CN115279899A - Cell populations with rationally designed edits - Google Patents

Abstract

The present disclosure provides compositions, automated multi-module instruments, and methods that increase the percentage of edited mammalian cells in a cell population when employing nucleic acid-guided editing.

Description

Cell populations with rationally designed edits

Cross Reference to Related Applications

This is International PCT application for USSN 16/740,420 filed on month 11 of 2020 and USSN 16/740,421 filed on month 11 of 2020.

Technical Field

The present disclosure relates to methods and compositions for increasing the percentage of edited mammalian cells in a cell population when using nucleic acid-guided editing, and automated multi-module instruments for performing these methods using the disclosed compositions.

Background

In the following discussion, certain articles and methods will be described for background and introductory purposes. Nothing contained herein is to be construed as an "admission" of prior art. The applicants expressly reserve the right to demonstrate, where appropriate, that the articles and methods cited herein do not constitute prior art according to applicable statutory provisions.

The ability to make precise, targeted changes to the genome of living cells has been a long-standing goal of biomedical research and development. Recently, various nucleases have been identified that allow manipulation of gene sequences and thus gene function. Nucleases include nucleic acid-guided nucleases that enable researchers to generate permanent editing in living cells. Of course, it is desirable to obtain as high an editing rate as possible in the cell population; however, in many cases, the percentage of edited cells resulting from nucleic acid-guided nuclease editing may be single-digit.

Accordingly, there is a need for improved methods, compositions, modules, and instruments for increasing editing efficiency in the field of nucleic acid-guided nuclease editing. The present disclosure addresses this need.

Summary of The Invention

This summary is provided to introduce a selection of concepts in a simplified form that are further described below in the detailed description. This summary is not intended to identify key or essential features of the claimed subject matter, nor is it intended to be used to limit the scope of the claimed subject matter. Other features, details, utilities, and advantages of the claimed subject matter will be apparent from the following written detailed description, including those aspects set forth in the accompanying drawings and defined in the appended claims.

In certain aspects, the present disclosure relates to methods, compositions, modules, and automated multi-module cell processing instruments that increase the efficiency of nucleic acid-guided editing in a cell population (e.g., a mammalian cell population). Thus, the methods presented herein include methods of increasing targeted editing rates using non-homologous end joining (NHEJ) repair, base editing, micro-homology directed repair (MMEJ), and/or Homology Directed Repair (HDR).

In some aspects, the disclosure provides methods for improving nuclease-directed cell editing using enrichment means to identify cells that have received editing components required to perform a desired editing operation. Enrichment can be performed directly or using an alternative (e.g., a cell surface handle co-introduced with one or more of the editing components).

In particular aspects, the disclosure provides methods for improving nuclease-directed cell editing using enrichment means to identify cells that have received editing components required to perform a desired editing operation.

In some aspects, enrichment processes and methods can be based on positive and negative signals for the surrogate. In other aspects, the enrichment methods can be based on a threshold level of the surrogate, e.g., a high level of the enrichment handle and a low level of the enrichment handle or a level where the enrichment handle is not present.

In some aspects, the disclosure provides methods for improving nuclease-directed editing rates by enriching mammalian cells that have received HDR donors, e.g., identifying cells that are more likely to have received editing mechanisms (editing artifacts) and designs encoding an enrichment handle.

In particular aspects, the disclosure provides methods for improving nuclease-guided mammalian cell editing using enrichment means to identify mammalian cells that have received HDR donors, guide nucleic acids, and/or nucleases. Such enrichment may include a single enrichment method for the HDR donor, guide nucleic acid, and nuclease, or two or more separate enrichment events for one or more of these elements. The HDR donor and guide nucleic acid may be introduced separately or covalently linked, as disclosed in, for example, USPN 9,982,278.

In some aspects, the disclosure provides methods of increasing the efficiency of target region editing in a population of cells, the methods comprising contacting a population of two or more cells with an editing mechanism (editing machinery) comprising (a) one or more editing cassettes comprising a nucleic acid encoding a gRNA sequence targeted to a first target region, wherein the gRNA is covalently attached to a region homologous to the first target region, the region comprising an expected sequence change relative to the target region, (b) one or more editing cassettes comprising a nucleic acid encoding a gRNA sequence targeted to a second target region, wherein the gRNA is covalently attached to a region encoding a selectable marker, and (c) a nuclease compatible with the gRNA sequence, exposing the population of cells to conditions that allow the cells to edit at the first target region and the second target region; and enriching for cells expressing the selectable marker from the population, wherein the selectable marker serves as a surrogate for determining editing of the first target region in cells enriched in the population of cells; and wherein the editing of the first target region enriches for cells expressing the selectable marker compared to cells in the population that do not express the selectable marker.

In some aspects, the disclosure provides methods of increasing the efficiency of editing of a population of cells, the methods comprising contacting a population of two or more cells with an editing mechanism comprising (a) one or more editing cassettes comprising a nucleic acid encoding a gRNA sequence targeted to a first target, wherein the gRNA is covalently attached to a region homologous to the first target, the region comprising an expected sequence change relative to the target, (b) one or more editing cassettes comprising a nucleic acid encoding a gRNA sequence targeted to a second target, wherein the gRNA is covalently attached to a region encoding a selectable marker, and (c) a nucleic acid encoding a nuclease compatible with the gRNA sequence, exposing the population of cells to conditions that allow the cells to edit at the first target and the second target, and enriching the population for cells expressing the selectable marker, wherein the selectable marker serves as a surrogate for determining editing of first target enrichment in cells in the population of cells.

In certain aspects, cell enrichment uses physical enrichment of cells expressing a selectable marker. Examples of this include fluorescence activated cell sorting selection, magnetic activated cell sorting selection, antibiotic selection, and the like.

In certain aspects, cell enrichment uses computational enrichment (computational enrichment) based on the presence of a selectable marker.

In some aspects, the editing cartridge that targets the first target zone further comprises a barcode. In particular aspects, the method further comprises incorporating a site-specific genomic barcode that enables tracing individual edited cells within the population.

In a particular aspect of the invention, HDR is improved using fusion proteins that retain certain properties of RNA-guided nucleases (e.g., binding specificity and ability to cleave one or more DNA strands), and also utilize other enzymatic activities, e.g., replication inhibition, reverse transcriptase activity, transcription enhancing activity, and the like. These nuclease fusion proteins can be used for nuclease-directed editing using the disclosed methods, whether or not enrichment methods as disclosed herein are used. The HDR donor and guide nucleic acid may be introduced separately or covalently linked, as disclosed in, for example, USPN 9,982,278.

In a particular aspect of the invention, HDR is improved using fusion proteins that retain the binding function and nickase activity of RNA-guided nucleases and also utilize other enzymatic activities, e.g., replication inhibition, reverse transcriptase activity, transcription enhancing activity, etc. These nickase fusion proteins can be used for RNA-guided nickase editing using the disclosed methods, whether or not enrichment methods as disclosed herein are used. The HDR donor and guide nucleic acid can be introduced separately or covalently linked as disclosed, for example, in USPN 9,982,278. In addition, the nicking enzyme may be introduced separately with DNA encoding the nicking enzyme or covalently linked to the donor DNA and the guide DNA, or separately in the form of a protein.

In particular aspects, the editing methods include the use of fusion proteins with nucleic acids having a guide RNA covalently attached to a region homologous to a target region, the region comprising one or more alterations of a native target sequence, and preferably using at least one enrichment mechanism, a physical enrichment mechanism, or a computational enrichment mechanism. Such methods may use a single guide RNA construct, or use two or more guide RNA constructs to target different genomic locations. In some aspects, the nucleic acid comprises more than one guide RNA covalently attached to different target regions within the genome.

In particular aspects, the editing methods comprise using a nickase fusion protein with a nucleic acid having a guide RNA covalently attached to a region homologous to a target region, the region comprising one or more alterations of a native target sequence, and using at least one of an enrichment mechanism, a physical enrichment mechanism, or a computational enrichment mechanism.

The use of fusion proteins and enrichment for editing methods may include a single enrichment method for HDR donors, guide nucleic acids, and nucleases, or two or more separate enrichment events for one or more of these editing mechanism elements.

In particular aspects, cells from a recipient HDR donor can be enriched using an initial enrichment step (e.g., using antibiotic selection or fluorescence detection), followed by an enrichment step using enrichment of cells that receive and express the co-introduced cell surface antigens.

A number of enrichment handles can be used in the methods and apparatus of the present disclosure, including but not limited to various cell surface molecules linked to a tag (e.g., hemagglutinin (HA) tag, FLAG tag, SBP tag, etc.). In certain aspects, the tagged cell surface markers are modified to alter their activity, including but not limited to Δ Tetherin-HA, Δ Tetherin-FLAG, Δ Tetherin-SBP, and the like.

In some aspects, the enrichment handle can bind an affinity ligand (e.g., engineered to comprise an HA tag, FLAG tag, SBP tag, etc.). In some aspects, the enrichment handle can be a heterologous cell surface receptor (a cell surface receptor not normally present in the cell type to be edited) or an autologous cell surface antigen with an engineered epitope tag. In particular aspects, the method uses edit selection cassettes, e.g., GFP to BFP edit cassettes.

The present disclosure also includes automated multi-module cell editing instruments having enrichment modules that perform enrichment methods, including the enrichment methods described herein, to increase the overall editing efficiency of a population of cells (e.g., mammalian cells).

An exemplary automated multi-module cell editing instrument of the present disclosure includes a housing configured to house all or some of the modules, a container configured to receive a cell, one or more containers configured to receive a nucleic acid, an editing mechanism introduction module configured to introduce a nucleic acid and/or protein into a cell, a recovery module configured to allow the cell to recover after the editing mechanism introduction, an enrichment module for enriching a cell that has received an editing nucleic acid and/or nuclease, an editing module configured to allow the introduced nucleic acid to edit the nucleic acid in the cell, and a processor configured to operate the automated multi-module cell editing instrument based on user input and/or selection of a preprogrammed script.

An exemplary automated multi-module cell editing instrument of the present disclosure includes a housing configured to house all or some of the modules, a container configured to receive the cells and edit nucleic acids, an editing mechanism introduction module configured to introduce nucleic acids into the cells, a recovery module configured to allow the cells to recover after the editing mechanism introduction, an enrichment module for enriching cells that have received editing nucleic acids and/or nucleases, an editing module configured to allow the introduced nucleic acids to edit nucleic acids in the cells, and a processor configured to operate the automated multi-module cell editing instrument based on user input and/or selection of preprogrammed scripts.

An exemplary automated multi-module cell editing instrument of the present disclosure includes a housing configured to house some or all of the modules, a container configured to receive a cell, at least one container configured to receive a nucleic acid for editing, a growth module configured to grow the cell, an editing mechanism introduction module including a flow-through electrical perforator to introduce the editing nucleic acid into the cell, an enrichment module to enrich the cell that has received the editing nucleic acid and/or nuclease, an editing module configured to allow editing of the nucleic acid in the nucleic acid editing cell, and a processor configured to operate the automated multi-module cell editing instrument based on user input and/or selection of a preprogrammed script.

An exemplary automated multi-module cell editing instrument of the present disclosure includes a housing configured to house some or all of the modules, a receptacle configured to receive a cell and edit nucleic acids, a growth module configured to grow the cell, a filtration module configured to concentrate the cell and render the cell electrically receptive, an editing mechanism introduction module including a flow-through electroporator to introduce the edit nucleic acids into the cell, an enrichment module to enrich the cell that has received the edit nucleic acids, an editing module configured to allow the cell to recover after electroporation and allow the nucleic acids to edit the cell, and a processor configured to operate the automated multi-module cell editing instrument based on user input.

Optionally, the nucleic acid and/or cell is contained within a cartridge that is introduced into the container of the instrument. Such cartridges for use in the present disclosure are described in, for example, USPN10,376,889, USPN10,478,822, and USPN10,406,525, which are incorporated herein by reference for all purposes.

The methods described herein enable users to obtain cell populations with much higher proportions of cells with precise intended editing and fewer unedited and/or unedited cells. The methods of the invention can produce 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90% or more of the desired edits within a population of cells.

Accordingly, in some aspects, the present disclosure provides a library of cells created using the editing methods described herein in the present disclosure.

In some aspects, the disclosure provides a cell library created using an automated editing system for nickase-directed genome editing, wherein the system comprises a housing, a container configured to receive a cell and one or more rationally designed nucleic acids comprising a sequence that facilitates a nickase-directed genome editing event in the cell, a transformation unit for introducing the one or more nucleic acids into the cell, an editing unit for allowing the nickase-directed genome editing event to occur in the cell, an enrichment module, and a processor-based system configured to operate an instrument based on user input, wherein the nickase-directed genome editing event created by the automated system produces a cell library comprising individual cells with rationally designed edits.

In some aspects, the present disclosure provides a cell library created using an automated editing system for nickase-directed genome editing, wherein the system comprises a housing, a cell container configured to receive cells, a nucleic acid container configured to receive one or more rationally designed nucleic acids comprising a sequence that facilitates nickase-directed genome editing events in the cells, a transformation unit for introducing the one or more nucleic acids into the cells, an editing unit for allowing the nickase-directed genome editing events to occur in the cells, and a processor-based system configured to operate the instrument based on user input, wherein the nickase-directed genome editing events created by the automated system produce a cell library comprising individual cells with rationally designed edits.

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

Detailed Description

All functions described in connection with one embodiment of the methods, devices, or apparatuses described herein are intended to apply to an additional embodiment of the methods, devices, and apparatuses described herein, unless explicitly stated or the features or functions are incompatible with the additional embodiment. For example, where 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 the feature or function may be deployed, utilized, or implemented in connection with an alternative embodiment unless the feature or function is incompatible with the alternative embodiment.

The practice of the techniques described herein may employ, unless otherwise indicated, conventional techniques and descriptions of molecular biology (including recombinant techniques), cell biology, biochemistry and genetic engineering techniques, which are within the skill of those of skill in the art. Such conventional techniques and descriptions can be found in standard Laboratory manuals such as Green and Sambrook, molecular Cloning A Laboratory Manual, 4 th edition, cold Spring Harbor Laboratory Press, cold Spring Harbor, N.Y., (2014); current Protocols in Molecular Biology, ausubel et al, (2017); neumann et al, electroposition and Electrofusion in Cell Biology, plenum Press, new York,1989; and Chang et al, guide to Electrodisplacement and Electrofusion, academic Press, california (1992), all of which are incorporated herein by reference in their entirety for all purposes. Nucleic acid-guided nuclease technology can be found, for example, in Genome Editing and Engineering from TALENs and CRISPRs to Molecular Surgery, appasani and Church (2018); and CRISPRs, methods and Protocols, lindgren and charpietier, 2015; both of these documents are incorporated herein by reference in their entirety for all purposes.

Note that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a cell" is a reference to one or more cells, and reference to "the system" includes reference to equivalent steps, methods, and devices known to those skilled in the art, and so forth. In addition, it should be understood that terms such as "left," "right," "top," "bottom," "front," "back," "side," "height," "length," "width," "upper," "lower," "inner," "outer," and the like as may be used herein merely describe reference points and do not necessarily limit embodiments of the present disclosure to any particular orientation or configuration. Moreover, terms such as "first," "second," "third," and the like, merely identify one of many parts, components, steps, operations, functions, and/or reference points as disclosed herein, and as such do not necessarily limit embodiments of the present disclosure to any particular configuration or orientation.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. All publications mentioned herein are incorporated by reference for all purposes, including but not limited to describing and disclosing devices, formulations and methodologies which might be used in connection with the invention described herein.

Where a range of values is provided, it is understood that each intervening value, to the extent that there is no such stated, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges, and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where a stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

In the following description, numerous specific details are set forth in order to provide a more thorough understanding of the present invention. It will be apparent, however, to one skilled in the art, that the present invention may be practiced without one or more of these specific details. In other instances, features and procedures well known to those skilled in the art have not been described in order to avoid obscuring the present invention. The terms used herein are intended to have plain and ordinary meanings as understood by those of ordinary skill in the art.

The term "complementary" as used herein refers to Watson-Crick base pairing between nucleotides, and in particular to nucleotides that form hydrogen bonds with each other, wherein a thymine or uracil residue is linked to an adenine residue by two hydrogen bonds, and a cytosine and guanine residue are linked by three hydrogen bonds. Typically, a nucleic acid comprises a nucleotide sequence that is described as having a "percent complementarity" or a "percent homology" with a specified second nucleotide sequence. For example, the nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10 nucleotides, 9 of 10 nucleotides, or 10 of 10 nucleotides of the sequence are complementary to the specified second nucleotide sequence. For example, the nucleotide sequence 3'-TCGA-5' and the nucleotide sequence 5'-AGCT-3' are 100% complementary; and the nucleotide sequence 3'-TCGA-5' is 100% complementary to the region of the nucleotide sequence 5 '-TTAGCTGG-3'.

The term DNA "control sequences" refers collectively to promoter sequences, polyadenylation signals, transcription termination sequences, upstream regulatory domains, origins of replication, internal ribosome entry sites, nuclear localization sequences, enhancers, and the like, which collectively provide for the replication, transcription, and translation of a coding sequence in a recipient cell. Not all of these types of control sequences need be present so long as the selected coding sequence is capable of being replicated, transcribed and (for some components) translated in an appropriate host cell.

As used herein, the term "donor DNA" or "donor nucleic acid" refers to a nucleic acid designed to introduce a DNA sequence modification (insertion, deletion, substitution) into a locus (e.g., a target genomic DNA sequence or a cellular target sequence) by homologous recombination using a nucleic acid-guided nuclease. For homology-directed repair, the donor DNA must have sufficient homology to the "cleavage site" in the genomic target sequence or to the region flanking the site to be edited. The length of one or more homology arms will depend, for example, on the type and size of modification being made. In many cases, and preferably, the donor DNA will have two regions of sequence homology (e.g., two homology arms) with the genomic target locus. Preferably, an "insert" region or "DNA sequence modification" region (nucleic acid modification of a genomic target locus that is desired to be introduced into a cell) will be located between two homologous regions. The DNA sequence modification may alter one or more bases of the target genomic DNA sequence at a particular site or sites. Alterations may include altering 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 a genomic target sequence. The deletion or insertion can be of 1, 2, 3, 4,5, 10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or more base pairs of the genomic target sequence.

The term "guide nucleic acid" or "guide RNA" or "gRNA" refers to a polynucleotide comprising: 1) A guide sequence capable of hybridizing to a genomic target locus and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid-guided nuclease.

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

The term "nickase" as used herein refers to a nuclease that cleaves one strand of double-stranded DNA at a specific recognition nucleotide sequence.

"operably linked" refers to an arrangement of elements wherein the components so described are configured to perform their usual function. Thus, a control sequence operably linked to a coding sequence can affect the transcription of the coding sequence and, in some cases, the translation of the coding sequence. Control sequences need not be contiguous with the coding sequence, so long as the control sequences function to direct the expression of the coding sequence. Thus, for example, an intervening sequence that is not translated but transcribed 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 necessarily reside on the same contiguous DNA molecule (i.e., chromosome) and may still have interactions that cause regulatory changes.

As used herein, the terms "protein" and "polypeptide" are used interchangeably. The protein may or may not consist entirely of amino acids.

A "promoter" or "promoter sequence" is a DNA regulatory region capable of binding to an RNA polymerase and initiating transcription of a polynucleotide or polypeptide coding sequence, such as messenger RNA, ribosomal RNA, small nuclear RNA (small nuclear RNA) or nucleolar RNA (small nuclear RNA), guide RNA or any kind of RNA transcribed by any RNA polymerase I, II or III of any class. Promoters may be constitutive or inducible.

The term "selectable marker" as used herein refers to a gene introduced into a cell that confers a trait suitable for artificial selection. Generally used selectable markers are well known to those of ordinary skill in the art. For example, selectable markers may use a means of depleting the cell population to enrich for edits, and include ampicillin/carbenicillin, kanamycin, chloramphenicol, nourseothricin, N-acetyltransferase, erythromycin, tetracycline, gentamicin, bleomycin, streptomycin, puromycin, hygromycin, blasticidin, and G418, or other selectable markers may be employed. In addition, selective markers include physical markers that confer a phenotype that can be used for physical or computational cell enrichment, for example, optically selective markers such as fluorescent proteins (e.g., green fluorescent protein, blue fluorescent protein) and cell surface handgrips.

The term "specific binding" as used herein includes having from about 10 between two molecules (e.g., an engineered peptide antigen and a binding target)^-7M, about 10^-8M, about 10^-9M, about 10^-10M, about 10^-11M, about 10^-12M, about 10^-13M, about 10^-14M or about 10^-15The dissociation constant of M indicates the binding affinity interaction.

The term "target genomic DNA sequence," "cellular target sequence," "target sequence," or "genomic target locus" refers to any locus in a nucleic acid (e.g., genome or episome) of a cell or population of cells at which it is desired to alter at least one nucleotide using a nucleic acid-guided nuclease editing system in vitro or in vivo. The target sequence may be a genomic locus or an extrachromosomal locus.

The term "variant" may refer to a polypeptide or polynucleotide that differs from a reference polypeptide or polynucleotide, but retains essential properties. Typical variants of a polypeptide differ in amino acid sequence from another reference polypeptide. Typically, the differences are limited such that the sequences of the reference polypeptide and the variant are very similar overall and are identical in many regions. The amino acid sequences of the variant and reference polypeptides may differ by one or more modifications (e.g., substitutions, additions, and/or deletions). The variant of the polypeptide may be a conservatively modified variant. The substituted or inserted amino acid residue may or may not be an amino acid residue encoded by the genetic code (e.g., a non-natural amino acid). Variants of a polypeptide may be naturally occurring, such as allelic variants, or variants of a polypeptide may be variants that are known not to occur naturally.

A "vector" is any of a variety of nucleic acids comprising a desired sequence or sequences to be delivered to and/or expressed in a cell. The vector is usually composed of DNA, but an RNA vector is also usable. Vectors include, but are not limited to, plasmids, F cosmids (fosmid), phagemids, viral genomes, synthetic chromosomes, and the like. In the present disclosure, the term "editing vector" comprises the coding sequence of a nuclease, the gRNA sequence to be transcribed, and the donor DNA sequence. However, in other embodiments, two vectors can be used — an engine vector comprising the coding sequence for the nuclease, and an editing vector comprising the gRNA sequence to be transcribed and the donor DNA sequence.

Brief Description of Drawings

The foregoing and other features and advantages of the invention will be more fully understood from the following detailed description of illustrative embodiments thereof, taken in conjunction with the accompanying drawings, in which:

1A-1C depict an automated multi-module instrument and its components by which a recursive editing method as taught herein is practiced.

Figure 2A depicts one embodiment of a spin growth flask for use with the cell growth modules described herein. Figure 2B illustrates a perspective view of one embodiment of a rotating growth flask in a cell growth module. Figure 2C depicts a cross-sectional view of the cell growth module from figure 2B. Figure 2D illustrates the cell growth module of figure 2B coupled with an LED, a detector, and a temperature regulation component.

Figure 3A is a model of tangential flow filtration used in the TFF device presented herein. Figure 3B depicts a top view of the lower member (lower member) of one embodiment of an exemplary TFF device. Fig. 3C depicts top views of the upper member (upper member) and lower member and membrane of an exemplary TFF device. Figure 3D depicts bottom views of the upper and lower members and membrane of an exemplary TFF device. Figures 3E-3K depict various views of yet another embodiment of a TFF module having fluidically coupled reservoirs. Fig. 3L is an exemplary pneumatic architecture diagram of the TFF module described in connection with fig. 3E-3K.

Fig. 4A shows an exemplary flow-through electroporation device (here, there are six such devices connected in common). Figure 4B is a top view of an embodiment of an exemplary flow-through electroporation device. Figure 4C depicts a top view of a cross-section of the electroporation device of figure 4C. Figure 4D is a cross-sectional side view of the lower portion of the electroporation device of figures 4C and 4D.

Fig. 5A and 5B depict the structure and components of one embodiment of a reagent cartridge.

