridges 510 are sequentially used and replaced. While the reagent cartridge 510 and flush cartridge 504 are shown in FIG. 5A as separate cartridges, the contents of the wash cartridge 504 may be included in the reagent cartridge 510. The reagent cartridge 510 and the flush cartridge 504 may be identical except for the consumables inserted into them (reagents or other components contained within different inserts). It should be noted that in this embodiment, the transformation module 530 is contained within the reagent cartridge 510; however, in alternative embodiments, transformation module 530 is contained within its own module, or may be part of another module, such as a growth module.

[0162] In some implementations, the wash and reagent cartridges 504 and 510 comprise disposable kits (one or more different inserts and reagents) provided for use in the automated multi-module cell processing/editing tool 500. For example, a user may open and install each of a reagent cartridge 510 and a wash cartridge 504 containing various desired inserts and reagents into the chassis of the automated multi-module cell editing tool 500 prior to activating cell processing.

[0163] Also in FIG. 5A illustrates an automated fluid handling system 558 including a gantry 502 and an air displacement pipettor 532. In some examples, the automated system 558 may include automated fluid handling systems manufactured by Tecan Group Ltd. of Mannedorf, Switzerland, by the Hamilton Company of Reno, Nevada (see, for example, patent document WO2018015544A1), or by Beckman Coulter, Inc. ., Fort Collins, Colorado (see, for example, US20160018427A1). Pipette tips may be provided in a tip kit (not shown) for use with the 532 air displacement pipettor.

[0164] The inserts or components of the flush cartridge and the reagent cartridge 504, 510 are in some implementations marked with a machine readable code (not shown), such as a bar code, for recognition by the automated system 558. For example, the robotic fluid handling system 558 may scan one or more inserts into each of the flush cartridge and the reagent cartridge 504, 510 to confirm the contents. In other implementations, machine-readable indicia may be applied to each of the wash and reagent cartridges 504, 510, and the processing system (not shown, but see element 526 in FIG. 5B) of the automated multi-module cell editing tool 500 may identify a map of stored materials on basis of these machine-readable characters. The exemplary automated multi-module cell processing tool 500 shown in FIG. 5A further includes a cell growing module 534. (All modules briefly mentioned here will be described in more detail below). In the embodiment illustrated in FIG. 5A, cell growth module 534 includes two cell growth flasks 518, 520 (described in more detail below with reference to FIGS. 6A-6D), as well as a cell concentration module 522 (described in detail with reference to FIGS. 7A-7H). In alternative embodiments, the cell concentration module 522 may be separate from the cell culture module 534, such as in a separate, dedicated module. Also as part of the automated multi-module cell processing tool 500 in FIG. 5A shows a singulation module 540 operated by, for example, a robotic fluid handling system 558 and an air displacement pipettor 532. Also seen is an optional nucleic acid assembly/desalting module 514 containing a reaction chamber or vial container (not shown) and a magnet 516 for purifying nucleic acids using, for example, Magnetic Solid Phase Reversible Immobilization (SPRI) beads (Applied Biological Materials Inc., Richmond, British Columbia The Cell Growth Module, Cell Concentration Module, Transformation Module, Reagent Cartridge, and Nucleic Acid Assembly Module are described in more detail below, and an exemplary singulation module is detailed above with reference to Figures 3A-3E and 4A-4BB.

[0165] FIG. 5B shows a plan view of the exemplary multi-module cell processing tool 500 shown in FIG. 5A. Cartridge-based starting materials (eg, in reagent cartridge 510), for example, can be located at designated locations on platform 502 of tool 500 for access by a robotic arm (not shown in this figure). As illustrated in FIG. 5B, the platform may include a spillway so that contaminants spilling, dripping, or overflowing from any of the tool modules 500 are contained within the spillway collar. In addition to the reagent cartridge 510, in FIG. 5B, the wash cartridge 504, the singulation module 540, and part of the growth module 534 are also visible. Also visible in this image is the touch screen 550, conversion module controls 538, electronics rack 536, and processing system 526.

[0166] FIG. 5C-5D illustrate side and front views, respectively, of a multi-module cell processing tool 500 comprising a chassis 590 for use in desktop versions of the automated multi-module cell editing tool 500. For example, chassis 590 may be approximately 24-48 inches wide, approximately 24-48 inches high, and approximately 24-48 inches deep. Chassis 590 can be, and preferably is, designed to accommodate all modules and consumables used in automated cell processing, as well as to perform all necessary processes without human intervention (i.e., chassis 590 is configured to provide an integrated stand-alone automated multi-module cell processing tool). The chassis 590 may be equipped with a robotic fluid handling system 558 to move materials between modules. As illustrated in FIG. 5C, chassis 590 includes a lid 552 having a handle 554 and a hinge 556a (hinges 556b and 556c visible in FIG. 5D) to raise the lid 552 and gain access to the inside of the chassis 590. A cooling grille 564 (FIG. 5C) allows air to pass through internal fan (not shown). In addition, the chassis 590 is raised by adjustable legs 570a, 570c (legs 570, 570b are shown in Fig. 5D). Adjustable legs 570a-570c, for example, may provide additional airflow under the chassis 590. A control button 566 in some embodiments provides one-button automatic start and/or stop of cell processing in the automated multi-module cell processing tool 500.

[0167] Within the chassis 590, in some implementations, the robotic fluid handling system 558 is located along the portal 502 above the flush cartridge 504 (the reagent cartridge 510 is not shown in these drawings). The control circuitry, fluid handling tubes, air pump controls, valves, thermal units (eg, heating and cooling units), and other controls are in some embodiments located under the chassis platform 590, in the control unit area 568. Also in FIG. 5C and 5D, a singulation device or module 540 is seen. Nucleic acid assembly module 514 containing magnet 516 is seen in FIG. 5D.

[0168] Although not illustrated, in some embodiments, the display screen may be located on the front surface of the chassis 590, such as covering part of the cover (eg, see display 550 in Fig. 5B). The display screen may provide the user with information about the processing status of the automated multi-module cell editing tool 500. In another example, the display screen may receive user input to perform cell processing.

Rotary cultivation module

[0169] FIG. 6A depicts one embodiment of a rotating growth flask 600 for use with the cell culture module described herein. The rotating growth vial 600 is an optically transparent container having an open end 604 for receiving liquid media and cells, a central region 606 that defines the main cell growth container, a tapering region 618 that defines at least one light path 610, a closed end 616, and a drive engagement mechanism 612. The rotating growth vial 600 has a central longitudinal axis 620 about which it rotates, and the light path 610 is generally perpendicular to the longitudinal axis of the vial. The first light path 610 is located in the lower compressed portion of the tapering region 618. Optionally, some embodiments of the rotating growth bottle 600 have a second light path 608 in the conical portion of the tapering region 618. Both light paths in this embodiment are located in that region of the rotating growth bottle which is constantly filled with cell culture (cells+growth media) and which is not affected by the speed of the growth flask. The first light path 610 is shorter than the second light path 608, allowing sensitive measurements of optical density (OD) values when cell culture OD values in the vial are at a high level (eg, later in the cell growth process), while the second the light path 608 allows sensitive measurements of OD values when the OD values of the cell culture in the vial are at a lower level (eg, earlier in the cell growth process).

[0170] The drive engagement mechanism 612 engages with a motor (not shown) to rotate the vial. In some embodiments, the motor drives the drive engagement mechanism 612 so that the rotating growth vial 600 rotates in one direction only, and in other embodiments, the rotating growth vial 600 rotates in the first direction for a first period of time or at a first frequency, then rotates in a second direction (i.e., in the opposite direction) for a second period of time or at a second periodicity, and this process can be repeated so that the rotating growth vial 600 (and the cell culture contained therein) is subjected to oscillatory motion. In addition, whether the culture is subject to fluctuations, and the frequency of these fluctuations can be set by the user. The first amount of time and the second amount of time may be the same or different from each other. 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, at an early stage of cell growth, the spinning growth flask 600 may oscillate at a first frequency (e.g., every 60 seconds), and then at a later stage of cell growth, the spinning growth flask 600 may oscillate at a second frequency (e.g., every 60 seconds). every second) different from the first periodicity.

[0171] Rotating growth vial 600 can be reusable or, preferably, disposable. In some embodiments, the rotating growth vial is disposable and is provided to the user pre-filled with growth medium and sealed with foil at the open end 604. The medium-filled, rotating growth vial so packaged may be part of a kit for use with a stand-alone cell culture device, or with a cell cultivation module, which is part of a multi-module automated cell processing system. In order to inject cells into a tube, the user only needs to pipette the desired volume of cells and inject it into the vial by piercing the foil with the tip of the pipette. The open end 604 may optionally include an elongated lip 602 for overlapping and interacting with the cell growing device. In automated systems, the rotating growth vial 600 may be labeled with a barcode or other identification means that can be read by a scanner or camera (not shown) that is part of the automated system.

[0172] The volume of the spinner 600 for growth and the volume of the cell culture (including the growth medium) can vary greatly, but the volume of the spinner 600 for growth must be large enough to produce a given total number of cells. In practice, the volume of the rotating growth vial 600 may be 1-250 ml, 2-100 ml, 5-80 ml, 10-50 ml, or 12-35 ml. Likewise, the volume of cell culture (cells+growth media) must be appropriate in order to ensure proper aeration and mixing in the rotating culture flask 600. Proper aeration promotes uniform cellular respiration in the culture medium. Thus, the cell culture volume should be approximately 5-85% of the volume of the growth flask, or 20-60% of the volume of the growth flask. For example, for a 30 ml vial, the cell culture volume may be from about 1.5 ml to about 26 ml, or from 6 ml to about 18 ml.

[0173] The rotating growth vial 600 is preferably made from a biocompatible optically transparent material, or at least the part of the vial that contains the light path(s) is transparent. In addition, the material from which the rotating growth flask is made must be able to be cooled to about 4°C or less and heated to about 55°C or more to allow for both temperature-based cell analysis and long-term storage at low temperatures. In addition, the material used to make the vial must be able to withstand temperatures up to 55°C without deformation during rotation. Suitable materials include cyclic olefin copolymer (COC), glass, polyvinyl chloride, polyethylene, polyamide, polypropylene, polycarbonate, polymethyl methacrylate (PMMA), polysulfone, polyurethane, as well as copolymers of these and other polymers. Preferred materials include polypropylene, polycarbonate or polystyrene. In some embodiments, the rotating growth vial is inexpensively manufactured, such as by injection molding or extrusion.

[0174] FIG. 6B is a perspective view of one embodiment of a cell growing apparatus 630. Fig. 6C is a sectional view of the cell culture apparatus 630 shown in FIG. 6b. In both drawings, the rotating growth bottle 600 is located within the main body 636 with the extended lip 602 of the rotating growth bottle 600 extending above the main body 636. In addition, both drawings show the end shrouds 652, the lower body 632, and the flanges 634. Flanges 634 are used for attaching the cell growth device 630 to a heating/cooling means or other structure (not shown). Fig. 6C shows additional details. On FIG. 6C shows an upper bearing 642 and a lower bearing 640 located inside the main body 636. The upper bearing 642 and the lower bearing 640 support the vertical load of the rotating growth bottle 600. The lower housing 632 includes a drive motor 638. The cell culture apparatus 630 shown in FIG. 6C includes two light paths: a primary light path 644 and a secondary light path 650. Light path 644 corresponds to light path 610 located in the compressed portion of the tapered portion of the rotating growth vial 600, and light path 650 corresponds to the light path 608 in the tapered portion of the tapered portion. rotating vial 6001 for cultivation. Light paths 610 and 608 are not shown in FIG. 6C but visible in FIG. 6A. In addition to the light paths 644 and 640, there is an emission board 648 for illuminating the light path(s) and a detector board 646 for detecting light after it has passed through the cell culture liquid in the rotating growth flask 600.

[0175] The motor 638 is engaged with the drive mechanism 612 and is used to rotate the rotating growth bottle 600. In some embodiments, motor 638 is a brushless DC motor with built-in controls that can be tuned to maintain a constant rotational speed from 0 to about 3000 rpm. Alternatively, other types of motors may be used, such as a stepper motor, a servo motor, a brushed DC motor, and the like. Optionally, the 638 motor can also be provided with direction control for reversing, as well as a tachometer to measure and report actual rotational speed. The motor is controlled by a processor (not shown) in accordance with, for example, standard protocols programmed into the processor and/or data entered by the user, and the motor can be configured to change the speed of rotation to cause axial precession of the cell culture, thereby improving mixing. , for example, to prevent cell clumping, increase aeration and optimize cellular respiration.

[0176] The main body 636, end shrouds 652, and bottom body 632 of the cell growth apparatus 630 may be made from any suitable rigid material, including aluminum, stainless steel, and other thermally conductive materials, including plastics. These structures, or parts of them, can be created using various techniques, such as metalworking, injection molding, the creation of structural layers and their subsequent fusion, etc. While in some embodiments, the rotating culture vial 600 is contemplated to be reusable, but preferably consumable, other components of the cell culture apparatus 630 are preferably reusable and function as a stand-alone desktop device or as a module in a multi-module cell processing system.

[0177] The processor (not shown) of the cell culture apparatus 630 may be programmed with information used as a "blank" or control for the cell culture being grown. A "blank" or control is a vessel containing only cell growth medium, which gives 100% transparency and zero OD, while a sample with cells will deflect light and have lower transparency and higher OD. As the cells grow in the medium and become denser, the transparency will decrease and the OD will increase. The processor (not shown) of cell growth apparatus 630 can be programmed to use control wavelengths appropriate for growth media commonly used for a particular cell culture (e.g., mammalian cells, bacterial cells, animal cells, yeast cells, and etc.). Alternatively, a second spectrophotometer and a vial may be included in the cell culture apparatus 630, where the second spectrophotometer is used to read a blank at predetermined intervals.

[0178] FIG. 6D illustrates a cell growing apparatus 630 as part of an assembly containing the cell growing apparatus 630 shown in FIG. 6B connected to a light source 690, a detector 692, and thermal components 694. The rotating culture vial 600 is inserted into the cell culture apparatus. Light source 690 and detector 692 components (eg, such as a gain controlled photodiode for 5-log coverage) are attached to the main body of the cell culture apparatus. Shown is the lower housing 632, which houses the motor that rotates the rotating growth vial 600, as well as one of the flanges 634, which secures the cell growth apparatus 630 to the assembly. In addition, thermal components 694 are shown, representing a Peltier device or a thermoelectric refrigerator. In this embodiment, thermal control is accomplished by connecting and electrically integrating cell growth apparatus 630 with thermal components 694 via flange 634 on base of bottom housing 632. Thermoelectric refrigerators are capable of "pumping" heat to either side of the connection, cooling or heating the surface depending on the direction of the electrical current. . In one embodiment, a thermistor is used to measure the temperature of the main body, and then using a standard electronic proportional-integral-derivative (PID) controller circuit, the temperature of the rotating growth vial 600 is controlled to an accuracy of approximately +/-0.5°C.

[0179] In use, the cells are inoculated (they can be pipetted, for example, from an automated fluid handling system or by the user) into a pre-filled growth medium 600 rotating flask by piercing the foil or film. The software of the cell culture apparatus 630 sets the control temperature for growth, typically 30° C., and then slowly rotates the rotating culture vial 600. The cell/media mixture slowly moves vertically up the wall due to centrifugal force, allowing the rotating growth flask 600 to expose a large surface area of the mixture to the normal oxygen environment. The growth monitoring system takes OD readings continuously or at predetermined or pre-programmed intervals. These measurements are stored internally and, if necessary, the software plots the measurements over time to display the growth curve. If improved mixing is required, for example to optimize growth conditions, the speed of rotation of the vial may be varied to cause axial precession of the liquid and/or reversal of rotation may be performed at programmed intervals. Growth monitoring can be programmed to automatically end the growth stage at a predetermined OD and then rapidly cool the mixture to a lower temperature to inhibit further growth.

[0180] One application of the cell culture device 630 is to continuously measure the absorbance of a growing cell culture. One advantage of the described cell culture apparatus is that optical density can be measured continuously (kinetic monitoring) or at specific time intervals; for example, 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 culture device 630 has been described in the context of measuring the optical density (OD) of a growing cell culture, one of skill in the art will appreciate from the present disclosure that other cell growth parameters may be measured in addition to the cell culture OD or instead of her. As with the optional measure of cell growth in connection with the solid wall device or module described above, spectroscopy using visible, UV, or near infrared (NIR) light allows monitoring of nutrient and/or waste concentrations in cell culture and other spectral measurements; that is, other spectral properties can be measured, for example, using dielectric impedance spectroscopy, visible fluorescence, fluorescence polarization or luminescence. In addition, the cell culture device 630 may include additional sensors to measure, for example, dissolved oxygen, carbon dioxide, pH value, electrical conductivity, and the like.

