Combinatorial gene editing and screening

Abstract

Compositions and methods are provided for assembling a multi-module gene editing construct. Methods of producing a combinatorial library comprising a plurality of multi-module gene editing constructs are also provided. The subject methods enable any type of genetic modification to be tested in combination with other genetic modifications.

Description

COMBINATORIAL GENE EDITING AND SCREENINGCROSS REFERENCE TO RELATED APPLICATIONS

[0001] This application claims benefit of U.S. Provisional Patent Application No. 63 / 548,743, filed February 1 , 2024, which application is incorporated herein by reference in its entirety.BACKGROUND OF THE INVENTION

[0002] Genetic screening systems have identified numerous drug targets across therapeutic systems, including small molecules, biologies, and cellular therapies. Technologies exist to screen numerous types of genetic modifications- gene knockouts, gene knockdowns, gene overexpression, synthetic gene knockins, individual gene domains, etc. However, a crucial block in these critical discovery systems is that each type of genetic modification must be screened separately - systems do not currently exist to allow say gene knockouts to be screened at the same time as synthetic gene knockins, or gene knockdowns to be screened alongside variable individual domains of genes. Furthermore, these different types of genetic manipulations cannot be screened combinatorially across categories, testing knockouts, knockins, overexpression, etc. in multiple combinations.SUMMARY OF THE INVENTION

[0003] Compositions and methods are provided for assembling a multi-module gene editing construct. Methods of producing a combinatorial library comprising a plurality of multi-module gene editing constructs are also provided. The subject methods enable any type of genetic modification to be tested in combination with other genetic modifications.

[0004] In one aspect, a method of a method of assembling a multi-module gene editing construct is provided, the method comprising: (a) providing a circular DNA or plasmid comprising a first recombinant polynucleotide, wherein the first recombinant polynucleotide comprises a first genome editing module and a first barcode to identify the first genome editing module, wherein the first genome editing module and the first barcode are separated by a first excisable stuffer sequence, wherein the first excisable stuffer sequence is flanked by a first excision-mediating site and a second excisionmediating site; (b) contacting the circular DNA or plasmid with an excising agent that binds to the first excision-mediating site and the second excision-mediating site, whereinthe first excisable stuffer sequence is excised from the first recombinant polynucleotide to produce a cleaved circular DNA or plasmid having the first genome editing module at a first cleaved end and the first barcode at a second cleaved end; (c) providing a second recombinant polynucleotide, wherein the second recombinant polynucleotide comprises a second genome editing module and a second barcode to identify the second genome editing module, wherein the second genome editing module and the second barcode are separated by a second excisable stuffer sequence, wherein the second excisable stuffer sequence is flanked by a third excision-mediating site and a fourth excision-mediating site; (d) ligating the second recombinant polynucleotide to the first cleaved end and the second cleaved end to produce a first ligation product, wherein the first genome module is connected to the second genome module, and wherein the second barcode is connected to the first barcode; (e) contacting the first ligation product with an excising agent that binds to the third excision-mediating site and the fourth excision-mediating site, wherein the second excisable stuffer sequence is excised from the first ligation product to produce a first cleaved ligation product having the first genome editing module connected to the second genome module at a first end of the first cleaved ligation product and the second barcode connected to the first barcode at a second end of the first cleaved ligation product; (f) providing a third recombinant polynucleotide, wherein the third recombinant polynucleotide comprises a third genome editing module and a third barcode to identify the third genome editing module, wherein the third genome editing module and the third barcode are separated by a third excisable stuffer sequence, wherein the third excisable stuffer sequence is flanked by a fifth excision-mediating site and a sixth excision-mediating site; (g) ligating the third recombinant polynucleotide to the first end of the first cleaved ligation product and the second end of the first cleaved ligation product to produce a second ligation product comprising the first genome module, the second genome module, the third genome module, the third excisable stuffer sequence flanked by the fifth excision-mediating site and the sixth excision-mediating site, and a 3’-barcode array comprising the third barcode, followed by the second barcode, followed by the first barcode; and (h) repeating steps (e) - (g) until assembly of the multi-module gene editing construct is completed.

[0005] In certain embodiments, each genome editing module in the multi-module gene editing construct can produce a different sequence-specific genetic perturbation.

[0006] In certain embodiments, the multi-module gene editing construct can produce at least 3, at least 4, at least 5, or at least 6 sequence-specific genetic perturbations selectedfrom a gene knockin, a gene knockout, a gene knock-down, a gene upregulation, an insertion of a synthetic gene, an insertion of a non-coding domain, an insertion of a protein coding domain, a deletion of an endogenous protein coding domain, an insertion of a regulatory element, a deletion of a regulatory element, an epigenetic perturbation, and an insertion of a unique molecular identifier or cell tracing sequence.

[0007] In certain embodiments, the multi-module gene editing construct comprises a plurality of genome editing modules to knockin a plurality of full-length genes or coding or non-coding domains, knockdown expression of a plurality of genes, upregulate expression of a plurality of genes, knockout a plurality of genes, or a combination thereof.

[0008] In certain embodiments, at least one recombinant polynucleotide further comprises a constant 5’-adapter at the 5’-end of the recombinant polynucleotide and a constant 3’-adapter at the 3’-end of the recombinant polynucleotide.

[0009] In certain embodiments, at least one recombinant polynucleotide further comprises an intron comprising a 5’-splice donor site, a constant 5’-adapter, and a 3'- splice acceptor site, wherein the constant 5’-adapter is positioned between the 5’-splice donor site and the 3’-splice acceptor site, and wherein the constant 5’-adapter is removed by messenger RNA splicing.

[0010] In certain embodiments, each excisable stuffer sequence further comprises a constant 5’-adapter at the 5’-end of the excisable stuffer sequence and a constant 3'- adapter at the 3’-end of the excisable stuffer sequence.

[0011] In certain embodiments, each excisable stuffer sequence comprises or consists of the same sequence.

[0012] In certain embodiments, each excisable stuffer sequence further comprises a safety cut site.

[0013] In certain embodiments, each excisable stuffer sequence further comprises a sequencing primer binding site.

[0014] In certain embodiments, the method further comprises sequencing the 3’-barcode array using a sequencing primer that binds to the sequencing primer binding site.

[0015] In certain embodiments, the excising agent is a restriction enzyme or a sitespecific recombinase.

[0016] In certain embodiments, each excision-mediating site comprises a recognition site for the same restriction enzyme or site-specific recombinase. In some embodiments, the restriction enzyme is a type IIS restriction enzyme. Exemplary type IIS restriction enzymes include, without limitation, Bsal, Bbsl Bsgl, BsmAI, BsmBI-v2, BsmFI, Bsml,BspCNI, BspMI, BspQI, BsrDI, Bsrl, BtgZI, BtsCI, Btsl-v2, BtsIMutl, CspCI, Earl, Ecil, Esp3l, Paul, Fokl, Hgal, Hphl, HpyAV, Mboll, Mlyl, Mmel, Mnll, NmeAIII, PaqCI, Piel, Sapl, BspQI, and SfaNI. Exemplary site-specific recombinases include, without limitation, a Cre recombinase, which catalyzes site-specific recombination between two loxP sites, a flippase (FLP) recombinase, which catalyzes site-specific recombination between two flippase recognition target (FRT) sites, a phiC31 recombinase, which catalyze sitespecific recombination between two attachment (att) sites referred to as attB and attP, a DreO recombinase, which catalyzes site-specific recombination between two rox sites, or a Tre recombinase, which catalyzes site-specific recombination between two loxP sites that are modified with HIV long terminal repeats (loxLTR).