Figure 6 is a simplified block diagram of an embodiment of an exemplary automated multi-module cell processing instrument.

FIG. 7 is a diagram illustrating a first set of exemplary workflows for conducting the editing and selection scheme of the present disclosure.

FIG. 8 is a diagram illustrating a second set of exemplary workflows for conducting the editing and selection scheme of the present disclosure.

Fig. 9 is a diagram illustrating a first set of exemplary workflows for conducting the CREATE merge editing scheme of the present disclosure.

Fig. 10 is a diagram illustrating a second set of exemplary workflows for performing the CREATE fusion scheme of the present disclosure.

Figure 11 is a diagram showing the potential mechanism for editing in more than one cell cycle using a fusion protein with reverse transcriptase activity.

Fig. 12 is a diagram illustrating exemplary elements in a plasmid structure for GFP expression assays.

Fig. 13A and 13B are diagrams showing delivery of the nuclease-GFP expression cassette monitored by FACS.

Fig. 14A and 14B are graphs showing GFP-to-BFP conversion for phenotypic evaluation of NHEJ and HDR mediated editing.

Figure 15 is a graph showing the differential expression levels of the thy1.2 reporter expressed by GFP to BFP editing cassettes.

FIGS. 16A-16E are graphs showing enrichment of Thy1.2 by MACS^{High (a)}Series of plots of the effect at the cellular level.

Figure 17 is a bar graph showing enrichment of comparable cell populations with higher editing rates (NHEJ and HDR) by FACS or MACS.

Figure 18 is a bar graph showing edits exhibited by Δ Tetherin-HA editing cassette enrichment by cells sorted using FACS.

FIGS. 19A and 19B are graphs showing how MACS bead concentration during enrichment affects Thy1.2 isolated by enrichment^{Height of}And Thy1.2^{Is low in}Expression cellsFigures and tables of relative proportions of (a).

FIGS. 20A and 20B are a graph and table showing how MACS bead concentration during enrichment affects the relative proportion of Δ Tetherin-HA enriched cells.

Figure 21 is a bar graph showing the edit rate of cells enriched using different amounts of thy 1.2-specific MACS beads.

FIG. 22 is a bar graph showing post-enrichment analysis of cells expressing high levels of the Δ Tetherin-HA reporter in HAP 1.

Fig. 23 is a bar graph showing enrichment of FACS enrichment techniques for cells with higher knockin edit rates at the DNMT3b gene.

Fig. 24 shows the design of CFE editing constructs CFE2.1 and CFE 2.2.

Fig. 25 shows the design of various grnas containing a 13bp TY to SH editing or a 13bp second region complementary to the nicked EGFP DNA sequence.

Figure 26 is a diagram showing the basic scheme of editing using the CREATE fusion editing cassette in figure 25, as compared to direct nuclease editing.

Figures 27A-27D are graphs showing editing of GFP into BFP HEK293T cells using various protocols.

Figure 28 is a diagram showing the basic protocol for CREATE fusion editing in combination with FACS selection.

Figure 29 is a graph showing the levels of dsRed _ low and dsRed _ high cells resulting from editing with MAD7 nuclease and editing with CREATE fusion editing.

Figure 30 is a graph showing differential expression levels of dsRed in edited cell populations.

Figure 31 is a histogram of dsRed edited by MAD7 or CREATE fusion showing GFP-to-BFP time course of cells sorted using FACS.

Fig. 32 is a diagram showing the basic scheme of CREATE fusion editing using a single gRNA.

Fig. 33A-33C are histograms showing editing efficiency of the CREATE fusion constructs CFE2.1 and CFE2.2 using lentiviral delivery.

Fig. 34A and 34B are histograms comparing editing efficiency edited using the CREATE fusion construct CFE2.2 and MAD7, both using lentiviral delivery.

Fig. 35A and 35B are diagrams illustrating exemplary strategies using the CREATE merge editing system with traceback or recording techniques.

Summary of the invention

The present disclosure relates to methods and apparatus for improving precise editing in a cell population. A variety of cellular mechanisms can be used in the editing process, including non-homologous end joining (NHEJ) repair, base editing, micro-homology directed repair (MMEJ), and/or Homology Directed Repair (HDR).

In particular aspects, the methods and apparatus improve editing via homology-directed repair (HDR); thus, in a particular aspect, the present disclosure provides methods of improving HDR in mammalian cells. In a more particular aspect, the present disclosure provides methods of improving HDR in human cells. In certain particular aspects, the present disclosure provides methods of improving HDR in human pluripotent cells, such as induced pluripotent stem cells.

In certain aspects, the disclosure provides for enrichment for co-introduced nucleic acids for enriching cells that have received editing components necessary for nucleic acid-directed editing, e.g., using specific selection for cells that have been transfected with plasmids comprising nucleic acids encoding donor and/or guide nucleic acids and optionally nucleases.

More specifically, enrichment of the cell subpopulation with the highest reporter expression enriches the cell population undergoing gene editing at a higher rate than the non-enriched population or the subpopulation with relatively lower reporter expression levels.

In particular aspects, the disclosure relates to automated methods for increasing editing efficiency using nucleic acids and cell surface selection handles that co-introduce coding editing mechanisms. In particular aspects, the co-introduction of nucleic acids occurs in a multi-module automated instrument, as described in more detail herein.

In certain aspects, the disclosure provides methods of improving homology-directed repair (HDR) using proteins that combine RNA-guided nucleases and enzymatic activities (e.g., replication inhibition, reverse transcriptase activity, transcription enhancing activity, etc.) from different proteins. In a preferred aspect, these nuclease fusion proteins have a nicking enzyme function and thus nick on a single strand of DNA to be edited, rather than creating a double-strand break.

Editing nuclease fusion proteins can be used with editing nucleic acids, such as those found, for example, in U.S. patent No. 9,982,278 and related patents. Such nucleic acids encode a gRNA comprising a region complementary to a target region of the nucleic acid in one or more cells, the gRNA being covalently linked to an editing cassette comprising a region homologous to the target region in one or more cells, the region having a mutation of at least one nucleotide relative to the target region in one or more cells. These nucleic acids may optionally include a pro-spacer (protospacer) and/or a barcode. The editing method may include one or more sets of these nucleic acids, and two or more incisions are made in the target region for intended editing. Examples of such methods include, but are not limited to, those described in Liu et al (Nature, 12.2019; 576 (7785): 149-157).

In certain preferred embodiments, the method employs a novel method known as "CREATE fusion editing". "CREATE fusion editing" is a novel technique for facilitating editing using a nuclease editing enzyme with nickase activity in conjunction with one or more nucleic acids. In particular aspects, the CREATE fusion editing methods utilize editing a fusion protein (e.g., a protein having CRISPR-targeting activity and reverse transcriptase activity) and a nucleic acid encoding one or more grnas comprising a region complementary to a target region of the nucleic acid. One or more grnas are covalently linked to an editing cassette comprising a region homologous to the target region having a mutation of at least one nucleotide relative to the target region expected to be edited in one or more cells. Optionally, the nucleic acid may further comprise a Protospacer Adjacent Motif (PAM) mutation and/or a barcode indicative of a desired mutation in the target region. Further description of the use of such CREATE nucleic acids can be found, for example, in U.S. Pat. No. 9,982,278, which is incorporated herein by reference for all purposes.

The use of a single gRNA to achieve editing rates of 30% or higher has many benefits over the double-nicked system described in Liu et al (supra) which they teach are required to achieve such editing rates in mammalian cells. For example, eliminating the need for a second nick allows for multiplexed genome editing to be much more scalable, as only one editing construct is required per cell to target the site of intended editing. This also increases the number of sites available for editing in the genome of the cell, enhancing the potential design and coverage of editing vector libraries to be introduced into a population of cells. The use of a single gRNA described herein will also reduce the formation of indels (indels) compared to a double nicking system, and, for example, will be expected to reduce off-target effects due to the specificity created by the nicking enzyme activity.

In some aspects, edits in the nuclease binding seed region (nuclease binding seed region) can be used to render the site nuclease resistant, thereby preventing additional cleavage using a nuclease (e.g., a nuclease fusion protein comprising nicking activity).

In particular aspects, the CREATE fusion method can utilize fusion proteins with nickase activity and a single gRNA to achieve high efficiency editing that is two times or more higher than the techniques taught in Liu et al (supra). By creating a single incision in the target region, the methods of the present disclosure can achieve editing efficiencies in mammalian cells of over 20%, including precise editing rates of up to 45%, without enrichment. Thus, the single-incision system disclosed herein enables the previously described high editing efficiency levels with only a double-incision system.

Some of the workflows for performing the CREATE fuse editing are outlined in FIGS. 7 and 8. In certain preferred embodiments, these workflows are performed using automated systems or instruments (e.g., multi-module instruments) and are set forth in the present disclosure.

Without being bound by a particular mechanism, the editing mechanism may be allowed to persist for several cell divisions. As shown in fig. 9, this editing cycle in the cell population allowed for editing of a higher proportion of cells using the introduced CREATE fusion editing mechanism.

Nuclease-guided genome editing overview

The compositions and methods described herein are useful for performing nuclease-directed genome editing to introduce desired edits into a population of mammalian cells. In some embodiments, a single edit is introduced in a single round of editing. In some embodiments, more than one edit is introduced in a single round of editing using simultaneous edits, e.g., two or more edits on a single carrier are introduced. In some embodiments, recursive cell editing is performed, wherein editing is introduced in successive rounds of editing.

A nucleic acid-guided nuclease complexed in a cell with an appropriate synthetic guide nucleic acid can cleave the genome of the cell at a desired location. The guide nucleic acid facilitates recognition and cleavage of DNA at a particular target sequence by a nucleic acid-guided nuclease. By manipulating the nucleotide sequence of the guide nucleic acid, the nucleic acid guided nuclease can be programmed to target any DNA sequence for cleavage, provided that the appropriate Protospacer Adjacent Motif (PAM) is nearby. In certain aspects, a nucleic acid-guided nuclease editing system can use two separate guide nucleic acid molecules that act in combination to guide nucleic acids, such as CRISPR RNA (crRNA) and trans-activating CRISPR RNA (tracrRNA). In other aspects and preferably, the guide nucleic acid is a single guide nucleic acid construct comprising both: 1) A guide sequence capable of hybridizing to a genomic target locus, and 2) a scaffold sequence capable of interacting or complexing with a nucleic acid-guided nuclease.

Typically, a guide nucleic acid (e.g., a gRNA) can be complexed with a compatible nucleic acid-guided nuclease, and can then hybridize to a target sequence, thereby directing the nuclease to the target sequence. The guide nucleic acid may be DNA or RNA; alternatively, the guide nucleic acid may comprise both DNA and RNA. In some embodiments, the guide nucleic acid may comprise modified or non-naturally occurring nucleotides. Where the guide nucleic acid comprises RNA, the gRNA may be encoded by a DNA sequence on a polynucleotide molecule, such as a plasmid, linear construct, or the coding sequence may, and preferably does, be located within an editing cassette. For additional information about edit boxes, see, e.g., USPN10, 240,167; USPN10,266,849; USPN 9,982,278; USPN10,351,877; USPN10,364,442; and USPN10,435,715; and USSN 16/275,465, filed on 14.2.2019, all of which are incorporated herein by reference for all purposes.

The guide nucleic acid comprises a guide sequence, wherein the guide sequence is a polynucleotide sequence that has sufficient complementarity (i.e., homology) with the target sequence to hybridize to the target sequence and direct specific binding of the complexed nucleic acid-guided nuclease to the sequence of the target sequence. The degree of complementarity between a leader sequence and a corresponding target sequence is about or greater than about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99% or more when optimally aligned using a suitable alignment algorithm. Optimal alignment can be determined by using any suitable algorithm for aligning sequences. In some embodiments, the guide sequence is about or more than about 10,11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23,24, 25, 26, 27, 28, 29, 30, 35, 40, 45, 50, 75 or more nucleotides in length. In some embodiments, the leader sequence is less than about 75, 50, 45, 40, 35, 30, 25, 20 nucleotides in length. Preferably, the leader sequence is 10-30 or 15-20 nucleotides in length, or 15, 16, 17, 18, 19 or 20 nucleotides in length.

Typically, to produce edits in the target 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 may be any polynucleotide endogenous or exogenous to the mammalian cell, or any polynucleotide in vitro. For example, the target sequence may be a polynucleotide residing in the nucleus of a mammalian cell. The target sequence may be a sequence encoding a gene product (e.g., a protein) or a non-coding sequence (e.g., a regulatory polynucleotide, intron, PAM, control sequence, or "junk" DNA).

The guide nucleic acid may be, and preferably is, part of an editing cassette encoding a donor nucleic acid that targets a cellular target sequence. Alternatively, the guide nucleic acid may not be part of the editing cassette, but may be encoded on the editing carrier backbone. For example, the sequences encoding the guide nucleic acid may be first assembled or inserted into the vector backbone, followed by insertion of the donor nucleic acid into, for example, an editing cassette. In other cases, the donor nucleic acid, e.g., in an editing cassette, can be inserted or assembled into the vector backbone first, followed by insertion of the sequence encoding the guide nucleic acid. Preferably, the sequences encoding the guide nucleic acid and the donor nucleic acid are located together in a rationally designed editing cassette and are simultaneously inserted or assembled into a linear plasmid or vector backbone via gap repair to create an editing vector. In some aspects, PCR amplicons of the editing cassette can be used for editing.

The target sequence is associated with a prometalocytic mutation (PAM), which is a short nucleotide sequence recognized by the gRNA/nuclease complex. The exact preferred PAM sequence and length requirements for different nucleic acid-guided nucleases vary; however, a PAM is typically a 2-7 base pair sequence adjacent or close to the target sequence and, depending on the nuclease, may be 5 'or 3' to the target sequence. Engineering of the PAM interaction domain of a nucleic acid guided nuclease may allow for altering PAM specificity, improving target site recognition fidelity, reducing target site recognition fidelity, or increasing versatility of the nucleic acid guided nuclease.

In certain embodiments, genome editing of a cellular target sequence both introduces a desired DNA alteration into the cellular target sequence, e.g., genomic DNA of the cell, and removes a prometallar mutation (PAM) region in the cellular target sequence, mutating or inactivating the prometallar mutation (PAM) region in the cellular target sequence. Inactivating the PAM at the cellular target sequence precludes additional editing of the cellular genome at the cellular target sequence, for example, when subsequently exposed to a nucleic acid-guided nuclease complexed with a synthetic guide nucleic acid in subsequent rounds of editing. Thus, cells with PAM edited and altered by the desired cellular target sequence can be selected by using a nucleic acid guided nuclease complexed with a synthetic guide nucleic acid that is complementary to the cellular target sequence. Cells that have not undergone the first editing event will be cleaved, causing double-stranded DNA breaks, and thus will not survive. Cells containing the desired cellular target sequence editing and PAM alteration will not be cut because these edited cells will no longer contain the necessary PAM site and will continue to grow and multiply.

As for the nuclease component of the nucleic acid-guided nuclease editing system, the polynucleotide sequence encoding the nucleic acid-guided nuclease can be codon optimized for expression in a particular mammalian cell type, such as a stem cell. The choice of nucleic acid-guided nuclease to be employed depends on many factors, such as what type of editing is to be performed in the target sequence, and whether the appropriate PAM is located in the vicinity of the desired target sequence. Nucleases for use in the methods described herein include, but are not limited to, cas9, cas12/CpfI, MAD2 or MAD7 or other MAD enzymes (MADzyme). Like the guide nucleic acid, the nuclease is encoded by a DNA sequence on the vector and optionally under the control of an inducible promoter. In some embodiments, the promoter may be independent of, but the same as, the promoter controlling the transcription of the guide nucleic acid; that is, a separate promoter drives transcription of the nuclease and leader nucleic acid sequence, but the two promoters may be the same type of promoter. Alternatively, the promoter controlling expression of the nuclease may be different from the promoter controlling transcription of the guide nucleic acid; that is, for example, the nuclease may be under the control of, for example, the pTEF promoter, and the guide nucleic acid may be under the control of, for example, the pCYC1 promoter.

Another component of the nucleic acid-guided nuclease system is a donor nucleic acid that comprises homology to a cellular target sequence. The donor nucleic acid is on the same vector and even in the same editing cassette as the guide nucleic acid, and preferably (but not necessarily) under the control of the same promoter (i.e., a single promoter that drives transcription of both the editing gRNA and the donor nucleic acid) as the editing gRNA. The donor nucleic acid is designed to serve as a template for homologous recombination with a cellular target sequence that is nicked or cleaved by a nucleic acid-guided nuclease that is part of the gRNA/nuclease complex. The donor nucleic acid polynucleotide can have any suitable length, such as a length of about or more than about 20, 25, 50, 75, 100, 150, 200, 500, or 1000 nucleotides, and a length of up to 2kb, 3kb, 4kb, 5kb, 6kb, 7kb, 8kb, 9kb, 10kb, 11kb, 12kb, 13kb, and up to 20kb if combined with the dual gRNA architecture described in USSN 62/869,240 as filed on 7/1 of 2019. In certain preferred aspects, the donor nucleic acid may be provided as an oligonucleotide of between 20-300 nucleotides, more preferably between 50-250 nucleotides. The donor nucleic acid comprises a region (e.g., homology arm) that is complementary to a portion of the cellular target sequence. When optimally aligned, the donor nucleic acid overlaps (is complementary) with the cellular target sequence by, for example, about 20, 25, 30, 35, 40, 50, 60, 70, 80, 90 or more nucleotides. In many embodiments, the donor nucleic acid comprises two homology arms (regions complementary to cellular target sequences) flanking a mutation or difference between the donor nucleic acid and the cellular target sequence. The donor nucleic acid comprises at least one mutation or alteration, such as an insertion, deletion, modification, or any combination thereof, as compared to the cellular target sequence.

As described with respect to grnas, the donor nucleic acid can be provided as part of a rationally designed editing cassette that is inserted into the editing plasmid backbone, where the editing plasmid backbone can comprise a promoter to drive transcription of the editing gRNA and donor DNA when the editing cassette is inserted into the editing plasmid backbone. Furthermore, there can be more than one, e.g., two, three, four, or more editing gRNA/donor nucleic acids in a rationally designed editing cassette inserted into an editing vector; alternatively, a single rationally designed editing cassette can comprise two to several editing gRNA/donor DNA pairs, where each editing gRNA is under the control of a separate different promoter, a separate similar promoter, or where all gRNA/donor nucleic acid pairs are under the control of a single promoter. In some embodiments, the promoter that drives transcription of the editing gRNA and the donor nucleic acid (or that drives more than one editing gRNA/donor nucleic acid pair) is optionally an inducible promoter.

In addition to the donor nucleic acid, the editing cassette may comprise one or more primer sites. The primer sites can be used to amplify the editing cassettes by using oligonucleotide primers; for example, if the primer site flanks one or more other components of the editing cassette. Further, the editing pod may contain a barcode. Barcodes are unique DNA sequences that correspond to donor DNA sequences such that the barcode can identify edits made to the corresponding cellular target sequence. Barcodes typically comprise four or more nucleotides. In some embodiments, the editing cassette comprises a collection or library of editing grnas and donor nucleic acids, which represents, for example, a whole gene library or a whole genome library of editing grnas and donor nucleic acids. The library of editing cassettes is cloned into a vector backbone, where, for example, each different donor nucleic acid is associated with a different barcode. Furthermore, in preferred embodiments, the editing vector or plasmid encoding the components of the nucleic acid-guided nuclease system further encodes a nucleic acid-guided nuclease comprising one or more Nuclear Localization Sequences (NLSs), such as about or more than about 1, 2, 3, 4,5, 6,7, 8,9, 10, or more NLSs, particularly as an element of a nuclease sequence. In some embodiments, the engineered nuclease comprises an NLS at or near the amino-terminus, an NLS at or near the carboxy-terminus, or a combination.

Cells with stably integrated genomic copies of the GFP gene can be phenotypically detected for different classes of genome editing (NHEJ, HDR, no editing) by flow cytometry, fluorescent cell imaging, or genotypically detected by sequencing the genome integrated GFP gene. The lack of editing or complete repair of cleavage events in the GFP gene results in cells remaining GFP positive. Cleavage events repaired by the non-homologous end joining (NHEJ) pathway often result in nucleotide insertion or deletion events (indels). These Indel edits often result in frame shift mutations in the coding sequence, which results in the loss of GFP gene expression and fluorescence. Cleavage events repaired by the homology-directed repair (HDR) pathway lead to a shift in the fluorescence spectrum of the cells from the GFP fluorescence spectrum to the BFP fluorescence spectrum, using the GFP to BFP HDR donor as a repair template.

Editing box

The editing cassette used is a plasmid that mediates expression of grnas targeting nucleases to specific DNA sequences. The editing cassette plasmid may also have a DNA sequence (e.g., HDR donor) that provides a template for targeted insertions, deletions, or nucleotide exchanges near the nuclease targeted cleavage site. In one example, the editing cassette plasmid expresses a gRNA that targets a genomic copy of the stably integrated GFP gene and provides an HDR donor that mediates nucleotide exchanges that convert the amino acid coding sequence of GFP to the amino acid coding sequence of BFP.

RNA-guided nucleases (e.g., cas9, cpf1, MAD 7) can be delivered to cells by means of expression plasmids encoding nucleases, mRNA encoding nucleases, recombinant nuclease proteins, or by generating stable cell lines expressing nucleases. In this particular example, the MAD7 nuclease is delivered by means of an expression plasmid encoding the nuclease.

The editing cassette plasmid and nuclease can be delivered to the target cell by conventional mammalian cell transfection techniques.

Automated cell editing apparatus and modules for nucleic acid guided nuclease editing

Automated cell editing apparatus

Fig. 1A depicts an exemplary automated multi-module cell processing instrument 100, e.g., performing one of the exemplary workflows comprising a split-body (split) protein reporter system as described herein. For example, the instrument 100 may be and preferably is designed as a stand-alone bench-top instrument for use in a laboratory environment. The instrument 100 may include a mixture of reusable and disposable components for performing various integrated processes without human intervention in performing automated genomic lysis and/or editing in a cell. A gantry (gantry) 102 is illustrated, the gantry 102 providing an automated mechanical motion system (actuator) (not shown) that provides XYZ-axis motion control to, for example, an automated (i.e., robotic) liquid handling system 158, the automated liquid handling system 158 including, for example, an air displacement pipettor 132, which allows cell processing between multiple modules without human intervention. In some automated multi-module cell processing instruments, the air displacement pipettor 132 is moved by the rack 102 and the various modules and reagent cartridges remain stationary; however, in other embodiments, the liquid handling system 158 may remain stationary while the various modules and reagent cartridges move. Also included in the automated multi-module cell processing instrument 100 is a reagent cartridge 110, the reagent cartridge 110 including a reservoir 112 and an editing mechanism introduction module 130 (e.g., a flow-through electroporation device described in detail in connection with fig. 4A-4D), as well as a wash reservoir 106, a cell input reservoir 151, and a cell output reservoir 153. Wash reservoir 106 can be configured to hold large tubes, such as wash solution, or solutions commonly used throughout the iterative process. Although in fig. 1A, two reagent cartridges 110 comprise a wash reservoir 106, a wash reservoir may also be included in a wash cartridge, wherein a reagent cartridge and a wash cartridge are separate cartridges. In such a case, reagent cartridge 110 and wash cartridge 104 may be identical except for the consumables (reagents or other components contained in the various inserts) inserted therein. Note that an exemplary reagent cassette is illustrated in fig. 5A and 5B.

In some embodiments, the reagent cartridge 110 is a disposable kit containing reagents and cells for use in the automated multi-module cell processing/editing instrument 100. For example, prior to initiating a cell process, a user may open and position each reagent cartridge 110 containing various desired inserts and reagents within the housing (chassis) of the automated multi-module cell editing instrument 100. Further, each reagent cartridge 110 may be inserted into a container (receptacle) in the cabinet having different temperature zones suitable for the reagents contained therein.