Cell Concentration Module

[0181] In the automated multi-module cell processing tool shown in FIG. 5A-5D, a cell concentration module 522 is included. Fig. 7A-7H depict variations on one embodiment of a cell concentration/buffer exchange module that uses tangential flow filtration. The cell concentration module described herein operates using tangential flow filtration (TFF), also known as cross flow filtration, in which most of the nutrition flows tangentially across the filter surface, thereby reducing the formation of sediment (retained matter) compared to stagnant flow. filtration, in which power is supplied to the filter. Secondary flows to the main feed are also used to create shear forces that prevent filter cake formation and membrane fouling, thus maximizing particle recovery, as described below.

[0182] The TFF device described herein was designed with two main design considerations in mind. First, the geometry of the TFF device results in cell culture filtration over a large surface area to minimize processing time. Second, the design of the TFF device is designed to minimize filter clogging. Fig. 7A shows a general tangential flow filtration model 750. The TFF device operates using tangential flow filtration, also known as cross flow filtration. Fig. 7A shows cells flowing over a membrane 754 where the feed flow of the cells 752 in medium or buffer is parallel to the membrane 754. TFF differs from no-flow filtration where both feed flow and pressure drop are perpendicular to the membrane or filter.

[0183] FIG. 7B depicts a plan view of the lower element 720 of one embodiment of an exemplary TFF device/module providing tangential flow filtration. As can be seen in FIG. 7B, the bottom element 720 of the TFF device/module includes a channel structure 716 containing a flow channel through which the cell culture flows. Channel structure 716 contains a single flow channel 702b. (Note that the flow channel as a whole is labeled 702, the part of the flow channel in the upper element 722 of the TFF device is labeled 702a, and the part of the flow channel in the lower element 720 of the TFF device is labeled 702b). This particular embodiment comprises a channel configuration 714, for example, having an undulating serpentine geometry (i.e., small "fluctuations" in the flow channel 702) and a serpentine "zigzag" pattern in which the flow channel 702b intersects the bottom element 720 of the TFF device from one end to the left part of the device to the other end on the right side of the device. This serpentine pattern provides filtration over a large surface area relative to device size and total channel volume, while the undulation creates a secondary inertial flow that ensures effective membrane regeneration and prevents clogging. Although the undulating geometry is illustrated here and the serpentine pattern is illustrated here, other channel configurations 714 may be used if the flow channel 702 can be separated by a membrane, as discussed below, and if the channel configuration 714 allows cells to flow through the TFF module in alternate directions. Portals 704 and 706 are part of the channel structure 716 due to the work of the cells passing through the flow channel 702. Typically, portals 704 collect cells passing through a flow channel 702 on one side of the membrane (not shown) ("retained matter") and portals 706 collect media ("filtrate" or "permeate") passing through a flow channel 702 on the opposite side of the membrane. (not shown). In this embodiment, recesses 708 receive screws or other fasteners (not shown) that allow components of the TFF device to be attached to each other.

[0184] The length 710 and width 712 of the channel structure 716 may vary depending on the volume of the cell culture being grown and the optical density of the cell culture being concentrated. The length 710 of the channel structure 716 is typically 1 mm to 300 mm, or 50 mm to 250 mm, or 60 mm to 200 mm, or 70 mm to 150 mm, or 80 mm to 100 mm. The width 712 of the channel structure 716 is typically 1 mm to 120 mm, or 20 mm to 100 mm, or 30 mm to 80 mm, or 40 mm to 70 mm, or 50 mm to 60 mm. The cross-sectional configuration of the flow channel 702 may be circular, elliptical, oval, square, rectangular, trapezoidal, or irregular. If the cross section is square, rectangular, or otherwise shaped with straight sides, it may have a width of about 10 µm to 1000 µm, or 200 µm to 800 µm, or 300 µm to 700 µm, or 400 µm to 600 µm; and a height of about 10 µm to 1000 µm, or 200 µm to 800 µm, or 300 µm to 700 µm, or 400 µm to 600 µm. If the cross section of the flow channel 702 is generally circular, oval, or elliptical, the hydraulic radius of the channel may be from about 50 microns to 1000 microns, or from 5 microns to 800 microns, or from 200 microns to 700 microns, or from 300 microns to 600 microns. microns, or from about 200 to 500 microns.

[0185] In a plan view of the bottom element 720 of the TFF device/module in FIG. 7B, it can be seen that there are two retention portals 704 and two filtrate portals 706, with one portal of each type at both ends (eg, narrow edges) of the TFF device/module 700. In other embodiments, the retention and permeate portals may be on the same surface of the same element (eg, top element 722 or bottom element 720), or they may be located on the side surfaces of the assembly. Unlike other tangential flow filter devices that operate continuously, the TFF device/module described herein uses a variable flow method to concentrate cells. Fig. 7C is a plan view of the top (722) and bottom (720) members of an exemplary TFF module 700. The bottom of the flow channel 702b is visible on the top surface of the bottom member 720. Portals 704 and 706 are also visible. As noted above, recesses such as recesses 708 in FIG. 7B provide a means of securing the components (top member 722, bottom member 720, and membrane 724) of the TFF device/membrane 700 to each other during operation, by means of, for example, screws or other similar fasteners. However, in alternative embodiments, an adhesive, such as pressure-sensitive adhesive or ultrasonic welding or solvent bonding, may be used to join the upper member 722, the lower member 720, and the membrane 724 together. Indeed, those skilled in the art, having read this disclosure, may find other configurations for connecting the components of the TFF device 700, such as, for example, clips; mating fittings located on the upper (722) and lower (720) elements; a combination of adhesive, welding, solvent bonding and mating fittings; and other similar fasteners and connections.

[0186] It should be noted that in FIG. 7C shows one retention portal 704 and one filtrate portal 706 at each "end" (eg, narrow edges) of the TFF device/module 700. Retentate portals 704 and filtrate portals 706 on the left side of the TFF device/module 700 will collect cells (flow 760 from top element 722) and media (flow 770 from bottom element 720), respectively, as cells and vehicle flow from right to left in module 700 TFF. Similarly, retention portals 704 and filtrate portals 706 on the right side of the TFF device/module 700 will collect cells (stream 760) and media (stream 770), respectively, as cells and vehicle flow from left to right in the TFF device. In this embodiment, the retained material is collected from portals 704 on the top surface of the TFF device and the filtrate is collected from portals 706 on the bottom surface of the device. The cells are supported in the TFF flow channel 702a (shown in Fig. 7D) above the membrane 724 while the filtrate (medium) flows through the membrane 724 and then through the filtrate portals 706; thus, a configuration with upper retention portals 704 and lower filtrate portals 706 is practical. However, it should be understood that other configurations of retentate portals 704 and filtrate portals 706 may be implemented, such as placing both retentate portals 704 and filtrate portals 706 on the side surface (as opposed to the top and bottom surfaces) of the TFF device 700. On FIG. 7C, flow channel 702b is visible on bottom member 720 of TFF device 700. However, in other embodiments, the retention portals 704 and filtrate portals 706 may be on the same surface of the TFF device.

[0187] The general workflow for cell concentration using the TFF device/module 700 involves passing the cell culture or cell sample tangentially through the channel structure 716. The membrane 724 separating the flow channels 702 keeps the cells on one side of the membrane and allows undesired media or buffer flow through the membrane to the filtrate side (eg, lower element 720) of the TFF device 700. In this process, a fixed volume of cells in media or buffer is passed through the device until a cell sample is collected in one of the retention portals 704 and media/buffer that has passed through the membrane is collected in one or both of the portals. 706 for filtrate. All types of prokaryotic and eukaryotic cells - both adherent and non-adherent - can be concentrated in the TFF device. Adhering cells can be grown on beads or other cell scaffolds suspended in medium in a rotating growth flask and then passed through a TFF device.

[0188] In the cell concentration process, passing a cell sample through the TFF device and collecting cells in one of the retention portals 404 while collecting media in one of the filtrate portals 706 is considered "one pass" of the cell sample. The transfer between retention tanks "flips" the culture. The retentate and filtrate portals collecting cells and media, respectively, for a given passage are located at one end of the 700 TFF device/module with fluid connections arranged so that there are two separate flow layers (not shown) for the retentate and filtrate sides, but if the retention portal 404 is on the upper member 722 of the TFF device/module 700 (i.e., the cells move through the flow channel 702a (not shown) above the membrane and the filtrate (media) passes into the flow channel 702b part below the membrane), the portal 706 for the filtrate will be on the bottom element of the TFF device/module 700, and vice versa (i.e. if the cell sample passes through the flow channel 702b under the membrane, then the filtrate (medium) passes into the part of the flow channel 702a above the membrane). This configuration is shown more clearly in FIG. 7C-7D, where the retentate flow path 760 passes through the retentate portals 704 and the permeate flow path 770 passes through the permeate portals 706.

[0189] At the end of the "passage", a cell sample is collected as it passes through the retention portal 704 into a retention reservoir (not shown). To initiate another "pass", the cell sample is again passed through the TFF device 700, this time with the opposite direction of flow compared to the first pass. The cell sample is collected as it passes through the retention portal 704 into a retention reservoir (not shown) at the opposite end of the TFF device/module 700 from the retention portal 704 that was used to collect the cells during the first pass. Similarly, media/buffer that passes through the membrane in the second pass is collected through the filtrate portal 706 at the opposite end of the TFF device/module 700 from the filtrate portal 706 that was used to collect the filtrate during the first pass, or through both portals. This alternating process of passing the retained material (concentrated cell sample) through the TFF device/module 700 is repeated until the cells are concentrated to the desired volume and both filtrate portals 706 can be kept open during passes to reduce run time. Alternatively, buffer exchange can be done by adding the desired buffer (or fresh medium) to the cell sample in the retention reservoir prior to starting the next "pass" and repeating this process until the old medium or buffer has been diluted and filtered. and the cells will not be in fresh medium or buffer. It should be noted that buffer exchange and cell concentration can be (and usually are) performed simultaneously.

[0190] In FIG. 7C also shows the membrane or filter 724. Filters or membranes suitable for use in the 700 TFF device/module must be solvent resistant, free of contaminants during filtration, and able to retain cell types and sizes of interest. For example, to retain small cells such as bacterial cells, pore sizes may be as small as 0.2 µm, however, for other cell types, pore sizes may be 5 µm. In fact, pore sizes useful in the TFF device/module 700 include filters 724 with pore sizes of 0.20 µm, 0.21 µm, 0.22 µm, 0.23 µm, 0.24 µm, 0, 25 µm, 0.26 µm, 0.27 µm, 0.28 µm, 0.29 µm, 0.30 µm, 0.31 µm, 0.32 µm, 0.33 µm, 0.34 µm, 0, 35 µm, 0.36 µm, 0.37 µm, 0.38 µm, 0.39 µm, 0.40 µm, 0.41 µm, 0.42 µm, 0.43 µm, 0.44 µm, 0, 45 µm, 0.46 µm, 0.47 µm, 0.48 µm, 0.49 µm, 0.50 µm or more. Filters 724 can be made from any suitable inert material, including mixed cellulose ether (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 700 shown in FIG. 7C and 7D does not show a seat in the top 722 and bottom 720 elements where the filter 724 can be mounted or secured (eg, a half-thickness seat of the filter 724 in each of the top 722 and bottom 720 elements); however, such a socket is provided in some embodiments.

[0191] FIG. 7D is a bottom view of the top element 722 and bottom element 720 of the exemplary TFF module shown in FIG. 7C. Portals 704 and 706 are also visible. Note that there is one retainer portal 704 and one filtrate portal 706 at each end of the top member 722 and bottom member 720 of the TFF device/module 700 (not all portals are visible in this figure). On the left side of the TFF device 700, retentate portals 704 will collect cells (stream 760) and filtrate portals 706 will collect media (stream 770), respectively, for the same passage. Similarly, on the right side of the TFF device 700, retentate portals 704 will collect cells (stream 760) and filtrate portals 706 will collect media (stream 770), respectively, for the same passage. On FIG. 7D, the top of the flow channel 702a is visible on the bottom surface of the top member 722 of the TFF device 700. Thus, in FIG. 7C and 7D, there is a flow channel 702 in both the upper element 722 (flow channel 702a in FIG. 7D) and the lower element 722 (flow channel 702b in FIG. 7C) with membrane 724 between the upper 722 and lower 720 elements. Again, the flow channels 702a and 702b of the top 722 and bottom 720 elements mate to create a flow channel 702 with a membrane 724 positioned horizontally between the top and bottom elements of the TFF device/module, thereby separating the flow channel 702 (not shown).

[0192] Buffer exchange during cell concentration and/or cell competency is performed on the TFF device/module 700 by adding the desired buffer to the cells concentrated to the desired volume; for example, after the cells have been concentrated at least 20, 30, 40, 50, 60, 70, 80, 90, 100, 150, 200 times or more. The desired exchange medium or exchange buffer is added to the cells by adding to a retention reservoir (not shown) and the process of passing the cells through the TFF device 700 is repeated until the cells are concentrated to the desired volume in the exchange medium or buffer. This process can be repeated any desired number of times in order to achieve the desired level of buffer exchange and the desired cell volume. The exchange buffer may contain, for example, glycerol or sorbitol, thereby rendering the cells competent for transformation in addition to reducing the overall volume of the cell sample.

[0193] The 700 TFF can be made from any durable material in which flow channels can be milled, including stainless steel, silicon, glass, aluminum, or plastics, including cyclic olefin copolymer (COC), cycloolefin polymer (COP), polystyrene , polyvinyl chloride, polyamide, polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate, polyether ether ketone (PEEK), poly(methyl methacrylate) (PMMA), polysulfone and polyurethane, as well as copolymers of these and other polymers. If the TFF device/module 700 is disposable, it is preferably made from plastic. In some embodiments, the material used to make the TFF device/module 700 is thermally conductive so that the cell culture can be heated or cooled to the desired temperature. In some embodiments, the TFF device 700 is formed by precision machining, laser machining, EDM (for metal devices); wet or dry etching (for silicon devices); dry or wet etching, powder or sandblasting, photostructuring (for glass products); or thermoforming, injection molding, hot stamping or laser processing (for plastic devices) using the materials mentioned above, which are amenable to these mass production methods.

[0194] FIG. 7E is an exploded perspective view of one exemplary embodiment of a TFF module in fluid communication with the retention, filtrate, and exchange buffer reservoirs. In this configuration, 790 indicates the top or lid of the TFF device having three holes 766 where the pipette tip 768 is located in the leftmost hole 766. The top 790 of the TFF device is in operation associated with the combined reservoir and top member structure 764. The combined reservoir and top member structure 764 comprises a top surface that, in operation, is adjacent to the top or cover 790 of the TFF device, a bottom surface that contains the top member 722 of the TFF device, where the top member 722 of the TFF device defines the top of the tangential flow channel (not shown). The combined structure 764 reservoir and upper element contains two reservoirs 772 for retained substances and reservoir 774 for the buffer or environment. Retentate reservoirs 772 are fluidly coupled to the top of the flow channel, and buffer or media reservoir 774 is fluidly coupled to retained fluid reservoirs 772. Also visible in this exploded view of the TFF device is the lower element 720, which, as previously described, comprises on its upper surface the lower part of the tangential flow channel 702b (seen on the upper surface of the lower element 720), where the upper and lower parts of the upper element flow channel 702 722 and lower member 720, respectively, form a single flow channel 702 when connected (the membrane that is located between the upper member 722 and the lower member 720 in operation is not shown).

[0195] Beneath the lower element 720 is a seal 792 which, in operation, is positioned between the lower element 720 and the filtrate (or permeate) reservoir 762. In operation, the top 790, the combined reservoir and top member structure 764, the membrane (not shown), the bottom member 720, the seal 792, and the filtrate reservoir 762 are connected and secured together so as to be liquid and air tight. On FIG. 7E shows fasteners that can be used to connect various structures (top 790, combined reservoir and top element structure 764, membrane (not shown), bottom element 720, seal 792, and filtrate reservoir 762) together. However, as an alternative to screws or other similar fasteners, various designs of the TFF device may be connected using an adhesive such as a pressure-sensitive adhesive; ultrasonic welding; or compounds with a solvent. In addition, a combination of fasteners, adhesives, and/or different types of welding can be used to bind different structures of the TFF device. One skilled in the art, having read this disclosure, may find other configurations for connecting the components of a TFF device, such as, for example, clips; mating fittings and other similar fasteners.

[0196] FIG. 7F depicts a combined reservoir and top element structure 764 comprising two retainer reservoirs 772 and a buffer or media reservoir 774, as well as a top element 722 that is located below the combined reservoir and top element structure 764. The upper element 722 of the TFF device defines the upper part of the tangential flow channel (not shown) located on the lower surface of the combined structure 764 of the reservoir and the upper element. Fig. 7G is a plan view of the top surface 778 of the combined reservoir and top member structure 764 showing the top 780 of the retainer reservoirs 772 and the top 782 of the buffer or media reservoir 774. Retentate reservoirs 772 are fluidly coupled to the top of a flow channel (not shown) and buffer or media reservoir 774 is fluidly coupled to retention reservoirs 772. Fig. 7H is a bottom view of the bottom surface of the combined reservoir and top member structure 764 showing the top member 722 with the top of the tangential flow channel 702a located on the bottom surface of the top member 722. The flow channel 702a located on the bottom surface of the top member 722 is mated in operation with the bottom of the tangential flow channel 702b located on the top surface of the bottom member 720 (not shown in this figure, see FIG. 7E), where the top and bottom of the flow channels 702a and 702b, respectively, mate to form a single flow channel 702 with a membrane or filter (not shown) between the top 702a and bottom 702b of the flow channel.