[0017] In certain embodiments, the excisable stuffer sequence comprises: a first recognition site for binding the restriction enzyme, wherein the restriction enzyme cuts at the first excision site; a second recognition site for binding the restriction enzyme, wherein the restriction enzyme cuts at the second excision site; a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site positioned between the safety cut site and the second recognition site for binding the restriction enzyme; a constant 5’-adapter positioned at the 5’-end of the excisable stuffer sequence; and a constant 3’-adapter positioned at the 3’-end of the excisable stuffer sequence.

[0018] In certain embodiments, at least one recombinant polynucleotide comprises a genome editing module comprising a sequence encoding a full-length gene, a coding domain, a non-coding domain, a regulatory RNA, or a guide RNA (gRNA). In some embodiments, the regulatory RNA comprises a short hairpin RNA (shRNA). In some embodiments, the shRNA is a microRNA (miR)-embedded shRNA, wherein endogenous microRNA processing of the miR-embedded shRNA within a cell transfected with the multi-module gene editing construct results in excision of the shRNA such that the shRNA can inhibit expression of a target gene. Exemplary miR-embedded shRNAs include, without limitation, the miR-30-embedded shRNA and the miR-E-embedded shRNA.

[0019] In certain embodiments, at least one recombinant polynucleotide further comprises a multicistronic element, wherein the multicistronic element is positioned between the sequence encoding the full-length gene or the coding domain and the excisable stuffer sequence. In some embodiments, the multicistronic element is a 2A multicistronic element.

[0020] In certain embodiments, at least one recombinant polynucleotide further comprises an intron spacer sequence positioned between the constant 5’-adapter sequence at the 5’-end of the recombinant polynucleotide and the sequence encoding the full-length gene or the coding domain.

[0021] In certain embodiments, at least one recombinant polynucleotide comprises: a constant 5’-adapter sequence at the 5'-end of the recombinant polynucleotide; a genome editing module comprising a sequence encoding a full-length gene; an intron spacer sequence, wherein the intron spacer sequence is positioned between the constant 5’- adapter sequence at the 5’-end of the recombinant polynucleotide and the sequence encoding the full-length gene; an excisable stuffer sequence flanked by a first excision site and a second excision site, wherein the excisable stuffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a constant 5’-adapter positioned at the 5’-end of the excisable stuffer sequence, and a constant 3’-adapter positioned at the 3’-end of the excisable stuffer sequence; a 2A multicistronic element, wherein the 2A multicistronic element is positioned between the sequence encoding the full-length gene and the excisable stuffer sequence to separate an open reading frame of the full-length gene from any other open reading frame; a barcode for identification of the genome editing module comprising the sequence encoding the full-length gene, wherein the barcode is separated from the genome editing module by the excisable stuffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.

[0022] In certain embodiments, at least one recombinant polynucleotide comprises: a genome editing module comprising a sequence encoding a coding domain or a noncoding domain; an intron spacer sequence positioned before the sequence encoding the coding domain or the non-coding domain, wherein the intron comprises a 5’-splice donor site, a constant 5’-adapter, and a 3’-splice acceptor site, wherein the constant 5’-adapter is positioned within the 5’-splice donor site or between the 5’-splice donor site and the 3'- splice acceptor site, and wherein the constant 5’-adapter is removed by messenger RNA splicing; an excisable stuffer sequence flanked by a first excision site and a secondexcision site, wherein the excisable stutter sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a constant 5’-adapter positioned at the 5’-end of the excisable stuffer sequence, and a constant 3’-adapter positioned at the 3’-end of the excisable stuffer sequence; a barcode for identification of the genome editing module comprising the sequence encoding the coding domain or the non-coding domain, wherein the barcode is separated from the genome editing module by the excisable stuffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.

[0023] In certain embodiments, a scar region generated by assembly of the multi-module gene editing construct is contained within the intron spacer sequence, wherein the intron spacer sequence containing the scar region is removed by messenger RNA splicing.

[0024] In certain embodiments, at least one recombinant polynucleotide comprises: a genome editing module comprising a sequence encoding a full-length gene or a protein coding domain; a stop codon operably linked to the sequence encoding the full-length gene or protein coding domain, wherein the stop codon terminates translation of the sequence encoding the full-length gene or protein coding domain; a 3’-untranslated region following the stop codon, wherein the 3’-untranslated region comprises: a mRNA stabilizing element, a sequence encoding a regulatory RNA, a first 5’-adapter positioned between the mRNA stabilizing element and the sequence encoding the regulatory RNA; an excisable stuffer sequence flanked by a first excision site and a second excision site, wherein the excisable stuffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a second constant 5’-adapter positioned at the 5’-end of the excisable stuffer sequence, and a first constant 3’-adapter positioned at the 3’-end of the excisable stuffer sequence;a barcode for identification of the genome editing module comprising the sequence encoding the regulatory RNA, wherein the barcode is separated from the sequence encoding the regulatory RNA by the excisable staffer sequence; and a constant 3’- adapter sequence at the 3’-end of the recombinant polynucleotide. In some embodiments, the regulatory RNA comprises a short hairpin RNA (shRNA). In some embodiments, the shRNA is a microRNA (miR)-embedded shRNA, wherein endogenous microRNA processing of the miR-embedded shRNA within a cell transfected with the multi-module gene editing construct results in excision of the shRNA such that the shRNA can inhibit expression of a target gene. In some embodiments, the miR-embedded shRNA is a miR-30-embedded shRNA or a miR-E-embedded shRNA. In some embodiments, the mRNA stabilizing element is a triplex stabilizer.

[0025] In certain embodiments, the 3’-untranslated region further comprises one or more additional sequences encoding one or more additional regulatory RNAs. In some embodiments, the one or more additional regulatory RNAs are shRNAs or miR- embedded shRNAs.

[0026] In certain embodiments, at least one recombinant polynucleotide comprises: a genome editing module comprising a sequence encoding a full-length gene or a protein coding domain; a stop codon operably linked to the sequence encoding the full-length gene or protein coding domain, wherein the stop codon terminates translation of the sequence encoding the full-length gene or protein coding domain; a 3’-untranslated region following the stop codon, wherein the 3’-untranslated region comprises: a mRNA stabilizing element, a sequence encoding a guide RNA (gRNA) for an RNA-guided nuclease, a first 5’-adapter positioned between the mRNA stabilizing element and the sequence encoding the gRNA; an excisable stuffer sequence flanked by a first excision site and a second excision site, wherein the excisable stuffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a second constant 5’-adapter positioned at the 5’-end of the excisable stuffer sequence, and a first constant 3’-adapter positioned at the 3’-end of the excisable stuffer sequence; a barcode for identification of the genome editing modulecomprising the sequence encoding the gRNA, wherein the barcode is separated from the sequence encoding the gRNA by the excisable stutter sequence; and a constant 3'- adapter sequence at the 3’-end of the recombinant polynucleotide. In some embodiments, the mRNA stabilizing element is a triplex stabilizer.

[0027] In certain embodiments, the RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease. In some embodiments, the Cas nuclease is Cas9, Cas12a, Cas12d, Cas13a, Cas13b, Cas13d, or a dead Cas9 (dCas9).

[0028] In certain embodiments, the gRNA forms a complex with the RNA-guided nuclease such that the gRNA directs the RNA-guided nuclease to a genomic target locus in a cell, wherein the RNA-guided nuclease creates a double-stranded break in the genomic DNA at the genomic target locus, wherein DNA repair of the double-stranded break by non-homologous end joining creates an insertion or deletion (indel) resulting in gene knockout at the genomic target locus.