Also illustrated in fig. 1A is a robotic liquid handling system 158, including rack 102 and air displacement pipettor 132. In some examples, the robotic manipulation system 158 may include an automated liquid manipulation system such as those manufactured by Tecan Group ltd, by Mannedorf, switzerland, hamilton Company, reno, NV (see, e.g., WO2018015544 A1) or Beckman Coulter, inc, by Fort Collins, co. (see, e.g., US20160018427 A1). Pipette tips may be provided in a pipette transfer tip supply (not shown) for use with air displacement pipettes 132.

In some embodiments, the inserts or components of the reagent cartridge 110 are marked with machine-readable indicia (not shown), such as a barcode, for identification by the robotic manipulation system 158. For example, the robotic liquid handling system 158 may scan one or more inserts within each reagent cartridge 110 to confirm the contents. In other embodiments, machine-readable indicia may be marked on each reagent cartridge 110, and a processing system (not shown, but see element 137 of fig. 1B) of the automated multi-module cell editing instrument 100 may identify the stored material map based on the machine-readable indicia. In the embodiment illustrated in fig. 1A, the cell growth module includes a cell growth vial 118 (described in more detail below in connection with fig. 2A-2D). Also seen is TFF module 122 (described in detail below in conjunction with fig. 3A-3L). Also seen is an enrichment module 140. The placement of three heat sinks 155 should also be noted.

Fig. 1B is a simplified illustration of the contents of the exemplary multi-module cell processing instrument 100 depicted in fig. 1A. For example, cassette-based source material (such as in reagent cassette 110) may be positioned in a designated area on a platform (deck) of instrument 100 for access by air displacement pipettor 132. The platform of the multi-module cell processing instrument 100 may include a protective well such that contaminants that spill (spill), drip, or overflow (overflow) from any module of the instrument 100 are contained within the edge (lip) of the protective well. Also seen is a reagent cartridge 110, which is shown provided with a thermal assembly 111, which thermal assembly 111 can create temperature zones that fit different zones. Note that one of the reagent cartridges also includes a flow-through electroporation device 130 (FTEP), supplied by a FTEP interface (e.g., manifold arm) and an actuator 131. It is also seen that there is a TFF module 122 adjacent to the thermal assembly 125, where the TFF module is supplied by a TFF interface (e.g., manifold arm) and an actuator 133. Thermal components 125, 135, and 145 include thermoelectric devices, such as Peltier devices, as well as heat sinks, fans, and coolers. The growth bottles 118, 120 are rotated within a growth module 134, wherein the growth module is supplied by two thermal assemblies 135. An enrichment module is seen at 140, where the enrichment module is supplied by a selection interface (e.g., manifold arm) and an actuator 147. Also seen in this view are a touch screen display 101, display actuators 103, illumination 105 (one on either side of the multi-module cell processing instrument 100), and a camera 139 (one illumination means on either side of the multi-module cell processing instrument 100). Finally, components 137 include electronic devices such as circuit control boards, high voltage amplifiers, power supplies, and power supply inputs; and pneumatic devices (pneumatics) such as pumps, valves and sensors.

Figure 1C illustrates a front perspective view (with the door open) of the multi-module cell processing instrument 100 used as a desktop version of the automated multi-module cell editing instrument 100. For example, the chassis 190 may have a width of about 24-48 inches, a height of about 24-48 inches, and a depth of about 24-48 inches. The cabinet 190 may be, and preferably is, designed to house all of the modules and disposable supplies used in automated cell processing and to perform all of the processes required without human intervention; that is, the chassis 190 is configured to provide an integrated, self-contained, automated multi-module cell processing instrument. As illustrated in fig. 1C, the chassis 190 includes the touch screen display 101, a cooling grille 164, the cooling grille 164 allowing air to flow via internal fans (not shown). The touch screen display provides information to the user regarding the processing state of the automated multi-module cell editing instrument 100 and accepts input from the user for cell processing. In this embodiment, chassis 190 is elevated by adjustable legs 170a, 170b, 170C, and 170d (legs 170a-170C are shown in this FIG. 1C). For example, the adjustable feet 170a-170d allow for additional airflow under the chassis 290.

Inside the housing 190 are, in some embodiments, most or all of the components described in connection with fig. 1A and 1B, including the robotic liquid handling system disposed along the rack, the reagent cartridge 110 including the flow-through electroporation device, the rotating growth vials 118, 120 in the cell growth module 134, the tangential flow filtration module 122, the enrichment module 140, and the interfaces and actuators for the various modules. In addition, the cabinet 190 houses control circuitry, liquid handling tubes, air pump controllers, valves, sensors, thermal components (e.g., heating and cooling units), and other control mechanisms. For examples of multi-module cell editing instruments, see USPN10,253,316 issued on 9/4/2019; USPN10,329,559 issued on 25.6.2019; USPN10,323,242 issued on 18/6/2019; USPN10,421,959 issued on 24 months 9 and 2019; USPN10,465,185 issued on 5.11.2019; and USSN 16/412,195, filed 5, month 14, 2019; USSN 16/571,091 filed on 9, 14, 2019; and USSN 16/666,964, filed 2019, 10,29, all of which are hereby incorporated by reference in their entirety for all purposes.

Rotating cell growth module

Fig. 2A shows one embodiment of a rotary growth flask 200 for use with the cell growth apparatus described herein, which is configured to grow a variety of cell types, including microbial and mammalian cell lines, as well as primary or produced stem cells (e.g., induced pluripotent stem cells, hematopoietic stem cells, embryonic stem cells, etc.). The rotating growth flask is an optically clear container having an open end 204 for receiving liquid culture medium and cells, a central flask region 206 defining a main container for growing cells, a tapered-to-constricted region 218 defining at least one light path 210, a closed end 216, and a drive engagement mechanism 212. The rotating growth vial has a central longitudinal axis 220, the vial rotates about the central longitudinal axis 220, and the light path 210 is generally perpendicular to the longitudinal axis of the vial. The first light path 210 is positioned at a lower constriction tapered to a constriction region 218. Optionally, some embodiments of the rotating growth bottle 200 have the second light path 208 in a tapered region that tapers to the constriction region 218. Both optical paths in this embodiment are positioned in a region of the rotating growth flask that is constantly filled with cell culture (cells + growth medium) and is not affected by the rotation speed of the growth flask. The first optical path 210 is shorter than the second optical path 208, allowing for sensitive measurement of the OD value when the OD value of the cell culture in the vial is at a high level (e.g., later in the cell growth process), while the second optical path 208 allows for sensitive measurement of the OD value when the OD value of the cell culture in the vial is at a low level (e.g., earlier in the cell growth process). Also shown is a rim 202, rim 202 allowing the rotary growth bottle to be located in a growth module (not shown) and also allowing easy handling by the user.

In some configurations of the rotary growth bottle, the rotary growth bottle has two or more "paddles" or internal features disposed within the rotary growth bottle extending from an inner wall of the rotary growth bottle toward the center of the central bottle region. In some aspects, the width of the paddle or feature varies with the size or volume of the rotating growth bottle, and may range from 1/20 to just greater than 1/3 of the diameter of the rotating growth bottle, or from 1/15 to 1/4 of the diameter of the rotating growth bottle, or from 1/10 to 1/5 of the diameter of the rotating growth bottle. In some aspects, the length of the paddle varies with the size or volume of the rotary growth bottle, and may range from 4/5 to 1/4 of the length of the rotary growth bottle body, or from 3/4 to 1/3 of the length of the rotary growth bottle body, or from 1/2 to 1/3 of the length of the rotary growth bottle body. In other aspects, there may be concentric rows of raised features disposed on the inner surface of the body of a horizontally or vertically arranged rotating growth flask; and in other aspects, a raised feature spiral configuration may be provided on the inner surface of the body of the rotating growth flask. In alternative aspects, a concentric row or spiral configuration of raised features may be provided on the post or central structure of the rotating growth flask. Although described above as having two paddles, the rotary growth bottle may include 3, 4,5, 6, or more paddles, and up to 20 paddles. The number of paddles depends on, for example, the size or volume of the rotating growth flask. The paddles may be symmetrically arranged as a single paddle extending from the inner wall of the bottle to the interior of the bottle, or the paddles may be symmetrically arranged to extend from the inner wall of the bottle to the interior of the bottle in groups of 2, 3, 4 or more paddles (e.g., one pair of paddles opposite the other pair of paddles). In another embodiment, the paddles may extend from the middle of the rotating growth flask outward toward the wall of the rotating growth flask, such as from posts or other support structures inside the rotating growth flask.

The drive engagement mechanism 212 engages a motor (not shown) to rotate the bottle. In some embodiments, the motor drives the drive engagement mechanism 212 such that the rotary growth bottle rotates in only one direction, and in other embodiments, the rotary growth bottle rotates in a first direction for a first amount of time or periodically, rotates in a second direction (i.e., opposite direction) for a second amount of time or periodically, and the process may be repeated such that the rotary growth bottle (and cell culture contents) experience an oscillating motion. The first amount of time and the second amount of time may be the same or may be different. The amount of time may be 1 second, 2 seconds, 3 seconds, 4 seconds, 5 seconds or more, or may be 1 minute, 2 minutes, 3 minutes, 4 minutes or more. In another embodiment, the rotating growth flask may be oscillated with a first periodicity (e.g., every 60 seconds) during an early stage of cell growth, and the rotating growth flask may then be oscillated with a second periodicity (e.g., every second) different from the first periodicity during a later stage of cell growth.

The rotating growth flask 200 may be specifically tailored for the growth of a particular cell type. For example, O may be monitored or controlled exclusively₂And/or CO₂And the rotating growth flask can be designed and OD measurements modified to be compatible with the use of specific carrier substrates (substrates) for adherent cell growth.

The rotating growth bottle 200 may be reusable or, preferably, the rotating growth bottle is consumable. In some embodiments, the spun growth bottle is consumable and is pre-filled with a growth medium provided to the user, wherein the bottle is sealed with a foil seal at the open end 204. The media-filled spin growth flasks packaged in this manner may be part of a kit for use with a stand-alone cell growth apparatus or with a cell growth module as part of an automated multi-module cell processing instrument. To introduce cells into the vial, the user need only pipette out the desired volume of cells and use the pipette tip to pierce the foil seal of the vial. Open end 204 may optionally include an extended rim 202 to overlap and engage a cell growth device (not shown). In an automated system, the rotating growing bottles 200 may be tagged with a barcode or other identifying means that can be read by a scanner or camera (not shown) that is part of the automated system.

The volume of the rotating growth flask 200 and the volume of the cell culture (including growth medium) can vary widely, but the volume of the rotating growth flask 200 must be large enough to allow proper aeration of the cell culture in the growth flask as the flask rotates. In practice, the volume of the spinner flask 200 may range from 1-250ml, 2-100ml, 5-80ml, 10-50ml, or 12-35ml. Also, the volume of cell culture (cells + growth medium) should be appropriate to allow proper aeration in the rotating growth flask. Thus, the volume of the cell culture should be about 10% -85% of the volume of the growth flask or 20% -60% of the volume of the growth flask. For example, for a 35ml growth flask, the volume of the cell culture will be about 4ml to about 27ml or 7ml to about 21ml.

The rotating growth flask 200 is preferably made of a biocompatible optically transparent material, or at least a portion of the flask including one or more optical paths is transparent. In addition, the material from which the rotating growth flask is made should be capable of being cooled to about 4 ℃ or less, and heated to about 55 ℃ or more, to accommodate both temperature-based cell assays and long-term storage at low temperatures. Furthermore, the material used to make the bottle must be able to withstand temperatures up to 55 ℃ without deforming on rotation. Suitable materials include glass, polyvinyl chloride, polyethylene, polyamide, polyethylene, polypropylene, polycarbonate, poly (methyl methacrylate) (PMMA), polysulfone, polyurethane, and copolymers of these and other polymers. Preferred materials include polypropylene, polycarbonate or polystyrene. In some embodiments, the spinner bottle is manufactured at low cost by, for example, injection molding or extrusion.

Fig. 2B-2D show embodiments of a cell growth module 250 including a rotating growth flask 200. Figure 2B is a perspective view of one embodiment of a cell growth device 250. Fig. 2C depicts a cross-sectional view of the cell growth device 250 from fig. 2B. In both figures, the rotary bottle 200 is seen positioned within the main housing 226 with the extended rim 202 of the rotary bottle 200 extending above the main housing 226. Additionally, the end housing 222, lower housing 232, and flange 224 are shown in both figures. Flange 224 is used to attach the cell growth device to a heating/cooling device or other structure (not shown). Fig. 2C depicts additional details. In fig. 2C, upper bearing 242 and lower bearing 230 are shown positioned in main housing 226. The upper bearing 242 and lower bearing 230 support vertical loading of the rotating growth flask 200. The lower housing 232 houses a drive motor 236. The cell growth apparatus of fig. 2C includes two optical paths: a first optical path 234 and a second optical path 230. The optical path 234 corresponds to the optical path 210 positioned in the taper-to-constriction portion of the rotating growth flask, and the optical path 230 corresponds to the optical path 208 in the taper-to-constriction portion of the rotating growth flask. Light path 210 and light path 208 are not shown in fig. 2C, but can be seen, for example, in fig. 2A. In addition to the optical paths 234 and 230, there is an emitter plate 228 that illuminates the optical paths, and a detector plate 246 that detects light after it has passed through the cell culture fluid in the rotating growth flask.

In some embodiments, the motor 236 for rotating the rotary growing bottle 200 is a brushless DC type drive motor with an in-line drive controller that can be set to maintain a constant Revolutions Per Minute (RPM) between 0RPM and about 3000 RPM. Alternatively, other motor types may be used, such as step (stepper), servo (servo), brushed DC, and the like. Optionally, the motor 206 may also have directional control that allows for reversal of the direction of rotation and a tachometer that senses and reports the actual RPM. The motor is controlled by a processor (not shown) according to standard protocols and/or user inputs, for example programmed into the processor, and the motor may be configured to vary the RPM to cause axial precession of the cell culture to enhance mixing, for example to prevent cell clumping, increase aeration, and optimize cell respiration.

The main housing 226, end housing 222, and lower housing 232 of the cell growth device 250 may be made of any suitable robust material, including aluminum, stainless steel, and other thermally conductive materials, including plastics. These structures, or portions thereof, may be produced by various techniques, such as metal fabrication, injection molding, creating a fused structural layer, and the like. While it is contemplated in some embodiments that the rotating growth flask is reusable, preferably consumable, other components of the cell growth device 250 are preferably reusable and may function as a stand-alone desktop device or as a module in a multi-module cell processing system as described herein.

The processor (not shown) of the cell growth system may be programmed with information to be used as a "blank" or control for growing cell cultures. A "blank" or control is a container that contains only cell growth medium, resulting in 100% transmission and 0OD, while the cell sample will deflect light and will have a lower percentage of transmission and a higher OD. As the cells grow in the medium and become denser, the transmittance will decrease and the OD will increase. The processor of the cell growth system can be programmed to use a wavelength value of a blank commensurate with growth media typically used in mammalian cell culture. Optionally, a second spectrophotometer and container may be included in the cell growth system, wherein the second spectrophotometer is used to read the blank at specified intervals.

Fig. 2D illustrates the cell growth apparatus as part of an assembly, including the cell growth apparatus of fig. 2B coupled with a light source 290, a detector 292, and a thermal component 294. The rotating growth flask 200 is inserted into a cell growth apparatus. Components of the light source 290 and detector 292, such as, for example, photodiodes with gain control covering 5-log, are coupled to the main housing of the cell growth apparatus. A lower housing 232 is illustrated that houses a motor that rotates the spinner flask, and one of the flanges 224 that secures the cell growth device to the assembly. A Peltier device or thermoelectric cooler 294 is also illustrated. In this embodiment, thermal control is achieved by attaching cell growth device 200 to thermal device 294 via flange 204 on the base of lower housing 232 and electrically integrating with thermal device 294. Thermoelectric coolers can "pump" heat to either side of a junction (junction), cooling or heating a surface depending on the direction of the current flow. In one embodiment, the temperature of the main housing is measured using a thermistor and then the rotating growth flask 200 is controlled to about +/-0.5 ℃ by a standard electronic Proportional Integral Derivative (PID) controller loop.

In some embodiments, the rear mounted power input module contains a safety fuse and an on-off switch that, when powered on, powers internal AC and DC power supplies (not shown) to start the processor. Measurements of Optical Density (OD) were done at programmed time intervals using 600nm Light Emitting Diodes (LEDs) (not shown) that had been arranged in columns (columnated) by optics (optical) into the lower constriction of a rotating growth flask containing the cells of interest. The light continues through the collection optics to the detection system, which consists of a (digital) gain controlled silicon photodiode. In general, optical density is generally shown as the absolute value of the base 10 logarithm of the power transmission factor (power transmission factor) of an optical attenuator: OD = -log10 (power off/on). Since OD is a measure of optical attenuation, i.e. the sum of absorption, scattering and reflection, the cell growth device OD measurement records the total power transmission, so as the cell growth and population becomes dense, the OD (signal loss) also increases. The OD system is pre-calibrated against OD standards, and these values are stored in onboard memory accessible by a measurement program.

In use, cells are seeded (cells can be removed from, for example, an automated liquid handling system or by a user) into the pre-filled growth medium of a spinning growth flask by piercing the foil seal. The programmed software of the cell growth apparatus sets a control temperature for growth, typically 30 ℃, and then slowly starts the rotation of the rotating growth flask. The cell/growth medium mixture slowly moves vertically upward to the wall due to centrifugal forces, allowing the rotating growth flask to expose a large surface area of the mixture to a normal oxygen environment. The growth monitoring system takes continuous readings of OD or OD measurements at preset or pre-programmed time intervals. These measurements are stored in internal memory and the software plots the measurements against time, if necessary, to demonstrate a growth curve. If enhanced mixing is required, for example to optimise growth conditions, the speed of rotation of the bottle may be varied to cause axial precession of the liquid and/or a complete change of direction may be made at programmed intervals. Growth monitoring can be programmed to automatically terminate the growth phase at a predetermined OD, and then the mixture is rapidly cooled to a lower temperature to inhibit further growth.

One application of the cell growth apparatus 250 is to constantly measure the optical density of a growing cell culture. One advantage of the described cell growth device is that the 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 growth device has been described in the context of measuring the Optical Density (OD) of a growing cell culture, those skilled in the art, in view of the teachings of this specification, will appreciate that other cell growth parameters may be measured in addition to, or instead of, the cell culture OD. For example, spectroscopy using visible, UV or near infrared light (NIR) allows monitoring the concentration of nutrients and/or waste in the cell culture. In addition, spectroscopic measurements can be used to quantify multiple chemicals simultaneously. Asymmetric chemicals can be quantified by identifying characteristic absorption features in the NIR. In contrast, symmetric chemicals can be easily quantified using raman spectroscopy. Many key metabolites, such as glucose, glutamine, ammonia and lactate, have different spectral features in the IR, such that they can be easily quantified. The amount and frequency of light absorbed by the sample may be related to the type and concentration of chemical species present in the sample. Each of these measurement types provides particular advantages. FT-NIR provides the greatest depth of light penetration and can be used for thicker samples. FT-mid-IR (MIR) provides information that can be more easily distinguished as specific to certain analytes, as these wavelengths are closer to the fundamental IR absorption. FT-raman is advantageous when the interference caused by water is minimized. Other spectral characteristics may be measured via, for example, dielectric impedance spectroscopy, visible fluorescence, fluorescence polarization, or luminescence. In addition, the cell growth apparatus may comprise further sensors for measuring, for example, dissolved oxygen, carbon dioxide, pH, conductivity, etc.

Cell concentration module

Fig. 3A-3K depict variations of one embodiment of a cell concentration/buffer exchange cassette and module that utilizes tangential flow filtration and is configured for all cell types, including immortalized cell lines, primary cells, and/or stem cells. One embodiment of the cell concentration device described herein operates using Tangential Flow Filtration (TFF), also known as cross-flow filtration (crossflow filtration), in which most of the feed flows tangentially across the filter surface, thereby reducing the formation of a filter cake (retentate) compared to dead-end filtration (dead-end filtration) in which the feed flows into the filter. Secondary flow relative to the primary feed is also utilized to generate shear forces to prevent filter cake formation and membrane fouling, thereby maximizing particle recovery, as described below.

The TFF devices described herein are designed to take into account two major design factors. First, the geometry of the TFF device results in filtration of the cell culture over a large surface area to minimize processing time. Second, the design of the TFF device is configured to minimize filter fouling. FIG. 3A is a general model of tangential flow filtration. The TFF device is operated using tangential flow filtration, also known as cross-flow filtration. Fig. 3A shows a system 390 with cells flowing on a membrane 394 where the feed stream of cells 392 in a culture medium or buffer is parallel to the membrane 394.TFF is distinct from dead-end filtration, in which both the feed stream and the pressure drop are perpendicular to the membrane or filter.

Figure 3B depicts a top view of the lower member of one embodiment of a TFF device/module that provides tangential flow filtration. As can be seen in the embodiment of the TFF device of fig. 3B, the TFF device 300 includes a channel structure 316, the channel structure 316 including a flow channel 302B through which the cell culture flows. The channel structure 316 includes a single flow channel 302b that is horizontally bifurcated by a membrane (not shown) through which buffer or culture medium can flow, but through which cells cannot flow. This particular embodiment includes a wavy serpentine geometry 314 (i.e., small "wiggles" in the flow channel 302) and a serpentine "zig-zag" pattern in which the flow channel 302 intersects the device from one end on the left side of the device to the other end on the right side of the device. The serpentine pattern allows filtration over a high surface area relative to the device size and total channel volume, while the undulating effect creates a secondary inertial flow to achieve effective membrane regeneration, preventing fouling of the membrane. Although an undulating geometry and serpentine pattern are illustrated herein, other channel shapes may be used provided that the channels may be bifurcated by the membrane and provided that the channel configuration provides flow in alternating directions through the TFF module. In addition to flow channel 302b, ports 304 and 306, as part of channel structure 316, and recess 308 can be seen. Port 304 collects cells passing through the channel ("retentate") on one side of the membrane (not shown), and port 306 collects media passing through the channel ("filtrate" or "permeate") on the opposite side of the membrane (not shown). In this embodiment, the recess 308 receives a bolt or other fastener (not shown) that allows the components of the TFF device to be secured to one another.

The length 310 and width 312 of the channel structure 316 may vary depending on the volume of cell culture to be grown and the optical density of the cell culture to be concentrated. The length 310 of the channel structure 316 is typically 1mm to 300mm, or 50mm to 250mm, or 60mm to 200mm, or 70mm to 150mm, or 80mm to 100mm. The width of the channel structure 316 is typically 1mm to 120mm, or 20mm to 100mm, or 30mm to 80mm, or 40mm to 70mm, or 50mm to 60mm. The cross-sectional shape of the flow channel 102 may be circular, elliptical, oval, square, rectangular, trapezoidal, or irregular. If square, rectangular, or another shape having substantially straight sides, the cross-section may be about 10 μm to 1000 μm wide, or 200 μm to 800 μm wide, or 300 μm to 700 μm wide, or 400 μm to 600 μm wide; and about 10 μm to 1000 μm high, or 200 μm to 800 μm high, or 300 μm to 700 μm high, or 400 μm to 600 μm high. If the cross-section of the flow channel 302 is substantially circular, oval or elliptical, the radius of the channel may be about 50 μm to 1000 μm in hydraulic radius, or 5 μm to 800 μm in hydraulic radius, or 200 μm to 700 μm in hydraulic radius, or 300 μm to 600 μm wide in hydraulic radius, or about 200 μm to 500 μm in hydraulic radius.