Nucleic acid assembly module

[0197] Some embodiments of the automated multi-module cell editing tools of the present invention optionally include a nucleic acid assembly module. The nucleic acid assembly module is configured to receive and assemble the nucleic acids necessary to facilitate the desired genome editing events. The term "vector" generally refers to a nucleic acid molecule capable of transporting into a cell the desired nucleic acid to which it is associated. 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 have no free ends (eg, cyclic); nucleic acid molecules that include DNA, RNA, or both; as well as other varieties of polynucleotides known in the art. One type of vector is a "plasmid", which refers to a circular double-stranded DNA into which additional DNA segments can be inserted, for example, using standard molecular cloning techniques. Another type of vector is a viral vector, in which DNA or RNA sequences of viral origin are present in the vector for packaging into a virus (eg, 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. Some vectors are capable of autonomous replication in the host cell into which they are introduced (eg, bacterial vectors having a bacterial origin of replication and mammalian episomal vectors). Other vectors (eg, non-episomal mammalian vectors) integrate into the genome of a host cell when introduced into it, and thereby replicate along with the host genome. In addition, some 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". General expression vectors useful in recombinant DNA techniques are often in the form of plasmids. Additional vectors include fosmids, phagemids and synthetic chromosomes.

[0198] Recombinant expression vectors may include a nucleic acid in a form suitable for transcription, and for some nucleic acid sequences, for translation and expression of the nucleic acid in a host cell, which means that recombinant expression vectors include one or more regulatory elements, which can be selected based on the host cells used for expression, which are operably linked to the expressed nucleic acid sequence. Within a recombinant expression vector, "operably linked" means that the nucleotide sequence of interest is linked to the regulatory element(s) in a manner that allows 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 the host cell when the vector is introduced into the host cell). Suitable recombination and cloning methods are disclosed in US Publication No. 2004/0171156, the contents of which are incorporated herein by reference in its entirety.

[0199] In some embodiments, the regulatory element is operably linked to one or more elements of the target nuclease system to direct transcription, and for some nucleic acid sequences, to translate and express one or more components of the target nuclease system.

[0200] In addition, a polynucleotide sequence encoding a nucleic acid-directed nuclease can be codon optimized for expression in specific cells, such as prokaryotic or eukaryotic cells. Eukaryotic cells can be yeast, fungal, algae, plant, animal, or human cells. Eukaryotic cells can be cells or derived from a particular organism, such as a mammal, including, but not limited to, a human, mouse, rat, rabbit, dog, or non-human mammal, including non-human primates. Additionally or alternatively, the vector may include a regulatory element operably linked to a polynucleotide sequence that, when transcribed, forms a guide RNA.

[0201] The nucleic acid assembly module can be configured to perform a wide variety of different automated nucleic acid assembly techniques. Nucleic acid assembly techniques that can be performed in the nucleic acid assembly module of the disclosed automated multi-module cell editing tools include, but are not limited to, assembly methods that use restriction endonucleases, including PCR, BioBrick assembly (US Pat. No. 9,361,427), type IIS cloning (e.g. GoldenGate assembly, EP 2395087 A1), and ligase cycle reaction (de Kok, ACS Synth Biol., 3(2):97-106 (2014); Engler, et al., PLoS One , 3(11):e3647 (2008); and US Pat. No. 6,143,527). In other embodiments, nucleic acid assembly techniques performed by the disclosed multi-module automated cell editing tools rely on overlap between adjacent nucleic acid portions, such as Gibson Assembly®, CPEC, SLIC, ligase cycle, 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); patents US Nos. 5,888,732 and 6,277,608), and topoisomerase-mediated cloning (Udo, PLoS One, 10(9): e0139349 (2015); and US Pat. No. 6,916,632). These and other nucleic acid assembly techniques are described, for example, in Sands and Brent, Curr Protoc Mol Biol., 113:3,26.1-3,26.20 (2016).

[0202] The nucleic acid assembly module is controlled by temperature depending on the type of nucleic acid assembly used in the automated multi-module cell editing tool. For example, when a PCR is used in a nucleic acid assembly module, the module includes a thermal cycling capability allowing temperature cycling between denaturation, annealing, and extension steps. When single temperature assembly techniques (eg, isothermal assembly techniques) are used in the nucleic acid assembly module, the module provides the ability to reach and maintain a temperature that optimizes the particular assembly process being performed. These temperatures and the duration of maintaining these temperatures can be determined by a pre-programmed set of parameters executed by a script, or controlled manually by the user using the processing system of an automated multi-module cell editing tool.

[0203] In one embodiment, the nucleic acid assembly module is an assembly module using a single isothermal reaction. Some isothermal assembly methods can combine up to 15 nucleic acid fragments simultaneously based on sequence identity. The assembly method, in some embodiments, assembles nucleic acids that include an overlap of about 20-40 bases with adjacent nucleic acid fragments. The fragments are mixed with a cocktail of three enzymes - exonuclease, polymerase and ligase along with buffer components. Because the process is isothermal and can be performed in a one or two step process using a single reactor, isothermal assembly reactions are ideal for use in an automated multi-module cell editing tool. The one-step method allows up to five different fragments to be assembled using a one-step isothermal process. Fragments and the master mix of enzymes are combined and incubated at 50°C for one hour. To create more complex constructs containing up to fifteen fragments, or to include fragments from 100 to 10,000 base pairs, a two-step method is usually used, in which a two-step reaction requires two separate additions of the master mix; the first for the exonuclease and annealing step, and the second for the polymerase and ligation step.

Transformation module

[0204] FIG. 8A-8E depict variations of one embodiment of a cell transformation module (in this case a flow-through electroporation device) that can be incorporated into a cell growth/concentration/transformation tool. Fig. 8A and 8B are top and bottom perspective views, respectively, of six connected electroporation flow devices 850. Fig. 8A depicts six electroporation flow units 850 arranged on a single substrate 856. Each of the six electroporation flow units 850 has inlet settlers 852 that define cell sample inlets and outlet settlers 854 that define cell sample outlets. Fig. 8B is a bottom perspective view of the six connected electroporation flow devices shown in FIG. 8A, also showing six electroporation flow units 850 arranged on a single substrate 856. Six inlet settlers 852 are visible, one for each electroporation flow unit 850, and one exit settler 854 (leftmost electroporation flow unit 850 outlet settler). Additionally, in Fig. 8B, inlet 802, outlet 804, flow channel 806, and two electrodes 808 on either side of the constriction in flow channel 806 of each electroporation flow block 850 are visible. Once six electroporation flow modules 850 are made, they can be separated from each other (eg, "split") and used one at a time, or alternatively in such embodiments where two or more electroporation flow blocks 850 can be used in parallel without separation.

[0205] Electroporation flow devices 850 achieve high efficiency cell electroporation with low toxicity. The electroporation flow devices 850 of the present invention provide for particularly easy integration with automated fluid handling equipment that is commonly used in automated systems such as air displacement pipettors. Such automated apparatus includes, but is not limited to, standard automated fluid handling systems from Tecan (Mannedorf, Switzerland), Hamilton (Reno, Nevada), Beckman Coulter (Fort Collins, Colorado), etc.

[0206] Generally speaking, microfluidic electroporation—using cell suspension volumes of less than about 10 ml and up to 1 μl—allows for more precise control of the transfection or transformation process and allows for flexible integration with other cell processing tools compared to benchtop electroporation devices. Microfluidic electroporation thus provides unique advantages, for example, for transformation, processing and analysis of single cells; configurations of multiblock electroporation devices; as well as integrated automatic multi-module cell processing and analysis.

[0207] In specific embodiments of the electroporation flow devices 850 of the present invention, the level of transformation toxicity 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% of viable cells after transformation, depending on the cell type and nucleic acids introduced into the cells.

[0208] The electroporation flow device 850 described with reference to FIG. 8A-8E comprises a housing with an electroporation chamber, a first electrode and a second electrode configured to be connected to an electrical pulse generator. In some embodiments, the electroporation devices are autoclavable and/or disposable, and may be packaged with reagents in a reagent cartridge. The electroporation device 850 can be configured to electroporate a cell sample with a volume of 1-2 ml, 10 μl - 1 ml, 25 μl - 750 μl, or 50 μl - 500 μl. Cells that can be electroporated with the disclosed electroporation devices 850 include mammalian cells (including human cells), plant cells, yeast, other eukaryotic cells, bacteria, protozoa, and other cell types.

[0209] In one exemplary embodiment, FIG. 8C is a plan view of an electroporation flow device 850 having an inlet 802 for introducing cells and an exogenous reagent electroporated into cells ("cell sample") and an outlet 804 for cell sample after electroporation. Electrodes 808 are inserted through electrode channels (not shown in Fig. 8C) in the device. Fig. 8D shows a top sectional view of an electroporation flow device 850 with inlet 802, outlet 804, and electrodes 808 positioned relative to the constriction in flow channel 806. A side sectional view of the bottom of the electroporation flow device 850 in FIG. 8E illustrates that the electrodes 808 in this embodiment are positioned in the electrode channels 810 perpendicular to the flow channel 806 such that the cell sample flows from the inlet channel 812 through the flow channel 806 to the outlet channel 814, and in the process the cell sample flows into the electrode channels 810 so to be in contact with the electrodes 808. In this aspect, the input channel 812, the output channel 814 and the electrode channels 810 start from the top flat side of the device; however, the flow electroporation architecture depicted in FIG. 8C-8E is just one architecture useful with the reagent cartridges described herein. Additional electrode architectures are described, for example, in US patent documents No. 16/147120, filed September 24, 2018; 16/147865 filed September 30, 2018; and 16/147871, filed September 30, 2018.

Sample Workflows

[0210] FIG. 9 is a simplified block diagram of one embodiment of an exemplary automated multi-module cell processing tool comprising a singulation or essential singulation/growing/editing and normalization or selection module for enrichment or selection of edited cells. The cell processing tool 900 may include a housing 944, a reservoir 902 for cells to be transformed or transfected, and a culture module 904 (cell culture device). Transformable cells are transferred from the reservoir to the growth module for culturing until the cells reach the target OD. Once the cells have reached the target OD, the growth module may refrigerate or freeze the cells for later processing, or the cells may be transferred to a cell concentration module 930 where they are made electrocompetent and concentrated to an optimum volume for cell transformation. After concentration, the cells are transferred to an electroporation device 908 (eg, a transformation/transfection module, with one exemplary module described above with reference to FIGS. 8A-8E). Exemplary electroporation devices used in automated multi-module cell processing instruments include flow-through electroporation devices such as those described in US Patent Documents No. 16/147,120, filed September 28, 2018; 16/147353 filed September 28, 2018; 16/147865 filed September 30, 2018; and 16/147871, filed September 30, 2018, all of which are incorporated herein by reference in their entirety.

[0211] In addition to the cell storage reservoir 930, the automated multi-module cell processing tool 900 may include a reservoir for storing editing oligonucleotide cassettes 916 and a reservoir 918 for storing an expression vector backbone. Both the editing oligonucleotide cassettes and the expression vector backbone are transferred from the reagent cartridge to the nucleic acid assembly module 920, where the editing oligonucleotide cassettes are inserted into the expression vector backbone. The collected nucleic acids may be transferred to an optional purification module 922 for desalting and/or other purification and/or concentration procedures necessary to prepare the collected nucleic acids for transformation. Alternatively, pre-assembled nucleic acids, such as an editing vector, may be stored in reservoir 916 or 918. After completion of the processes performed by the purification module 922, the collected nucleic acids are transferred, for example, to an electroporation device 908, which already contains a cell culture grown to the target OD and made electrocompetent by cell concentration module 930. In the electroporation device 908, the collected nucleic acids are injected into the cells. After electroporation, the cells are transferred to the combined recovery/selection module 910. Examples of multi-module cell editing tools can be found in US Patent Documents Nos. 16/024816 and 16/024831, filed June 30, 2018; 16/147865 filed September 30, 2018; and 16/147871, filed September 30, 2018, all of which are incorporated herein by reference in their entirety.

[0212] After recovery and optional selection, the cells are transferred to the singulation or essential singulation, editing and growth module 940, where the cells are diluted and separated into compartments so that there is an average of one cell per compartment. Once they are substantially or substantially singulated, the cells are allowed to grow to achieve a predetermined number of doublings. Once these initial colonies are formed, editing is induced and the edited cells are grown to form colonies that grow to size limit (eg, colonies normalize). In some embodiments, editing is induced by one or more editing components under the control of an inducible promoter. In some embodiments, the inducible promoter is activated by raising the temperature and deactivated by lowering the temperature. Similarly, in those embodiments in which a solid wall device containing a filter forming the bottom of a microwell, the solid wall device can be transferred to a plate (e.g., an agar plate or even liquid medium) containing a medium with a component that activates or induces editing, and then transfer to an environment that deactivates editing. In solid wall and solid bottom devices, induction of editing or deactivation of editing can be done by changing the medium. When the colonies grow to the maximum size, they unite. Again, singulation or significant singulation overcomes growth bias in favor of unedited cells, as well as growth bias resulting from the fitness effects of various changes.

[0213] All restoration, selection and singulation/induction/editing and normalization modules can be separate, can be arranged and combined as shown in FIG. 9, or may be located and combined in other configurations. In some embodiments, all of the steps of repair, selection, singulation or significant singulation, growth, editing, and normalization are performed in a single module, such as on a nutrient agar substrate, or on a solid wall device described with reference to FIG. 3A-3E and 4A-4AA. Alternatively, recovery, selection and dilution, if necessary, are performed in a liquid medium in a separate vessel (module), and then transferred to the singulation/growth/induction/editing and normalization module.

[0214] After pooling the normalized cell colonies, the cells can be stored, for example, in a storage unit or module 912, where the cells can be stored, for example, at a temperature of 4°C until the cells are needed for further research 914. Alternatively, the cells can be used in another edit cycle. The multi-module cell processing tool 900 is controlled by the processor 942 based on user input, one or more scripts, or a combination of user input and a script. The processor 942 may control the timing, duration, temperature, and operations of the various modules of the instrument 900, as well as the distribution of reagents. For example, processor 942 may cool cells after transformation until it is time for editing, when the temperature may be raised to a level conducive to genome editing and cell growth. The processor may be programmed with standard protocol parameters from which the user may select, the user may define one or more parameters manually, or one or more scripts associated with the reagent cartridge may define one or more operations and/or reaction parameters. In addition, the processor may notify the user (for example, via an application on a smartphone or other device) that the cells have reached the target OD, as well as keep the user updated on the progress of the cells in various modules in the multi-module system.

[0215] The automated multi-module cell processing tool 900 is a nuclease genome editing system and can be used in single editing systems (eg, introducing one or more changes to the cellular genome in a single editing process). The system described below, shown in FIG. 10 is configured to perform serial editing, for example, using different nuclease systems sequentially to provide two or more genome changes in a cell; and/or recursive editing, for example, using a single nuclease system to sequentially introduce two or more genome changes into a cell.

[0216] FIG. 10 illustrates another embodiment of an automated multi-module cell processing tool configured to perform singulation or substantial singulation of cells, growth, induction of editing, and normalization of cell colonies. This embodiment depicts an exemplary system that performs recursive gene editing on a population of cells. As with the embodiment shown in FIG. 9, cell processing tool 1000 may include a housing 1044, a reservoir 1002 for storing cells to be transformed or transfected, and a cell culture module 1004 (comprising, for example, a rotating culture vial). The transformable cells are transferred from the reservoir to the cell growth module for culturing until the cells reach the target OD. Once the cells have reached the target OD, the growth module may refrigerate or freeze the cells for later processing, or the cells may be transferred to the cell concentration module 1060 where they are buffer exchanged and made electrocompetent and cell volume can be substantially reduced. When the cells are concentrated to a suitable volume, they are transferred to the electroporation device 1008. In addition to the cell storage reservoir, the multi-module cell processing tool 1000 includes a reservoir for storing the vector pre-assembled with the editing oligonucleotide cassettes 1052. The pre-assembled nucleic acid vectors are transferred to the electroporation device 1008, which already contains the cell culture grown to the target OD. . In the electroporation device 1008, nucleic acids are electroporated into cells. Following electroporation, the cells are transferred to an optional recovery module 1056, where the cells are allowed to briefly recover from transformation.