[0029] In certain embodiments, the multi-module gene editing construct further comprises a genome editing module comprising a sequence encoding a donor polynucleotide, wherein the gRNA can form a complex with the RNA-guided nuclease such that the gRNA directs the RNA-guided nuclease to a genomic target locus in a cell, wherein the RNA-guided nuclease creates a double-stranded break in the genomic DNA at the genomic target locus, wherein a donor polynucleotide is integrated at the genomic target locus by homology directed repair (HDR).

[0030] In certain embodiments, the multi-module gene editing construct further comprises a genome editing module comprising a sequence encoding the RNA-guided nuclease.

[0031] In certain embodiments, the method further comprises sequencing the barcodes in the multi-module gene editing construct to identify the genome editing modules in the multi-module gene editing construct.

[0032] In certain embodiments, the method further comprises adding a barcode module comprising one or more additional barcodes to the circular DNA or plasmid comprising the multi-module gene editing construct. In some embodiments, the barcode module is added by a method comprising: contacting the multi-module gene editing construct with an excising agent, wherein an excisable stuffer sequence is excised from the multimodule gene editing construct to produce a cleaved circular DNA or plasmid comprising the genome editing modules of the multi-module gene editing construct at a first cleaved end and the 3’-barcode array comprising the barcodes of the multi-module gene editingconstruct at a second cleaved end; and ligating a polynucleotide comprising the barcode module comprising the one or more additional barcodes to the first cleaved end and the second cleaved end, wherein the barcode module comprising the one or more additional barcodes is added to the 3’-barcode array comprising the barcodes of the genome editing modules of the multi-module gene editing construct. In some embodiments, the one or more additional barcodes comprise a cell tracing sequence, a sequence unique to a specific cell donor, or unique molecular identifier sequence. For example, the cell tracing sequence may identify a clone, cell lineage, donor, or condition.

[0033] In certain embodiments, the method further comprises sequencing the one or more additional barcodes to identify the clone, the cell lineage, or the donor from which a genetically modified cell comprising the multi-module gene editing construct was derived.

[0034] In certain embodiments, the method further comprises performing single-cell RNA sequencing on a genetically modified cell comprising the multi-module gene editing construct.

[0035] In certain embodiments, the method further comprises integrating the multimodule gene editing construct into a target locus in the genome of a host cell. In certain embodiments, the multi-module gene editing construct is integrated into a target locus in the genome of a host cell using a CRISPR system.

[0036] In certain embodiments, the method further comprises cloning the multi-module gene editing construct into a vector. In some embodiments, the vector is a plasmid or a viral vector. Exemplary viral vectors include, without limitation, a lentivirus vector, retrovirus vector, or adeno-associated virus vector.

[0037] In certain embodiments, the method further comprises transfecting a host cell with the vector or the multi-module gene editing construct. In some embodiments, the host cell is transfected transiently or stably. In some embodiments, transfecting comprises performing electroporation, nucleofection, or lipofection.

[0038] In certain embodiments, at least one genome editing module in the multi-module gene editing construct produces a chimeric antigen receptor (CAR) gene knockin in a T cell.

[0039] In certain embodiments, at least one genome editing module in the multi-module gene editing construct produces a T cell receptor (TCR) gene knockout in the T cell.

[0040] In certain embodiments, at least one genome editing module in the multi-module gene editing construct produces a knockout of a gene encoding an alloantigen. In someembodiments, the alloantigen is a major histocompatibility complex (MHC) class I alloantigen or an MNS blood group alloantigen. In some embodiments, the alloantigen is CD1 , CD2, CD3, CD4, CD7, CD8, Ly-6, Qa-2, RT6, CD19, CD22, CD56, CD58 (LFA-3), CD59, or CDw90 (Thy 1 ).

[0041] In certain embodiments, at least one genome editing module in the multi-module gene editing construct produces a knockout of a CD5, CD52, CD70, BATF, LCK, PD-1 , LAG-3, CTLA-4, 2-B2M, PD-1 , HLA-I, Fas, TGFBR2, PDCD-1 , DGK, EZH2, PAX5, or LDLR gene.

[0042] In certain embodiments, at least one genome editing module in the multi-module gene editing construct engineers the T cell to further comprise a binding-triggered transcriptional switch that regulates expression of the chimeric antigen receptor or activation of the T cell. In some embodiments, the binding-triggered transcriptional switch comprises a synthetic notch receptor, a modular extracellular sensor architecture (MESA), or a synthetic intramembrane proteolysis receptor (SNIPR).

[0043] In certain embodiments, the binding-triggered transcriptional switch comprises a synthetic notch receptor comprising i) an extracellular ligand-binding domain that specifically binds to a second target antigen on the target cell, and ii) an intracellular domain, wherein binding of the extracellular ligand-binding domain to the second target antigen results in cleavage of the intracellular domain to release a transcription factor from the intracellular domain, wherein the transcription factor that is released from the intracellular domain induces expression of the chimeric antigen receptor on the CAR-T cell.

[0044] In another aspect, a multi-module gene editing construct produced by a method, described herein, is provided.

[0045] In another aspect, a combinatorial library comprising two or more different multimodule gene editing constructs produced by a method, described herein, is provided.

[0046] In another aspect, a vector comprising a multi-module gene editing construct, described herein, is provided. In some embodiments, the vector is a plasmid or a viral vector.

[0047] In another aspect, a host cell comprising a multi-module gene editing construct, described herein, is provided. In some embodiments, the host cell is a mammalian host cell. For example, the host cell may be from a mammal such as, but not limited to, human and non-human mammals such as non-human primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits,hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; farm animals such as sheep, goats, pigs, horses, and cows.

[0048] In certain embodiments, the mammalian host cell is an immune cell. For example, the immune cell may include, without limitation, a T cell, a B cell, a natural killer cell, a neutrophil, an eosinophil, a mast cell, a basophil, a monocyte, a macrophage, or a dendritic cell. In some embodiments, the T cell is a helper CD4+T cell, a cytotoxic CD8+T cell, a natural killer T cell, or a gamma delta T cell.

[0049] In another aspect, a method of producing a genetically modified host cell is provided, the method comprising introducing a multi-module gene editing construct, described herein, into a host cell, wherein the multi-module gene editing construct genetically modifies the genome of the host cell to generate a genetically modified host cell.

[0050] In certain embodiments, the method further comprises introducing an RNA-guided nuclease into the host cell.

[0051] In certain embodiments, the RNA-guided nuclease is provided by a vector, or as an mRNA sequence encoding the RNA-guided nuclease along with co-delivery of a guide RNA or plasmid expressing a guide RNA, or as a ribonucleoprotein complex of the RNA- guided nuclease with a guide RNA.

[0052] In certain embodiments, the RNA-guided nuclease is provided by the multi-module gene editing construct.

[0053] In another aspect, a method of simultaneously screening the effects of a plurality of sequence-specific genetic perturbations is provided, the method comprising: introducing a multi-module gene editing construct, described herein, into a host cell, wherein the multi-module gene editing construct genetically modifies the genome of the host cell to generate a genetically modified host cell comprising the plurality of sequencespecific genetic perturbations; and detecting an effect of the plurality of sequence-specific genetic perturbations on the genetically modified host cell.