When looking at the top view of the TFF device/module of fig. 3B, note that there are two retentate ports 304 and two filtrate ports 306, with one type of port at each end (e.g., narrow side) of the device 300. In other embodiments, the retentate port and the filtrate port may be on the same surface of the same member (e.g., upper member or lower member), or they may be disposed on a side surface of the assembly. Unlike other TFF devices that operate continuously, the TFF devices/modules described herein use an alternating approach to concentrating cells. The general workflow for cell concentration using TFF devices/modules involves flowing a cell culture or cell sample through a channel structure tangentially. The membrane that bifurcates the flow channel retains the cells on one side of the membrane and allows unwanted media or buffer to flow across the membrane into the filtrate side of the device (e.g., lower member 320). In the process, a fixed volume of cells in culture medium or buffer is driven through the device until a cell sample is collected in a retentate port 304, and culture medium/buffer that has passed through the membrane is collected through one or two filtrate ports 306. All types of prokaryotic and eukaryotic cells (both adherent and non-adherent) can be grown in TFF devices. Adherent cells may be grown on beads or other cell scaffolds suspended in media flowing through the TFF device.

During cell concentration, the cell sample is passed through the TFF device and cells are collected in a retentate port 304 while media is collected in a filtrate port 306, which is considered a "one-pass" of the cell sample. Transfer between retentate reservoirs "flips" the culture. For a given pass, the retentate and filtrate ports that collect cells and media, respectively, reside at the same end of TFF device/module 300, the fluidic connections are arranged such that there are two distinct flow layers on the retentate and filtrate sides, but if the retentate port 304 resides on the upper member of device/module 300 (i.e., cells are driven through the channel above the membrane and filtrate (media) through the portion of the channel below the membrane), the filtrate port 306 would reside on the lower member of device/module 100, and vice versa (i.e., if a cell sample is driven through the channel below the membrane, filtrate (media) through the portion of the channel above the membrane). This configuration can be seen more clearly in fig. 3C-3D, where retentate flows 360 from retentate port 304 and filtrate flows 370 from filtrate port 306.

At the end of the "pass" of the growth concentration process, the cell sample is collected through retentate port 304 and into a retentate reservoir (not shown). To initiate another "pass," the cell sample is again passed through the TFF device, this time in the opposite direction to the first pass. The cell sample is collected through retentate port 304 and into a retentate reservoir (not shown) located on the opposite end of the device/module from retentate port 304, retentate port 304 being used to collect cells during the first pass. Likewise, the media/buffer that passes through the membrane in the second pass is collected through filtrate port 306, or through both ports, the filtrate port 306 being located on the opposite end of the device/module from the filtrate port 306, the filtrate port 306 being used to collect filtrate during the first pass. This alternating process of passing the retentate (concentrated cell sample) through the device/module is repeated until the cells have concentrated to the desired volume, and both filtrate ports can be opened during the pass to reduce the operating time. Furthermore, buffer exchange can be achieved by: the desired buffer (or fresh medium) is added to the cell sample in the retentate reservoir, then another "pass" is initiated, and the process is repeated until the old medium or buffer is diluted and filtered off and the cells reside in the fresh medium or buffer. Note that buffer exchange and cell concentration can (and usually do) occur simultaneously.

Fig. 3C depicts a top view of the upper member (322) and lower member (320) of an exemplary TFF module. Also seen are port 304 and port 306. As explained above, the recesses (such as recess 308 seen in fig. 3B) provide a means to secure the components of the TFF device/membrane (upper member 322, lower member 320, and membrane 324) to one another via, for example, bolts or other similar fasteners during operation. However, in alternative embodiments, an adhesive such as a pressure sensitive adhesive, or ultrasonic welding, or solvent bonding, may be used to couple the upper member 322, the lower member 320, and the membrane 324 together. Indeed, other configurations for coupling components of a TFF device may be found by those of ordinary skill in the art, such as, for example, clamps (clamps); fitting fittings provided on the upper member and the lower member; a combination of adhesives, welding, solvent bonding, and mating fittings; and other such fasteners and couplings (couplings).

Note that there is one retentate port and one filtrate port at each "end" (e.g., narrow side) of the TFF device/module. The retentate port and the filtrate port on the left side of the device/module would collect the cells (flow path at 360) and media (flow path at 370), respectively, in the same pass. Likewise, the retentate port and the filtrate port on the right side of the device/module would collect the cells (flow path at 360) and media (flow path at 370), respectively, in the same pass. In this embodiment, the retentate is collected from port 304 on the top surface of the TFF device and the filtrate is collected from port 306 on the bottom surface of the device. The cells are maintained in the TFF flow channel above the membrane 324 while the filtrate (media) flows through the membrane 324 and then through the port 306; thus, the configuration of the top/retentate and bottom/filtrate ports is practical. However, it should be appreciated that other configurations of retentate and filtrate ports may be implemented, such as having both retentate and filtrate ports positioned on the sides (opposite the top and bottom surfaces) of the TFF device. In fig. 3C, channel structure 302b on the lower member 320 of TFF device 300 can be seen. However, in other embodiments, the retentate port and the filtrate port may reside on the same end of the TFF device.

Also seen in fig. 3C is a membrane or filter 324. Filters or membranes suitable for use in TFF devices/modules are those that are solvent resistant, non-contaminating during filtration, and capable of retaining the type and size of cells of interest. For example, the pore size may be a minimum of 0.2 μm, whereas for other cell types the pore size may be a maximum of 5 μm. In fact, pore sizes that may be used in TFF devices/modules include filters having 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, and larger. The filter may be made of any suitable non-reactive material, including cellulose mixed ester (nitrocellulose and cellulose acetate) (CME), polycarbonate (PC), polyvinylidene fluoride (PVDF), polyethersulfone (PES), polytetrafluoroethylene (PTFE), nylon, glass fibers, or metal substrates as in the case of laser or electrochemical etching. The TFF devices shown in fig. 3C and 3D do not show seats in the upper and lower members 312, 320 where the filter 324 may seat or be secured (e.g., a half thickness seat for the filter in each of the upper and lower members 312, 320); however, such seats are contemplated in some embodiments.

Fig. 3D depicts a bottom view of the upper and lower members of the exemplary TFF module shown in fig. 3C. Fig. 3D depicts a bottom view of the upper member (322) and the lower member (320) of an exemplary TFF module. Also, port 304 and port 306 are seen. Also note that there is one retentate port and one filtrate port at each end of the device/module. The retentate port and the filtrate port on the left side of the device/module would collect the cells (flow path at 360) and media (flow path at 370) respectively, in the same pass. Likewise, the retentate port and the filtrate port on the right side of the device/module would collect the cells (flow path at 360) and media (flow path at 370), respectively, in the same pass. In fig. 3D, channel structure 302a on upper member 322 of TFF device 300 can be seen. Thus, looking at fig. 3C and 3D, note that there are channel structures 302 (302 a and 302 b) in both the upper and lower members, with a membrane 324 between the upper and lower portions of the channel structures. The channel structures 302 (302 a and 302b, respectively) of the upper member 322 and the lower member 320 cooperate to create a flow channel with the membrane 324 positioned horizontally between the upper and lower members of the flow channel, thereby bifurcating the flow channel.

Media exchange (during cell growth) or buffer exchange (during cell concentration or rendering cells competent) was performed on the TFF device/module by: adding fresh medium to the growing cells, or adding the desired buffer to the cells concentrated to the desired volume; for example, after the cells are concentrated at least 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, 150-fold, 200-fold or more. The desired exchange medium or exchange buffer is added to the cells by addition to the retentate reservoir or through the membrane from the filtrate side, and the process of passing the cells through the TFF device 300 is repeated until the cells are grown to the desired optical density or concentrated to the desired volume in the exchange medium or buffer. This process may be repeated any number of desired times to achieve the desired buffer exchange level and desired cell volume. The exchange buffer may contain, for example, glycerol or sorbitol, in order to render the cells competent for transformation, in addition to reducing the total volume of the cell sample.

May be made of any robust material in which the channel (and channel branches) may be milled including: stainless steel, silicon, glass, aluminum, or plastics including Cyclic Olefin Copolymer (COC), cyclic Olefin Polymer (COP), polystyrene, polyvinyl chloride, polyethylene, polyamide, polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate, polyetheretherketone (PEEK), poly (methyl methacrylate) (PMMA), polysulfone, and polyurethane, as well as copolymers of these and other polymers. If the TFF device/module is disposable, it is preferably made of plastic. In some embodiments, the materials used to fabricate the TFF device/module are thermally conductive such that the cell culture can be heated or cooled to a desired temperature. In certain embodiments, the TFF device is formed using the materials mentioned above as being suitable for such mass production techniques by: precision machining, laser machining, electrical discharge machining (for metal devices); wet or dry etching (for silicon devices); dry or wet etching, powder or sand blasting, photostructuring (for glass devices); or thermoforming, injection molding, hot embossing, or laser machining (for plastic devices).

Fig. 3E-3K depict alternative embodiments of Tangential Flow Filtration (TFF) devices/modules. Fig. 3E depicts the configuration of an upper (retentate) member 3022 (on the left), a membrane or filter 3024 (middle), and a lower (permeate/filtrate) member 3020 (on the right). In the configuration shown in fig. 3E-3, retentate member 3022 is no longer "upper" and permeate/filtrate member 3020 is no longer "lower" because retentate member 3022 and permeate/filtrate member 3020 are side-to-side coupled as seen in fig. 3J and 3K. In fig. 3E, retentate member 3022 comprises tangential flow channel 3002, tangential flow channel 3002 having a serpentine configuration that traverses from one lower corner of retentate member 3022 (specifically, at retentate port 3028) and up and then down and across retentate member 3022, terminating at a second retentate port 3028 at the other lower corner of retentate member 3022. Also seen on retentate member 3022 is an energy director 3091, energy director 3091 surrounding the area where membrane or filter 3024 is located. In this embodiment, the energy director 3091 mates with an energy director component on the permeate/filtrate member 3020 and is used via it to facilitate ultrasonic welding or bonding of the retentate member 3022 to the permeate/filtrate member 3020. Membrane or filter 3024 is also seen to have a through hole of retentate port 3028 configured to be positioned within the perimeter of energy director 3091 between retentate member 3022 and permeate/filtrate member 3020. In addition to energy director 3091, permeate/filtrate member 3020 also includes through holes for retentate port 3028 at each bottom corner (which mate with the through holes of retentate port 3028 and retentate port 3028 at the bottom corner of membrane 3024 in retentate member 3022), and tangential flow channels 3002 and a single permeate/filtrate port 3026 located at the top and center of permeate/filtrate member 3020. The tangential flow channel 3002 structure in this embodiment has a serpentine configuration and a corrugated geometry, although other geometries may be used. In some aspects, the tangential flow channel has a length of 10mm to 1000mm, 60mm to 200mm, or 80mm to 100mm. In some aspects, the channel structure has a width of 10mm to 120mm, 40mm to 70mm, or 50mm to 60mm. In some aspects, the tangential flow channel 1202 is rectangular in cross-section. In some aspects, the cross-section of the tangential flow channel 1202 is 5 μm to 1000 μm wide and 5 μm to 1000 μm high, 300 μm to 700 μm wide and 300 μm to 700 μm high, or 400 μm to 600 μm wide and 400 μm to 600 μm high. In other aspects, the tangential flow channel 1202 is circular, elliptical, trapezoidal, or oval in cross-section and has a hydraulic radius of 100 μm to 1000 μm, a hydraulic radius of 300 μm to 700 μm, or a hydraulic radius of 400 μm to 600 μm.

Fig. 3F is a side perspective view of the reservoir assembly 3050. In the embodiment of fig. 3F, the retentate component is separate from the reservoir assembly. The reservoir assembly 3050 comprises a retentate reservoir 3052 on either side of a single permeate reservoir 3054. During cell concentration and/or growth, the retentate reservoir 3052 is used to hold cells and media as the cells are transferred through the cell concentration/growth device or module and into the retentate reservoir. The permeate/filtrate reservoir 3054 is used to collect filtrate removed from the cell culture during cell concentration, or to collect old buffer or culture medium during cell growth. In the embodiments depicted in fig. 3E-3L, the buffer or culture medium is supplied to the permeate/filtrate member from an agent reservoir separate from the device module. Additionally seen in fig. 3F are a recess 3032 that houses a pneumatic port (not visible), a permeate/filtrate port 3026 and a retentate port throughbore 3028. The retentate reservoir is fluidly coupled to a retentate port 3028, which retentate port 3028 is in turn fluidly coupled to a portion of a tangential flow channel disposed in a retentate member (not shown). The permeate/filtrate reservoir is fluidly coupled to a permeate/filtrate port 3026, which permeate/filtrate port 3026 is in turn fluidly coupled to a portion of a tangential flow path disposed in a permeate/filtrate member (not shown), wherein the portion of the tangential flow path is bifurcated by a membrane (not shown). In embodiments including this embodiment, up to 120mL of cell culture may be grown and/or filtered, or up to 100mL, 90mL, 80mL, 70mL, 60mL, 50mL, 40mL, 30mL, or 20mL of cell culture may be grown and/or concentrated.

Fig. 3G depicts a top view of the reservoir assembly 3050 shown in fig. 3F, fig. 3H depicts a lid 3044 for the reservoir assembly 3050 shown in fig. 3F, and fig. 3I depicts a seal 3045 disposed in operation on the lid 3044 of the reservoir assembly 3050 shown in fig. 3F. Fig. 3G is a top view of the reservoir assembly 3050 showing two retentate reservoirs 3052, one on each side of the permeate reservoir 3054. Also seen is a recess 3032 that mates with a pneumatic port (not shown), and a fluid channel 3034 that resides at the bottom of the retentate reservoir 3052, the fluid channel 3034 fluidly couples the retentate reservoir 3052 with the retentate port 3028 (not shown) via the permeate/filtrate member 3024 and the through-holes of the retentate port in the membrane 3024 (also not shown). Fig. 3H depicts a lid 3044 configured to be disposed on top of the reservoir assembly 3050. The lid 3044 has circular openings at the top of the retentate reservoir 3052 and the permeate/filtrate reservoir 3054. Likewise, a fluid channel 3034 can be seen at the bottom of the retentate reservoir 3052, wherein the fluid channel 3034 fluidly couples the retentate reservoir 3052 with a retentate port 3028 (not shown). Three pneumatic ports 3030 for each retentate reservoir 3052 and permeate/filtrate reservoir 3054 are also shown. Fig. 3I depicts a seal 3045 configured to be disposed on the lid 3044 of the reservoir assembly 3050. Seen are three fluid transfer ports 3042 for each retentate reservoir 3052 and for permeate/filtrate reservoir 3054. Likewise, three pneumatic ports 3030 are shown for each retentate reservoir 3052 and for the permeate/filtrate reservoir 3054.

Fig. 3J depicts an embodiment of an assembled TFF module 3000. Note that in this embodiment of the TFF module, retentate member 3022 is no longer "upper" and permeate/filtrate member 3020 is no longer "lower" because retentate member 3022 and permeate/filtrate member 3020 are coupled side-to-side with membrane 3024 sandwiched between retentate member 3022 and permeate/filtrate member 3020. In addition, a retentate member 3022, a membrane member 3024 and a permeate/filtrate member 3020 are coupled side-to-side with the reservoir assembly 3050. Two retentate ports 3028 are seen, which couple the tangential flow channels 3002 in the retentate member 3022 to two retentate reservoirs (not shown), and one permeate/filtrate port 3026, which couples the tangential flow channels 3002 in the permeate/filtrate member 3020 to a permeate/filtrate reservoir (not shown). Also seen are tangential flow channels 3002 formed by mating retentate members 3022 and permeate/filtrate members 3020, with membranes 3024 sandwiched between the tangential flow channels 3002 and bifurcating the tangential flow channels 3002. Also seen is an energy director 3091, which in this fig. 3J, energy director 3091 is used to ultrasonically weld or couple retentate member 3022 and permeate/filtration member 3020 around membrane 3024. A lid 3044 is visible on top of the reservoir assembly 3050, and a seal gasket 3045 is disposed on the lid 3044. The gasket 3045 engages and provides a fluid tight and pneumatic connection with the fluid transfer port 3042 and the pneumatic port 3030, respectively.

Fig. 3K depicts on the left an exploded view of the TFF module 3000 shown in fig. 3J. The following components are seen: a reservoir assembly 3050, a lid 3044 to be disposed on the reservoir assembly 3050, a seal 3045 to be disposed on the lid 3044, a retentate member 3022, a membrane or filter 3024, and a permeate/filtrate member 3020. Also seen are permeate/filtrate ports 3026 which mate with permeate/filtrate ports 3026 on the permeate/filtrate reservoir 3054, and two retentate ports 3028 which mate with retentate ports 3028 on the retentate reservoir 3052 (where only one retentate reservoir 3052 can be clearly seen in this fig. 3K). Also seen are through-holes for the membrane 3024 and the retentate port 3028 in the permeate/filtrate member 3020. Fig. 3K depicts on the left an assembled TFF module 3000 showing the length, height and width dimensions. The assembled TFF device 3000 is typically 50mm to 175mm in height, or 75mm to 150mm in height, or 90mm to 120mm in height; a length of 50mm to 175mm, or a length of 75mm to 150mm, or a length of 90mm to 120mm; and a depth of 30mm to 90mm, or a depth of 40mm to 75mm, or a depth of about 50mm to 60mm. An exemplary TFF device is 110mm in height, 120mm in length, and 55mm in depth.

Similar to other embodiments described herein, the TFF device or module depicted in fig. 3E-3K can constantly measure cell culture growth, and in some aspects, cell culture growth is measured via the Optical Density (OD) of the cell culture in one or both retentate reservoirs and/or flow channels of the TFF device. The optical density may be measured continuously (kinetic monitoring) or at specific time intervals (e.g., every 5 seconds, 10 seconds, 15 seconds, 20 seconds, 30 seconds, 45 seconds, or 60 seconds, or every 1 minute, 2 minutes, 3 minutes, 4 minutes, 5 minutes, 6 minutes, 7 minutes, 8 minutes, 9 minutes, or 10 minutes, etc.). In addition, the TFF module may adjust the growth parameters (temperature, aeration) to bring the cells to the desired optical density at the desired time.

Fig. 3L is an exemplary pneumatic block diagram suitable for the TFF module depicted in fig. 3E-3K. The pump is connected to two solenoid valves (SV 5 and SV 6) that provide either positive (P) or negative (V) pressure. Two solenoid valves SV5 and SV6 couple the pump to the manifold, and two solenoid valves SV1 and SV2 are connected to the outlet reservoirs (RR 1 and RR 2). There are also two solenoid valves (SV 3 and SV 4) in the reserve. There are proportional valves (PV 2 and PV 2), flow meters (FM 1 and FM 2) and pressure sensors (pressure sensors 1 and 2) located between the respective solenoid valves SV1 and SV2, connecting the pump to the system and the solenoid valves SV1 and SV2 to the reservoir. The pressure sensor and proportional valve work in concert in a feedback loop to maintain the desired pressure.

As an alternative to the TFF module described above, a cell concentration module comprising a hollow filter may be employed. Examples of filters suitable for use in the present invention include membrane filters, ceramic filters, and metal filters. The filter may be used in any shape; the filter may be cylindrical or substantially flat, for example. Preferably, the filter used is a membrane filter, preferably a hollow fiber filter. The term "hollow fiber" means a tubular membrane. The inner diameter of the tube is at least 0.1mm, more preferably at least 0.5mm, most preferably at least 0.75mm, and the inner diameter of the tube is preferably at most 10mm, more preferably at most 6mm, most preferably at most 1mm. Filter modules containing hollow fibers are commercially available from a number of companies, including g.e. life Sciences (Marlborough, MA) and InnovaPrep (Drexel, MO). Specific examples of hollow fiber filtration systems that may be used, modified or retrofitted in the present methods and systems include, but are not limited to, USPN 9,738,918; USPN 9,593,359; USPN 9,574,977; USPN 9,534,989; USPN 9,446,354; USPN 9,295,824; USPN 8,956,880; USPN 8,758,623; USPN 8,726,744; USPN 8,677,839; USPN 8,677,840; USPN 8,584,536; USPN 8,584,535; and USPN 8,110,112.

Editing mechanism lead-in module

In addition to modules for cell growth and cell concentration, fig. 4A-4E depict variations of one embodiment of a module for introducing an editing mechanism into a cell. The method of introduction can be tailored to the cell type and nature of the mechanism (e.g., nucleic acid or protein) to be introduced.

In some aspects, the module is configured to transform a mammalian cell. In some aspects, the editing cassette plasmid and nuclease can be delivered to the target cell by conventional mammalian cell transfection techniques. Examples include lipid-mediated transfection, calcium phosphate-mediated transfection, electroporation, cationic peptides, cationic polymers or nuclear transfection. Proteins, such as RNA-guided nucleases, can also be delivered into cells by various mechanisms. For example, a shuttle vector (such as those described in USPN 9,982,267 and USPN 9,738,687, which are incorporated herein by reference for all purposes) can be used to introduce RNA-guided nucleases into mammalian cells.

In certain embodiments, transformations are used to introduce some or all of the mechanisms necessary for editing. Fig. 4A is a perspective view of six commonly connected flow-through electroporation devices 450. Figure 4A depicts six flow-through electroporation units 450 disposed on a single substrate 456. Each of the six flow-through electroporation units 450 has an aperture 452 defining a cell sample inlet and an aperture 454 defining a cell sample outlet. After the six flow-through electroporation units 450 are made, they may be separated from each other (e.g., "fractured" and used one at a time), or alternatively, in embodiments, two or more flow-through electroporation units 450 may be used in parallel without being separated.

The flow-through electroporation device achieves efficient cell electroporation with low toxicity. The flow-through electroporation devices of the present disclosure allow for particularly easy integration with robotic liquid handling instruments commonly used in automated systems, such as air displacement pipettes. Such automated instruments include, but are not limited to, off-the-shelf automated liquid handling systems from Tecan (mannidorf, switzerland), hamilton (Reno, NV), beckman Coulter (Fort Collins, CO), and the like.

Generally speaking, microfluidic electroporation (using cell suspension volumes of less than about 10ml and as low as1 μ l) allows for more precise control of the transfection or transformation process and allows for flexible integration with other cell processing tools compared to small electroporation devices. Microfluidic electroporation is thus exemplified by single cell transformation, processing and analysis; a multi-cell electroporation device configuration; and integrated, automated, multi-modular cell processing and analysis provide unique advantages.

In particular embodiments of the flow-through electroporation devices of the present disclosure, the toxicity level of transformation results in greater than 10% viable cells after electroporation, preferably greater than 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 70%, 75%, 80%, 85%, 90% or even 95% viable cells after transformation, depending on the cell type and the nucleic acid introduced into the cell.

The flow-through electroporation devices described in connection with fig. 4A-4D include a housing having an electroporation chamber, a first electrode and a second electrode configured to engage with an electrical pulse generator, the electrical contact being engaged with an electrode of the electroporation device by the electrical pulse generator. In certain embodiments, the electroporation devices are autoclavable and/or disposable and can be packaged with reagents in a reagent cartridge. The electroporation device may be configured to electroporate cell sample volumes between 1 μ l and 2ml, 10 μ l and 1ml, 25 μ l and 750 μ l, or 50 μ l and 500 μ l.

In an exemplary embodiment, fig. 4B depicts a top view of a flow-through electroporation device 450 having an inlet 402 for introducing cells and an exogenous reagent to be electroporated into the cells ("cell sample") and an outlet 404 for the electroporated cell sample. The electrodes 408 are introduced through electrode channels (not shown) in the device. Fig. 4C shows a cross-sectional view from the top of the flow-through electroporation device 450, where the inlet 402, outlet 404, and electrode 408 are positioned relative to the constriction in the flow channel 406. The side cross-sectional view of the bottom of the flow-through electroporation device 450 in fig. 4D illustrates that the electrodes 408 in this embodiment are positioned in the electrode channels 410 and perpendicular to the flow channels 406 such that the cell sample flows from the inlet channels 412 through the flow channels 406 to the outlet channels 414 and, in the process, the cell sample flows into the electrode channels 410 to contact the electrodes 408. In this regard, the inlet channel, outlet channel, and electrode channel all originate from the top planar side of the device; however, the flow-through electroporation structures depicted in fig. 4B-4D are but one architecture that can be used for the reagent cartridges described herein. Additional electrode architectures USSN 16/147,120, submitted, for example, on 24 months 9 and 2018; USSN 16/147,865, filed on 30/9/2018 and USSN 16/147,871, filed on 30/9/2018.