[0217] After recovery, the cells can be transferred to a storage unit or module 1012, where the cells can be stored, for example, at 4°C for later processing, or the cells can be diluted and transferred to the selection, singulation or significant singulation module 1058 / growth/induction/editing and normalization with a solid wall. In this 1058 multi-process module, cells are lined up so that there is an average of one cell per microwell. The cells to be lined up may be in a selection medium to select those cells that have been transformed or transfected with the editing vector(s). Once they are substantially or largely singulated, the cells grow to 2-50 doublings and form colonies. After colony formation, editing is induced by the conditions (eg, temperature, addition of stimulating or inhibitory chemicals) necessary to induce editing. After editing, the cells are allowed to grow to size limits (eg, colony normalization) in the microwells, after which they can be washed out of the microwells and pooled, and then transferred to the cell extraction unit 1014, or can be returned back to the growth module 1004 for another editing cycle. . Between pooling and transfer to the growth module, one or more additional steps may take place, such as cell recovery, medium exchange, cell concentration, etc., such as tangential flow filtration. It should be noted that the selection/singulation or essential singulation/growth/induction/editing and normalization modules may be the same module where all processes are performed in a solid wall device, or selection and/or dilution may take place in a separate vessel before how the cells will be transferred to the solid wall singulation or essential singulation/growth/induction/editing and normalization module (solid wall device). Once the putatively edited cells are pooled, they can be subjected to another round of editing, starting with growth, cell concentration and processing to render them electrocompetent, and transformation with another donor nucleic acid in another editing cassette via the electroporation module 1008.

[0218] In the electroporation device 1008, cells selected from the first editing cycle are transformed with a second set of editing oligonucleotides (or other type of oligonucleotides), and the cycle is repeated until the cells have been transformed and edited with the desired number of, for example, editing cassettes. The multi-module cell processing tool 1000 illustrated in FIG. 10 is controlled by the processor 1042 based on user input or based on one or more scripts, including at least one script associated with the reagent cartridge. The processor 1042 may control the timing, duration and temperature of various processes, the distribution of reagents, and other operations of the various modules of the automated multi-module cell processing tool 1000. For example, a script or processor may control the distribution of cells, reagents, vectors, and editing oligonucleotides; which editing oligonucleotides are used to edit cells and in what sequence; time, temperature and other conditions used in the recovery and expression module; the wavelength at which the OD is measured in the cell growth module, the target OD to which cells are grown, and the target time for cells to reach the target OD. In addition, the processor may be programmed to inform the user (eg, via an application on a smartphone or other device) of the progress of the cells in the automated multi-module cell processing instrument.

[0219] A person skilled in the art in view of the present disclosure should be obvious that the described process can be recursive and multiplexed; that is, cells can go through the workflow described with reference to FIG. 10, and then the resulting edited culture may go through one more (or several, or many) additional editing cycles (eg, recursive editing) with different edit vectors. For example, cells from Edit Run 1 can be diluted and an aliquot of cells edited with Edit Vector A can be combined with Edit Vector B, an aliquot of cells edited with Edit Vector A can be combined with Edit Vector C, an aliquot of cells edited with Edit Vector C, an aliquot of cells edited with Edit Vector A, can be combined with edit vector D, and so on for the second edit cycle. After the second cycle, an aliquot of each of the doubly edited cells may be subjected to a third editing cycle in which, for example, aliquots of each of the AB-, AC-, AD-edited cells are combined with additional edit vectors such as the X, Y, and Z edit vectors. That is, the double-edited AB cells can be combined (and edited) with the X, Y, and Z vectors to produce the triple-edited ABX, ABY, and ABZ cells; double-edited AC cells can be combined (and edited) with the X, Y, and Z vectors to produce triple-edited ACX, ACY, and ACZ cells; and the double-edited AD cells can be combined (and edited) with the X, Y, and Z vectors to produce triple-edited ADX, ADY, and ADZ cells, and so on. Many permutations and combinations of editing can be performed in this process, resulting in very diverse cell populations and cell libraries. In any recursive process, it is beneficial to "heal" the previous motor vectors and edit vectors (or one motor vector + edit vector in a one-vector system). "Treatment" is a process in which one or more of the vectors used in the previous editing cycle is eliminated from the transformed cells. Treatment can be achieved, for example, by cleaving the vector(s) using a treatment plasmid, rendering the editing vector and/or motor vector (or one combined vector) non-functional; diluting the vector(s) in a population of cells through cell growth (i.e., the more growth cycles the cells go through, the fewer daughter cells the edit vector or motor vector will retain), or, for example, by using a temperature-sensitive replication source on the edit vector or vector -engine (or combined vector). Conditions for treatment will depend on the mechanism used for treatment; that is, in this example, how the treatment plasmid cleaves off the editing plasmid and/or the motor plasmid.

Production of cell libraries using automated editing methods, modules, tools and systems

[0220] In one aspect, the present disclosure provides methods, modules, automated editing tools, as well as automated multi-module cell editing tools for creating a cell library in which the expression, levels and/or activity of RNA and/or proteins of interest vary in different cell types. using various editing strategies, as described in more detail in this document. Accordingly, the present disclosure is intended to cover libraries of edited cells generated by the automated editing methods and automated multi-module cell editing tools of the present invention. These cell libraries can have a variety of targeted changes including, but not limited to, gene knockouts, gene insertions, insertions, deletions, single nucleotide changes, short tandem repeat editing, frameshifts, triplet codon expansion, and the like. in the cells of various organisms. These changes may be directed to coding or non-coding regions of the genome and are preferably rational.

[0221] In other aspects, the present disclosure provides automated editing methods and automated multi-module cell editing tools for creating a cell library in which DNA-related processes vary. For example, a cell library may include individual cells having alterations in DNA binding sites to prevent DNA binding of regulatory elements that modulate the expression of selected genes. In addition, cell libraries may include changes in genomic DNA that affect cellular processes such as heterochromatin formation, class switch recombination, and VDJ recombination.

[0222] In certain aspects, cell libraries are created using multiplexed editing of individual cells within a cell population, with multiple cells within the cell population being edited in a single edit cycle, i.e., multiple changes within the library cells are performed in a single automated operation. Libraries that can be created in one multiplex automated operation can contain 500 Edit Cells, 1000 Edit Cells, 2000 Edit Cells, 5000 Edit Cells, 10,000 Edit Cells, 50,000 Edit Cells, 100,000 Edit Cells, 200,000 Edit Cells, 300,000 Edit Cells, 400000 отредактированных клеток, 500000 отредактированных клеток, 600000 отредактированных клеток, 700000 отредактированных клеток, 800000 отредактированных клеток, 900000 отредактированных клеток, 1000000 отредактированных клеток, 2000000 отредактированных клеток, 3000000 отредактированных клеток, 4000000 отредактированных клеток, 5000000 отредактированных клеток, 6000000 отредактированных клеток, 7000000 отредактированных cells, 8000000 edited cells, 9000000 edited cells, 10000000 edited cells or more.

[0223] In other specific aspects, cell libraries are created using recursive editing of individual cells within a population of cells, with editing changes added to individual cells in two or more editing cycles. The use of recursive editing results in combining two or more changes targeting two or more sites in the genome in individual cells in the library. Libraries that can be created in an automated recursive operation can contain 500 Edited Cells, 1000 Edited Cells, 2000 Edited Cells, 5000 Edited Cells, 10000 Edited Cells, 50000 Edited Cells, 100000 Edited Cells, 200000 Edited Cells, 300000 Edited Cells, 400000 отредактированных клеток, 500000 отредактированных клеток, 600000 отредактированных клеток, 700000 отредактированных клеток, 800000 отредактированных клеток, 900000 отредактированных клеток, 1000000 отредактированных клеток, 2000000 отредактированных клеток, 3000000 отредактированных клеток, 4000000 отредактированных клеток, 5000000 отредактированных клеток, 6000000 отредактированных клеток, 7000000 отредактированных клеток , 8000000 edited cells, 9000000 edited cells, 10000000 edited cells or more.

[0224] Examples of manual editing strategies that can be modified based on the present disclosure to use automated systems can be found, for example, in U.S. Patent Nos.

[0225] In specific aspects, recursive editing can be used to first create the phenotype of the cell, and then later editing cycles can be used to completely change the phenotype and/or enhance other properties of the cell.

[0226] In some aspects, the cell library contains changes to create unnatural amino acids in the cell.

[0227] In specific aspects, the present disclosure provides libraries of edited cells having changes in one or more regulatory elements created using the automatic editing methods and automated multi-module cell editing tools of the present invention. The term "regulatory element" refers to nucleic acid molecules that can affect the transcription and/or translation of an operably linked coding sequence in a particular environment and/or context. The term includes all elements that maintain or regulate transcription and RNA stability, including promoters, basic elements required for the basic interaction of RNA polymerase and transcription factors, precursor elements, enhancers, and response elements (see, e.g., Lewin, " Genes V" (Oxford University Press, Oxford) pages 847-873). Exemplary prokaryotic regulatory elements include, but are not limited to, promoters, operator sequences, and ribosome binding sites. Regulatory elements that are used in eukaryotic cells may include, but are not limited to, promoters, enhancers, insulators, splicing signals, and polyadenylation signals.

[0228] Preferably, the edited cell library includes edits rationally designed based on predictions of protein structure, expression, and/or activity in a particular cell type. For example, a rational design can be based on a system-wide biophysical model of genome editing with a specific nuclease and gene regulation to predict how various editing parameters, including nuclease expression and/or binding, growth conditions, and other experimental conditions, collectively control the dynamics of nuclease editing. See, for example, Farasat and Salis, PLoS Comput Biol., 29:12(1):e1004724 (2016).

[0229] In one aspect, the present disclosure provides for the creation of a library of edited cells with various rationally designed regulatory sequences generated using the automated editing apparatus, systems, and methods of the present invention. For example, a library of edited cells may include prokaryotic cell populations generated using a set of constitutive and/or inducible promoters, enhancer sequences, operator sequences, and/or ribosome binding sites. In another example, a library of edited cells may include eukaryotic sequences designed using a set of constitutive and/or inducible promoters, enhancer sequences, operator sequences, and/or various Kozak sequences to express proteins of interest.

[0230] In some aspects, the present disclosure provides libraries of cells, including cells with rationally designed changes containing one or more classes of changes in sequences of interest in the genome of an organism. In specific aspects, the present disclosure provides cell libraries comprising intelligently designed alteration cells containing one or more classes of alterations in sequences of interest in a subset of the genome. For example, a cell library may include cells with rationally designed alterations containing one or more classes of alterations in sequences of interest in the exome, for example, in each or most of the open reading frames of the genome. For example, a cell library may include intelligently designed alteration cells containing one or more classes of alterations in sequences of interest in the kinome. In yet another example, a cell library may include intelligently designed alteration cells containing one or more classes of alterations in secretome sequences of interest. In other aspects, a cell library may include cells with intelligently designed alterations designed to analyze different isoforms of proteins encoded in the exome, and cell libraries may be designed to direct the expression of one or more specific isoforms, for example, for transcriptome analysis.

[0231] It is important to note that in some aspects, cell libraries may contain changes using randomized sequences, such as randomized promoter sequences, to reduce the similarity between the expression of one or more proteins in individual cells within the library. In addition, the promoters in the cell library can be constitutive, inducible, or both to ensure strong and/or titratable expression.

[0232] In other aspects, the present disclosure provides automated editing methods and automated multi-module cell editing tools for creating a cell library containing changes to identify optimal expression of a selected target gene. For example, the production of biochemicals by metabolic engineering often requires the expression of pathway enzymes, and the best production yield is not always achieved by maximizing the number of target pathway enzymes in the cell, but rather by fine tuning the expression levels of individual enzymes and associated regulatory proteins and/or pathways. . Similarly, expression levels of heterologous proteins can sometimes be experimentally adjusted to obtain optimal yields.

[0233] The most obvious way transcription affects gene expression levels is the rate of Pol II initiation, which can be modulated by a combination of promoter or enhancer strength and trans-activating factors (Kadonaga, et al., Cell, 116(2):247-57 (2004 )). In eukaryotes, elongation rate may also determine gene expression patterns by influencing alternative splicing (Cramer et al., PNAS USA, 94(21):11456-60 (1997)). Unsuccessful gene termination can disrupt the expression of subsequent genes by reducing promoter availability for Pol II (Greger, et al., PNAS USA, 97(15):8415-20 (2000)). This process, known as transcriptional interference, is especially relevant in lower eukaryotes because they often have closely spaced genes.

[0234] In some embodiments, the implementation of the present disclosure provides methods for optimizing the transcription of cellular genes. Gene transcription is the result of several different biological events, including transcription initiation (RNA-p recruitment and transcription complex formation), elongation (synthesis/strand extension), and transcriptional termination (RNA-p cleavage and termination).

Site directed mutagenesis

[0235] Cell libraries can be created using automated editing methods, modules, tools and systems using site-directed mutagenesis, i.e. where the amino acid sequence of a protein or other genomic feature can be changed deliberately and accurately by mutating a protein or genomic feature. These cell lines can be useful for a variety of purposes, such as determining the function of a protein within a cell, identifying enzymatic active sites within cells, and designing new proteins. For example, site-directed mutagenesis can be used in a multiplex manner to replace one amino acid in a protein sequence with another amino acid with different chemical properties. This allows one to determine the effect of a rationally designed or randomly generated mutation on individual cells in a cell population. See, for example, Berg, et al. Biochemistry, Sixth Ed. (New York: W. H. Freeman and Company) (2007).

[0236] In another example, changes can be made to individual cells within a cell library to change amino acids at binding sites, such as changing one or more amino acids at a protein binding site to interact within a protein complex, or changing one or more amino acids in enzyme pockets that may contain a cofactor or ligand. This class of changes allows the creation of specific protein manipulations to measure specific properties of one or more proteins, including interactions with other cofactors, ligands, and so on. within the protein complex.

[0237] In other examples, various types of editing can be performed on individual cells in a cell library using site-directed mutagenesis to study quantitative expression trait loci (eQTLs). The eQTL is a locus that explains part of the genetic variation in the expression phenotype of a gene. The libraries of the present invention may be useful for assessing and linking eQTL to actual disease states.

[0238] In specific aspects, the changes introduced into the cell libraries of the present invention can be created using a rational design based on known or predicted protein structures. See, for example, Chronopoulou and Labrou, Curr Protoc Protein Sci.; Chapter 26:Unit 26.6 (2011). Such site-directed mutagenesis can provide individual cells within a library with one or more site-directed changes, and preferably two or more site-directed changes (eg, combinatorial changes) in a population of cells.

[0239] In other aspects, the cell libraries of the present invention are created using site-directed codon mutation, "scanning" all or substantially all of the codons in the coding region of a gene. Thus, individual changes in specific codons can be examined for loss or gain of function based on specific polymorphisms in one or more codons of a gene. These libraries can be a powerful tool for determining which genetic changes are latent or responsible for a particular phenotype in a cell or cell population. Codon changes may be randomly generated or may be rationally designed based on known polymorphisms and/or mutations that have been identified in the gene under analysis. In addition, the use of these techniques for two or more genes separately in the same pathway in a cell can identify potential protein:protein interactions or redundancy in cellular functions or pathways.

et al., Nucleic Acids Research, Volume 25(2):447-448 (1997). Аланин часто используется в этой методике сканирования кодонов из-за его небольшой, химически инертной метильной функциональной группы, которая может имитировать предпочтения вторичной структуры, которыми обладают многие другие аминокислоты. Сканирование кодонов может также использоваться для определения того, играет ли боковая цепь определенного остатка значительную роль в функции и/или активности клетки. Иногда другие аминокислоты, такие как валин или лейцин, могут использоваться для создания библиотек клеток для сканирования кодонов, если необходимо сохранение размера мутированных остатков. [0240] For example, an alanine scan can be used to determine the contribution of a particular residue to the stability or function of a given protein. See, for example, the publication

et al., Nucleic Acids Research, Volume 25(2):447-448 (1997). Alanine is frequently used in this codon scanning technique due to its small, chemically inert methyl functional group, which can mimic the secondary structure preferences that many other amino acids possess. Codon scanning can also be used to determine if the side chain of a particular residue plays a significant role in cell function and/or activity. Occasionally, other amino acids, such as valine or leucine, can be used to create cell libraries for codon scanning if the size of the mutated residues is to be preserved.

[0241] In other specific aspects, cell libraries can be created using the automated editing methods and automated multi-module cell editing tools of the present invention to determine the active site of a protein, such as an enzyme or hormone, and elucidate the mechanism of action of one or more of these proteins in cell library. Site-directed mutagenesis, associated with molecular modeling studies, can be used to determine the structure of the enzyme's active site and hence its mechanism of action. Analysis of these cell libraries can provide insight into the role played by certain amino acid residues in protein active sites, in contacts between subunits of protein complexes, in intracellular migration and protein stability/half-life in various genetic backgrounds.