[0054] In certain embodiments, detecting an effect of the plurality of sequence-specific genetic perturbations on the genetically modified host cell comprises detecting a change in cell morphology, cell growth, cell proliferation, gene expression, biological activity of a protein, or any combination thereof in the genetically modified host cell compared to an unmodified host cell.

[0055] In certain embodiments, the method further comprises: contacting the genetically modified host cell with a test agent; and detecting an effect of the plurality of sequencespecific genetic perturbations on activity of the test agent.

[0056] In another aspect, a method of simultaneously screening the effects of a plurality of sequence-specific genetic perturbations is provided, the method comprising: providing a combinatorial library comprising a plurality of genetically modified host cells, wherein each genetically modified host cell comprises a different multi-module gene editing construct produced according to a method described herein, wherein the different multimodule gene editing constructs generate different sequence-specific genetic perturbations in each genetically modified host cell; and detecting effects of the different sequence-specific genetic perturbations in the plurality of genetically modified host cells; and sequencing the 3’-barcode arrays of the multi-module gene editing constructs in one or more genetically modified host cells of interest out of the plurality of genetically modified host cells to identify the genome editing modules present in the multi-module gene editing constructs in the one or more genetically modified host cells of interest.

[0057] In certain embodiments, detecting the effects of the different sequence-specific genetic perturbations in the plurality of genetically modified host cells comprises detecting a change in cell morphology, cell growth, cell proliferation, gene expression, biological activity of a protein, or any combination thereof in the plurality of genetically modified host cells compared to an unmodified host cell.

[0058] In certain embodiments, the method further comprises contacting the plurality of genetically modified host cells with a test agent; and detecting effects of the different sequence-specific genetic perturbations in the plurality of genetically modified host cells on activity of the test agent.

[0059] In another aspect, a composition comprising a genetically modified host cell comprising a multi-module gene editing construct, described herein, and a pharmaceutically acceptable excipient or carrier is provided.

[0060] In another aspect, a method of performing cellular therapy is provided, the method comprising administering to a subject a therapeutically effective amount of a composition comprising a genetically modified host cell comprising a multi-module gene editing construct, described herein.

[0061] In certain embodiments, the genetically modified host cell is autologous or allogeneic.

[0062] In certain embodiments, the genetically modified host cell is an immune cell. In some embodiments, the immune cell is a T cell, a B cell, a natural killer cell, a neutrophil, an eosinophil, a mast cell, a basophil, a monocyte, a macrophage, or a dendritic cell. In some embodiments, the T cell is a helper CD4+T cell, a cytotoxic CD8+T cell, a natural killer T cell, or a gamma delta T cell.

[0063] In certain embodiments, the T cell comprises a multi-module gene editing construct that comprises a genome editing module that produces a gene knockin of a gene encoding a chimeric antigen receptor (CAR) that specifically binds to a target antigen.

[0064] In certain embodiments, the T cell comprises a multi-module gene editing construct that further comprises a genome editing module that produces a gene knockout of a gene encoding a T cell receptor (TCR) protein chain.

[0065] In certain embodiments, the TCR protein chain is a T cell receptor alpha chain, a T cell receptor beta chain, a T cell receptor delta chain, or a T cell receptor gamma chain.

[0066] In certain embodiments, the CAR comprises a transmembrane domain linked to an extracellular antigen binding domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to the target antigen.

[0067] In certain embodiments, the extracellular antigen binding domain comprises a single chain variable fragment (scFv), an antigen-binding fragment (Fab), a nanobody, a heavy chain variable (VH) domain, a light chain variable (VL) domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, a diabody, or a functional fragment thereof that binds specifically to the antigen.

[0068] In certain embodiments, the intracellular signaling domain is a CD3-zeta intracellular signaling domain or a ZAP-70 intracellular signaling domain.

[0069] In certain embodiments, the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

[0070] In certain embodiments, the CAR further comprises a costimulatory domain.

[0071] In certain embodiments, the costimulatory domain is a 4-1 BB, CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, or HVEM costimulatory domain.

[0072] In certain embodiments, the transmembrane domain is a CD8, Megfl O, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, integrin subunit P5, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, or CD86 transmembrane domain.

[0073] In certain embodiments, the target antigen is on a cancer cell, a tumor cell, an activated fibroblast, an autoreactive immune cell, a pathogen, or a diseased cell.

[0074] In certain embodiments, the target antigen is a tumor antigen or a tumor- associated antigen.

[0075] In certain embodiments, the pathogen is a virus, a bacterium, a fungus, or a parasite.

[0076] In certain embodiments, the target antigen is a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen.

[0077] In certain embodiments, the autoreactive immune cell is an autoreactive T cell or B cell.

[0078] In certain embodiments, the target antigen is an antigen on the autoreactive T cell or B cell.

[0079] In another aspect, a recombinant polynucleotide is provided, the recombinant polynucleotide comprising: a constant 5’-adapter sequence at the 5’-end of the recombinant polynucleotide; a genome editing module comprising a sequence encoding a genetic perturbation; an excisable staffer sequence flanked by a first excision-mediating site and a second excision-mediating site, wherein the excisable staffer comprises: a first recognition site for binding a restriction enzyme, wherein the restriction enzyme cuts at the first excision site, a second recognition site for binding the restriction enzyme, wherein the restriction enzyme cuts at the second excision site, and a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the first recognition site for binding the restriction enzyme and the second recognition site for binding the restriction enzyme; a barcode associated with the genetic perturbation, wherein the barcode is separated from the genome editing module by the excisable staffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.

[0080] In certain embodiments, the genetic perturbation is a gene knockin, a gene knockout, a gene knock-down, a gene upregulation, an insertion of a synthetic gene, an insertion of a non-coding domain, an insertion of a domain, a deletion of a domain, an insertion of a regulatory element, a deletion of a regulatory element, an epigenetic perturbation, or an insertion of a unique molecular identifier or cell tracing sequence.

[0081] In certain embodiments, the genome editing module comprises a sequence encoding a full-length gene, a coding domain, a non-coding domain, or a regulatory RNA.

[0082] In certain embodiments, the recombinant polynucleotide further comprises an intron comprising a 5’-splice donor site, a constant 5’-adapter, and a 3’-splice acceptor site, wherein the constant 5’-adapter is positioned between the 5'-splice donor site and the 3’-splice acceptor site, and wherein the constant 5’-adapter is removed by messenger RNA splicing.

[0083] In another aspect, a recombinant polynucleotide is provided, the recombinant polynucleotide comprising: a constant 5’-adapter sequence at the 5’-end of the recombinant polynucleotide; a genome editing module comprising a sequence encoding a full-length gene; an intron spacer sequence, wherein the intron spacer sequence is positioned between the constant 5’-adapter sequence at the 5’-end of the recombinant polynucleotide and the sequence encoding the full-length gene; an excisable stuffer sequence flanked by a first excision site and a second excision site, wherein the excisable stuffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a constant 5’-adapter positioned at the 5’-end of the excisable stuffer sequence, and a constant 3'- adapter positioned at the 3’-end of the excisable stuffer sequence; a 2A multicistronic element, wherein the 2A multicistronic element is positioned between the sequence encoding the full-length gene and the excisable stuffer sequence to separate an open reading frame of the full-length gene from any other open reading frame; a barcode for identification of the genome editing module comprising the sequence encoding the full- length gene, wherein the barcode is separated from the genome editing module by the excisable stuffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.