Reagent box

Fig. 5A depicts an exemplary combined reagent cartridge and electroporation device 500 ("cartridge") that may be used in an automated multi-module cell processing instrument. The cartridge 500 includes a body 502, and a reagent container or reservoir 504. In addition, the cassette 500 includes a device for introducing nucleic acids and/or proteins into cells, e.g., an electroporation device 506 (exemplary embodiments of which are described in detail in conjunction with fig. 4A-4D). The cassette 500 may be disposable or may be configured to be reusable. Preferably, the cartridge 500 is disposable. Cassette 500 can be made of any suitable material, including stainless steel, aluminum, or plastics, including polyvinyl chloride, cyclic Olefin Copolymer (COC), polyethylene, polyamide, polyethylene, polypropylene, acrylonitrile butadiene, polycarbonate, polyetheretherketone (PEEK), poly (methyl methacrylate) (PMMA), polysulfone, and polyurethane, as well as copolymers of these and other polymers. If the cartridge is disposable, it is preferably made of plastic. Preferably, the material used to make the cartridge is thermally conductive, as in certain embodiments, the cartridge 500 contacts a thermal device (not shown) that heats or cools the reagent in the reagent container or reservoir 504. In some embodiments, the thermal device is a Peltier device or a thermoelectric cooler. The reagent container or reservoir 504 may be a container into which individual reagent tubes are inserted as shown in fig. 5A, a container into which one or more commonly connected tubes are inserted, or the reagent container may contain a reagent without an inserted tube, the reagent being dispensed directly into the container or reservoir. In addition, the containers in the reagent cartridge can be configured for any combination of tubes, commonly connected tubes, and direct filling of reagents.

In one embodiment, the reagent containers or reservoirs 504 of the reagent cartridge 500 are configured to accommodate tubes of various sizes, including, for example, 250ml tubes, 25ml tubes, 10ml tubes, 5ml tubes, and Eppendorf tubes or microcentrifuge tubes. In yet another embodiment, all containers can be configured to hold tubes of the same size, e.g., 5ml tubes, and the reservoir insert can be used to hold smaller tubes in the reagent reservoir. In yet another embodiment, particularly where the reagent cartridge is disposable, the reagent reservoir holds the reagent without an interposed tube. In this disposable embodiment, the reagent cartridge may be part of a kit, wherein the reagent cartridge is pre-filled with reagents and the container or reservoir is sealed with, for example, foil, heat-seal acrylic, etc., and presented to the consumer, and the reagent cartridge may then be used in an automated multi-module cell processing instrument. The reagents contained in the reagent cartridge will vary according to the workflow; that is, the reagents will vary according to the process that the cells undergo in the automated multi-module cell processing instrument.

FIG. 5B depicts an exemplary matrix configuration 140 of reagents contained in the reagent cartridge of FIG. 5A; wherein the matrix embodiment is a4 x 4 reagent matrix. By way of a matrix configuration, a user (or programmed processor) can locate the appropriate reagents for a given procedure. That is, reagents such as cell samples, enzymes, buffers, nucleic acid vectors, expression cassettes, reaction components (such as, for example, mgCl₂Dntps, isothermal nucleic acid assembly reagents, gap repair reagents, etc.), wash solutions, ethanol, and magnetic beads for nucleic acid purification and isolation, etc., at known locations in the matrix 540. For example, the reagent is located at position A1 (510), position A2 (511), position A3 (512), position A4 (513), position B1 (514), position B2 (515), etc., in this embodiment, up to position D4 (525). FIG. 5A is labeled to show the location of several banks 504 corresponding to matrix 540: see containers 510, 513, 521 and 525. Although the reagent cartridge 500 of fig. 5A and the matrix configuration 540 of fig. 5B illustrate a4 × 4 matrix, the matrix of reagent cartridges and electroporation devices may be any configuration, such as 2 × 2,2 × 03, 2 × 4, 2 × 5, 2 × 6,3 × 3,3 × 5,4 × 6, 6 × 7, or any other configuration including an asymmetric configuration, or two or more different matrices of reagents depending on the desired workflow. Note that in FIG. 4A, the matrix configuration is a 5 × 3+1 matrix.

In the preferred embodiment of the reagent cartridge 500 shown in fig. 5A, the reagent cartridge includes a script (not shown) readable by a processor (not shown) for dispensing reagents via a liquid handling device (not shown) and controlling an electroporation device contained within the reagent cartridge 500. Further, the reagent cartridge 500, which is a component of the automated multi-module cell processing instrument, may include a script that specifies two, three, four, five, ten, or more processes performed by the automated multi-module cell processing instrument, or even all processes performed by the automated multi-module cell processing instrument. In certain embodiments, the reagent cartridge is disposable and prepackaged with reagents tailored to perform a particular cellular processing protocol (e.g., genome editing or protein production). Because the reagent cartridges differ in content and the components of the automated multi-module cell processing instrument may not be changed, the scripts associated with a particular reagent cartridge are matched to the reagents used and the cell process being performed. Thus, for example, a reagent cartridge can be prepackaged with reagents for genome editing and scripts specifying processing steps for performing genome editing in an automated multi-module cell processing instrument (such as described in connection with fig. 1A-1D). For example, a reagent cartridge may include scripts for: pipetting the electroconductively infected cells from reservoir A2 (511), transferring the cells to electroporation device 506, pipetting the nucleic acid solution containing the editing support from reservoir C3 (520), transferring the nucleic acid solution to the electroporation device, initiating the electroporation process for a specified period of time, and then moving the punctured cells to reservoir D4 (525) in a reagent cartridge, or to another module such as a spin bottle (118 or 120 in fig. 1A) in the automated multi-module cell processing apparatus of fig. 1A. In another example, a reagent cartridge may include scripts for: the nucleic acid solution comprising the vector is pipetted from reservoir C3 (520), the nucleic acid solution comprising the editing oligonucleotide cassettes in reservoir C4 (521), and the isothermal nucleic acid assembly reaction mixture from A1 (510) to the isothermal nucleic acid assembly/desalting reservoir (414 in fig. 4A). The script may also specify process steps to be performed by other modules in the automated multi-module cell processing instrument. For example, the script may specify that the isothermal nucleic acid assembly/desalination module is heated to 50 ℃ for 30 minutes to produce an assembled isothermal nucleic acid product; and desalting the assembled isothermal nucleic acid product via magnetic bead-based nucleic acid purification involving a series of pipetting transfers, and mixing the magnetic beads in reservoir B2 (515), the ethanol wash in reservoir B3 (516), and the water in reservoir C1 (518) to the isothermal nucleic acid assembly/desalting reservoir (114 in fig. 1A).

Enrichment module

The present disclosure also includes automated multi-module cell editing instruments with enrichment modules that perform enrichment methods, including the enrichment methods described herein, to increase overall editing efficiency in a population of cells (e.g., mammalian cells).

As will be apparent to those skilled in the art upon reading this disclosure, the enrichment module can be designed to accommodate a particular enrichment method, and is preferably (but not required to be) connected to the remaining modules of the multi-module instrument, e.g., via an automated liquid handling system or other cell transfer device.

In certain embodiments, the enrichment module can be a module used outside of the instrument, wherein the resulting enriched cell population is introduced back into the integrated instrument, or alternatively, into an auxiliary instrument that completes the editing and recovery cycle. In such cases, the enrichment module functions independently of the automated multi-module instrument, but is included in the overall workflow. Thus, a workflow may require coordination of two or more processors responsible for different portions of the workflow.

In some embodiments, the enrichment module is in fluid communication with an automated multi-module instrument and is integrated with a liquid handling system and controlled by a single processor.

In some modules, enrichment is a positive enrichment module that enriches cells containing the introduced selectable marker. In some aspects, enrichment is based on negative selection, e.g., antibiotic selection, that lacks a selective marker or depletes cells in the absence of a feature due to the particular enrichment method used.

In some aspects of the disclosure, the selection process may be performed computationally, and expression of selective markers may be monitored and used for future data analysis to determine the edit rate of the cell population.

Certain selection methods that can be used with the methods of the present disclosure provide fluorescent or bioluminescent selection as a read of correctly edited cells. Correctly edited cells can be sorted from unedited or incorrectly edited cells via methods such as Fluorescence Activated Cell Sorting (FACS) and Magnetic Activated Cell Sorting (MACS), and the modules for making such selections can be incorporated into an automated multi-module cell processing instrument (see, e.g., 140 of fig. 1A). Using FACS or MACS, heterogeneous mixtures of living cells can be sorted into different populations based on the expression markers that have been expressed due to the presence of editing mechanisms for introducing the selection method and the intended editing of the target region.

FACS can separate cells based on intracellular staining or intracellular protein expression and allow for purification of individual cells based on size, granularity, and fluorescence. The cells in suspension pass in the form of a stream of droplets, wherein each droplet contains a single cell of interest. The droplet passes in front of the laser. The optical detection system detects the cells of interest based on a predetermined optical parameter (e.g., a fluorescence or bioluminescence parameter). The instrument applies a charge to the droplets containing the cells of interest, and the electrostatic deflection system facilitates collection of the charged droplets into appropriate tubes or wells. The sorting parameters can be adjusted according to the requirements of purity and yield.

MACS^TM(Miltenyi Biotec) is a method for separating various cell populations according to their surface antigens. This selection method relies on the co-introduction of cell surface markers that are not otherwise present on the cell surface to be edited.

Use of an automated multi-module mammalian cell processing instrument

FIG. 6 illustrates one embodiment of a multi-module cell processing instrument. This embodiment depicts an exemplary system for recursive gene editing of a population of mammalian cells. Cell processing instrument 600 can include a housing, a reservoir 604 for storing cells to be transformed or transfected, and a cell growth module and/or concentration module (including, for example, a spinner flask) 608. Cells to be transformed are transferred from the reservoir to the cell growth module for culture until the cells reach the target OD. After the cells reach the target OD, the growth module may cool or freeze the cells for continued processing for cell concentration, where the cells are buffer exchanged and rendered electrically receptive, and the volume of the cells may be substantially reduced. After the cells are concentrated to the appropriate volume, the cells are transferred to an editing mechanism introduction module 610, such as the flow-through electroporation device described above. In addition to the reservoir 604 for storing cells, the multi-module cell processing instrument further comprises a reservoir 606 for storing editing vectors pre-assembled with editing oligonucleotide cartridges. The pre-assembled nucleic acid vector is transferred to the editing mechanism introduction module 610, which already contains the cell culture grown to the target OD. In addition, the instrument may include a reservoir 602 for storing an engine vector comprising a coding sequence for a nucleic acid-guided nuclease. The engine vector may be transferred to the editing mechanism introduction module 610 and transformed at the same time as the editing vector is transformed, or the engine vector may be transformed into the cell before or after the editing vector is transformed into the cell. In the editing mechanism introduction module 610, nucleic acids are, for example, electroporated into cells. Following electroporation, the cells are transferred to an optional recovery module (not shown) where the cells recover shortly after transformation.

After optional recovery, the cells may be transferred to a storage module (also not shown) where the cells may be stored, for example, at 4 ℃ for subsequent processing. Further, selection may optionally be made in a separate module between the editing mechanism lead-in module and the editing module, or selection may be made in the editing module. In this case, selection refers to selecting a cell that has been correctly transformed with a vector comprising a selectable marker, thereby ensuring that the cell may have received both the vector for nucleic acid-guided nuclease editing and for reporting correct editing. After selection, the cells may optionally be diluted and transferred to editing module 612. Conditions are then provided so that editing occurs. For example, if one or more editing components (e.g., one or more of a nucleic acid-guided nuclease, gRNA, or donor DNA) are under the control of an inducible promoterThen conditions are provided to activate the one or more inducible promoters. After editing has occurred, cells are selected in the enrichment module 614 (where they are selected), e.g., using FACS or MACS^TMAnd (5) sorting. In the enrichment module, cells expressing the selectable marker are separated from cells not expressing the expression marker and optionally prepared for another round of editing. The multi-module cell processing instrument is controlled by processor 616, and processor 616 is configured to operate the instrument based on user input (e.g., as directed by one or more scripts, or as a combination of user input or scripts). Processor 616 can control the timing, duration, temperature, and operation of the various modules of instrument 600 and the dispensing of reagents from the reagent cartridges. The processor may be programmed with standard protocol parameters from which a user may select, the user may manually specify one or more parameters, or one or more scripts associated with the reagent cartridge may specify one or more operational and/or reaction parameters. In addition, the processor may notify the user (e.g., via a smartphone or other device application) that the cells have reached the target OD, make them competent or concentrated, and/or update the user with the progress of the cells in the various modules in the multi-module system.

It will be apparent to those of ordinary skill in the art in view of this disclosure that the described process can be recursive and multiplexed; that is, the cell may undergo the workflow described in connection with fig. 6, and the resulting edited culture may then undergo another round (or several or many rounds) of additional editing (e.g., recursive editing) using a different editing carrier. For example, cells from a first round of editing may be diluted and an aliquot of editing cells edited by editing carrier a may be combined with editing carrier B, an aliquot of editing cells edited by editing carrier a may be combined with editing carrier C, an aliquot of editing cells edited by editing carrier a may be combined with editing carrier D, and so on, for a second round of editing. After the second round, aliquots of each of the double-edited cells can be subjected to a third round of editing, in which, for example, aliquots of each of the AB-edited, AC-edited, AD-edited cells are combined with additional editing carriers (such as editing carriers X, Y, and Z). That is, double-edited cell AB can be combined with and edited by vectors X, Y, and Z to produce triple-edited cells ABX, ABY, and ABZ; the double-edited cell AC can be combined with and edited by vectors X, Y and Z to produce triple-edited cells ACX, ACY and ACZ; and the double-editing cell AD can be combined with and edited by vectors X, Y and Z to produce triple-editing cells ADX, ADY and ADZ, and so on. In this process, many permutations and combinations of editing can be performed, resulting in a very diverse population of cells and cell libraries. In any recursive process, "treat" (cure) both the previous engine carrier and the edit carrier (or single engine + edit carrier in a single carrier system) are advantageous. "treating" is a process in which one or more vectors used in a previous round of editing are eliminated from transformed cells.

Treatment may be accomplished by: for example, using a treatment plasmid to lyse one or more vectors so as to render the editing vector and/or engine vector (or single, two-in-one engine/editing vector) non-functional; dilution of one or more vectors in a cell population via cell growth (i.e., the more growth cycles the cell undergoes, the fewer daughter cells that retain the editing or engine vector), or by, for example, utilizing a heat-sensitive origin of replication on the editing or engine vector (or engine + editing vector in one). The conditions for treatment will depend on the mechanism used for treatment; that is, in this example, how the plasmid cleaves the editing and/or engine vector is treated.

Editing and selection workflow for higher editing efficiency

The combination of nucleic acid-guided nuclease editing methods with selection procedures (computational or physical, as further described herein) results in a significant increase in editing efficiency compared to editing methods without such selection methods.

In the first set of workflows illustrated in fig. 7 and 8, the editing workflow consists of: nucleases (e.g., RNA-guided nucleases, such as cas-9, cpf-1, MAD7, etc.) and one or more selection events are used to increase the rate of editing in cells, including increasing the rate of editing in mammalian cells.

Figure 7 shows an exemplary workflow in which the coding sequence for the editing mechanism and the RNA-guided nuclease are delivered to the cell in two separate vectors. The workflow includes designing a gRNA that targets a region of the genome to be edited, the gRNA being covalently attached to a homology arm 702 that comprises one or more desired edits. In particular aspects, the editing comprises editing that renders the target site resistant to further nuclease cleavage, e.g., a mutation in the PAM site and/or spacer. These gRNA-HA constructs are introduced into an editing vector 704, which editing vector 704 comprises a promoter for expression of the nucleic acid, and optionally a barcode or other mechanism to trace back specific edits. Optionally, the promoter used to drive the editing mechanism is inducible.

The coding sequence for the RNA-guided nuclease (e.g., cas-9, cpf-1, MAD 7) is introduced into the second set of vectors 708 to create the engine vectors. The engine vector has a coding sequence for a nuclease under a promoter independent of the editing vector. The independent promoter of the engine vector may be the same or different from the promoter used to edit the vector, and is optionally inducible.

The engine vector and editing vector are introduced into cell 710, for example, using transformation, transfection, or other mechanisms that will be apparent to those skilled in the art upon reading the present disclosure. The cell is then provided with conditions 712 for editing the cell and allowed to edit.

After editing, cell selection 714 is performed using techniques such as those described herein to obtain cells enriched for editing. Such techniques may use computational selection means to further analyze the edited cell population, as well as physical selection using negative selection and/or positive selection, such as selection of selective markers (e.g., cell surface markers that can be used as handles for physical enrichment of putative edited cells).

Steps 710-714 (or in some cases, steps 712-714 if there are enough editing and/or engine carriers in the cell population to not need to be added again) may optionally be repeated 716 to increase the editing efficiency of the cell population.

Figure 8 shows an exemplary workflow for introducing coding sequences for editing nucleic acids and nucleases into a population of cells to be edited using a single vector system. The workflow includes designing a gRNA that targets a region of the genome to be edited, the gRNA covalently attached to a homology arm 802 that comprises one or more desired edits. In particular aspects, the editing comprises editing that renders the target site resistant to further nuclease cleavage, e.g., a mutation in the PAM site and/or spacer.

The constructs of these gRNA-HA and the coding sequence of a nuclease (e.g., an RNA-guided nuclease) are introduced 804 into the same vector to create a single vector that comprises one or more promoters for expressing the nucleic acid and nuclease. The individual carriers optionally contain a barcode or other mechanism to trace back specific edits. The vector may contain a single promoter for expression of both the gRNA-HA construct and the coding sequence for the nuclease, or the gRNA-HA construct and the coding sequence for the nuclease may be under the control of different promoters in the same vector. Optionally, the one or more promoters used to drive the coding of the editing mechanism and/or nuclease are inducible.

The vector is introduced into cell 810, for example, using transformation, transfection, or other mechanisms that will be apparent to those of skill in the art upon reading this disclosure. The cell is then provided with conditions 812 for editing the cell and allowed to edit.

After editing, using techniques such as those described herein, a cell selection 814 is performed to obtain cells enriched for editing. Such techniques may use computational selection means to further analyze the edited cell population, as well as physical selection using negative selection and/or positive selection, such as selection of selective markers (e.g., cell surface markers that can be used as handles for physical enrichment of putative edited cells).

Steps 810-814 (or in some cases, steps 812-814 if there are enough editing and/or engine carriers in the cell population to not need to be added again) may optionally be repeated 816 to increase the editing efficiency of the cell population.

Figure 9 shows an exemplary workflow in which the coding sequences for the editing mechanism and the RNA-guided nuclease are delivered to the cell in two separate vectors. The workflow includes designing a gRNA that targets a region of the genome to be edited, the gRNA being covalently attached to a homology arm 902 that comprises one or more desired edits. In particular aspects, the editing comprises editing that renders the target site resistant to further nuclease cleavage, e.g., a mutation in the PAM site and/or spacer. These gRNA-HA constructs are introduced into an editing vector 904, the editing vector 904 comprising a promoter for expression of the nucleic acid, and optionally a barcode or other mechanism to trace back specific edits. Optionally, the promoter used to drive the editing mechanism is inducible.

The coding sequences of the fusion vectors of the RNA-guided nuclease (e.g., cas-9, cpf-1, MAD 7) and the enzyme region with the desired function (e.g., reverse transcriptase activity) are introduced into a second set of vectors 908 to create engine vectors. The engine vector has a coding sequence for a nuclease under a promoter separate from the editing vector. The individual promoters of the engine vector may be the same as or different from the promoter used to edit the vector, and are optionally inducible.

The engine vector and editing vector are introduced into the cell 910, for example, using transformation, transfection, or other mechanisms that will be apparent to those of skill in the art upon reading this disclosure. The cells are then provided with conditions for editing the cells 912 and allowed to edit.

After editing, using techniques such as those described herein, a cell selection 914 is performed to obtain cells enriched for editing. Such techniques may use computational selection means to further analyze the edited cell population, as well as physical selection using negative selection and/or positive selection, such as selection of selective markers (e.g., cell surface markers that can be used as handles for physical enrichment of putative edited cells).

Steps 910-914 (or in some cases, steps 912-914 if there are enough editing and/or engine carriers in the cell population to not need to be added again) may optionally be repeated 916 to increase the editing efficiency of the cell population.

Figure 10 illustrates an exemplary workflow for introducing coding sequences for editing nucleic acids and nucleases into a population of cells to be edited using a single vector system. The workflow includes designing a gRNA that targets a region of a genome to be edited, the gRNA being covalently attached to a homology arm 1002 that comprises one or more desired edits. In particular aspects, the editing comprises editing that renders the target site resistant to further nuclease cleavage, e.g., a mutation in the PAM site and/or spacer.

The coding sequences of these gRNA-HA constructs and fusion vectors of RNA-guided nucleases (e.g., cas-9, cpf-1, MAD 7) and enzyme regions with desired functions (e.g., reverse transcriptase activity) are introduced 1004 into the same vector to create a single vector containing one or more promoters for expression of the nucleic acids and fusion proteins. The individual carriers optionally contain a barcode or other mechanism to trace back specific edits. The vector may contain a single promoter for expression of both the coding sequences of the gRNA-HA construct and the fusion protein, or the coding sequences of the gRNA-HA construct and the fusion protein may be under the control of different promoters in the same vector. Optionally, one or more promoters used to drive the coding of the editing mechanism and/or fusion protein are inducible.

The vector is introduced into the cell 1010, for example, using transformation, transfection, or other mechanisms that will be apparent to those skilled in the art upon reading this disclosure. The cell is then provided with conditions for editing the cell 1012 and allowed to edit.

After editing, cell selection 1014 is performed using techniques such as those described herein to obtain cells enriched for editing. Such techniques may use computational selection means to further analyze the edited cell population, as well as physical selection using negative selection and/or positive selection, such as selection of selective markers (e.g., cell surface markers that can be used as handles for physical enrichment of putative edited cells).

Steps 1010-1014 (or in some cases, steps 1012-1014 if there are enough editing and/or engine carriers in the cell population to not need to be added again) may optionally be repeated 1016 to increase the editing efficiency of the cell population.

Cell libraries created using automated editing methods, modules, instruments, and systems

In one aspect, the present disclosure provides editing methods, modules, instruments, and automated multi-module cell editing instruments for creating cell libraries that alter the expression, level, and/or activity of RNAs and/or proteins of interest in various cell types using various nicking enzyme-based editing strategies, including CREATE fusions, as described in more detail herein. Accordingly, the present disclosure is intended to encompass edited cell libraries created by the automated editing methods, automated multi-module cell editing instruments of the present disclosure. These cell libraries may have different targeted edits including, but not limited to, gene knockouts, gene knockins, insertions, deletions, single nucleotide edits, short tandem repeat edits, frame shifts, triplet codon expansions, and the like in the cells of various organisms. These edits may relate to coding or non-coding regions of the genome and are preferably rationally designed.

In some aspects, the present disclosure provides automated editing methods, automated multi-module cell editing instruments for creating cell libraries that alter DNA-related processes. For example, a cell library can include individual cells having edits in the DNA binding site that interfere with DNA binding of regulatory elements that regulate expression of a selected gene. In addition, cell libraries may include edits in genomic DNA that affect cellular processes such as heterochromatin formation, switch class recombination, and VDJ recombination.