Saturating mutagenesis

[0242] In some aspects, cell libraries generated using the automated editing methods and automated multi-module cell editing tools of the present invention may be saturation mutagenesis libraries in which a single codon or set of codons is randomized to produce all possible amino acids at the position of a particular gene or genes of interest. These cell libraries can be particularly useful for generating variants, for example for directed evolution. See, for example, Chica, et al., Current Opinion in Biotechnology 16 (4): 378-384 (2005); and Shivange, Current Opinion in Chemical Biology, 13 (1): 19-25 (2009).

[0243] In some aspects, changes containing various degenerate codons can be used to encode sets of amino acids in individual cells in libraries. Since some amino acids are encoded by more codons than others, the exact ratio of amino acids cannot be equal. In some aspects, more limited degenerate codons are used. "NNK" and "NNS" have the advantage that they encode all 20 amino acids, but still encode a stop codon 3% of the time. Alternative codons such as "NDT", "DBK" completely avoid stop codons and code for a minimal set of amino acids that still covers all major biophysical types (anionic, cationic, aliphatic hydrophobic, aromatic hydrophobic, hydrophilic, small).

[0244] In certain aspects, non-redundant saturation mutagenesis is used, in which a commonly used codon for a particular organism is used in the editing process of saturation mutagenesis.

Exchanges and ladders of promoters

[0245] One mechanism for analyzing and/or optimizing the expression of one or more genes of interest is to create a "promoter exchange" cell library in which cells contain genetic modifications in which specific promoters are linked to one or more genes of interest. Accordingly, cell libraries generated using the methods and automated multi-module cell editing tools of the present invention can be promoter exchange cell libraries that can be used, for example, to increase or decrease the expression of a gene of interest in order to optimize the metabolic or genetic pathway. In some aspects, a promoter-exchange cell library can be used to identify an increase or decrease in the expression of a gene that affects cell survival or viability, for example, a gene encoding a protein that affects the growth rate or overall health of cells. In some aspects, a promoter exchange cell library can be used to create cells having dependencies and logic between promoters to create synthetic gene networks. In some aspects, promoter exchanges can be used to direct cell-to-cell communication between cells of both homogeneous and heterogeneous (complex tissue) populations in nature.

[0246] Libraries of cells can use any given number of promoters that have been grouped together based on the expression range of expression strength and any given number of target genes. A ladder of promoter sequences varies the expression of at least one locus under at least one condition. This ladder is then systematically applied to a group of genes in an organism using the automated editing methods and automated multi-module cell editing tools of the present invention.

[0247] In specific aspects, a cell library formed using the automated editing processes, modules, and systems of the present invention includes individual cells that are representative of a given promoter, operably linked to one or more target genes of interest in an otherwise identical genetic background. . Examples of manual editing strategies that can be modified to use automated systems can be found, for example, in US Patent No. 9988624.

[0248] In specific aspects, a promoter exchange cell library is produced by editing a set of target genes to be operably linked to a preselected set of promoters that act as a "promoter ladder" for expression of the genes of interest. For example, cells are edited so that one or more of the individual genes of interest are edited to be functionally linked to different promoters in the promoter ladder. When an endogenous promoter does not exist, its sequence is unknown, or it has been previously altered in some way, individual promoter ladder promoters can be inserted before the genes of interest. These resulting cell libraries contain single cells with an individual ladder promoter operably linked to one or more target genes in an otherwise identical genetic context.

[0249] Promoters are typically chosen to result in variable expression at different loci, and may include inducible promoters, constitutive promoters, or both.

[0250] The set of target genes edited using the promoter ladder may include all or most of the open reading frames (ORFs) in the genome or a selected subset of the genome, such as the kinome or secretome ORF. In some aspects, target genes may include coding regions for different gene isoforms, and cell libraries can be designed to express one or more specific isoforms, such as for transcriptome analysis using different promoters.

[0251] A set of target genes can also be genes known or suspected to be involved in a particular cellular pathway, such as a regulatory pathway or a signaling pathway. The set of target genes can be open reading frames related to function related to previously demonstrated beneficial changes (previous promoter exchanges or previous SNP exchanges), algorithmic selection based on epistatic interactions between previously created changes, other selection criteria based on hypotheses regarding benefits for ORF targets, or randomly selected. In specific embodiments, target genes may contain genes that do not code for proteins, including non-coding RNAs.

[0252] Editing of other functional genetic elements, including insulator elements and other elements of genomic organization, can also be used to systematically change the expression level of a set of target genes and can be introduced using the methods and automated multi-module cell editing tools of the present invention. In one aspect, a population of cells is edited using a ladder of enhancer sequences, either alone or in combination with selected promoters or a promoter ladder, to create a cell library having various changes in these enhancer elements. In another aspect, a population of cells is edited using a ladder of ribosome binding sequences, either alone or in combination with selected promoters or a promoter ladder to create a cell library having various changes in these ribosome binding sequences.

[0253] In another aspect, the cell population is edited to allow the attachment of various mRNA and/or protein stabilizing or destabilizing sequences to the 5' or 3' end or anywhere else in the transcript or protein.

[0254] In some aspects, a cell population of a previously established cell line can be edited using the automated editing methods, modules, tools, and systems of the present invention to create a cell library to improve cell function, health, and/or viability. For example, many commercial strains currently used for large scale production have been developed using random mutagenesis processes iteratively over many years, sometimes decades. Unwanted neutral and deleterious mutations were introduced into strains along with beneficial changes, and over time this resulted in strains with insufficient overall reliability and key characteristics such as growth rate. In another example, mammalian cell lines continue to mutate over a period of time, and similarly, these cell lines can become unstable and acquire undesirable traits. The automated editing methods and automated multi-module cell editing tools of the present invention may use editing strategies such as SNP and/or STR exchange, insert-delete generation, or other techniques to remove or modify unwanted genome sequences and/or introduce new genome sequences to eliminate deficiencies while maintaining the desired properties of cells.

[0255] When recursive editing is used, editing in individual cells in a library of edited cells may include the inclusion of "landing sites" in the ectopic region of the genome (eg, CarT locus) to optimize expression, stability and/or control.

[0256] In some embodiments, each resulting library having individual cells containing one or more changes (inserts or deletions) is cultured and analyzed for one or more criteria (eg, to obtain a chemical or product of interest). Cells that have specific criteria are then associated or correlated with one or more specific changes in the cell. Thus, the effect of a given change on any number of genetic or phenotypic traits of interest can be determined. The identification of multiple changes associated with specific criteria or improved functionality/reliability can lead to cells with highly desirable characteristics.

Knockout or injection libraries

[0257] In some aspects, the present disclosure provides automated editing methods, modules, tools, and systems for generating a library of cells having "knockout" (KO) or "introduce" (KI) changes to various genes of interest. Thus, the present invention is intended to encompass libraries of edited cells generated by the automated editing methods and automated multi-module cell editing tools of the present invention that have one or more mutations that remove or reduce the expression of selected genes of interest, in order to investigate the impact of these changes. on the function of a gene in individual cells within a cell library.

[0258] Cell libraries can be created using a targeted KO gene (eg, by insertion/deletion) or multiple KOs (eg, by homology-targeted repair). For example, double-strand breaks are often repaired through the path of repair by joining the non-homologous ends of the DNA. Such repair is known to be error prone, and thus insertions and deletions can be introduced that can disrupt the function of the gene. Preferably, the changes are rationally designed to specifically affect the genes of interest, and individual cells can be designed to have the KI or KI of one or more of the loci of interest. Cells having KO or KI of two or more loci of interest can be generated using the automated recursive editing of the present invention.

[0259] In specific aspects, KO or KI cell libraries are created using simultaneous multiplexed editing of cells within a cell population, and multiple cells within a cell population are edited in a single edit cycle, i.e., multiple changes within the library cells are performed in a single automated operation. In other specific aspects, cell libraries are created using recursive editing of individual cells within a population of cells, resulting in the combination of multiple changes of two or more regions in the genome in single cells.

SNP or short tandem repeat substitutions

[260] In one aspect, cell libraries are created using the automated editing methods and automated multi-module cell editing tools of the present invention by systematically introducing or substituting single nucleotide polymorphisms ("SNPs") into the genomes of individual cells in order to create a cell library with "SNP exchange". In some embodiments, the SNP exchange methods of the present invention include both the addition of beneficial SNPs and the removal of detrimental and/or neutral SNPs. SNP exchanges may be for coding sequences, non-coding sequences, or both.

[0261] In another aspect, a cell library is generated using the automated editing methods, modules, tools, and systems of the present invention by systematically introducing or replacing short tandem repeats ("STRs") into the genomes of individual cells in order to create a cell library with " STR exchange. In some embodiments, the STR exchange methods of the present invention include both the addition of beneficial STRs and the removal of detrimental and/or neutral STRs. STR exchanges may be for coding sequences, non-coding sequences, or both.

[0262] In some embodiments, the SNP and/or STR replacement used to create a cell library is multiplexed and multiple cells within a cell population are edited in a single edit cycle, i.e., multiple changes to cells in the cell library are performed in a single automated operation. In other embodiments, the SNP and/or STR replacement used to create a cell library is recursive, resulting in the pooling of multiple beneficial sequences and/or deletion of detrimental sequences in single cells. Multiple changes can either be a specific set of defined changes, or a partially randomized, combinatorial library of mutations. Removing deleterious mutations and pooling beneficial mutations can provide immediate improvements in various cellular processes. Removing the genetic burden, or combining beneficial changes into a strain without the genetic burden, also provides a new reliable starting point for additional random mutagenesis that can lead to further improvements.

[0263] SNP exchange overcomes the fundamental limitations of random mutagenesis approaches, as it is not a random approach, but rather the systematic introduction or removal of individual mutations in cells.

Editing a splice site

[0264] RNA splicing is a process during which introns are excised and exons are spliced together to create mRNA that is translated into protein. Accurate recognition of splicing signals by cellular mechanisms is critical to this process. Accordingly, in some aspects, a population of cells is edited using systematic editing for known and/or predicted donor and/or acceptor splice sites at various loci to create a library of splice site variants of various genes. Such editing may help elucidate the biological significance of different gene isoforms in a cellular context. Sequences for rationally constructing splice sites of various coding regions, including actual or predicted mutations associated with various mammalian diseases, can be predicted using analysis methods such as those described in Nalla and Rogan, Hum Mutat, 25:334-342 (2005); Divina, et al., Eur J Hum Genet, 17:759-765 (2009); Desmet, et al., Nucleic Acids Res, 37:e67 (2009); Faber, et al., BMC Bioinformatics, 12(suppl 4):S2 (2011).

Start-stop codon exchanges and incorporation of nucleic acid analogues

[0265] In some aspects, the present disclosure provides for the creation of cell libraries using the methods, modules, tools, and automated editing systems of the present invention, where the libraries are created by changing start and end codon variants throughout the genome of an organism or for a selected subset of coding regions in the genome, for example, kinoma or secretoma. Within this cell library, individual cells will have one or more start and end codons that replace the native start or end codon for one or more genes of interest.

[0266] For example, typical start codons used by eukaryotes are ATG (AUG), while prokaryotes most commonly use ATG (AUG), followed by GTG (GUG) and TTG (UUG). This cell library may include individual cells having native start codon substitutions for one or more genes of interest.

[0267] In some aspects, the present disclosure provides for the automated creation of a cell library by changing the initial ATG to TTG codons prior to selected genes of interest. In other aspects, the present disclosure provides for the automated creation of a cell library by changing the initial codons from ATG to GTG. In other aspects, the present disclosure provides for the automated creation of a cell library by changing the initial codons of GTG to ATG. In other aspects, the present disclosure provides for the automated creation of a cell library by changing the initial codons of GTG to TTG. In other aspects, the present disclosure provides for the automated generation of a cell library by changing the initial TTG to ATG codons. In other aspects, the present disclosure provides for the automated creation of a cell library by changing the initial TTG codons to GTG.

[0268] In other examples, typical stop codons for S. cerevisiae and mammals are TAA (UAA) and TGA (UGA), respectively. A typical stop codon for monocot plants is TGA (UGA), while insects and E. coli commonly use TAA (UAA) as the stop codon (Dalphin et al., Nucl. Acids Res., 24: 216-218 (1996 )). This cell library may include individual cells having native stop codon substitutions for one or more genes of interest.

[0269] In some aspects, the present disclosure provides automated generation of a cell library by replacing TAA stop codons with TAG. In other aspects, the present disclosure provides for the automated generation of a cell library by replacing TAA with TGA stop codons. In other aspects, the present disclosure provides automated generation of a cell library by replacing TGA with TAA stop codons. In other aspects, the present disclosure provides for the automated creation of a cell library by replacing TGA with TAG stop codons. In other aspects, the present disclosure provides for the automated creation of a cell library by changing stop codons from TAG to TAA. In other aspects, the present invention provides for the automated generation of a cell library by changing stop codons from TAG to TGA.

Terminator exchanges and ladders

[0270] One mechanism for determining optimal termination of a pre-spliced mRNA of one or more genes of interest is to create a "terminator swap" cell library in which cells contain genetic alterations having specific terminator sequences associated with one or more genes of interest. Accordingly, cell libraries constructed using the methods, modules, tools, and systems of the present invention may be terminator-swapped cell libraries that can be used, for example, to influence mRNA stability by releasing transcripts from synthesis sites. In other embodiments, a library of terminator-swapped cells can be used to identify an increase or decrease in the efficiency of transcription termination and thus accumulation of unspliced pre-mRNA (see, for example, West and Proudfoot, Mol Cell.; 33(3-9); 354 -364 (2009)) and/or 3' end treatments (see, for example, West, et al., Mol Cell. 29(5):600-10 (2008)). In the case when a gene is associated with several termination regions, it is possible to edit a combination of edits for several terminators associated with the gene during editing. Additional amino acids can also be added to the ends of proteins to determine the effect of protein length on terminators.

[0271] These cell libraries can use any given number of terminator revisions that have been selected for the terminator ladder based on the range of activity exhibited, and any given number of target genes. The ladder of termination sequences varies the expression of at least one locus under at least one condition. This ladder is then systematically applied to a group of genes in an organism using the automated editing methods, modules, tools and systems of the present invention.

[0272] In some aspects, the present disclosure provides for the creation of cell libraries using the automated editing methods, modules, tools, and systems of the present invention, where the libraries are created to edit terminator signals in one or more regions of the genome in individual cells of the library. Transcription termination in eukaryotes is mediated by terminator signals, which are recognized by protein factors associated with RNA polymerase II. For example, a cell library may contain individual eukaryotic cells with alterations in genes encoding polyadenylation specificity factor (CPSF) and cleavage-stimulating factor (CstF) and/or in genes encoding proteins recruited by CPSF and CstF to termination sites. In prokaryotes, two major mechanisms, termed Rho-independent and Rho-dependent termination, mediate transcriptional termination. For example, a cell library may contain individual prokaryotic cells with changes in protein-coding genes that affect the binding, efficiency, and/or activity of these termination pathways.

[0273] In some aspects, the present disclosure provides methods for selecting termination sequences ("terminators") with optimal properties. For example, in some embodiments, the present disclosure provides methods for introducing and/or editing one or more terminators and/or generating variants of one or more terminators within a host cell that exhibit a range of activity. A particular combination of terminators can be grouped together as a ladder of terminators, and the cell libraries of the present disclosure include single cells that represent terminators operably linked to one or more target genes of interest in an otherwise identical genetic background. Examples of manual editing strategies that can be modified to use automated tools can be found, for example, in US Pat.

[0274] In specific aspects, a cell library with terminator exchange is produced by editing a set of target genes to be operably linked to a preselected set of terminators that act as a "terminator ladder" to express the genes of interest. For example, cells are edited so that an endogenous promoter is operably linked to individual genes of interest edited by different promoters in the promoter ladder. When an endogenous promoter does not exist, its sequence is unknown, or it has been previously altered in some way, individual promoter ladder promoters can be inserted before the genes of interest. These resulting cell libraries contain single cells with an individual ladder promoter operably linked to one or more target genes in an otherwise identical genetic context. The terminator ladder in question is then associated with that gene of interest.

[0275] The terminator ladder can be used to more generally affect the termination of all or most of the ORFs in the genome, or in a selected subset of the genome, such as a kinome or secretome ORF. The set of target genes may also be genes known or suspected to be involved in a particular cellular pathway, such as a regulatory pathway or a signaling pathway. The set of target genes can be open reading frames related to function related to previously demonstrated beneficial changes (previous promoter exchanges or previous SNP exchanges), algorithmic selection based on epistatic interactions between previously created changes, other selection criteria based on hypotheses regarding benefits for ORF targets, or randomly selected. In specific embodiments, target genes may contain genes that do not code for proteins, including non-coding RNAs.

EXAMPLES

[0276] The following examples are provided to provide those skilled 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 authors consider their invention to be, nor do they imply, nor do not mean that the experiments below are all or the only experiments performed. It is assumed that other equivalent methods, steps and compositions are included in the scope of the present invention. Every effort has been made by the authors to ensure the accuracy of the numbers used (eg quantities, temperatures, etc.), but some experimental errors and deviations still need to be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weight is weight average molecular weight, temperature is in degrees Celsius, and pressure is atmospheric or near atmospheric.