[0084] In another aspect, a recombinant polynucleotide is provided, the recombinant polynucleotide comprising: a genome editing module comprising a sequence encoding a coding domain or a non-coding domain; an intron spacer sequence positioned before the sequence encoding the coding domain or the non-coding domain, wherein the intron comprises a 5'-splice donor site, a constant 5’-adapter, and a 3’-splice acceptor site, wherein the constant 5’-adapter is positioned within the 5’-splice donor site or betweenthe 5’-splice donor site and the 3’-splice acceptor site, and wherein the constant 5'- adapter is removed by messenger RNA splicing; an excisable stutter sequence flanked by a first excision site and a second excision site, wherein the excisable stutter sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a constant 5’-adapter positioned at the 5’-end of the excisable stuffer sequence, and a constant 3’-adapter positioned at the 3'- end of the excisable stuffer sequence; a barcode for identification of the genome editing module comprising the sequence encoding the coding domain or the non-coding domain, wherein the barcode is separated from the genome editing module by the excisable stuffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.

[0085] In another aspect, a recombinant polynucleotide is provided, the recombinant polynucleotide comprising: a genome editing module comprising a sequence encoding a full-length gene or a protein coding domain; a stop codon operably linked to the sequence encoding the full-length gene or protein coding domain, wherein the stop codon terminates translation of the sequence encoding the full-length gene or protein coding domain; a 3’-untranslated region following the stop codon, wherein the 3’-untranslated region comprises: a mRNA stabilizing element, a sequence encoding a regulatory RNA, a first 5'-adapter positioned between the mRNA stabilizing element and the sequence encoding the regulatory RNA; an excisable stuffer sequence flanked by a first excision site and a second excision site, wherein the excisable stuffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a second constant 5’-adapter positioned at the 5’-end of the excisable stuffer sequence, and a first constant 3’-adapter positioned at the 3’-end of theexcisable staffer sequence; a barcode for identification of the genome editing module comprising the sequence encoding the regulatory RNA, wherein the barcode is separated from the sequence encoding the regulatory RNA by the excisable staffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide. In some embodiments, the mRNA stabilizing element is a triplex stabilizer.

[0086] In certain embodiments, the regulatory RNA comprises a short hairpin RNA (shRNA). In some embodiments, the shRNA is a microRNA (miR)-embedded shRNA, wherein endogenous microRNA processing of the mi R-em bedded shRNA within a cell transfected with the recombinant polynucleotide results in excision of the shRNA such that the shRNA can inhibit expression of a target gene. In some embodiments, the miR- embedded shRNA is a miR-30-embedded shRNA or a miR-E-embedded shRNA.

[0087] In certain embodiments, the 3’-untranslated region further comprises one or more additional sequences encoding one or more additional regulatory RNAs. In some embodiments, the one or more additional regulatory RNAs are shRNAs or miR- embedded shRNAs.

[0088] In another aspect, a recombinant polynucleotide is provided, the recombinant polynucleotide comprising: a genome editing module comprising a sequence encoding a full-length gene or a protein coding domain; a stop codon operably linked to the sequence encoding the full-length gene or protein coding domain, wherein the stop codon terminates translation of the sequence encoding the full-length gene or protein coding domain; a 3’-untranslated region following the stop codon, wherein the 3’-untranslated region comprises: a mRNA stabilizing element, a sequence encoding a guide RNA (gRNA) for an RNA-guided nuclease, a first 5’-adapter positioned between the mRNA stabilizing element and the sequence encoding the gRNA; an excisable staffer sequence flanked by a first excision site and a second excision site, wherein the excisable staffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a second constant 5’-adapter positioned at the 5’-end of the excisable staffer sequence, and a first constant 3’-adapter positioned at the 3’-end of the excisable staffer sequence; a barcode foridentification of the genome editing module comprising the sequence encoding the gRNA, wherein the barcode is separated from the sequence encoding the gRNA by the excisable stuffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide. In some embodiments, the mRNA stabilizing element is a triplex stabilizer.

[0089] In certain embodiments, the RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease. In some embodiments, the Cas nuclease is Cas9, Cas12a, Cas12d, Cas13a, Cas13b, Cas13d, or a dead Cas9 (dCas9).

[0090] In certain embodiments, the gRNA forms a complex with the RNA-guided nuclease such that the gRNA directs the RNA-guided nuclease to a genomic target locus in a cell, wherein the RNA-guided nuclease creates a double-stranded break in the genomic DNA at the genomic target locus, wherein DNA repair of the double-stranded break by non-homologous end joining creates an insertion or deletion (indel) resulting in gene knockout at the genomic target locus.

[0091] In certain embodiments, the 3’-untranslated region further comprises a sequence encoding a donor polynucleotide, wherein the gRNA can form a complex with the RNA- guided nuclease such that the gRNA directs the RNA-guided nuclease to a genomic target locus in a cell, wherein the RNA-guided nuclease creates a double-stranded break in the genomic DNA at the genomic target locus, wherein a donor polynucleotide is integrated at the genomic target locus by homology directed repair (HDR).BRIEF DESCRIPTION OF THE DRAWINGS

[0092] FIG. 1. CRISPR-AII: A Unified Genetic Perturbation Language. CRISPR-AII screening involves a single standardized module architecture), with each module representing a different type of genetic perturbation (full length gene, gene knockout, gene knockdown, coding or non-coding domains, cell tracing barcodes). These modules are each barcoded using a standardized barcode length and placement, encompasses a cloning and expression architecture that allows any number of modules to be used individually or in combination. Barcodes for each of the modules used in the screen are successively built up at the 3’ end of the full length gene construct, allowing for simple amplicon sequencing of the full barcode array, as well as compatibility with existing off- the-shelf single cell sequencing methods. A library of gene knockout modules can be screened at the same time as a library of gene knockin modules. A library of geneknockdowns can be screened combinatorially against a library of gene domains, or a library of modules representing all types of genetic manipulations can be screened simultaneously and / or in combination. The final construct library comprising all of the combinations of modules used in a given screen is integrated into target cells using a targeted gene integration (e.g. CRISPR / Cas9). We have demonstrated CRISPR-AII screening with primary human T cells but it is applicable across any eukaryotic cell type.

[0093] FIG. 2. CRISPR-AII Modular Construction. All CRISPR-AII modules have a standardized architecture, with constant adaptor sequences encoded at the 5’ and 3’ ends of the enclosed DNA sequence encoding the specific genetic perturbation desired. These enclosed DNA sequences regardless of genetic perturbation type have three standard sequence elements: 1 ) a sequence encoding the desired genetic perturbation (in this case the coding sequence of a full-length gene); 2) a standardized “Internal Stuffer” sequence containing constant restriction enzyme and primer binding sites; and 3) a unique 1 1 bp barcode associated with the genetic perturbation made. The internal stuffer and barcode elements are standardized across CRISPR-AII component modules. CRISPR-AII modules can be placed into the genome at any site, including within exons, introns, or nontranslated regions, with the internal stuffer and barcode components able to be integrated into and sequenced off of a protein coding frame, a 3’ untranslated region, or within nontranscribed genomic DNA. The Internal Stuffer of any module can be opened using restriction digestions, and an additional module can be ligated in its place, creating a seamless connection of different genetic perturbation modules at the 5’ end of the construct, and a seamless connection of their corresponding barcodes at the 3’ end. Every added module contains a new Internal Stuffer sequence, enabling additional module to be continuously added, with the DNA sequences encoding the genetic perturbations continuously building at the 5’ end, and all barcodes being connected at the 3’ end of the construct. The 3’ barcode array can be sequenced using amplicon sequencing, with a constant primer binding site included within the Internal Stuffer region.