In particular aspects, a cell library is created using multiplexed, nickase-directed editing of individual cells within a cell population, wherein more than one cell within the cell population is edited in a single round of editing, i.e., more than one change within a cell of the cell library is edited in a single automated operation. A library that can be created in a single multiplexed automated operation can include up to 500 cells with an expected edit, which can be the same incoming edit in a cell or two or more discrete edits in different cells. The library may also include one or more of the following prospective edits (same or different): 1000 edited cells, 2000 edited cells, 5000 edited cells, 10,000 edited cells, 50,000 edited cells, 100,000 edited cells, 200,000 edited cells, 300,000 edited cells, 400,000 edited cells, 500,000 edited cells, 600,000 edited cells, 700,000 edited cells, 800,000 edited cells, 900,000 edited cells, 1,000,000 edited cells, 2,000,000 edited cells, 3,000,000 edited cells, 4,000,000 edited cells, 5,000,000 edited cells, 6,000,000 edited cells, 7,000,000 edited cells, 8,000,000 edited cells, 9,000,000 edited cells, 10,000 edited cells, or more edited cells.

In other particular aspects, nickase-directed recursive editing is used on individual cells within a population of cells to create a library of cells, wherein the editing is added to the individual cells in two or more rounds of editing. Using recursive editing results in the merging of two or more edits targeting two or more sites in the genome of individual cells in the library. A library that can be created in a single multiplexed automated operation can include up to 500 cells with an expected edit, which can be the same incoming edit in a cell or two or more discrete edits in different cells. The library may also include one or more of the following prospective edits (same or different): 1000 edited cells, 2000 edited cells, 5000 edited cells, 10,000 edited cells, 50,000 edited cells, 100,000 edited cells, 200,000 edited cells, 300,000 edited cells, 400,000 edited cells, 500,000 edited cells, 600,000 edited cells, 700,000 edited cells, 800,000 edited cells, 900,000 edited cells, 1,000,000 edited cells, 2,000,000 edited cells, 3,000,000 edited cells, 4,000,000 edited cells, 5,000,000 edited cells, 6,000,000 edited cells, 7,000,000 edited cells, 8,000,000 edited cells, 9,000,000 edited cells, 10,000 edited cells, or more edited cells.

Examples of non-automated editing strategies that may be modified based on the present description to take advantage of automated systems may be found, for example, in Liu et al (supra).

In particular aspects, recursive editing may be used to first create a cell phenotype, and then subsequent rounds of editing are used to reverse the phenotype and/or promote other cellular characteristics.

In some aspects, the cell library includes edits used to create unnatural amino acids in the cell.

In particular aspects, the disclosure provides edited cell libraries created using the disclosed editing methods, the automated multi-module cell editing instruments of the disclosure, having edits in one or more regulatory elements. The term "regulatory element" refers to a nucleic acid molecule capable of affecting the transcription and/or translation of an operably linked coding sequence in a particular environment and/or context. The term is intended to include all elements that promote or regulate transcription as well as RNA stability, including the core elements, upstream elements, enhancers and response elements required for the basic interaction of the promoter, RNA polymerase and transcription factors (see, e.g., lewis, "Genes V" (Oxford University Press, oxford) pages 847-873). Exemplary regulatory elements in prokaryotes include, but are not limited to, promoters, operator sequences, and ribosome binding sites. Regulatory elements used in eukaryotic cells may include, but are not limited to, promoters, enhancers, insulators (insulators), splicing signals, and polyadenylation signals.

Preferably, the edited cell library comprises rationally designed edits designed based on predictions of protein structure, expression and/or activity in particular cell types. For example, rational design can be based on a system-wide biophysical model of genome editing using specific nucleases and gene regulation to predict how different editing parameters including nuclease expression and/or binding, growth conditions, and other experimental conditions collectively control the kinetics of nuclease editing. See, e.g., farasat and Salis, PLoS Compout biol.,29 (1): e1004724 (2016).

In one aspect, the disclosure provides for the creation of libraries of edited cells with various rationally designed regulatory sequences created using the nickase methods of the disclosure (including automated methods using the disclosed instruments). For example, an edited cell library can include a prokaryotic cell population created using a set of constitutive and/or inducible promoters, enhancer sequences, operator sequences, and/or ribosome binding sites. In another example, the edited cell library may include eukaryotic sequences created using a set of constitutive and/or inducible promoters, enhancer sequences, operator sequences, and/or different Kozak sequences for expression of the protein of interest.

In some aspects, the disclosure provides a cell library comprising cells with rationally designed edits comprising one or more types of edits in a sequence of interest throughout the genome of an organism. In particular aspects, the disclosure provides a cell library comprising cells with rationally designed edits comprising one or more types of edits in a sequence of interest throughout a subset of a genome. For example, a cell library can include cells with rationally designed edits including one or more types of edits in a sequence of interest throughout an exome (e.g., each or most open reading frames of a genome). For example, a cell library can include cells with rationally designed edits including one or more types of edits in a sequence of interest throughout a kinase set. In yet another example, the cell library can include cells with rationally designed edits including one or more types of edits in the sequence of interest throughout the secretory component. In still other aspects, a cell library can include cells with rationally designed edits created to analyze various isoforms of proteins encoded within an exome, and a cell library can be designed to control the expression of one or more particular isoforms, e.g., for transcriptome analysis.

Importantly, in certain aspects, a cell library can include edits using randomized sequences (e.g., randomized promoter sequences) to reduce similarity between expression of one or more proteins in individual cells within the library. In addition, the promoters in the cell library may be constitutive, inducible, or both, to achieve strong and/or titratable expression.

In other aspects, the disclosure provides nicking enzyme-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments for creating edited cell libraries that include identifying optimal expression of selected gene targets. For example, production of biochemicals by metabolic engineering often requires expression of pathway enzymes, and optimal production yields are not always achieved by the highest amount of target pathway enzymes in the cell, but 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 adjusted experimentally for optimal yield.

The most obvious way in which transcription affects gene expression levels is by the rate of Pol II initiation, which can be regulated by a combination of promoter or enhancer strength and transactivator (Kadonaga et al, cell,116 (2): 247-57 (2004)). In eukaryotes, elongation rate (elongation rate) can also determine the gene expression pattern by affecting selective splicing (Cramer et al, PNAS USA,94 (21): 11456-60 (1997). Failure of gene termination can impair expression of downstream genes by reducing promoter accessibility to Pol II (Greger et al, 2000PNAS USA,97 (15): 8415-20 (2000)). This process is called transcriptional interference, and is particularly significant in lower eukaryotes because they typically have closely spaced genes.

Site-directed mutagenesis

Site-directed mutagenesis (i.e., where the amino acid sequence of a protein or other genomic feature can be altered by deliberate and precise mutagenesis of the protein or genomic feature) can be employed to create cell libraries using nickase-based editing methods, modules, instruments, and systems. These cell lines can be useful for a variety of purposes (e.g., for determining protein function within a cell, identifying enzymatic active sites within a cell, and designing novel proteins). For example, site-directed mutagenesis can be used in a multiplexed fashion to exchange a single amino acid in a protein sequence for another amino acid having a different chemical property. This allows one to determine the effect of rationally designed or randomly generated mutant genes in individual cells within a population of cells. See, e.g., berg et al Biochemistry, sixth edition, (New York: W.H.Freeman and Company) (2007).

In another example, individual cells within a cell library may be edited to replace an amino acid in a binding site, such as replacing one or more amino acids in a protein binding site for interaction within a protein complex, or replacing one or more amino acids in an enzyme pocket that may accommodate a cofactor or ligand. Such editing allows the creation of specific manipulations of the protein to measure certain properties of one or more proteins, including interactions with other cofactors, ligands, etc. within the protein complex.

In yet another example, site-specific mutagenesis can be used to perform various editing types on individual cells within a cell library for the study of expression quantitative trait loci (eQTLs). eQTL is a locus that accounts for a portion of the genetic variance of a gene expression phenotype. The libraries of the present invention will be useful for evaluating eQTLs and relating the eQTLs to actual disease states.

In particular aspects, rational design based on known or predicted structure of proteins can be used to create edits that are introduced into the cell libraries of the present disclosure. See, e.g., chronooulouu EG and Labrou, curr protocol Protein sci; chapter 26 unit 26.6 (2011). Such site-directed mutagenesis can provide one or more site-directed edits to individual cells within the library, and preferably two or more site-directed edits within a population of cells (e.g., combinatorial edits).

In other aspects, site-directed codon mutagenesis is used to "scan" all codons or substantially all codons in the coding region of a gene to create a library of cells of the present disclosure. In this way, a single edit of a particular codon can be examined for loss of function or gain of function based on a particular polymorphism in one or more codons of the gene. These libraries can be powerful tools for determining which genetic changes are silent or causative of a particular phenotype in a cell or population of cells. The editing of codons can be randomly generated or can be rationally designed based on known polymorphisms and/or identified mutations in the gene to be analyzed. In addition, using these techniques for two or more genes in a single pathway in a cell, potential protein-protein interactions or redundancies in cell function or pathway can be determined.

For example, alanine scanning can be used to determine the contribution of a particular residue to the stability or function of a given protein. See, e.g., lef [ e ] vre et al, nucleic Acids Research, vol.25 (2): 447-448 (1997). Alanine is often used in this codon scanning technique because of its non-bulky, chemically inert methyl functionality, which can mimic the secondary structure preference possessed by many other amino acids. Codon scanning can also be used to determine whether the side chain of a particular residue plays a significant role in cell function and/or activity. Sometimes, if it is desired to maintain the size of the mutated residue, other amino acids, such as valine or leucine, may be used in creating the codon-scanned cell library.

In other particular aspects, cell libraries can be created using the nicking enzyme-based editing methods of the present disclosure, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments to determine the active sites of proteins (such as enzymes or hormones) and elucidate the mechanism of action of one or more of these proteins in the cell library. Site-directed mutagenesis in connection with molecular modeling studies can be used to discover the active site structure of an enzyme and hence the mechanism of action of the enzyme. Analysis of these cell libraries can provide insight into: specific amino acid residues at the active site of a protein, in contact between subunits of a protein complex, play a role in intracellular trafficking and protein stability/half-life in various genetic backgrounds.

Saturation mutagenesis

In some aspects, cell libraries created using nickase-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments are 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 gene of interest. These cell libraries may be particularly useful for generating variants (e.g., for directed evolution). See, e.g., chica et al, current Opinion in Biotechnology 16 (4): 378-384 (2005); and Shivange, current Opinion in Chemical Biology,13 (1): 19-25.

In some aspects, edits comprising different degenerate codons can be used to encode a set of amino acids in individual cells in a library. Because some amino acids are encoded by more codons than others, the exact ratios of amino acids cannot be equal. In certain aspects, more restricted degenerate codons are used. 'NNK' and 'NNS' have the advantage of encoding all 20 amino acids, but also encode a stop codon 3% of the time. Alternative codons such as 'NDT', 'DBK' avoid stop codons completely and encode a minimal set of amino acids that still cover all major biophysical types (anionic, cationic, aliphatic hydrophobic, aromatic hydrophobic, hydrophilic, small).

In particular aspects, non-redundant saturation mutagenesis is used in a saturation mutagenesis editing process, wherein codons most commonly used by a particular organism are used.

Promoter exchange Ladder (Ladder)

One mechanism for analyzing and/or optimizing the expression of one or more genes of interest is by creating a library of "promoter swap" cells, wherein the cells contain genetic edits with specific promoters linked to one or more genes of interest. Thus, cell libraries created using nicking enzyme-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments can be promoter swap cell libraries, which can be used, for example, to increase or decrease expression of a gene of interest to optimize a metabolic or genetic pathway. In some aspects, a promoter swap cell library can be used to identify increased or decreased expression of genes (e.g., genes encoding proteins that affect cell growth rate or overall health) that affect cell viability (viability) or viability (viability). In some aspects, a library of promoter-swapped cells can be used to create cells with dependencies and logic between promoters to create a synthetic gene network. In some aspects, promoter swapping can be used to control cell-to-cell communication between cells of both homogeneous and heterogeneous (complex tissue) populations in nature.

Cell libraries can utilize any given number of promoters grouped together based on expression of a range of expression intensities and any given number of target genes. The promoter ladder alters expression of at least one locus under at least one condition. The ladder is then systematically applied to a set of genes in an organism using the automated editing methods, automated multi-module cell editing instruments of the present disclosure.

In particular aspects, a library of cells formed using a nicking enzyme-based editing method includes individual cells representing a given promoter operably linked to one or more target genes of interest that are otherwise genetically identical in context. An example of a non-automated editing strategy that can be modified to utilize an automated system can be found, for example, in U.S. patent No. 9,988,624.

In particular aspects, a promoter swap cell library is created by editing a set of target genes to be operably linked to a set of pre-selected promoters that serve as a "promoter ladder" for expression of a gene of interest. For example, an editing cell allows one or more individual genes of interest to be edited to operably link to different promoters in a promoter ladder. The individual promoters in the promoter ladder may be inserted in front of the gene of interest when the endogenous promoter is not present, its sequence is unknown, or it has been altered in some way previously. These generated cell libraries have individual cells with individual promoters in the ladder operably linked to one or more target genes that are otherwise genetically identical in background. Promoters are typically selected to produce variable expression between different loci, and may include inducible promoters, constitutive promoters, or both.

A set of target genes edited using a promoter ladder may include all or most Open Reading Frames (ORFs), e.g., ORFs of a kinase set or a secretory set, of a genome or a selected subset of a genome. In some aspects, the target gene may include coding regions of various subtypes of the gene, and the cell library may be designed to express one or more specific subtypes, e.g., for transcriptome analysis using various promoters.

The set of target genes may also be genes known or suspected to be involved in a particular cellular pathway (e.g., a regulatory pathway or a signaling pathway). The set of target genes can be ORFs that are functionally related by association with previously displayed beneficial edits (previous promoter exchanges or previous SNP exchanges), by algorithmic selection based on epistatic interactions between previously generated edits, based on other selection criteria on the assumption of beneficial ORFs for the target, or by random selection. In particular embodiments, the target gene may comprise a non-protein coding gene, including non-coding RNA.

Editing of other functional genetic elements (including insulator elements and other genomic tissue elements) can also be used to systematically alter the expression levels of a set of target genes and can be introduced using the methods of the present disclosure, automated multi-module cell editing instruments. In one aspect, ladders of enhancer sequences are used to edit cell populations, either alone or in combination with selected promoters or promoter ladders, to create cell libraries with various edits in these enhancer elements. In another aspect, ladders of ribosome binding sequences are used to edit cell populations, either alone or in combination with selected promoters or promoter ladders, to create cell libraries having various edits in these ribosome binding sequences.

In another aspect, the cell population is edited to allow for the attachment of various mRNA and/or protein stabilizing or destabilizing sequences to the 5 'end or 3' end or any other location of the transcript or protein.

In certain aspects, cell populations of previously established cell lines may be edited using the automated editing methods, modules, instruments, and systems of the present disclosure to create cell libraries to improve the function, health, and/or viability of the cells. For example, many of the industrial strains currently used for large-scale production are developed over a period of years (sometimes decades) using an iterative random mutagenesis method. Unwanted neutral and deleterious mutations are introduced into strains with beneficial changes, and over time this leads to strains that are deficient in overall robustness and key traits (such as growth rate). In another example, mammalian cell lines continue to mutate through a period of cell passage, and as such, these cell lines may become unstable and acquire undesirable traits. Automated editing methods, automated multi-module cell editing instruments of the present disclosure may use editing strategies (such as SNP and/or STR swapping, indel creation, or other techniques) to remove or alter undesired genomic sequences and/or introduce new genomic sequences to address the defect while preserving the desired properties of the cell.

When recursive editing is used, edits in individual cells in the edited cell library can incorporate the inclusion of a "landing pad" in an ectopic site (e.g., the CarT locus) in the genome to optimize expression, stability, and/or control.

In some embodiments, each library generated with individual cells comprising one or more edits (introduced or removed) is cultured and analyzed under one or more criteria (e.g., production of a chemical or product of interest). Cells with specific criteria are then associated or correlated with one or more specific edits in the cell. In this manner, the effect of a given edit on any number of genetic or phenotypic traits of interest can be determined. Identifying more than one edit associated with a particular standard or enhanced function/robustness can result in cells with highly desirable characteristics.

Knock-out or knock-in libraries

In certain aspects, cell libraries created using nicking enzyme-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments may be "knock-out" (KO) or "knock-in" (KI) edits of various genes of interest. Accordingly, the present disclosure is intended to encompass edited cell libraries created by nicking enzyme-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments, having one or more mutations that remove or reduce the expression of a selected gene of interest, to study the effect of these edits on gene function in individual cells within the cell library.

Cell libraries may be created using targeted genes KO (e.g., via insertion/deletion) or KI (e.g., via homology-directed repair). For example, double-stranded breaks are often repaired via non-homologous end-joining DNA repair pathways. This repair is known to be error prone and may therefore introduce insertions and deletions that disrupt gene function. Preferably, the edits are rationally designed to specifically affect a gene of interest, and individual cells of a KO or KI having one or more loci of interest can be created. Automated recursive editing of the present disclosure may be used to create cells having a KO or KI with two or more loci of interest.

In particular aspects, a KO or KI cell library is created using simultaneous multiplexed editing on cells within a cell population, and more than one cell within the cell population is edited in a single round of editing, i.e., more than one change within a cell of the cell library is edited in a single automated operation. In other particular aspects, recursive editing is used on individual cells within a population of cells to create a library of cells and results in more than one edit at two or more respective sites in the genome being incorporated into an individual cell.

SNP exchange or short tandem repeat exchange

In one aspect, cell libraries created using nickase-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments can be generated for systematically introducing or substituting single nucleotide polymorphisms ("SNPs") into the genome of individual cells to create "SNP swap" cell libraries. In some embodiments, the SNP swapping methods of the present disclosure include both the addition of beneficial SNPs as well as the removal of deleterious and/or neutral SNPs. SNP swapping may target coding sequences, non-coding sequences, or both.

In another aspect, cell libraries are created using nickase-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments for systematically introducing or substituting short tandem repeat sequences ("STRs") into the genome of individual cells to create "STR swap" cell libraries. In some embodiments, the STR swap methods of the present disclosure include both the addition of beneficial STRs and the removal of harmful and/or neutral STRs. STR shuffling can target coding sequences, non-coding sequences, or both.

In some embodiments, the SNP exchanges and/or STR exchanges used to create the cell library are multiplexed and more than one cell within the population of cells is edited in a single round of editing, i.e., more than one change within a cell of the cell library is edited in a single automated operation. In other embodiments, the SNP and/or STR exchanges used to create the cell library are recursive and result in the incorporation of more than one beneficial sequence and/or the removal of deleterious sequences into a single cell. More than one alteration can be either a specific defined set of alterations or a partially randomized combinatorial mutation library. Removal of deleterious mutations and incorporation of beneficial mutations can provide immediate improvements in various cellular processes. Removing the genetic burden or incorporating beneficial changes into strains without genetic burden also provides a new, robust starting point for additional random mutagenesis that can further achieve improvements.

SNP swapping overcomes fundamental limitations of random mutation methods, as SNP swapping is not a random method, but rather is systematically introduced or removed by individual mutations throughout the cell.

Splice site editing

RNA splicing is the processing during which introns are excised and exons spliced together to produce mRNA processing that is translated into protein. The precise identification of splicing signals by cellular machinery is critical to this process. Thus, cell libraries of the present disclosure include cell libraries created using nickase-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments for systematically introducing alterations to known and/or predicted splice donor and/or acceptor sites in individual genomes to create libraries of splice site variants of individual genes. Such editing may help elucidate the biological importance of various subtypes of genes in a cellular environment. Rationally designed sequences of splice sites of various coding regions, including actual or predicted mutations associated with various mammalian disorders, can be predicted using analytical techniques such as those found in: nalla and Rogan, hum Mutat,25 (2005); divina et al, eur J Hum Genet, 17; desmet et al, nucleic Acids Res,37 (2009); faber et al, BMC Bioinformatics,12 (supplement 4): S2 (2011).

Initiation/termination codon exchange and incorporation of nucleic acid analogs

In some aspects, the disclosure provides for the creation of cell libraries created using nicking enzyme-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments, for exchanging start codon and stop codon variants throughout the genome of an organism or in a selected subset of coding regions in the genome (e.g., a kinase set or a secretion set). In a cell library, individual cells will replace the start codon or stop codon of one or more genes of interest with one or more start codons or stop codons.

For example, the typical initiation codon used by eukaryotes is ATG (AUG), and most commonly used by prokaryotes is ATG (AUG), followed by GTG (GUG) and TTG (UUG). The cell library may include individual cells having a substitution of the native start codon for one or more genes of interest.

In some aspects, the disclosure provides for the creation of cell libraries by replacing the ATG initiation codon with TTG in front of a selected gene of interest. In other aspects, the disclosure provides automated creation of cell libraries by replacing the ATG start codon with GTG. In other aspects, the disclosure provides automated creation of cell libraries by replacing the GTG start codon with ATG. In other aspects, the disclosure provides automated creation of cell libraries by replacing the GTG start codon with TTG. In other aspects, the disclosure provides automated creation of cell libraries by replacing the TTG start codon with ATG. In other aspects, the disclosure provides automated creation of cell libraries by replacing the TTG start codon with GTG.

In other examples, typical stop codons for saccharomyces cerevisiae (s. Cerevisiae) and mammals are TAA (UAA) and TGA (UGA), respectively. Typical stop codons for monocotyledons are TGA (UGA), whereas insects and e.coli (e.coli) generally use TAA (UAA) as stop codon (Dalphin et al, nucleic acids res., 24. The cell library can include individual cells having a substitution of a natural stop codon for one or more genes of interest.

In some aspects, the disclosure provides automated creation of cell libraries by replacing the TAA stop codon with TAG. In other aspects, the disclosure provides automated creation of cell libraries by replacing the TAA stop codon with TGA. In other aspects, the disclosure provides automated creation of cell libraries by replacing the TGA stop codon with TAA. In other aspects, the disclosure provides automated creation of cell libraries by replacing the TGA stop codon with TAG. In other aspects, the disclosure provides automated creation of cell libraries by replacing the TAG stop codon with TAA. In other aspects, the invention teaches automated creation of cell libraries by replacing the TAG stop codon with TGA.

Terminator exchange and ladder

One mechanism for identifying the optimal termination of pre-splicing mRNA of one or more genes of interest is by creating a library of "terminator swap" cells, wherein the cells include genetic edits with specific terminator sequences linked to one or more genes of interest. Thus, the cell libraries of the present disclosure include terminator-swapped cell libraries created using nicking enzyme-based editing methods, modules, instruments and systems employing automated editing methods, and/or automated multi-module cell editing instruments. The terminator swap cell library can be used to affect mRNA stability, for example, by releasing transcripts from the synthesis site. In other embodiments, a library of terminator exchange cells can be used to identify increases or decreases in transcription termination efficiency, and thus accumulation of unspliced pre-mRNA (e.g., west and Proudfoot, mol cell.;33 (3-9); 354-364 (2009)) and/or 3' end processing (e.g., west et al, mol cell.29 (5): 600-10 (2008)). Where a gene is linked to more than one termination site, the edit may edit a combination of edits of more than one terminator associated with the gene. Additional amino acids may also be added to the ends of the protein to determine the effect of the terminator on the length of the protein.

Cell libraries can utilize any given number of terminator edits selected for the terminator ladder based on the performance of the activity ranges and any given number of target genes. The terminator ladder alters expression of at least one locus under at least one condition. The ladder is then systematically applied to a set of genes in an organism using the automated editing methods, modules, instruments, and systems of the present disclosure. In some aspects, the present disclosure provides for the creation of cell libraries using the automated editing methods, modules, instruments, and systems of the present disclosure, wherein the libraries are created to edit terminator signals in one or more regions in the genome of individual cells of the library. Transcription termination in eukaryotes is operated by a terminator signal recognized by a protein factor associated with RNA polymerase II. For example, a cell library can comprise individual eukaryotic cells with edits in the genes encoding polyadenylation specific factor (CPSF) and cleavage stimulating factor (CstF) and/or the genes encoding proteins recruited to the termination site by CPSF and CstF factors. In prokaryotes, two major mechanisms, termed Rho-independent termination and Rho-dependent termination, mediate transcriptional termination. For example, a cell library can comprise individual prokaryotic cells with edits in the genes encoding proteins that affect the binding, efficiency, and/or activity of these termination pathways.