Example 1: Evaluation of the suitability of a model inducible system

[0277] The main components of the model system were evaluated. The model system contained E. coli cells transformed with a motor vector, where the motor vector contained the coding sequence for the MAD nuclease (i.e., the MAD 4 or MAD 7 nuclease) under the control of the thermoinducible pL promoter, the chloramphenicol resistance marker, and the λ Red recombination system. under the control of the pBAD promoter (induced by adding arabinose to the nutrient medium). E. coli cells were also transformed with an editing vector containing an editing oligonucleotide, which in this model system was a library of editing oligonucleotides configured to inactivate galK, where successful editing produces white (versus red) colonies when plated on agar agar. McConkie containing galactose as the sole carbon source. In addition, the editing vector contained an gRNA coding sequence driven by the temperature-inducible pL promoter, a carbenicillin resistance marker, and a sequence to remove, mutate, or otherwise inactivate the PAM region in the target sequence. Fig. 11A and 11B depict an exemplary engine vector (FIG. 11A) and an edit vector (FIG. 11B) that can be used in the exemplary editing processes described herein. On FIG. 11A, an exemplary engine vector 1110 (p197) contains a replicator 1112 and a promoter 1114 driving the expression of a gene encoding repressor 1116 c1857 that drives the pL promoter. The first pL promoter in this exemplary embodiment drives the transcription of gRNA on the editing vector as described with reference to FIG. 11B below, and the second promoter pL 1118 on the motor vector drives the expression of nuclease 1120. The promoter that drives nuclease 1120 can be an inducible or constitutive promoter; however, the tightest regulation of the nucleic acid-directed nuclease system is achieved by using an inducible promoter to drive nuclease expression as well as a target nucleic acid. Again, the same inducible promoters and different inducible promoters can be used to direct the transcription of the guide nucleic acid and the nuclease. The pL promoter can be regulated (eg downregulated or induced) by the c1857 (1116) thermolabile repressor, which is active at permissive temperatures (eg 30°C) and inactive at higher temperatures (eg 42°C). The regulation of the pL promoter is shown in more detail in FIG. 1c above.

[0278] The motor vector in FIG. 11A additionally contains the pBAD (1140) promoter driving the expression of components of the λ Red (1142) recombination system. The pBAD promoter, like the pL promoter, is an inducible promoter; however, the pBAD promoter is regulated (induced) by the addition of arabinose to the growth medium. It should be noted that in this exemplary bacterial editing system, a recombination system, such as the λ Red recombination system, is provided as a component of a nucleic acid-directed nuclease-based editing system to repair DNA breaks that occur during editing. In some embodiments, however, the edited cells may already contain a recombination system (eg, episomal, integrated into the cellular genome, or naturally). In addition, while the λ Red recombination system is illustrated here, it should be understood that other recombination systems may be used. In addition, cells - such as yeast, plant and animal cells - do not need a recombination system equivalent to the λ Red recombination system to repair DNA breaks resulting from editing. Thus, nucleic acid-directed nuclease editing components for, for example, yeast, plant, and animal cells need not include a heterologous recombination system. Finally, an exemplary motor vector 1110 contains a promoter 1150 driving the expression of the chloramphenicol selectable marker 1152. In addition, the motor vector and all other vectors or constructs used in the disclosed method contain suitable control elements (eg, polyadenylation signals, enhancers) operably linked to components of the nucleic acid-directed nuclease editing system.

[0279] FIG. 11B depicts an exemplary edit vector 1130 (pLFORGE). Editing vector 1130 contains a replicator 1132 and a pL promoter 1134 that directs the expression of editing cassette 1136. The editing cassette contains the 1144 gRNA coding sequence, 1146 spacer and 1158 donor DNA (containing the homologous arms flanking the desired change in the target sequence). Editing vector 1130 also contains a promoter 1154 that controls the expression of selectable marker 1156 carbenicillin. It should be noted that the pL-inducible promoter 1134 is encoded on the backbone of the vector and is not part of the editing cassette 1136; however, in other embodiments, an inducible promoter directing gRNA transcription may be part of an editing cassette. Fig. 11A and 11B depict an exemplary engine and editing vector, respectively, but one of ordinary skill in the art will appreciate from the present disclosure that all elements of a nucleic acid-directed nuclease editing system may be contained on a single plasmid, or elements shown on a vector. -motor and edit vector in FIG. 11A and 11B may be on a different vector than shown. For example, the pBAD promoter and the λ Red recombination system may reside on an edit vector rather than a drive vector; similarly, the gene for the c1857 repressor may be on an editing vector rather than on an engine vector.

[0280] FIG. 12 is a bar graph showing the transformation efficiency observed for galK gRNA targeting cassettes under various promoters. The transformation efficiency of uninduced promoters is an indicator of promoter leakage. If there is high basal gRNA expression (e.g. "leakage"), then the CFU count will be low. If there is low basal gRNA expression (eg, tight regulation), then the number of CFUs will be high. The non-targeted gRNA experiment was performed with a pool of cassettes designed to target inactive PAM sequences that cannot promote gRNA and nuclease binding and cleavage. For each of the promoters shown (pL, pBAD and sigma32), a galK-targeted cassette with TTTC PAM and a spacer configured to insert a stop codon at amino acid D70 were cloned after the promoter. Plasmid concentrations were normalized to 50 ng/µl and transformed into equal cell volumes. The CFU of the serial dilution inoculations were used to determine the transformation efficiency, expressed in CFU/µg. The J23119 promoter is a constitutive promoter, the pL promoter is induced by high temperature, the pBAD promoter is induced by the presence of arabinose in the growth medium, and the sigma32 promoter is induced in stationary phase cells. For the MAD7 nuclease, the sigma32 promoter exhibits leaky expression, while the pL and pBAD promoters are heavily repressed.

[0281] FIG. 13 is a graph showing the effect of thermal induction on cell viability. In this experiment, a pool of cassettes was designed to target an inactive PAM sequence. Cells were diluted 1/50 by volume in microtiter plates containing 200 μl of LB+chloramphenicol/carbenicillin. The diluted cultures were induced for 1 hour at the indicated temperatures before returning to 30° C. with continuous shaking in an Infinite M Nano+ plate reader. T ₀ corresponds to the time of the stage of induction, and growth was monitored by measuring OD600 at 10-minute intervals. It should be noted that high temperature (42°C) induction does not affect cell viability or growth rate.

[0282] FIG. 14 shows the results of pooled induction experiments using a sequence verified editing cassette that targets the galK locus and introduces a stop codon at amino acid D70. After three hours of growth, cells were either induced at 42°C for 15 min or kept at 30°C before seeding for serial dilutions. The CFU/ml value was calculated based on the inoculation volumes. For reference, the dotted line in Fig. 14 shows a typical CFU value obtained in an equivalent experiment using the J23119 constitutive promoter (data not shown). As can be seen, the absence of induction (i.e., the absence of editing) ensures high transformation efficiency (10-100 fold) due to the absence of toxic DNA double-strand breaks.

[0283] FIG. 15 shows the growth profiles of randomly selected variants from the PAM silent mutation (SPM) library. This 500-element library targets regions throughout the E. coli body and combines synonymous mutations that do not have the expected fitness effects. Colonies were selected from agar plates with non-induced transformed cells. Cells were taken from an agar plate and grown in 200 μl LB+chloramphenicol/carbenicillin overnight in a 96-well microtiter plate. Then, 10 μl of the contents of the wells of the original microtiter plate were transferred to two replicas of daughter microtiter plates that were not inducible (top) or gRNA and nuclease induction (via the pL-inducible promoter) for 1 hour at 42°C (bottom). Well maps show relative OD at 6 hours. The insets show examples of complete growth curves for the indicated wells for reference. Replicate wells represent the growth observed for cassettes of the same construct with or without gRNA induction. While most wells for the non-induction plate show normal growth profiles, the induction plate shows that most of the gRNA constructs are still active when induced, as indicated by the long delay before the cells reach exponential growth. Thus, actively edited cells have reduced viability due to DNA damage so that many of them die, and edited cells that survive grow slowly from the start as the cellular machinery works to restore editing. This characteristic of edited cells can be used for high throughput active editing screening.

Example 2: Amplification and Assembly of Editing Cassette and Backbone

[0284] Preparing the Edit Cassette: 5 nM oligonucleotides synthesized on the chip were amplified using polymerase Q5 in volumes of 50 μl. PCR conditions were as follows: 95°C for 1 min; 8 cycles at 95°C for 30 s/60°C for 30 s/72°C for 2.5 min; with a final hold at 72°C for 5 min. After amplification, the PCR products were subjected to SPRI purification, where 30 µl of the SPRI mixture was added to 50 µl of the PCR reaction and incubated for 2 min. The tubes were subjected to a magnetic field for 2 minutes, the liquid was removed and the beads were washed 2 times with 80% ethanol with a 1 minute break between washes. After the final wash, the beads were allowed to dry for 2 minutes, 50 μl of 0.5x TE pH 8.0 was added to the tubes and the beads were shaken to mix. This suspension was cultured at room temperature for 2 min and then subjected to a magnetic field for 2 min. Then the eluate was removed and the DNA was quantified.

[0285] After quantification, a second amplification procedure was performed using a dilution of the eluate after SPRI purification. PCR was performed under the following conditions: 95°C for 1 min; 18 cycles at 95°C for 30 s/72°C for 2.5 min; with a final hold at 72°C for 5 min. The amplicons were tested on a 2% agarose gel and the pools with the purest yield(s) were identified. Amplification products containing heterodimers or chimeras were not used.

[0286] Preparing the main circuit: A 10-fold serial dilution series of the purified backbone was performed, and each of the backbone dilution series was amplified under the following conditions: 95° C. for 1 min; then 30 cycles at 95°C for 30 s/60°C for 1.5 min/72°C for 2.5 min; with a final hold at 72°C for 5 min. After amplification, the amplified backbone was subjected to the SPRI purification described above for the cassettes. The backbone was eluted in 100 µl of distilled demineralized water and quantified prior to Gibson Assembly® assembly.

[0287] Gibson Assembly: 150 ng of DNA backbone was combined with 100 ng of cassette DNA. An equal volume of 2x Gibson Master Mix was added and the reaction mixture was incubated for 45 min at 50°C. After assembly, the assembled main chain and cassettes were subjected to SPRI cleaning as described above.

Example 3: Transformation of the Editing Vector Library into E cloni®

[0288] Transformation: 20 µl of the prepared Gibson Assembly Editing Vector Reaction Mix was added to 30 µl of chilled water along with 10 µl of E cloni® maximally competent cells (Lucigen, Middleton, Wisconsin). Aliquots of transformed cells were spot-plated to test for transformation efficiency, with more than 100x coverage required to continue. Transformed E cloni® cells were grown in 25 ml SOB + 100 μg/ml carbenicillin. Initial glycerol mixtures were prepared from a saturated culture by adding 500 μl of 50% glycerol to 1000 μl of a saturated overnight culture. These initial mixtures were frozen at -80°C. This step is optional and provides the finished raw material of the cloned editing library. Alternatively, Gibsonian or other assembly of the editing cassettes and the vector backbone can be performed prior to each editing experiment.

Example 4 Preparation of Competent Cells

[0289] A 1 ml aliquot of a fresh overnight culture of EC1 cells transformed with a motor vector was added to a 250 ml flask containing 100 ml LB/SOB+25 μg/ml chloramphenicol medium. Cells were grown to OD 0.4-0.7 and cell growth was stopped by transferring the culture to ice for 10 minutes. Cells were pelleted at 8000 g in a JA-18 rotor for 5 min, washed 3 times with 50 ml of ice-cold distilled demineralized water or 10% glycerol, and pelleted at 8000 g in a JA-18 rotor for 5 min. Washed cells were resuspended in 5 ml ice-cold 10% glycerol and divided into 200 μl aliquots. Optionally, at this point, the glycerin raw material could be stored at -80°C for later use.

Example 5: Creation of a New Cell Line Transformed with a Motor Vector

[0290] Transformation: 1 µl of motor vector DNA (containing the coding sequence for MAD7 nuclease under the control of pL-inducible promoter, chloramphenicol resistance gene and λ Red recombination system) was added to 50 µl of E. coli strain EC1. Transformed cells were seeded on LB plates with 25 μg/ml chloramphenicol and cultured overnight to accumulate clonal (or essentially clonal) isolates. The next day, a colony was selected, grown overnight in LB+25 μg/ml chloramphenicol, and glycerol stock was prepared from a saturated overnight culture by adding 500 μl of 50% glycerol to 1000 μl of culture. The EC1 feed containing the motor vector was frozen at -80°C.

Example 6: High Performance Clone Editing

[0291] The following protocols address the issue of cell growth/survival bias due to double-stranded DNA breaks:

[0292] Transformation: 100 ng of the cloned editing vector library or Gibson Assembly® editing vector reaction mixture was transformed by electroporation into 100 μl of competent EC1 cells containing the motor vector. The electroporator was set to 2400 V in a 2 mm cuvette. After transformation, cells were allowed to recover for 3 hours in SOB medium. A series of 10-fold dilutions of the recovered cells (in H ₂ O) were spotted and the resulting CFU/dilution ratios were used to calculate the transformation efficiency.

[0293] Seeding and lining up colonies: 100 μl of a suitable dilution of cells were seeded on LB medium + 25 μg/ml chloramphenicol and grown at 30°C overnight. Colonies were selected and grown overnight to saturation at 30° C. in 96-well microtiter plates.

[0294] Induced Cutting, Editing, and Edit Validation: The replicator was used to transfer colonies from overnight plate wells to 200 µl of fresh SOB+1% arabinose in 96-well microtiter replica plates. The replicator transfers approximately 1-2 µl per well resulting in a 100-fold dilution for growth. The microtiter plates were incubated with shaking at 250 rpm for 3-4 hours to allow the cells to reach the mid-logarithmic phase. The plates were then transferred to a 42°C static incubator and incubated for 2 hours, and then placed in a 30°C incubator for 1 hour to recover. At this point, cell replicas were ready for genomic preparation and were verified by sequencing analysis. As has been described with reference to FIG. 2B, additional replica plates of fresh SOB free of arabinose can be used to provide functional deconvolution of inactive slicing designs from the population.

Example 7: Error Correction: Realignment and pooled editing using inducible gRNAs

[0295] Transformation: 100 ng of the cloned editing vector library or Gibson Assembly® reaction was transformed by electroporation into 100 μl of competent EC1 cells containing the motor vector. The electroporator was set to 2400 V in a 2 mm cuvette. After transformation, cells were allowed to recover for 3 hours in SOB medium. A series of 10-fold dilutions of the recovered cells (in H ₂ O) were spotted and the resulting CFU/dilution ratios were used to calculate the transformation efficiency.

[0296] Seeding and lining up colonies: 100 μl of a suitable dilution of cells were seeded on LB medium + 25 μg/ml chloramphenicol and grown at 30°C overnight. Colonies were selected and grown overnight to saturation in 1 ml SOB+25 μg/ml chloramphenicol/100 μg/ml carbenicillin at 30° C. in 96-well microtiter plates.

[0297] Identification of active cassettes and realignment: The replicator was used to transfer colonies from the wells of overnight plates into 200 µl of fresh SOB without arabinose in 96-well microtiter plates for replicas. The replicator transfers approximately 1-2 µl per well resulting in a 100-fold dilution for growth. Replicate microtiter plates were incubated with shaking at 250 rpm for 3-4 hours to allow the cells to reach mid-log growth. The plates were then transferred to a 42°C static incubator and incubated for 2 hours, and then placed in a 30°C incubator for 1 hour to recover. The replicator was then used to transfer cells from the replicate microtiter plates to agar plates with LB+25 μg/ml chloramphenicol/100 μg/ml carbenicillin medium, after which the cells were allowed to grow overnight. Wells with active gRNAs were identified and 50 µl of cells from these colonies from the original 96-well microtiter plate were re-arranged into "functionally corrected" plates (eg, cells identified as having functional gRNAs were transferred (selected) to the 96-well plate).

[0298] Pooled editing using pooled cassettes identified as active: 100 μl of cells identified as having functional gRNAs were transferred to 5 ml of SOB medium and grown until the culture was saturated. 1% arabinose was added to the tube and the tube was incubated for 2 hours at 30°C. Cutting/editing was induced by transferring the tube to a 42°C shaking water bath for 2 hours. The tube was then removed from the water bath and left to reconstitute for 2 hours at 30°C in an incubator shaken at 250 rpm. A diluted culture (approximately 10 ^-4 -10 ^-5 ) was plated on LB+25 μg/ml chloramphenicol/100 μg/ml carbenicillin medium, resulting in isolated clonal colonies. Editing was evaluated/validated by target or whole genome sequencing.