[0094] FIG. 3. CRISPR-AII Module: Full Length Gene. Example schematic of a CRISPR- AII Full Length Gene module, which enables any endogenous or synthetic protein to be expressed as part of a CRISPR-AII construct. Constant adaptor sequences at the 5’ and 3’ ends enclose the full length gene, standardized internal stuffer, and unique barcode. For full length gene modules, a multicistronic element (e.g. a 2A sequence) is included before the internal stuffer to separate the full length gene’s open reading frame from any other potential proteins expressed as part of the CRISPR-AII construct or endogenousgenes expressed if the CRISPR-AII construct has been integrated into an endogenous gene and is being expressed on the same mRNA transcript by an endogenous promoter.

[0095] FIG. 4. CRISPR-AII Module: Domains. Example schematic of CRISPR-AII domain module, which enables any coding or non-coding domains to be expressed as part of a CRISPR-AII construct. A protein coding domain is represented. Protein coding domain modules contain a short intron sequence before the beginning of the coding domain, enabling the 5’ adaptor sequence to be included within the intron, and thus not part of the final protein coding sequence after mRNA splicing (FIG. 5). The standardized internal stuffer sequence and unique barcode follow the end of the domain sequence.

[0096] FIG. 5. Short Introns for Scarless Assembly of Multiple Domain Modules. As CRISPR-AII modules are combinatorially combined with each other using restriction digestion cloning, a short DNA scar sequence is created at the junction of each set of modules from the overlapping “sticky end” necessary for the ligation of the second module. These cloning scars (often 4 bps) must be included in the final DNA sequence of the CRISPR-AII construct, which would potentially require addition of non-desired amino acids to a protein coding frame if the cloning scar fell in between two protein coding domains (or two full length genes). By including a short intron in front of every protein coding domain, the cloning scar can be integrated into the intronic sequence, enabling the same modular cloning and arbitrary combination of domains, but with the intervening introns and cloning scars spliced out by endogenous mRNA splicing machinery inside the cell, resulting in total control over the final protein coding sequence without cloning scars regardless of what modules are combined together.

[0097] FIG. 6. Demonstration of Short Intron Inclusion in Multiple Domain CRISPR-AII Syntax. Demonstration of successful expression of example fluorescent proteins constructed using CRISPR-AII modular construction from two component protein coding domains each containing a short intron, protein coding domain, internal stuffer, and barcode. Both GFP and mCherry fluorescent proteins are functionally expressed even when including intervening introns and trailing internal stuffer and barcode sequences.

[0098] FIG. 7. CRISPR-AII Module: Gene Knockdowns. Example schematic of CRISPR- AII knockdown module. CRISPR-AII knockdown and knockout modules are placed into the 3’ untranslated region of the inserted DNA sequence, and are assembled into the CRISPR-AII construct after any protein coding full length genes or domains. Embedded between constant 5’ and 3’ adaptors necessary for modular construction are: 1 ) an shRNA sequence; 2) standardized internal stuffer sequence; and 3) unique barcode. The shRNAsequence is contained within a microRNA architecture (such as miR-E) that enables human cell’s endogenous microRNA processing machinery to excise the shRNA sequence and functionally process it for targeted gene knockdowns. Multiple knockdown or knockout modules can be iteratively added to the 3’ UTR region of the CRISPR-AII construct to enable multiplexed and / or combinatorial gene knockdowns and knockouts. The excision of knockdown (shRNA) modules and / or knockout (gRNA) modules in the 3’UTR results in a separation of the protein coding reading frame of the mature mRNA from its polyA tail, which can destabilize the mature mRNA sequence. Inclusion of a stabilizing element (e.g. triplex stabilizer sequence) in between the CRISPR-AII constructs protein coding modules (full length genes, protein coding domains) and the knockdown / knockout modules in the 3’ UTR allows for continued stabilization of the mature mRNA even if it has been separated from its polyA tail.

[0099] FIG. 8. Demonstration of Successful Inclusion of CRISPR-AII Components in 3’ UTRs. Example CRISPR-AII construct containing one full length gene module encoding GFP, and a second knockdown module containing one of four different shRNAs, targeting either B2M or CD47. Demonstration flow cytometry plots show successful knockdown of either B2M or CD47 respectively in the GFP+ cells, demonstrating functional integration of CRISPR-AII modules into the 3’ UTR of the full CRISPR-AII construct.

[0100] FIG. 9. CRISPR-AII Module: Gene Knockouts. Example schematic of CRISPR-AII knockout module. Similar to the knockdown module, knockout modules are placed into the 3’ UTR of a CRISPR-AII construct. Embedded between constant 5’ and 3’ adaptors necessary for modular construction are: 1 ) a guide RNA sequence; 2) standardized internal stuffer sequence; and 3) unique barcode. The guide RNA sequences used are excisable gRNAs that can be removed from the mRNA template to serve as a functional gRNA to target a separate site in the genome in order to generate a gene knockout (if targeting a protein coding site). For example, the CRISPR enzyme Cas12a can excise its gRNA targets from an mRNA template and then use that gRNA to edit another genomic locus.

[0101] FIG. 10. CRISPR-AII Module: Cell Tracing Barcodes. Example schematic of CRISPR-AII cell tracing module. Additional DNA barcodes can be integrated into CRISPR- AII constructs using the same modular, iterative cloning as gene, domain, knockdown, and knockout modules. Inclusion of additional barcodes enables tracing of individual cellular clones, marking individual donors or experimental conditions, addition of unique molecular identifiers, or any other application requiring additional barcoding of DNA constructs.These additional barcode are added into the Barcode array at the 3’ end of the CRISPR- All construct, along with all of the barcodes from the other included modules.

[0102] FIG. 11. Arbitrarily Combinatorial Genetic Perturbations using CRISPR-AII. Due to the modular, iterative nature of CRISPR-AII constructs, any arbitrary number of modules can be included in a single construct, and any combination of modules can be included together, allowing for arbitrarily combinatorial genetic perturbations across perturbation type to be made, each encoded by a uniquely barcoded single DNA construct. Thus, any desired combination of perturbations (full length gene expression, protein domains, gene knockdowns, gene knockouts) can be made together in the same pooled experiment, and all types and combinations of perturbations can be uniquely identified by sequencing of their standardized barcode arrays.

[0103] FIG. 12. Demonstration of CRISPR-AII Combinatorial Syntax - Five Full Length Genes. Combinatorial CRISPR-AII construct showing expression of five full length genes constructed using modular cloning of five iterative additions of full-length gene modules (two fluorescent proteins, two selection markers, and a chimeric antigen receptor). After gene editing of human T cells with the x5 Gene CRISPR-AII construct, flow cytometry analysis reveals successful expression of the entire set of included fluorescent proteins and expression markers.

[0104] FIG. 13. Demonstration of CRISPR-AII Combinatorial Syntax - One Full Length Gene plus Two Knockouts. Combinatorial CRISPR-AII construct showing expression of one full length gene (GFP) along with two knockout modules containing gRNAs targeting the cell surface receptors CD47 and B2M. After gene editing of human T cells with the Gene + KO + KO CRISPR-AII construct, flow cytometry analysis showed that in successfully edited cells expressing the knocked in gene (GFP), successful knockout of both targeted genes (B2M, CD47) was observed in the majority of edited cells, whereas in unedited cells not expressing GFP, the vast majority of cells showed no knockout of either surface receptor.