In certain aspects, the present disclosure provides methods of selecting a termination sequence ("terminator") with optimal properties. For example, in some embodiments, the present disclosure teaches methods for introducing and/or editing one or more terminators and/or producing variants of one or more terminators within a host cell that exhibit a range of activities. Particular combinations of terminators may be grouped together as a terminator ladder, and cell libraries of the present disclosure include individual cells representing terminators operably linked to one or more target genes of interest that are otherwise genetically identical in context. An example of a non-automated editing strategy that can be modified to utilize automated instrumentation can be found, for example, in U.S. Pat. No. 9,988,624 to Serber et al, entitled "microbiological strain improvement by a HTP genetic engineering platform".

In particular aspects, a terminator swap cell library is generated by editing a set of target genes to be operably linked to a set of preselected terminators, which serve as "terminator ladders" for expression of the gene of interest. For example, the cell is edited such that the endogenous terminator is operably linked to the individual gene of interest that is edited with a different terminator in the terminator ladder. When an endogenous terminator is not present, its sequence is unknown, or it has been altered in some way previously, a single terminator in the terminator ladder can be inserted behind the gene of interest. These generated cell libraries have individual cells in which individual terminators in the ladder are operably linked to one or more target genes that are otherwise genetically identical. The terminator ladder in question is then associated with a given gene of interest.

The terminator ladder can be used to more generally affect the termination of all or most ORFs (e.g., ORFs of the kinase or secretory group) in a genome or a selected subset of a genome. The set of target genes may also be genes known or suspected to be involved in a particular cellular pathway (e.g., a regulatory pathway or a signaling pathway). The set of target genes can be ORFs that are functionally related by association with previously displayed beneficial edits (previous promoter exchanges or previous SNP exchanges), by algorithmic selection based on epistatic interactions between previously generated edits, based on other selection criteria on the assumption of beneficial ORFs for the target, or by random selection. In particular embodiments, the target gene may comprise a non-protein coding gene, including non-coding RNA.

Examples

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the present invention, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent or imply that the experiments below are all or the only experiments performed. It will be appreciated by persons skilled in the art that numerous variations and/or modifications may be made to the invention as shown in the specific aspects without departing from the spirit or scope of the invention as broadly described. The present aspects are, therefore, to be considered in all respects as illustrative and not restrictive.

Example I: full-automatic single RGN-guided editionEditing runs

Single-plex automated genome editing using MAD7 nuclease was successfully performed with an automated multi-module instrument as described in the following: for example, U.S. patent nos. 9,982,279; and USSN16/024,831 filed on 30/6/2018; USSN16/024,816 filed on 30/6/2018; USSN 16/147,353 filed on 28/9/2018; USSN 16/147,865, filed on 30/9/2018; and USSN 16/147,871 filed on 30/6/2018.

Isothermal nucleic acid assembly module via Gibson in an automated instrument

The ampR plasmid backbone and lacZ _ F172 × editing cassette were assembled into an "editing vector". lacZ _ F172 functionally knocks out the lacZ gene. "lacZ _ F172" indicates that editing occurred at residue 172 in the lacZ amino acid sequence. After assembly, the product was desalted using AMPure beads in an isothermal nucleic acid assembly module, washed with 80% ethanol, and eluted with buffer. The assembled editing vector and the recombineering ready electrocompetent cells are transferred to an editing mechanism introduction module for electroporation. Cells and nucleic acids were combined and allowed to mix for 1 minute and electroporation was performed for 30 seconds. The parameters of the puncture pulse are: voltage, 2400V; length, 5ms; interval, 50ms; the number of pulses, 1; polarity, +. The parameters of the transfer pulse are: voltage, 150V; length, 50ms; interval, 50ms; pulse number, 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 after 1 hour and the cells were allowed to recover for an additional 2 hours. After recovery, the cells were maintained at 4 ℃ until retrieval by the user.

After automated processing and recovery, aliquots of cells were plated on MacConkey agar supplemented with lactose (as sugar substrate), chloramphenicol, and carbenicillin, and allowed to grow until colonies appeared. White colonies represent functionally edited cells and purple colonies represent unedited cells. All liquid transfers were performed by automated liquid handling devices of an automated multi-module cell processing instrument.

The result of the automated process was a total of about 1.0E⁰³Individual cells were transformed (comparable to the conventional desktop results) and the editing efficiency was 83.5%. lacZ _172 editing in the white colonies was confirmed by sequencing the edited regions of the cell genome. Further, the steps of automated cell processing are remotely observed by a webcam, and a text message is sent to update the status of the automated processing program.

Example II: full automated recursive edit execution

Recursive editing was successfully achieved using an automated multi-module cell processing system. Isothermal nucleic acid assembly modules included in an automated system via Gibson

The ampR plasmid backbone and lacZ _ V10 × editing cassette were assembled into an "editing vector". Like the lacZ _ F172 editing, lacZ _ V10 editing functionally knocks out the lacZ gene. "lacZ _ V10" indicates that editing occurs at amino acid position 10 in the lacZ amino acid sequence. After assembly, the product was desalted using AMPure beads in an isothermal nucleic acid assembly module, washed with 80% ethanol, and eluted with buffer. The first assembled editing vector and the electrically competent e.coli cells ready for recombinant engineering are transferred to the editing mechanism lead-in module for electroporation. Cells and nucleic acids were combined and allowed to mix for 1 minute and electroporation was performed for 30 seconds. The parameters of the puncture pulse are: voltage, 2400V; length, 5ms; interval, 50ms; pulse number, 1; polarity, +. The parameters of the transfer pulse are: voltage, 150V; length, 50ms; interval, 50ms; pulse number, 20; polarity, +/-. After electroporation, the cells were transferred to a recovery module (another growth module) and allowed to recover in SOC medium containing chloramphenicol. After 1 hour carbenicillin was added to the medium and the cells were grown for an additional 2 hours. The cells were then transferred to a centrifuge module and then media exchanged. Resuspending the cells in TB containing chloramphenicol and carbenicillin and growing the cells in this TB to OD600 was 2.7, and the cells were then concentrated and rendered electrocompetent.

During cell growth, a second editing vector is prepared in an isothermal nucleic acid assembly module. The second editing vector contains the kanamycin resistance gene and the editing cassette contains the galK Y145 x edit. If successful, galK Y145 edits confer to the cell the ability to uptake and metabolize galactose. The edit resulting from the galK Y154 x cassette introduces a stop codon at amino acid residue 154, changing the tyrosine amino acid to a stop codon. This editing renders the galK gene product non-functional and inhibits the ability of the cell to metabolize galactose. After assembly, the second editing vector product was desalted using AMPure beads in an isothermal nucleic acid assembly module, washed with 80% ethanol, and eluted with buffer. The assembled second editing vector and electrocompetent cells (transformed with and selected for the first editing vector) are transferred to the editing mechanism introduction module for electroporation using the same parameters as detailed above. After electroporation, the cells were transferred to a recovery module (another growth module) and allowed to recover in SOC medium containing carbenicillin. After recovery, cells were kept at 4 ℃ until retrieval, after which aliquots of cells were plated on LB agar supplemented with chloramphenicol and kanamycin. To quantify both lacZ and galK editing, replicate plaque plates were generated on two media types: 1) MacConkey agar base supplemented with lactose (as sugar substrate), chloramphenicol, and kanamycin, and 2) MacConkey agar base supplemented with galactose (as sugar substrate), chloramphenicol, and kanamycin. All liquid transfers were performed by automated liquid handling devices of an automated multi-module cell processing system.

In this recursive editing experiment, 41% of the screened colonies had both lacZ and galK editing, with results comparable to the dual editing efficiency obtained using the "desktop" or manual methods.

Cells were transfected with an editing cassette plasmid that mediated expression of gene-specific grnas with or without DNA sequences that mediated precise genome editing (HDR donors). The plasmid also expresses handles (cell surface receptors, fluorescent proteins, antibiotic resistance genes) that enable enrichment of cells that have been functionally transfected with the editing cassette plasmid. Cells are also co-transfected with nucleases (plasmids, mRNA, proteins) that, when paired with gene-specific grnas, can mediate DNA sequence-specific endonuclease activity at the genomic target.

After delivery of the enrichment-capable editing cassettes, the enrichment handle must be expressed to a level that supports a specific positive selection of transfected cells while allowing depletion of cells that do not receive an enrichment-capable editing cassette. In certain instances, the expression level of the enrichment reporter may enable enrichment of a subpopulation having significantly higher or lower levels of the enrichment reporter.

Surface reporter expressing cells can be specifically labeled with fluorophore conjugated antibodies and then sorted into different populations (receptor negative, high or low) using a Fluorescence Activated Cell Sorter (FACS). By electronically gating cells with different levels of fluorescence intensity, one can specifically enrich for subpopulations that take up relatively more or less copies of the editing cassette. As observed in GFP-to-BFP analysis of enriched versus non-enriched populations, certain enriched cell subpopulations exhibit higher edit rates as determined by the relative percentages of GFP-positive, BFP-positive, and double-negative cells. Enrichment via cell surface displayed receptors or affinity ligands was also performed using antibody-coupled magnetic beads.

Example III: development of GFP expression assay

Using the RNA-guided nuclease-GFP expression cassette an editing detection assay was developed that accelerated the genome editing workflow of single cell clones from initial nuclease screening to the final stage. The vector also contained a U6-gRNA cassette, creating a single vector system for CRISPR/nuclease delivery and expression (fig. 10).

Two systems were developed to help enrich for the desired genome-edited cell population, e.g., using cell sorting. The first system uses a single vector, in which an RNA-guided nuclease (e.g., cas9 nuclease or MAD7 nuclease) is co-expressed with GFP from the same mRNA, and a dual plasmid system, in which the RNA-guided nuclease is expressed on separate vectors. The single vector system described herein comprises a T7 promoter for nuclease-GFP mRNA in vitro transcription (figure 10).

The ability to detect and enrich via GFP expression significantly reduces the labor and costs associated with single cell cloning and genotyping in genome editing applications. The following data set illustrates how our single vector system can be used for expression monitoring and FACS enrichment for low and high levels of cleavage. In particular, the single plasmid GFP format ensures that all of the required CRISPR/nuclease components (e.g., MAD7 and gRNA coding sequences) are efficiently delivered to GFP positive cells.

Cell fractions were divided into low, medium and high pools based on GFP expression and observed for corresponding increases in indel activity. For grnas targeting the KRAS locus, a 4-fold increase in indel activity was observed when comparing the unsorted population to the first 2% of cells with the highest GFP expression (see fig. 13A and 13B). When nuclease screening is initially performed against gene targets, not all targeted gRNA designs produce detectable indel activity, and current gRNA design rules cannot predict activity based on sequence content or genomic context. gRNA design for CCR5 initially failed to produce detectable indels, and indel activity could be detected in the medium and high GFP fractions when they were split into low, medium and high GFP fractions.

The GFP reporter allows for rapid detection of transfection efficiency, saving time and costs associated with downstream quantitative expression assays. The assay also allows for rapid elimination of plasmid delivery and expression problems associated with specific cell types. Promoter/cell type incompatibility can be circumvented using nuclease-GFP mRNA if GFP expression and nuclease indel activity are not observed in a particular cell type despite repeated attempts.

Example IV: GFP to BFP conversion assay

A GFP to BFP reporter cell line was created using mammalian cells with stably integrated genomic copies of the GFP gene (HEK 293T-GFP). These cell lines enable phenotypic detection of different classes of genome editing (NHEJ, HDR, no editing) by a variety of different mechanisms including flow cytometry, fluorescent cell imaging, and genotyping by sequencing of the genome-integrated GFP gene. Lack of editing or complete repair of the cleavage event of the GFP gene results in the cells remaining GFP positive. Cleavage events repaired by the non-homologous end joining (NHEJ) pathway often result in nucleotide insertion or deletion events (indels) leading to frame shift mutations in the coding sequence, resulting in the loss of GFP gene expression and fluorescence. Cleavage events for repair by the homology-directed repair (HDR) pathway using GFP to BFP HDR donors as repair templates lead to a shift in the fluorescence spectrum of the cells from the GFP fluorescence spectrum to the BFP fluorescence spectrum. Examples of GFP and BFP fluorescence before and after gene editing as measured by FACS are shown in fig. 14A and 14B.

Example V: thy1.2-mediated enrichment for editing cassette uptake using FACS

Cells with stably integrated copies of the GFP gene (HEK 293T-GFP) were co-nuclear transfected with a plasmid expressing MAD7 nuclease and a plasmid expressing GFP to the BFP editing cassette, which also drives expression of the cell surface ligand thy 1.2. Thy1.2 is a cell surface protein expressed on mouse thymocytes and absent on any human cell. Thus, thy1.2 is a unique reporter for identifying human cells that have received the editing machinery necessary to provide thy1.2 expression.

Briefly, using the program CM-130 on a 4D nuclear transfectator X unit (Lonza, morristown, NJ), 2X 10 pairs of the 200ng MAD7 expression plasmid and 200ng GFP-to-BFP editing cassette expressing Thy1.2 were used in 20. Mu.L nucleocuvette⁵Individual cells were subjected to nuclear transfection.

24 hours after nuclear transfection, cells were labeled with anti-Thy1.2 antibody conjugated to Fluorophora hemoglobin (PE). Antibody-labeled cells were then enriched using Fluorescence Activated Cell Sorting (FACS) analysis on FACS Melody (Becton Dickenson, franklin Lakes, NJ) to isolate thy1.2 negative cells from cells expressing low or high amounts of thy1.2 (fig. 15). FACS sorted subpopulations, as well as unenriched control samples, were plated in individual wells of 24-well tissue culture dishes and allowed to undergo gene editing. Cells receiving precise HDR-mediated two base exchange exhibited a GFP to BFP conversion phenotype.

120 hours after transfection, the expression levels of GFP or BFP were analyzed by FACS sorting cell subpopulations enriched for thy1.2 expression. The percentage of cell counts in the GFP positive (wild type or no editing), GFP negative (NHEJ mediated insertion or deletion frameshifting), or BFP positive (HDR mediated precise conversion of GFP to BFP sequence) quadrants of the FACS dot plots was quantified and compared between samples (figure 17). The unenriched population was 83% GFP-positive (WT), 17% GFP and BFP-Negative (NHEJ), and 1% BFP-positive (HDR). Cells enriched for editing cassette uptake and thy1.2 expression by FACS were 15% -68% gfp positive (WT), 30% -74% gfp and BFP Negative (NHEJ) and 2% -10% BFP positive (HDR), depending on whether low or high expressing populations were specifically enriched.

Example VI: thy1.2-mediated enrichment for editing cassette uptake using MACS

The enrichment method as described in example V above using the Magnetic Activated Cell Sorting (MACS) assay showed very similar efficiencies. Cells with stably integrated copies of the GFP gene (HEK 293T-GFP) were co-nuclear transfected with a plasmid expressing MAD7 nuclease and a plasmid expressing GFP to the BFP editing cassette, which also drives expression of the cell surface ligand thy1.2, as described above. Briefly, using the program CM-130 on a 4D nuclear transfectator X unit (Lonza, morristown, NJ), in 20. Mu.Lnucleocuvette, 2X 10 pairs of 200ng MAD7 expression plasmid and 200ng GFP-to-BFP editing cassette expressing Thy1.2 were used⁵Individual cells were subjected to nuclear transfection.

24h after nuclear transfection, cells were labeled with anti-Thy1.2 magnetic beads and purified on a MACS column (Miltenyi Biotec, sunnyvale, calif.) according to the manufacturer's protocol. Cell samples from MACS column flow-through, column wash and magnetically purified elution fractions, as well as pre-enrichment controls, were labeled with anti-thy 1.2-PE fluorescent antibodies and analyzed for thy1.2 expression levels by FACS. Under the conditions tested, MACS purification specifically enriched the cell subpopulation with the highest thy1.2 expression level, as measured by thy1.2-PE labeling (fig. 16A-16E). Cells from MACS purified flow-through, wash and elution fractions, as well as the unenriched controls were plated in individual wells of 24-well tissue culture dishes and allowed to undergo gene editing and GFP-to-BFP conversion.

120 hours after transfection, the expression levels of GFP or BFP were further analyzed by FACS for cell subsets enriched for Thy1.2 expression by MACS bead needle. The percentage of cell counts in the GFP positive (wild type or no editing), GFP negative (NHEJ mediated insertion or deletion frameshifting), or BFP positive (HDR mediated precise conversion of GFP to BFP sequence) quadrants of the FACS dot plots was quantified and compared between samples (figure 17). The non-enriched population was 80% GFP positive (WT), 17% GFP and BFP Negative (NHEJ), and 1% BFP positive (HDR). Cells taken up and Thy1.2 expressed by the MACS enrichment editing cassettes were 15% -35% GFP positive (WT), 57% -74% GFP and BFP Negative (NHEJ) and 8% -10% BFP positive (HDR).

The unique cell population with the highest thy1.2 expression level, whether enriched by FACS or MACS, had a significantly higher overall editing rate and a higher HDR to NHEJ ratio. In addition, unedited GFP positive cell populations have been significantly reduced. Examples IV and V the methods described herein enable a user to obtain a cell population with a much higher proportion of cells with expected edits and fewer unedited cells.

Example VII: delta Tetherin-HA mediated enrichment for editing cassette uptake using FACS

Cells with a stably integrated copy of the GFP gene (HEK 293T-GFP) were subjected to a corefection with a plasmid expressing MAD7 nuclease and a plasmid of GFP to BFP editing cassette (which also drives expression of the cell surface ligand Tetherin, which has been engineered to contain an additional His-tag and a deletion that renders the protein non-functional). The Δ Tetherin-HA used was a cell surface surrogate handle containing deletions that render the molecule non-functional.

Briefly, procedure CM-130 was used on a 4D nucleostainer X unit (Lonza, morristown, NJ) and expressed with 200ng MAD7 in 20. Mu.L nucleofuttePlasmid and 200ng of a GFP to BFP editing cassette pair expressing. DELTA.Tetherin-HA 2X 10⁵Individual cells were subjected to nuclear transfection.

24 hours after nuclear transfection, cells were labeled with anti-HA antibody conjugated to rhodopsin (PE) of P.fluorescens. The antibody-labeled cells were then enriched using FACS Melody (Becton Dickenson, franklin Lakes, NJ) to isolate Δ Tetherin-HA negative cells from cells expressing low or high amounts of Δ Tetherin-HA. FACS-sorted subpopulations, as well as unenriched control samples, were plated in individual wells of 24-well tissue culture dishes and allowed to undergo gene editing. Cells receiving precise HDR-mediated editing exhibit a GFP to BFP conversion phenotype.

120 hours after transfection, the expression levels of GFP or BFP were analyzed by FACS for subpopulations of cells enriched for delta Tetherin-HA expression by FACS sorting or MACS beads. The percentage of cell counts in the GFP positive (wild type or no editing), GFP negative (NHEJ mediated insertion or deletion frameshifting), or BFP positive (HDR mediated precise conversion of GFP to BFP sequence) quadrants of the FACS dot plots was quantified and compared between samples (figure 18). The unenriched population 42% GFP-positive (WT), 54% GFP and BFP-Negative (NHEJ), and 4% BFP-positive (HDR). Cells enriched for editing cassette uptake and Δ Tetherin-HA expression by FACS or MACS were 2% -23% gfp positive (WT), 70% -82% gfp and BFP Negative (NHEJ), and 7% -16% BFP positive (HDR), depending on whether low or high expression populations were specifically enriched. The unique cell population with the highest Δ teherin-HA expression level had a significantly higher overall editing rate and also had a higher HDR to NHEJ ratio. In addition, unedited GFP positive cell populations have been significantly reduced. This method enables the user to obtain a cell population with a much higher proportion of cells with the desired edits and fewer unedited cells.

Example VIII: receptor specificity for enriching cell subsets with higher reporter expression and editing rates Titration of magnetic beads

Cells with stably integrated copies of the GFP gene (HEK 293T-GFP or HAP 1-GFP) were edited with plasmids expressing the MAD7 nuclease and GFP to BFPCassette plasmids (which also drive expression of cell surface ligands Δ Tetherin-HA or Thy1.2) were subjected to cotenomic transfection. Briefly, program CM-130 was used for HEK293T or DS-120 was used for HAP1-GFP on 4D nucleofecter X unit (Lonza, morristown, NJ), and 2X 10 pairs of BFP editing cassettes in 20. Mu.L nucleofectte with 200ng MAD7 expression plasmid and 200ng GFP to BFP editing cassette expressing. DELTA.Tetherin-HA or Thy1.2⁵Individual cells were subjected to nuclear transfection.

24 hours after nuclear transfection, cells were labeled with increasing amounts of anti-Thy1.2 magnetic beads or anti-HA magnetic beads and purified on a Magnetically Activated Cell Sorting (MACS) column (Miltenyi) according to the manufacturer's protocol. When the amount of MACS beads was increased by 9. Mu.L beads per 100. Mu.L total enrichment reaction volume, the relative amount of purified cells with high and low receptor expression was changed. This phenomenon was observed for enrichment of both HEK293T-GFP cells expressing Thy1.2 (FIGS. 19A and 19B) and HAP1-GFP cells expressing Δ Tetherin-HA (FIGS. 20A and 20B).

HEK293T-GFP cells enriched for editing machinery uptake using varying amounts of thy 1.2-specific MACS beads were replated into 24-well tissue culture plates and allowed to undergo gene editing and GFP to BFP conversion. As the amount of beads increased, the proportion of cells with imprecise editing (GFP and BFP negative) and precise editing (BFP positive) increased accordingly (figure 21). We also specifically enriched HAP1 cells expressing high Δ Tetherin-HA levels using FACS. Similar to the thy1.2 reporter system, cells enriched for high Δ Tetherin-HA expression levels had relatively higher NHEJ-mediated editing rates (48%) and HDR-mediated editing rates (1%) relative to the unenriched control that exhibited 8% indels and no detected HDR (fig. 22).

Example ix. Enrichment for HDR mediated knock-in editing.

Cells with a stably integrated copy of the GFP gene (HEK 293T-GFP) were co-nucleofected with one plasmid expressing the MAD7 nuclease and the editing cassette that mediates insertion of six base pairs into the DNMT3b gene and a second plasmid with the GFP to BFP editing cassette that also drives expression of the cell surface ligand thy1.2 as described above.

In short,using the program CM-130 on a 4D nucleostainer X unit (Lonza, morristown, NJ), 2X 10 pairs of the 200ng MAD7 expression plasmid and 200ng GFP-to-BFP editing cassettes expressing Thy1.2 were used in 20. Mu.L nucleocovette⁵Individual cells were subjected to nuclear transfection.

24 hours after nuclear transfection, cells were labeled with anti-Thy1.2 magnetic beads and purified on a MACS column (Miltenyi Biotec, sunnyvale, calif.) according to the manufacturer's protocol. Cells were also labeled with anti-thy 1.2-PE fluorescent antibody and cells with high levels of thy1.2 expression were enriched by FACS. Cells from MACS-enriched or FACS-enriched or non-enriched controls were plated in individual wells of 24-well tissue culture dishes and allowed to undergo gene editing.