Example 8: Error Correction: Rebuild and Clone

[0299] Transformation: 100 ng of the cloned editing vector library or Gibson Assembly® reaction was transformed by electroporation into 100 μl of competent EC1 cells containing the motor vector. The electroporator was set to 2400 V in a 2 mm cuvette. After transformation, cells were allowed to recover for 3 hours in SOB medium. A series of 10-fold dilutions of the recovered cells (in H ₂ O) were spotted and the resulting CFU/dilution ratios were used to calculate the transformation efficiency.

[0300] Seeding and lining up colonies: 100 μl of a suitable dilution of cells were seeded on LB medium + 25 μg/ml chloramphenicol and grown at 30°C overnight. Colonies were selected and grown overnight to saturation in 1 ml SOB+25 μg/ml chloramphenicol and 100 μg/ml carbenicillin at 30° C. in 96-well microtiter plates.

[0301] Identification of active cassettes and realignment: The replicator was used to transfer colonies from the wells of overnight plates to 200 µl of fresh SOB without arabinose in 96-well microtiter replica plates. The replicator transfers approximately 1-2 µl per well resulting in a 100-fold dilution for growth. Replicate microtiter plates were incubated with shaking at 250 rpm for 3-4 hours to allow the cells to reach mid-log growth. The plates were then transferred to a 42°C static incubator and incubated for 2 hours, and then placed in a 30°C incubator for 1 hour to recover. The replicator was then used to transfer cells from replica microtiter plates to LB+25 μg/ml chloramphenicol/100 μg/ml carbenicillin LB+ medium plates, after which the cells were allowed to grow overnight. Wells with active gRNAs were identified and 50 µl of cells from these colonies from the original 96-well microtiter plate were re-arranged into "functionally corrected" plates (eg, cells identified as having functional gRNAs were transferred to the 96-well plate).

[0294] Re-amplification, cloning and library validation: Cells identified as having functional gRNAs were pooled and DNA was extracted and isolated. Serial dilutions were made from isolated DNA and amplification was performed with primers designed to amplify editing cassettes. PCR was performed under the following conditions: 95°C for 1 min; 18 cycles at 95°C for 30 s/60°C for 30 s/72°C for 2.5 min; with a final hold at 72°C for 5 min. The amplicons were tested on a 1% agarose gel and the pools with the purest yield(s) were identified. The amplified cassettes were mini-prepared and eluted in 50 µl of distilled demineralized water. A Gibson Assembly® reaction was then performed containing 150 ng of co DNA backbone per 100 ng of cassette inserts. An equal volume of 2x Master Mix was added to the backbone and insert, and the reaction mixture was incubated for 45 min at 50°C, then dialyzed for 30 min in a sessile droplet using a 0.25 µm disc filter. 100 ng of the Gibson Assembly® reaction was electroporated into competent EC1 cells containing the motor vector as described above. After transformation, cells were allowed to recover for 3 hours in SOB medium. A series of 10-fold dilutions of the recovered cells (in H ₂ O) were spotted and the resulting CFU/dilution ratios were used to calculate the transformation efficiency. 100 µl of a suitable cell dilution were seeded on LB+25 µg/ml chloramphenicol medium and grown at 30° C. overnight. Editing was evaluated/validated, for example, by sequencing.

Example 9: Enrichment of Edited Cells with Identification of Growth Retardation

[0303] Transformation: 100 ng of the cloned editing vector library or Gibson Assembly® reaction was transformed by electroporation into 100 μl of competent EC1 cells containing the motor vector. The electroporator was set to 2400 V in a 2 mm cuvette. After transformation, cells were allowed to recover for 3 hours in SOB medium. A series of 10-fold dilutions of the recovered cells (in H ₂ O) were spotted and the resulting CFU/dilution ratios were used to calculate the transformation efficiency.

[0304] Seeding and colony alignment: 100 μl of a suitable dilution of cells were seeded on LB agar medium + 25 μg / ml chloramphenicol + arabinose and grown at 30 ° C for 6-8 hours. Alternatively, cells can be grown in liquid culture in LB + 25 µg/ml chloramphenicol medium at 30°C until saturation and diluted to an appropriate concentration before plating on LB + 25 µg/ml chloramphenicol + arabinose agar medium, followed by growth at 30°C in within 6-8 hours. After 6-8 hours of growth, the plate temperature was adjusted to 42° C. and the plates were incubated for 2 hours. The temperature was then lowered to 30°C and the cells were allowed to recover overnight.

[0305] Identification of edited cells: Small colonies were identified. The small colony phenotype indicates that cell viability was compromised during the induced editing procedure. Efficient recovery of edited cells from the original pool was achieved by identifying and selecting small colonies and placing cells from these small colonies on a 96-well plate to create a library of edited cells, or cells from colonies were pooled to create highly edited cell populations for recursive editing. Editing was evaluated/validated by sequencing. It was found that 85% of the small colonies were edited cells.

Example 10: Managing a Standard Seeding Protocol

[0306] This protocol describes a standard seeding protocol for enriching nucleic acid-directed nuclease-edited bacterial cells by singulation or substantial singulation, growth, editing, and normalization. This protocol was used to amplify the inducible system for both nuclease and gRNA to ensure phenotypic differences in colonies. From the resulting agar plates, it was possible to select edited cells with a high degree of certainty (~80%). While it is clear that this protocol can be used to enrich edited cells, in the experiments described here, this "standard seeding protocol" or "SPP" was used to compare the efficiency of singulation or significant singulation, editing, and normalization with a solid wall device. Materials: In addition to standard molecular biology tools, you will need the following:

Table 3

Product Provider SOB Teknova LB Teknova LB agar plate with chloramphenicol/carbenicillin and 1% arabinose Teknova

[0307] Protocol: The inputs to this protocol are frozen electrocompetent cells and purified nucleic acid assembly product. Immediately after electroporation, the cell/DNA mixture was transferred to a culture tube containing 2.7 ml of SOB medium. Preparation of 2.7 ml aliquots in 14 ml culture tubes prior to electroporation allowed faster recovery of cells from the electroporation cuvette; the final extraction volume was 3 ml. All culture tubes were placed in a shaking incubator set at 250 rpm and 30° C. for 3 hours. While the cultures were being reconstituted, the required number of LB agar plates with chloramphenicol and carbenicillin + 1% arabinose were removed from the refrigerator and warmed to room temperature. Multiple dilutions were used for each inoculation in order to have countable and isolated colonies on the plates. Seeding suggestions:

Table 4

Sequencing type Suggested dilution Seeding volume SinglePlex 10 ^-1 -10 ^-3 300 µl Amplicon Not 300 µl (= 1/10 part reconstituted)

[0308] After 3 hours, the culture tubes were removed from the incubator. Amplicon sequencing was first seeded according to the table above. Seed balls were used to distribute the culture evenly over the agar-agar. These beads were removed from the plate and the plate was allowed to dry uncovered in a laminar flow cabinet. While the plates were drying, the remaining culture was used to make serial dilutions, where standard dilutions were 50 µl of culture per 450 µl of sterile 0.8% NaCl. The plate/tubes used for these dilutions (as well as for the initial culture) were kept at 4° C. in case additional dilutions were needed depending on the number of colonies. Seeding for SinglePlex was performed according to Table 4. More or fewer dilutions may be used based on knowledge of the library/competent cells. The cultures were spread evenly on agar-agar using sterile seed beads. These beads were then removed from the plate and the plates were allowed to dry uncovered in a laminar flow cabinet. While the plates were drying, the incubator was programmed according to the following settings: 30°C for 9 hours → 42°C for 2 hours → 30°C for 9 hours. The agar plates were placed in the set up incubator and after the temperature cycle was completed (~21 hours), the agar plates were removed from the incubator. The editing induction was successful, as indicated by the difference in the size of the resulting cell colonies.

Example 11 Culture Singulation E. coli in solid wall device

[0309] Electrocompetent E. coli cells were transformed with a cloned library, an isothermally assembled library, or a process control sgRNA plasmid (loss surrogate) as described in Examples 2-5 above. The E. coli strain carried the appropriate endonuclease and λ Red components as well as an editing induction system (eg, on a motor plasmid or integrated into the bacterial genome, or a combination of both). Transformations typically used 150 ng of plasmid DNA (or Gibson Assembly reaction mix) with 150 ng of pL sgRNA backbone DNA. After electroporation, the cells were allowed to recover in 3 ml of SOB, after which they were incubated at 30° C. with shaking for 3 hours. In parallel with the processing of the samples in the solid wall device, the samples were also processed according to the standard seeding protocol (see Example 10 above) to compare the "normalization" in the solid wall device with the standard laboratory process. Immediately prior to introducing the cells into the solid wall permeable bottom device, the 0.2 µm filter forming the bottom of the microwells was treated with 0.1% Tween to ensure proper cell distribution in the microwells of the solid wall device. Filters were placed in a Swinnex filter holder (47 mm, Millipore®, SX0004700) and 3 ml of a solution of 0.85% NaCl and 0.1% Tween was passed through a solid wall device and filtered using vacuum. Various concentrations of tween were evaluated and it was determined that for a 47 mm diameter solid wall device with a 0.2 µm microwell bottom filter, solid wall device + 0.1% tween filter pretreatment was preferred (data not shown) .

[0310] After a 3 hour reconstitution in SOB, the transformed cells were diluted and 3 ml of the diluted cells were processed with a tween-treated solid wall device and filter, again using vacuum. The number of successfully transformed cells was expected to be approximately 1.0E+06 to 1.0E+08 in order to load approximately 10,000 transformed cells into a current 47 mm solid wall permeable bottom device (having ~30,000 wells). Serial dilutions of 10 ^-1 , 10 ^-2 and 10 ^-3 were prepared, then 100 μl of each of these dilutions were combined with 3 ml of 0.85% NaCl and the samples were loaded into solid wall devices. Each solid wall permeable bottom device was then removed from the Swinnex filter holder and transferred to an LB agar plate containing carbenicillin (100 µg/mL), chloramphenicol (25 µg/mL) and arabinose (1% final concentration). Solid wall devices were placed metal side up so that the permeable bottom membrane touched the surface of the agar agar and the nutrients from the plate could pass up through the filter bottom of the wells. Solid wall devices on LB agar plates were incubated for 9 hours at 30°C, 2 hours at 42°C, then again at 30°C for 12-16 hours, and in another experiment for 36-40 hours.

[0311] At the end of the incubation, the perforated discs and filters (still assembled) were removed from the reference power source (in this case, an agar plate) and were photographed with a focused translucent light source so that the number and distribution of loaded microwells on the device could be assessed. solid wall (data not shown). To extract cells from a solid wall, permeable bottom device, the filter was transferred to a labeled sterile Petri dish 100 mm in diameter containing 15 ml of sterile NaCl at a concentration of 0.85%, then this Petri dish was placed in a shaking incubator set to 30° C/80 rpm to gently remove the cells from the filter and resuspend them in the NaCl solution. Cells were shaken for 15 min and then transferred to a sterile tube, for example, a 50 ml conical centrifuge tube. The OD600 value of the cell suspension was measured and at this stage the cells can be processed in different ways depending on the purpose of the study. For example, if the plasmids or libraries are designed to target a sugar pathway gene such as galK, then the resuspended cells can be plated on MacConkey agar plates containing galactose (at a final concentration of 1%) and appropriate antibiotics. On this differential medium, colonies that result from successfully edited cells are expected to be phenotypically white while unedited colonies are red. This red/white phenotype can then be used to evaluate the percentage of edited cells and the degree of normalization of edited and unedited cells. The results of one experiment are shown below in Table 5. In all repetitions, the transformed cells grew in solid wall devices for 9 hours at 30°C, 2 hours at 42°C, and overnight at 30°C.

Table 5

Twin? Dilution
counted Red colonies white colonies % edit Without tween ^10-4 72 5 6% Without tween ^10-4 89 3 3% Without tween ^10-3 64 5 7% Twin pretreatment ^10-4 71 5 7% Twin pretreatment ^10-3 443 29 6% Twin pretreatment ^10-3 149 12 7% Twin pretreatment ^10-3 83 21 twenty% Twin pretreatment ^10-2 318 112 26% Twin+twin pretreatment in cell loading buffer ^10-3 163 25 13% Twin+twin pretreatment in cell loading buffer ^10-4 132 ten 7% Twin+twin pretreatment in cell loading buffer ^10-4 31 9 23% Twin+twin pretreatment in cell loading buffer ^10-3 147 eighteen 10.9% Twin+twin pretreatment in cell loading buffer ^10-2 720 150 17% Twin+twin pretreatment in cell loading buffer ^10-3 55 fifteen 21%

[0312] FIG. 16 is a graph showing the extent of cell normalization (% cells edited) for various dilutions of transformed cells, no tween treatment, tween pretreatment, and tween+tween pretreatment in buffer when loading cells into microwells of the solid wall device. The standard seeding protocol (SPP) was performed in parallel with the solid wall singulation experiments as a guide (first bar on the left in the diagram). Of note, the percent change for the standard seeding protocol was approximately 27.5%, and the percent change for two replicates of cell dilution to 10 ^-3 with Tween pretreatment was approximately 20% and 26%, respectively.

Example 12 Singulation of Yeast Colonies in a Solid Wall Device

[0313] Electrocompetent yeast cells were transformed with a cloned library, an isothermally assembled library, or a process control sgRNA plasmid (loss surrogate) as described in Examples 2-5 above. Saccharomyces cerevisiae electrocompetent cells were prepared as follows: The day before the transformation was to take place, 10 ml of YPAD was inoculated with the selected Saccharomyces cerevisiae strain. This culture was shaken at 250 rpm and 30° C. overnight. The next day, 100 ml of YPAD was added to a 250 ml baffled flask and inoculated with overnight culture (approximately 2 ml of overnight culture) until the OD600 reached 0.3 +/- 0.05. This culture was placed in a 30° C. incubator shaking at 250 rpm and grown for 4-5 hours with the OD checked hourly. When the culture reached an OD600 of approximately 1.5, 50 ml each was poured into two 50 ml conical tubes which were then centrifuged at 4300 rpm for 2 minutes at room temperature. The supernatant was carefully discarded from the sediment from all conical tubes. 50 ml of lithium acetate/dithiothreitol solution was added to each conical tube and the pellet was carefully suspended. Both suspensions were transferred to a 250 ml flask and placed on a shaker; and then shaken at 30°C and 200 rpm for 30 min. After completion of the incubation, the suspension was transferred to two 50 ml conical tubes. These tubes were then centrifuged at 4300 rpm for 3 minutes, after which the supernatant was discarded.

[0314] After the lithium acetate/dithiothreitol treatment step, cold liquids were used and the cells were kept on ice until electroporated. 50 ml of 1 M sorbitol was added and the pellet was resuspended and then centrifuged at 4300 rpm for 3 min at 4° C. after which the supernatant was discarded. Washing with 1M sorbitol was repeated twice for a total of three washes. 50 µl of 1M sorbitol was added to one pellet, the cells were resuspended and then transferred to another tube to suspend the second pellet. The volume of the cell suspension was measured and adjusted to 1 ml with cold 1M sorbitol. At this point, cells were electrocompetent and could be transformed with a cloned library, an isothermally assembled library, or srRNA plasmids to control the process. Briefly, the required number of 2 mm gap electroporation cuvettes was prepared by labeling the cuvettes and then cooling on ice. The appropriate plasmid - or DNA mixture - was added to each respective cuvette and it was placed back on ice. 100 µl of electrocompetent cells were transferred to each labeled cuvette and each sample was electroporated using suitable electroporation conditions. Then, 900 µl of YPAD-sorbitol medium at room temperature was added to each cuvette. The cell suspension was transferred to a 14 ml culture tube and then shaken at 30° C. and 250 rpm for 3 hours. After 3 hours of recovery, 9 ml of YPAD containing a suitable antibiotic, such as geneticin or hygromycin B, was added.

[0315] At this point, transformed cells were processed in parallel in a solid wall device and in a standard seeding protocol (see Example 10 above) to compare "normalization" in a solid wall device with a standard laboratory process. Immediately prior to introducing the cells into the solid wall permeable bottom device, the 0.45 µm filter forming the bottom of the microwells was treated with 0.1% Tween to ensure proper cell distribution in the microwells of the solid wall device. Filters were placed in a Swinnex filter holder (47 mm, Millipore®, SX0004700) and 3 ml of a solution of 0.85% NaCl and 0.1% Tween was passed through a solid wall device and filtered using vacuum. Various concentrations of Tween were evaluated and it was determined that for a 47 mm solid wall device with a 0.45 µm microwell bottom filter, pretreatment of the solid wall device + 0.1% Tween filter was preferred (data not shown) .