[0105] FIG. 14. Demonstration of CRISPR-AII Combinatorial Syntax - One Full Length Gene plus One Knockout plus One Knockdown. Combinatorial CRISPR-AII construct showing expression of one full length gene (GFP) along with one knockout module (gRNAs targeting CD47) and one knockdown module (shRNA targeting B2M). After gene editing of human T cells with the Gene + KO + KD CRISPR-AII construct, flow cytometry analysis showed that in successfully edited cells expressing the knocked in gene (GFP), successful knockout CD47 and knockdown of B2M was observed in the majority of editedcells, whereas in unedited cells not expressing GFP, the vast majority of cells showed no knockout of CD47 or knockdown of B2M.

[0106] FIG. 15. Arbitrarily Combinatorial Genetic Perturbations in Human T Cells using CRISPR-AII.

[0107] FIG. 16. Single DNA Template Encoding of Complex Genetic Perturbations in Primary Human T Cells. A schematic is shown of an exemplary multi-module gene editing construct comprising a CAR gene knockin module, a tNGFR gene knockin module, two GFP domain modules, an mCherry-A domain module, and an mCherry-B domain module. Flow cytometry analysis shows the percentage of successfully edited cells.

[0108] FIG. 17. Single DNA Template Encoding of Complex Genetic Perturbations in Primary Human T Cells. A schematic is shown of an exemplary multi-module gene editing construct comprising a CAR gene knockin module, a GFP gene knockin module, a CD47 knockout module, a B2M knockout module, and a CD226 knockout module. Flow cytometry analysis shows the percentage of successfully edited cells.

[0109] FIG. 18. Single DNA Template Encoding of Complex Genetic Perturbations in Primary Human T Cells. A schematic is shown of an exemplary multi-module gene editing construct comprising a CAR gene knockin module, a tNGFR gene knockin module, a CD47 knockdown module, a B2M knockdown module, and a CD226 knockdown module. Flow cytometry analysis shows the percentage of successfully edited cells.

[0110] FIG. 19. Single DNA Template Encoding of Complex Genetic Perturbations in Primary Human T Cells. A schematic is shown of an exemplary multi-module gene editing construct comprising a CAR gene knockin module, a tNGFR gene knockin module, two GFP domain modules, a B2M knockdown module, and a CD47 knockout module. Flow cytometry analysis shows the percentage of successfully edited cells.

[0111] FIG. 20. CACTUS: CRISPR-AII Cell Therapy Universal Screening Library.

[0112] FIGS. 21A-21 D. CACTUS: CRISPR-AII Cell Therapy Universal Screening Library. FIG. 21 A. Meta-analysis of proposed T cell genetic perturbations agnostic of perturbation type. A CACTUS meta-library was generated for screening. The meta-library encompasses the vast majority of proposed CAR T cell modifying genetic edits, which can be screened simultaneously. FIG. 21 B. CACTUS meta-library proliferative performance in chronic stimulation exhaustion assay across 8 human donors. FIG. 21 C. Simultaneous pooled genetic screening across perturbation classes in primary human T cells. FIG. 21 D. Genetic screening across perturbation classes showing log 2-fold changes in expression.

[0113] FIGS. 22A-22B. Arbitrarily combinatorial pooled genetic perturbations. CRISPR- All allows screening of complex combinatorial arrangements of genetic perturbations regardless of perturbation type. FIG. 22A. Top performing individual genetic perturbations across classes. Iterative combinatorial construction using CRISPR-AII was performed to screen greater than 10,000 unique combinatorial perturbations. FIG. 22B. Combinatorial cross-perturbation class pooled screening in primary human T cells. Proliferative performance of T cells was screened in a chronic stimulation exhaustion assay. CRISPR-AII allowed new combinations of gene knockouts, gene knockdowns, gene overexpression, and signaling domains to be identified that together improved CAR T cell function in the tested exhaustion setting.DETAILED DESCRIPTION OF THE INVENTION

[0114] Compositions and methods are provided for assembling a multi-module gene editing construct. Methods of producing a combinatorial library comprising a plurality of multi-module gene editing constructs are also provided. The subject methods enable any type of genetic modification to be tested in combination with other genetic modifications.

[0115] Before the present devices, systems, software, and methods are described, it is to be understood that this invention is not limited to the particular devices, systems, software, and methods described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0116] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated 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 or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.

[0117] 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. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, some potential and preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and / or materials in connection with which the publications are cited. It is understood that the present disclosure supersedes any disclosure of an incorporated publication to the extent there is a contradiction.

[0118] As will be apparent to those of skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein has discrete components and features which may be readily separated from or combined with the features of any of the other several embodiments without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events recited or in any other order which is logically possible.

[0119] It must be noted 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" includes a plurality of such cells and reference to "the nucleic acid" includes reference to one or more nucleic acids and equivalents thereof, such as polynucleotides, known to those skilled in the art, and so forth.

[0120] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed.Definitions

[0121] The term "about," particularly in reference to a given quantity, is meant to encompass deviations of plus or minus five percent. As used herein, the term "immune cells" generally includes white blood cells (leukocytes) which are derived from hematopoietic stem cells (HSC) produced in the bone marrow.

[0122] “Biocompatible” or “cytocompatible” as used herein, refers to a property of a material that allows for prolonged contact with a cell or tissue without causing toxicity or significant damage.

[0123] The terms “engineered” or “recombinant” in reference to a T cell, gene, nucleic acid and / or protein as used herein, refer to a T cell, gene, nucleic acid and / or protein that has been altered through human intervention. Accordingly, the term “naturally occurring” as used herein in reference to a T cell, gene, nucleic acid and / or protein as used herein, refer to a T cell, gene, nucleic acid and / or protein existing in nature and without any human intervention. Exemplary human interventions comprise transfection with a heterologous polynucleotide, molecular cloning resulting in a deletion, insertion, modification and / or rearrangement with respect to a naturally occurring sequence such as a naturally occurring sequence in a T cell, gene, nucleic acid and / or protein herein described.

[0124] The term “unit dosage form,” as used herein, refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of the agents calculated in an amount sufficient to produce the desired effect in association with a pharmaceutically acceptable diluent, carrier or vehicle. The specifications for the unit dosage forms for use in the present invention depend on the particular compound employed and the effect to be achieved, the pharmacodynamics associated with each compound in the host, and the like.

[0125] The term “biological sample” encompasses a clinical sample, including, but not limited to, a bodily fluid, tissue obtained by surgical resection, tissue obtained by biopsy, cells in culture, cell supernatants, cell lysates, tissue samples, organs, bone marrow, blood, plasma, serum, fine needle aspirate, lymph node aspirate, cystic aspirate, a paracentesis sample, a thoracentesis sample, and the like.

[0126] The terms “obtained” or “obtaining” as used herein can also include the physical extraction or isolation of a biological sample (e.g., comprising immune cells) from a subject. Accordingly, a biological sample comprising immune cells can be isolated from a subject (and thus “obtained”) by the same person or same entity that subsequently isolates immune cells from the sample. When a biological sample is “extracted” or “isolated” from a first party or entity and then transferred (e.g., delivered, mailed, etc.) to a second party, the sample was “obtained” by the first party (and also “isolated” by the first party), and then subsequently “obtained” (but not “isolated”) by the second party.Accordingly, in some embodiments, the step of obtaining does not comprise the step of isolating a biological sample.

[0127] In some embodiments, the step of obtaining comprises the step of isolating a biological sample. Methods and protocols for isolating various biological samples (e.g., a blood sample, a biopsy sample, an aspirate, etc.) will be known to one of ordinary skill in the art and any convenient method may be used to isolate a biological sample.