Genomic DNA was purified 120 hours post-transfection from each subpopulation of enriched or non-enriched cells using Qiagen DNeasy blood and tissue kit (Velmo, netherlands). First, an 613 base pair fragment of the DNMT3b gene was amplified by PCR with primers outside the region spanning the 180 base pair homology arm region on the editing cassette plasmid. A second PCR reaction was performed to amplify a 180 base pair region of the DNMT3b gene, which region contained the region targeted by the MAD7-gRNA complex and the 6 base insertion targeted by the HDR donor on the editing cassette. These PCR amplicons were prepared using the Illumina TruSeq DNA sample preparation kit according to the manufacturer's instructions. Samples were sequenced using Illumina MiSeq using a 2x300 kit (Illumina, san Diego, CA). NGS analysis was performed using a custom NGS analysis and sequencing read alignment scheme (pipeline) to bin reads by sequence identity to DNMT3b (WT), DNMT3b with a full or partial targeted 6 base insertion (HDR _ full or HDR _ partial), or DNMT3b sequences containing insertions or deletions (Indel or NHEJ). Cells enriched for editing cassette uptake by FACS had 9.8% complete expected HDR-mediated knock-in editing, 1.1% partial HDR editing, and 73.9% indels (fig. 24). Cells enriched for cassette uptake by MACS have insertions or deletions (indels). Cells enriched for editing cassette uptake by MACS had 11.2% complete expected HDR-mediated knock-in edits, 1.3% partial HDR edits, and 78.4% indels. In contrast, cells that were not enriched in any showed 4.2% complete expected HDR-mediated knock-in editing, 0.5% partial HDR editing, and 51.8% indels. (FIG. 24).

The unique cell population with the highest level of thy1.2 uptake reporter expression, whether enriched by FACS or MACS, had a significantly higher overall edit rate and a higher ratio of HDR-mediated knock-in to NHEJ at the DNMT3b locus. In addition, unedited cell populations had decreased significantly (fig. 24).

Example X: CREATE fusion editing

CREATE fusion editing is a novel technique for using a nucleolytic enzyme fusion protein having reverse transcriptase activity with a nucleic acid encoding a gRNA that comprises a region complementary to a target region of the nucleic acid in one or more cells, the gRNA covalently linked to an editing cassette comprising a region homologous to the target region in one or more cells, the region having a mutation of at least one nucleotide and a prometallar region adjacent motif (PAM) mutation relative to the target region in one or more cells. To test the feasibility of the CREATE fusion editing in HEK293T cells, two editing vectors were designed, as shown in FIG. 25.

In the first design, nickases derived from type II CRISPR enzymes are fused at the C-terminus to an engineered Reverse Transcriptase (RT) and cloned downstream of the CMV promoter. In this case, the RT used is derived from Moloney murine leukemia virus (M-MLV). This design was designated CREATE fusion editor 2.1 (CFE 2.1) and allowed strong expression of nicking enzyme and M-MLV RT fusion protein. In CFE2.2, an enrichment handle (T2A-dsRed) was also added at the C-terminus of CFE 2.1. The enrichment handle allows selection of cells expressing nickase and RT fusion proteins.

The RNA guide was designed to be complementary to a single region near the EGFP to BFP editing site. The CREATE fusion gRNA was extended at the 3' end to include a 13bp region containing TY to SH edits and a second region of 13bp complementary to the nicked EGFP DNA sequence (FIG. 26). This allows the nicked genomic DNA to anneal to the 3' end of the gRNA, which can then be extended by RT to incorporate the edits into the genome. The second gRNA targets a region 86bp upstream of the editing site in the EGFP DNA sequence. This gRNA was designed such that it achieved nicking enzyme cleavage of the opposite strand of the fused gRNA relative to the CREATE. Both grnas were cloned downstream of the U6 promoter. Also included are multiple T sequences that terminate transcription of the gRNA.

A flow chart of an exemplary experimental procedure performed is shown in fig. 27.

Plasmids were transformed into NEB-stable escherichia coli (Ipswich, ny) and grown overnight in 2mL LB medium. The following day, plasmids were purified from E.coli using Qiagen Midi Prep kit (Venlo, netherlands). The purified plasmid was then treated with rnase a (ThermoFisher, waltham, mass) and purified again using a DNA cleaning and concentration kit (Zymo, irvine, CA).

HEK293T cells were cultured in DMEM medium supplemented with 10% FBS and 1 XPicillin and streptomycin. 100ng of total DNA (50 ng of gRNA plasmid and 50ng of CFE plasmid) was mixed with 1. Mu.l of PolyFect (Qiagen, venlo, netherlands) in 25. Mu.l of OptiMEM in 96-well plates. The complexes were incubated for 10 minutes and then 20,000 HEK293T cells resuspended in 100 μ L DMEM were added to the mixture. The resulting mixture was then subjected to CO reduction at 37 ℃ and 5%₂Incubate for 80 hours.

Cells were harvested from flat bottom 96 well plates using TrypLE Express reagent (ThermoFisher, waltham, mass) and transferred to v-bottom 96 well plates. The plates were then centrifuged at 500g for 5 minutes. The trypLE solution was then aspirated and the cell pellet resuspended in FACS buffer (1 XPBS, 1% FBS,1mM EDTA and 0.5% BSA). GFP +, BFP + and RFP + cells were then analyzed on an Attune NxT flow cytometer and data analyzed on FlowJo software.

The identified RFP + BFP + cells indicate the proportion of enriched cells that underwent an accurate or imprecise editing process. BFP + cells indicate cells that underwent a successful editing process and expressed BFP. GFP-cells indicate cells that have been inaccurately edited resulting in disruption of the GFP open reading frame and loss of expression.

The CREATE fusion editing process utilizes gRNAs covalently linked to regions homologous to the intended target sites in the genome. In this exemplary experiment, the edit is located immediately 3' to the gRNA, and the edited 3' is another region complementary to the nicked genome, although it is contemplated that the edit may also be present further 5' within the region homologous to the nicked genome. The nickase RT fusion enzyme creates a nick in the target site, and the nicked DNA anneals to its complementary sequence on the 3' end of the gRNA. RT then extends the DNA, incorporating the desired edits directly into the genome.

The effectiveness of the CREATE fusion edits was then tested in GFP + HEK293T cells. Successful precise editing in the designed assay system produced BFP + cells, while imprecisely edited cells made cells double negative for BFP and GFP. As shown in fig. 28A-28D, the CREATE fused gRNA in combination with either CFE2.1 or CFE2.2 gave-40% -45% BFP + cells, indicating that nearly half of the cell population had undergone successful editing. GFP-cells were 10% of the population. The use of a second nicked gRNA, as described in Liu et al (Nature, 12.2019; 576 (7785): 149-157), did not further improve the precision editing rate; in fact, it significantly increased the population of imprecisely edited GFP-negative cells, and the rate of editing was lower.

Previous literature has shown that double nicks on opposite strands (distance <90 bp) do result in double strand breaks, which tend to repair via NHEJ, leading to imprecise insertions or deletions. In summary, the results indicate that CREATE fusion editing produces predominantly precisely edited cells, and that the proportion of inaccurately edited cells is much lower.

Enrichment handles, in particular fluorescent Reporters (RFP) linked to nuclease expression (CFE 2.2) were included in this experiment as representatives of the cells receiving the editing mechanism. When RFP positive cells were analyzed (computationally enriched) only after 3-4 cell divisions, up to 75% of the cells were BFP + when tested with the CREATE fusion gRNA. This suggests that uptake or expression of linked reporters can be used to enrich for cell populations with higher CREATE fusion-mediated gene editing rates. In fact, the combined use of the CREATE fusion edit and the described enrichment method produced significantly improved expected edit rates.

Example XI: FACS enrichment was performed for CREATE fusion-mediated exact editing.

CREATE fusion editing in combination with physical selection using FACS was also performed in mammalian cells. The basic scheme is illustrated in fig. 29.

Cells with stably integrated copies of the GFP gene (HEK 293T-GFP) were nucleofected with either a plasmid expressing MAD7 nuclease and a GFP to BFP editing cassette plasmid (which also drives the expression of a fluorescent reporter (dsRed)) or a CREATE-fusion enzyme plasmid with an RFP reporter (figure 25, cpe 2.2) and a CREATE-fusion gRNA expression plasmid that drives nick editing of GFP to BFP (figure 26, GFP CREATE'). Briefly, 1X 10 of the 4D nuclear transfectant X unit (Lonza, morristown, NJ) was paired with 4ug of MAD7 GFP to BFP editing plasmid or 2ug of CTREATE-fusion enzyme plasmid and 2ug of CREATE-fusion gRNA plasmid in 100. Mu.L of nucleocovette using the procedure CM-130⁶Individual cells were subjected to nuclear transfection.

24 hours after nuclear transfection, cells were detached and fluorescence-based sorted using FACS Melody (Becton Dickenson, franklin Lakes, NJ) based on the dsRed reporter expression level of the cells. Cells that were nuclear transfected with either the MAD 7-based editing mechanism or the CREATE fusion editing mechanism were transfected with similar efficiency as reported by the percentage of dsRed positive cells 24h after transfection (fig. 30). Cells were sorted into three populations using electronic gating based on dsRed fluorescence intensity: dsRed _ all, dsRed _ Low, or dsRed _ high (FIG. 31). FACS-sorted subpopulations, as well as unenriched control samples, were plated in individual wells of 24-well tissue culture dishes and allowed to undergo gene editing. Cells receiving the knock-in edit exhibited a GFP to BFP conversion phenotype.

120 hours after nuclear transfection, the expression levels of GFP or BFP were analyzed by FACS sorting of cell subsets enriched for dsRed expression (which indicates enrichment for the presence of the CREATE fusion editing machinery). The percentage of cell counts in GFP-positive (wild-type or no editing), GFP-negative (NHEJ-mediated insertion or deletion frameshifting), or BFP-positive (HDR-mediated precise conversion of GFP to BFP sequence) quadrants of FACS dot plots were quantified and compared among samples (figure 32). For MAD 7-based editing, the unenriched population was 89% GFP positive (WT), 10% GFP and BFP Negative (NHEJ) and 1% BFP positive (HDR). Cells enriched for MAD7 linked dsRed expression were 14% -16% gfp positive (WT), 21% -78% gfp and BFP Negative (NHEJ) and 3% -9% BFP positive (HDR), depending on the dsRed subpopulation (dsRed _ total, dsRed _ low or dsRed _ high) selected for sorting. For edits based on CREATE fusion, the unenriched population was 87% GFP positive (WT), 3% GFP and BFP Negative (NHEJ) and 9% BFP positive (HDR). Cells enriched for MAD7 linked dsRed expression were 25% -55% gfp positive (WT), 4% -7% gfp and BFP Negative (NHEJ) and 25% -68% BFP positive (HDR), depending on the dsRed subpopulation (dsRed _ total, dsRed _ low or dsRed _ high) selected for sorting. These results indicate that for both the MAD7-CREATE and CREATE-fusion editing systems, enrichment for editing machinery uptake can produce cell populations with a higher proportion of cells containing precise edits.

Example XII: CREATE fusion editing using single gRNA

CREATE fusion editing is performed in mammalian cells using a single guide RNA covalently linked to a homology arm with the expected editing of the native sequence and an editing that disrupts nuclease cleavage at this site. The basic scheme is illustrated in fig. 32.

Briefly, lentiviral vectors were generated using the following protocol: in 6-well plates, 1000ng of lentiviral transfer plasmid containing the CREATE fusion cassette (FIGS. 23 and 24) was transfected into HEK293T cells using Lipofectamine LTX, along with 1500ng of lentiviral packaging plasmid (ViraSafe lentiviral packaging System, cell BioLabs). Lentiviral containing medium was collected 72hr after transfection. Two clones of the lentiviral CREATE fusion gRNA-HA design were selected and an empty lentiviral backbone was included as a negative control.

The day before transduction, 20,000 HEK293T cells were seeded in 6-well plates. Different volumes of CREATE' lentivirus (10. Mu.L to 1000. Mu.L) were added to HEK293T cells in 6-well plates along with 10. Mu.g/ml polybrene. After 48 hours of transduction, medium containing 15. Mu.g/ml blasticidin was added to the wells. Cells were maintained in selection for one week. After selection, the well with the lowest viable cell number (< 5% cells) was selected for subsequent experiments.

Constructs CFE2.1, CFE2.2 (as shown in figure 25) or wild-type SpCas9 were electroporated into HEK293T cells using the Neon transfection system (Thermo Fisher Scientific, waltham, MA). Briefly, 400ng of total plasmid DNA was mixed with 100,000 cells in buffer R in a total volume of 15. Mu.l. Cells were electroporated with 2 pulses of 20ms and 1150v using a 10 μ L Neon tip. 80hr after electroporation, cells were analyzed on a flow cytometer.

As shown in fig. 34A and 34B, up to 15% unenriched edit rate was achieved by single copy delivery of grnas.

However, when editing was combined with computational selection of RFP + cells, up to 30% enriched editing rates were achieved by single copy delivery of grnas. This enrichment by selecting cells that received the editing mechanism was shown to result in an increase in precise, complete expected editing by a factor of 2 (fig. 35). Two or more enrichment/delivery steps can also be used to achieve higher editing rates of CREATE fusion editing in an automated instrument (e.g., using modules for cell-handle enrichment and identification of cells with BFP expression). When the method enriches for cells with higher gRNA expression levels, the editing rate is increased even further, and thus the growth and/or enrichment module of the instrument can include gRNA enrichment.

Example XIII: traceable CREATE fusion editing double-box architecture

Combining the enhanced editing efficiency and reduced toxicity of the CREATE fusion system with retrospective or documenting techniques provides a novel method of performing retrospective tracing on large genomic libraries in massively parallel or combinatorial formats using CREATE fusion editing. Examples of such recording techniques useful for the methods of the present disclosure include those described in USPN10,017,760, USPN10,294,473, and USPN10,287,575, each of which is incorporated herein by reference for all purposes.

A simple example of how this method can be implemented is shown in fig. 35A and 35B. The CREATE fusion enzyme including nickase and RT activity was encoded on the same plasmid or amplicon as a dual CREATE cassette fusion system (figure 35A). CREATE cassette 1 encodes gRNA-HA targeting sequences that are necessary, after transcription into RNA, to direct nick-based translation editing at a functional site of interest in the chromosome. CREATE cassette 2 encodes a second set of gRNA-HA that targets an inert second site (e.g., as one possible location, 3' UTR of a pseudogene) to integrate a DNA barcode unique to each target site variant.

In this exemplary embodiment, covalently coupled gRNA-HA elements within each editing cassette function to co-localize RNA at each nick site for efficient reverse transcription to drive the editing process at each locus. At the same time, covalent coupling between the cassettes ensures that both edits are highly correlated at the single cell level. The unique identity of the barcode sequence encoded in CREATE cassette 2, when integrated, thus serves as a traceable genomic barcode that can report the identity of edits throughout the genome based on sequencing or other molecular reads of fixed chromosomal locations. This barcoding approach reduces the complexity of downstream population sequencing to a simple PCR amplicon sequencing assay.

As an additional example, this recording logic could be further extended to cover combinatorial editing within single cells by including additional creatate boxes (fig. 35B). Here, the recording sites and unique barcodes are maintained, but the editing sites encompass ≧ 2 targets within the same cell. In this case, the barcode now provides a report of combinatorial editing events at the single cell level and allows traceability and computational deconvolution adaptability to combinatorial edited cell populations using traceable barcode features.

While this invention is satisfied by embodiments in many different forms, as described in detail in connection with preferred embodiments of the invention, it is to be understood that the present disclosure is to be considered as an exemplification of the principles of the invention and is not intended to limit the invention to the specific embodiments illustrated and described herein. Many variations may be made by those skilled in the art without departing from the spirit of the invention. The scope of the invention is to be determined by the appended claims and their equivalents.The abstract and headings should not be construed as limiting the scope of the invention as they are intended to enable an appropriate organization and the general public to quickly ascertain the general nature of the invention. In the appended claims, unless the term "means" is used, none of the features or elements recited therein should be construed as being consistent with 35 u.s.c. § 112,

plus a functional limitation.

Claims (40)

1. A library of cells created using an automated editing system for nuclease-guided genome editing, wherein the system comprises:

a housing;

a container configured to receive a cell and one or more rationally designed nucleic acids comprising a sequence that facilitates a nickase-directed genome editing event in the cell;

a transformation unit for introducing the one or more nucleic acids into the cell;

an editing unit for allowing the nicking enzyme-directed genome editing event to occur in the cell,

an enrichment module; and

a processor-based system configured to operate the instrument based on user input;

wherein the nickase-directed genome editing events created by the automated system produce a cell library comprising individual cells with rationally designed edits.

2. The cell library of claim 1, wherein the nickase-directed genome editing events in the cells create a saturated mutagenized cell library.

3. The library of cells of claim 1, wherein the nickase-directed genome editing event in the cells creates a library of promoter swap cells.

4. The library of cells of claim 1, wherein the nickase-directed genome editing event in the cells creates a library of terminator swap cells.

5. The cell library of claim 1, wherein the nickase-directed genome editing events in the cells create a SNP swap cell library.

6. The library of cells of claim 1, wherein the nickase-directed genome editing events in the cells create a library of promoter-swapped cells.

7. The cell library of claim 1, wherein the nickase-directed genome editing is performed using an RNA-directed nickase.

8. The cell library of claim 1, wherein the nickase-directed genome editing is performed using a nickase fusion protein.

9. The cell library of claim 7, wherein the nickase-directed genome editing comprises the use of an RNA-directed nickase and a separate reverse transcriptase protein.

10. A library of cells created using an automated editing system for nickase-directed genome editing, wherein the system comprises:

a housing;

a cell container configured to receive cells;

a nucleic acid container configured to receive one or more rationally designed nucleic acids comprising sequences that facilitate nickase-directed genome editing events in the cell;

a transformation unit for introducing the one or more nucleic acids into the cell;

an editing unit for allowing the nicking enzyme-directed genome editing event to occur in the cell, an

A processor-based system configured to operate the instrument based on user input;

wherein the nickase-directed genome editing events created by the automated system produce a cell library comprising individual cells with rationally designed edits.

11. The cell library of claim 10, wherein the nickase-directed genome editing events in the cells create a saturated mutagenized cell library.

12. The library of cells of claim 10, wherein the nickase-directed genome editing events in the cells create a library of promoter-swapped cells.

13. The cell library of claim 10, wherein the nickase-directed genome editing event in the cells creates a terminator swap cell library.

14. The cell library of claim 10, wherein the nickase-directed genome editing event in the cells creates a SNP swap cell library.

15. The library of cells of claim 10, wherein the nickase-directed genome editing events in the cells create a library of promoter-swapped cells.

16. The cell library of claim 10, wherein the nickase-directed genome editing is performed using an RNA-directed nickase.

17. The cell library of claim 10, wherein the nicking enzyme-directed genome editing is performed using a nicking enzyme fusion protein.

18. The cell library of claim 10, wherein the nicking enzyme-directed genome editing comprises use of an RNA-directed nicking enzyme and a separate reverse transcriptase protein.

19. A library of cells created using an automated editing system for recursive nickase-directed genome editing, wherein the system comprises:

a housing;

a device that receives a cell and one or more rationally designed nucleic acids comprising sequences that facilitate nickase-directed genome editing in the cell;

means for introducing the one or more nucleic acids into the cell;

means for enriching the cells that receive the one or more nucleic acids;

means for allowing the nickase-directed genome editing event to occur,

means for edited cell growth;

means for concentrating the edited cells;

means for collecting the edited cells; and

means for configuring operation of the system based on user input;

wherein the nickase-directed genome editing event is repeated two or more times in the automated system to create a cell library comprising individual cells with two or more rationally designed edits.

20. The cell library of claim 19, wherein the automated system for creating the cell library further comprises a means for selecting the edited cells.

21. An automated multi-module cell editing instrument, comprising:

a housing configured to house all or some of the modules;

a container configured to receive cells;

one or more containers configured to receive nucleic acids and/or proteins, wherein the nucleic acids and/or proteins comprise an editing mechanism;

an editing mechanism introduction module configured to introduce the nucleic acid and/or protein into the cell;

an editing module configured to allow the introduced nucleic acid to edit the nucleic acid in the cell;

an enrichment module that enriches cells that receive the editing mechanism;

a processor configured to operate the automated multi-module cell editing instrument based on user input and/or selection of a preprogrammed script; and

an automated liquid handling system that moves cells from the container configured to receive cells to the editing mechanism introduction module, from the editing mechanism introduction module to the editing module, and from the editing module to the enrichment module; and moving nucleic acids and/or proteins to the editing mechanism introduction module, all without user intervention.

22. The automated multi-module cell editing instrument of claim 21, wherein the nucleic acids in the one or more containers comprise a scaffold and an editing cassette, the automated multi-module cell editing instrument further comprising a nucleic acid assembly module.

23. The automated multi-module cell editing instrument of claim 21, wherein the enrichment module enriches cells receiving the editing mechanism using FACS.

24. The automated multi-module cell editing instrument of claim 21, wherein the enrichment module enriches cells receiving the editing mechanism using MACS.

25. The automated multi-module cell editing instrument of claim 21, wherein the editing module further comprises a recovery module after introduction of the editing mechanism.

26. The automated multi-module cell editing instrument of claim 21, further comprising a growth module configured to grow the cells.

27. The automated multi-module cell editing instrument of claim 26, wherein the growth module measures optical density of growing cells.

28. The automated multi-module cell editing instrument of claim 27, wherein optical density is measured continuously.

29. The automated multi-module cell editing instrument of claim 26, wherein the processor is configured to adjust growth conditions in the growth module such that the cells reach a target optical density at a time requested by a user.

30. The automated multi-module cell editing instrument of claim 21, wherein the container configured to receive cells and the one or more containers configured to receive nucleic acids are contained within a reagent cartridge.

31. The automated multi-module cell editing instrument of claim 30, wherein some or all of the reagents required for cell editing are contained within the reagent cartridge.

32. The automated multi-module cell editing instrument of claim 31, wherein the reagents contained within the reagent cartridge are locatable by a script read by the processor.

33. The automated multi-module cell editing instrument of claim 32, wherein the reagent cartridge comprises reagents and the reagent cartridge is provided in a kit format.

34. The automated multi-module cell editing instrument of claim 21, wherein the editing mechanism introduction module comprises an electroporation device.

35. The automated multi-module cell editing instrument of claim 34, wherein the electroporation device is a flow-through electroporation device.

36. The automated multi-module cell editing instrument of claim 21, further comprising a filtering module configured to concentrate and render the cells electrocompetent.

37. An automated multi-module cell editing instrument, comprising:

a housing configured to house all or some of the modules;

a container configured to receive a cell, a nucleic acid, and/or a protein, wherein the nucleic acid and/or protein comprises an editing mechanism;

an editing mechanism introduction module configured to introduce the nucleic acid and/or protein into the cell;

an editing module configured to allow the introduced nucleic acids and/or proteins to edit nucleic acids in the cell;

an enrichment module that enriches cells that receive the editing mechanism;

a processor configured to operate the automated multi-module cell editing instrument based on user input and/or selection of a preprogrammed script; and

an automated liquid handling system that moves cells from the container configured to receive cells to the editing mechanism introduction module, from the editing mechanism introduction module to the editing module, and from the editing module to the enrichment module; and moving nucleic acids and/or proteins to the editing mechanism introduction module, all without user intervention.

38. The automated multi-module cell editing instrument of claim 37, further comprising at least one reagent cartridge containing reagents for performing cell editing in the automated multi-module cell editing instrument.

39. The automated multi-module cell editing instrument of claim 38, wherein the containers for cells and nucleic acids are disposed within the reagent cartridge.

40. An automated multi-module cell editing instrument comprising:

a housing configured to house some or all of the modules;

a container configured to receive cells;

at least one container configured to receive a nucleic acid, wherein the nucleic acid comprises an editing mechanism;

a growth module configured to grow the cells;

a filtration module configured to concentrate the cells and render the cells electrocompetent;

a transformation module comprising a flow-through electrical perforator that introduces the nucleic acid into the cell;

a combined recovery and editing module configured to allow the cell to recover following electroporation in the transformation module and to allow the nucleic acid to edit the cell;

an enrichment module that enriches cells that receive the editing mechanism;

a processor configured to operate the automated multi-module cell editing instrument based on user input and/or selection of a preprogrammed script; and

an automated liquid handling system that moves cells from the container configured to receive cells to the growth module; move from the growth module to the filtering module, move from the filtering module to the transformation module, move from the transformation module to the combined restoration and editing module, and move from the combined restoration and editing module to the enrichment module; and moving nucleic acid from the container configured to receive nucleic acid to the conversion module, all without user intervention.

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