[0316] After a 3 hour reconstitution in SOB, the transformed cells were diluted and 3 ml of the diluted cells were processed with a tween-treated solid wall device and filter, again using vacuum. The number of successfully transformed cells was expected to be approximately 1.0E+06 to 1.0E+08 in order to load approximately 10,000 transformed cells into a current 47 mm solid wall permeable bottom device (having ~30,000 wells). Serial dilutions of 10 ^-1 , 10 ^-2 and 10 ^-3 were prepared, then 100 μl of each of these dilutions were combined with 3 ml of 0.85% NaCl and the samples were loaded into solid wall devices. Each solid wall permeable bottom device was then removed from the Swinnex filter holder and transferred to an LB agar plate containing carbenicillin (100 µg/mL), chloramphenicol (25 µg/mL) and arabinose (1% final concentration). Solid wall devices were placed metal side up so that the permeable bottom membrane touched the surface of the agar agar and the nutrients from the plate could pass up through the filter bottom of the wells. Solid wall devices on YPD agar plates were incubated for 2-3 days at 30°C.

[0317] At the end of the incubation, the perforated discs and filters (still assembled) were removed from the reference power source (in this case an agar plate) and were photographed with a focused translucent light source so that the number and distribution of loaded microwells on the device could be assessed. solid wall (see Fig. 17A and 17B). To extract cells from a solid wall, permeable bottom device, the filter was transferred to a labeled sterile Petri dish 100 mm in diameter containing 15 ml of sterile NaCl at a concentration of 0.85%, then this Petri dish was placed in a shaking incubator set to 30° C/80 rpm to gently remove the cells from the filter and resuspend them in the NaCl solution. Cells were shaken for 15 min and then transferred to a sterile tube, for example, a 50 ml conical centrifuge tube. The OD600 value of the cell suspension was measured; at this stage, the cells can be processed in different ways depending on the purpose of the study. For example, if the ADE2 stop codon mutagenesis library is used, successfully edited cells should produce colonies with a red phenotype when resuspended cells are placed on YPD agar plates and allowed to grow for 4-7 days. This phenotypic difference allows one to quantify the percentage of edited cells and the degree of normalization of edited and unedited cells.

Example 13: Singulation, growth and editing of E.coli in SWIIN with 200K holes

[0318] Automated editing of the Singleplex genome using the MAD7 nuclease, a library with 94 different revisions in a single gene (yagP), and using a 200K cell singulation device such as illustrated in FIG. 4F-4R was completed successfully. The motor vector used was substantially similar to that shown in FIG. 11A (with MAD7 driven by the pL-inducible promoter) and the editing vector used was essentially similar to that shown in FIG. 11B, including the editing cassette driven by the pL inducible promoter and the λ Red recombination system driven by the inducible pBAD promoter, except that the editing cassette contained 94 yagP gene changes (donor DNA) and the appropriate corresponding gRNAs. Two SWIIN processes were compared and their performance compared to a standard seeding protocol (see Example 7). These SWIIN protocols differ from each other in that in one replica set, LB medium containing arabinose was used to distribute cells in SWIIN (arabinose was used to induce the λ Red recombination system (which allows repair of double-strand breaks in E. coli that are generated during editing). ), and in another set of replicates, arabinose-free SOB medium was used for cell distribution in SWIIN and for initial growth, replacing arabinose-free SOB medium with arabinose-containing SOB medium.Approximately 70,000 cells were loaded into SWIIN from 200K wells.

[0319] In all protocols (standard seeding, LB-SWIIN, and SOB-SWIIN), cells were allowed to grow at 30°C for 9 hours, and editing was induced by raising the temperature to 42°C for 2.5 hours, after which the temperature returned to 30°C and the cells were grown overnight. The results of this experiment are shown in Fig. 18 and in Table 6 below. It should be noted that similar editing efficiency was observed with four replicates of these two SWIIN processes, which means that the seeding efficiency of SWIIN with and without arabinose in the initial vehicle is similar. The percentage of editing in the standard seeding protocol was approximately 77%, in the liquid volume approximately 67%, and for the SWIIN replicas from approximately 63% to 71%. It should be noted that the percentage of unique edit cassettes divided by the total number of edit cassettes was the same for each protocol.

Table 6

Standard
Sowing SWIN
LB/arabinose
replica A SWIN
LB/arabinose replica B SWIN
SOB then
SOB/ arabinose
replica A SWIN
SOB then
SOB/arabinose
replica B 40006 edit calls/identified wells 0.777 0.633 0.719 0.663 0.695 Unique Editing Cassettes/Total
editing cassettes 0.49 0.49 0.43 0.50 0.51

Example 14: Fully automated run of RGN-guided Singleplex editing

[0320] Automated Singleplex genomic editing using the MAD7 nuclease has been successfully performed using an automated multi-module tool such as that shown in FIG. 5A-5D. See US Patent Nos. 9982279 issued May 29, 2018 and 10/240167 issued April 09, 2019

[0321] The ampR plasmid backbone and the lacZ_F172* editing cassette were assembled by Gibson Assembly® into an "editing vector" in an isothermal nucleic acid assembly module included in the automated tool. lacZ_F172 functionally knocks out the lacZ gene. "lacZ_F172*" means that the editing occurs at the 172nd residue in the lacZ amino acid sequence. After assembly, the product was desalted in an isothermal nucleic acid assembly module using AMPure beads, washed with 80% ethanol, and eluted in buffer. The assembled editing vector and electrocompetent E. coli cells ready for recombination were transferred to the transformation module for electroporation. Cells and nucleic acids were pooled and mixed for 1 min and electroporation was performed for 30 s. The parameters of the porous pulse were as follows: voltage 2400 V; duration 5 ms; interval 50 ms; number of pulses 1; polarity +. The transfer pulse parameters were as follows: voltage 150 V; duration 50 ms; interval 50 ms; number of pulses 20; polarity +/-. After 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 1 hour later and the cells were allowed to recover for another 2 hours. After reconstitution, the cells were stored at 4°C until they were claimed by the user.

[0322] Following an automated process and reconstitution, an aliquot of cells was plated on MacConkey agar with lactose (as the sugar substrate), chloramphenicol, and carbenicillin, and grown to form colonies. White colonies represented functionally edited cells, magenta colonies represented unedited cells. All fluid transfers were performed by the automated fluid handling device of the automated multi-module cell processing instrument.

[0323] As a result of automated processing, approximately 1.0E ^-03 cells were transformed in total (comparable to conventional laboratory results), and the editing efficiency was 83.5%. Editing of lacZ_172 in white colonies was confirmed by sequencing of the edited region of the cell genome. In addition, the automated cell processing steps were remotely monitored with a webcam and text messages were sent to update the status of the automated processing procedure.

Example 15: Fully automated recursive editing run

[0324] Recursive editing has been successfully achieved using a multi-module automated cell processing system. The ampR plasmid backbone and the lacZ_V10* editing cassette were assembled by a Gibson Assembly® into an "editing vector" in an isothermal nucleic acid assembly module included in the automated tool. Similar to the lacZ_F172 edit, the lacZ_V10 edit functionally knocks out the lacZ gene. "lacZ_V10" means that the editing occurs at amino acid position 10 in the lacZ amino acid sequence. After assembly, the product was desalted in an isothermal nucleic acid assembly module using AMPure beads, washed with 80% ethanol, and eluted in buffer. The first assembled editing vector and electrocompetent E. coli cells ready for recombination were transferred to the transformation module for electroporation. Cells and nucleic acids were pooled and mixed for 1 min and electroporation was performed for 30 s. The parameters of the porous pulse were as follows: voltage 2400 V; duration 5 ms; interval 50 ms; number of pulses 1; polarity +. The transfer pulse parameters were as follows: voltage 150 V; duration 50 ms; interval 50 ms; number of pulses 20; polarity +/-. After 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 1 hour later and the cells were grown for another 2 hours. The cells were then transferred to the centrifuge module and medium exchange was performed. The cells were resuspended in TB containing chloramphenicol and carbenicillin where the cells were grown to OD600=2.7 and then concentrated and made electrocompetent.

[0325] During cell growth, a second editing vector was prepared in an isothermal nucleic acid assembly module. The second editing vector contained the kanamycin resistance gene and the editing cassette contained the galK Y145* change. If successful, the change in galK Y145* gives the cells the ability to absorb and metabolize galactose. The editing performed by the galK Y154* cassette introduces a stop codon at the 154th amino acid location, changing the amino acid tyrosine to a stop codon. This editing renders the galK gene product non-functional and interferes with the uptake of galactose by the cells. After assembly, the second editing vector product was desalted in an isothermal nucleic acid assembly module using AMPure beads, washed with 80% ethanol, and eluted in buffer. The assembled editing vector and electrocompetent E. coli cells (which were transformed and selected for the first editing vector) were transferred to the transformation module for electroporation using the same parameters as described above. After electroporation, the cells were transferred to a recovery module (another growth module) and allowed to recover in SOC medium containing carbenicillin. After reconstitution, the cells were kept at 4° C. until retrieval, after which an aliquot of the cells was plated on LB agar with chloramphenicol and kanamycin. To quantify changes in lacZ and galK, replica plates were generated on two types of media: 1) MacConkey agar supplemented with lactose (as the sugar substrate), chloramphenicol, and kanamycin, and 2) MacConkey agar supplemented with galactose (as the sugar substrate), chloramphenicol and kanamycin. All fluid transfers were performed by the automated fluid handling device of the automated multi-module cell handling system.

[0326] In this recursive editing experiment, 41% of colonies tested had both lacZ and galK changes, which results were comparable to twice the editing efficiency obtained using a "laboratory" or manual approach.

[0327] While the present invention is embodied in many different forms, as detailed in connection with the preferred embodiments of the invention, it should be understood that the present disclosure is merely an example of the principles of the invention and is not intended to limit the present invention to particular embodiments. implementation illustrated and described in this document. Numerous variations can be made by those skilled in the art without departing from the spirit of the present invention. The scope of the present invention is defined by the appended claims, as well as their equivalents. The abstract and title should not be construed as limiting the scope of the present invention, as their purpose is to enable the relevant authorities, as well as the general public, to quickly determine the general nature of the present invention. In the following claims, if the term "means" is not used, none of the features or elements listed therein should be construed as a "means plus feature" limitation under applicable US patent law.

Claims (44)

1. An assembly for a module for cell separation on a solid wall, essential separation, growth, induction of editing, normalization and selection (SWIIN), designed to isolate cells, grow cells, create conditions for cell editing, and normalize or select small cell colonies, containing: a retaining element comprising an upper surface and a lower surface, which contains at least one retained substance distribution channel that crosses the retaining element from its upper surface to its lower surface by 70-95% of the length of the retaining element; in which the lower surface of the retaining element contains retaining ribs, between which there are guides for the flow of the retained substance; wherein the retaining member further comprises one or more retaining member openings configured to supply cells and fluid to the retaining member and remove cells and fluid from the retaining member; and in which the openings of the retaining element are hydraulically connected with the retained substance distribution channel and the retained substance flow guides; a perforated element with an upper surface and a lower surface, in which the upper surface of the perforated element is located below the lower surface of the retaining element, and in which the perforated element contains at least 25,000 holes; a filter with a top surface and a bottom surface, in which the upper surface of the filter is located below the lower surface of the perforated element, and in which the lower surface of the filter is located above the upper surface of the perforated element; a seal surrounding the perforated element and the filter; and a permeable element comprising a top surface and a bottom surface that includes at least one permeate distribution channel that traverses the permeable element from its bottom surface to its top surface for 70-95% of the length of the permeate element; in which the upper surface of the permeable element contains permeable ribs, between which are permeate flow guides; in which the permeable element further comprises one or more openings of the permeable element, configured to supply liquid to the permeable element and remove liquid from the permeable element; and wherein the orifices of the permeate member are in fluid communication with the permeate distribution channel and the permeate flow guides; and means for connecting the retaining element, the perforated element, the filter, the seal and the permeable element. 2. The assembly of claim. 1, in which the means for connecting the retaining element, the perforated element, the filter, the seal and the permeable element comprises ultrasonic welding. 3. The node according to claim 1, in which the perforated element contains at least 100,000 holes. 4. The node according to claim 3, in which the perforated element contains at least 200,000 holes. 5. An assembly according to claim 1 wherein the retaining and permeable elements are made of polycarbonate, cyclic olefin copolymer, or poly(methyl methacrylate). 6. The knot according to claim 1, wherein the retaining and permeable elements are 75 mm to 350 mm long, 50 mm to 250 mm wide and 2 mm to 15 mm thick. 7. An assembly according to claim 6, wherein the retaining and permeable elements are 150 mm to 250 mm long, 100 mm to 150 mm wide, and 4 mm to 8 mm thick. 8. The node according to claim 1, in which there are two channels for distributing the retained substance. 9. The assembly of claim 8, wherein the retention channels are approximately 150 mm long and 1 mm wide. 10. The node according to claim 1, in which there are two permeate distribution channels. 11. An assembly according to claim 10, wherein the permeate distribution channels are approximately 150 mm long and 1 mm wide. 12. The knot of claim 1 wherein the retaining ribs are approximately 0.5 mm high and 80 mm long. 13. The assembly of claim 1, wherein the retained material flow guides are approximately 5 mm wide. 14. The knot according to claim 1, wherein the permeable ribs are approximately 0.5 mm high and 80 mm long. 15. An assembly according to claim 1 wherein the permeate flow guides are approximately 5 mm wide. 16. Node according to claim 1, in which the volume of the node is from 15 ml to 40 ml. 17. The SWIIN module containing the node according to claim 1, additionally containing: tank assembly containing at least two tanks, in which the first reservoir is 1) in fluid communication with at least one reservoir opening through which fluids and/or cells enter the first reservoir from outside the SWIIN module, 2) in fluid communication with the reservoir/channel opening through which fluids and/or cells flow into one or more holes of the retaining element; and 3) pneumatically connected to a pressure source; the second reservoir is 1) in fluid communication with at least one reservoir opening through which fluids enter from outside the SWIIN module into the second reservoir, 2) in fluid communication with a reservoir/channel opening through which fluids flow into one or more permeable element apertures; and 3) pneumatically connected to a pressure source; and cover for the SWIIN module. 18. The SWIIN module according to claim 17, further comprising: two additional tanks, in which the first and third reservoirs are 1) hydraulically connected to at least two reservoir openings through which liquids and/or cells enter the first and third reservoirs from outside the SWIIN module, 2) hydraulically connected to the reservoir/channel opening through which liquids and/or the cells flow into at least two holes of the holding element; and 3) pneumatically connected to a pressure source; and the second and fourth reservoirs are 1) in fluid communication with at least two reservoir openings through which fluids enter the second and fourth reservoirs from outside the SWIIN module, 2) in fluid communication with a reservoir/channel opening through which fluids flow into at least two apertures permeable element; and 3) pneumatically connected to a pressure source. 19. The SWIIN module of claim 17, wherein the lid for the SWIIN module comprises a reservoir lid portion of the lid for the SWIIN module, wherein the reservoir lid portion comprises 1) at least two reservoir access holes that provide access to the reservoir openings, and 2) at least two pneumatic access openings that provide access to at least these two tanks and provide a pressure below and above atmospheric pressure in these at least two tanks. 20. Module for separation of cells on a solid wall, essential separation, growth, induction of editing, normalization and selection (SWIIN), designed to isolate cells, grow cells, create conditions for cell editing, and normalize or select small cell colonies, containing: node containing: a retaining element comprising an upper surface and a lower surface, which contains at least one retained substance distribution channel that crosses the retaining element from its upper surface to its lower surface by 70-95% of the length of the retaining element; in which the lower surface of the retaining element contains retaining ribs, between which there are guides for the flow of the retained substance; wherein the retaining member further comprises one or more retaining member openings configured to supply cells and fluid to the retaining member and remove cells and fluid from the retaining member; and in which the holes of the retaining element are hydraulically connected with the permeate distribution channel and the retained substance flow guides; a perforated element with an upper surface and a lower surface, in which the upper surface of the perforated element is located below the lower surface of the retaining element; a filter with a top surface and a bottom surface, in which the upper surface of the filter is located below the lower surface of the perforated element, and in which the lower surface of the filter is located above the upper surface of the perforated element; a seal surrounding the perforated element and the filter; and a permeable element comprising a top surface and a bottom surface that includes at least one permeate distribution channel that traverses the permeable element from its bottom surface to its top surface for 70-95% of the length of the permeate element; in which the upper surface of the permeable element contains permeable ribs, between which are permeate flow guides; in which the permeable element further comprises one or more openings of the permeable element, configured to supply liquid to the permeable element and remove liquid from the permeable element; and wherein the orifices of the permeate member are in fluid communication with the permeate distribution channel and the permeate flow guides; and means for connecting the retaining element, the perforated element, the filter, the seal and the permeable element; tank assembly containing at least two tanks, in which the first reservoir is 1) hydraulically connected to at least one reservoir opening through which fluids and/or cells enter the first reservoir from outside the SWIIN module, 2) hydraulically connected to at least one reservoir/channel opening through which liquids and/or cells flow into one or more holes of the retaining element; and 3) pneumatically connected to a pressure source; the second reservoir is 1) in fluid communication with at least one reservoir opening through which fluids enter from outside the SWIIN module into the second reservoir, 2) in fluid communication with a reservoir/channel opening through which fluids flow into one or more permeable element apertures; and 3) pneumatically connected to a pressure source; and cover for the SWIIN module.

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