[0128] “Isolated” refers to an entity of interest that is in an environment different from that in which it may naturally occur. “Isolated” is meant to include entities that are within samples that are substantially enriched for the entity of interest and / or in which the entity of interest is partially or substantially purified.

[0129] "Substantially" or "essentially" means nearly totally or completely, for instance, 95% or greater of some given quantity.

[0130] "Substantially purified" generally refers to isolation of a component of a sample (e.g., cell or substance), such that the component comprises the majority percent of the sample in which it resides. Typically in a sample, a substantially purified component comprises at least 70%, preferably at least 80%-85%, more preferably at least 90-99% of the sample.

[0131] The terms "individual," "subject," and "patient" are used interchangeably herein to refer to an individual to be treated by (e.g., administered) the compositions and methods of the present invention. Subjects include, but are not limited to, mammals, including human and non-human mammals such as non-human primates, including chimpanzees and other apes and monkey species; laboratory animals such as mice, rats, rabbits, hamsters, guinea pigs, and chinchillas; domestic animals such as dogs and cats; farm animals such as sheep, goats, pigs, horses and cows. In some cases, the methods of the invention find use in experimental animals, in veterinary application, and in the development of animal models for disease, including, but not limited to, rodents including mice, rats, and hamsters; primates, and transgenic animals. In the context of the disclosure, the term "subject" generally refers to an individual who will be administered or who has been administered one or more compositions described herein (e.g., cellular therapy with cells screened according to the methods described herein).

[0132] The terms "treatment", "treating", "treat" and the like are used herein to generally refer to obtaining a desired pharmacologic and / or physiologic effect. The effect can be prophylactic in terms of completely or partially preventing a disease or symptom(s) thereof and / or may be therapeutic in terms of a partial or complete stabilization or curefor a disease and / or adverse effect attributable to the disease. The term “treatment" encompasses any treatment of a disease in a mammal, particularly a human, and includes: (a) preventing the disease and / or symptom(s) from occurring in a subject who may be predisposed to the disease or symptom but has not yet been diagnosed as having it; (b) inhibiting the disease and / or symptom(s), i.e., arresting their development; or (c) relieving the disease symptom(s), i.e., causing regression of the disease and / or symptom(s). Those in need of treatment include those already inflicted as well as those in which prevention is desired, including those with a genetic predisposition or increased susceptibility to developing a disease.

[0133] A therapeutic treatment is one in which the subject is inflicted prior to administration and a prophylactic treatment is one in which the subject is not inflicted prior to administration. In some embodiments, the subject has an increased likelihood of becoming inflicted or is suspected of being inflicted prior to treatment. In some embodiments, the subject is suspected of having an increased likelihood of becoming inflicted.

[0134] A "therapeutically effective amount" or “therapeutic dose” is an amount sufficient to effect desired clinical results (i.e., achieve therapeutic efficacy). A therapeutically effective dose or amount can be administered in one or more administrations.

[0135] "Pharmaceutically acceptable excipient or carrier" refers to an excipient that may optionally be included in the compositions of the invention and that causes no significant adverse toxicological effects to the patient.

[0136] "Pharmaceutically acceptable salt" includes, but is not limited to, amino acid salts, salts prepared with inorganic acids, such as chloride, sulfate, phosphate, diphosphate, bromide, and nitrate salts, or salts prepared from the corresponding inorganic acid form of any of the preceding, e.g., hydrochloride, etc., or salts prepared with an organic acid, such as malate, maleate, fumarate, tartrate, succinate, ethylsuccinate, citrate, acetate, lactate, methanesulfonate, benzoate, ascorbate, para-toluenesulfonate, palmoate, salicylate and stearate, as well as estolate, gluceptate and lactobionate salts. Similarly salts containing pharmaceutically acceptable cations include, but are not limited to, sodium, potassium, calcium, aluminum, lithium, and ammonium (including substituted ammonium).

[0137] The terms "polynucleotide" and "nucleic acid," used interchangeably herein, refer to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, ormulti-stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases.

[0138] "Homology" refers to the percent identity between two polynucleotide or two polypeptide molecules. Two nucleic acid, or two polypeptide sequences are “substantially homologous” to each other when the sequences exhibit at least about 50% sequence identity, preferably at least about 75% sequence identity, more preferably at least about 80% 85% sequence identity, more preferably at least about 90% sequence identity, and most preferably at least about 95% 98% sequence identity over a defined length of the molecules. As used herein, substantially homologous also refers to sequences showing complete identity to the specified sequence.

[0139] In general, "identity" refers to an exact nucleotide to nucleotide or amino acid to amino acid correspondence of two polynucleotides or polypeptide sequences, respectively. Percent identity can be determined by a direct comparison of the sequence information between two molecules by aligning the sequences, counting the exact number of matches between the two aligned sequences, dividing by the length of the shorter sequence, and multiplying the result by 100. Readily available computer programs can be used to aid in the analysis, such as ALIGN, Dayhoff, M.O. in Atlas of Protein Sequence and Structure M.O. Dayhoff ed., 5 Suppl. 3:353 358, National biomedical Research Foundation, Washington, DC, which adapts the local homology algorithm of Smith and Waterman Advances in AppL Math. 2:482 489, 1981 for peptide analysis. Programs for determining nucleotide sequence identity are available in the Wisconsin Sequence Analysis Package, Version 8 (available from Genetics Computer Group, Madison, Wl) for example, the BESTFIT, FASTA and GAP programs, which also rely on the Smith and Waterman algorithm. These programs are readily utilized with the default parameters recommended by the manufacturer and described in the Wisconsin Sequence Analysis Package referred to above. For example, percent identity of a particular nucleotide sequence to a reference sequence can be determined using the homology algorithm of Smith and Waterman with a default scoring table and a gap penalty of six nucleotide positions.

[0140] Another method of establishing percent identity in the context of the present invention is to use the MPSRCH package of programs copyrighted by the University of Edinburgh, developed by John F. Collins and Shane S. Sturrok, and distributed by IntelliGenetics, Inc. (Mountain View, CA). From this suite of packages, the SmithWaterman algorithm can be employed where default parameters are used for the scoring table (for example, gap open penalty of 12, gap extension penalty of one, and a gap of six). From the data generated the “Match” value reflects "sequence identity." Other suitable programs for calculating the percent identity or similarity between sequences are generally known in the art, for example, another alignment program is BLAST, used with default parameters. For example, BLASTN and BLASTP can be used using the following default parameters: genetic code = standard; filter = none; strand = both; cutoff = 60; expect = 10; Matrix = BLOSUM62; Descriptions = 50 sequences; sort by = HIGH SCORE; Databases = non-redundant, GenBank + EMBL + DDBJ + PDB + GenBank CDS translations + Swiss protein + Spupdate + PIR. Details of these programs are readily available.

[0141] Alternatively, homology can be determined by hybridization of polynucleotides under conditions which form stable duplexes between homologous regions, followed by digestion with single stranded specific nuclease(s), and size determination of the digested fragments. DNA sequences that are substantially homologous can be identified in a Southern hybridization experiment under, for example, stringent conditions, as defined for that particular system. Defining appropriate hybridization conditions is within the skill of the art. See, e.g., Sambrook et al., supra', DNA Cloning, supra; Nucleic Acid Hybridization, supra.

[0142] "Recombinant" as used herein to describe a nucleic acid molecule means a polynucleotide of genomic, cDNA, viral, semisynthetic, or synthetic origin which, by virtue of its origin or manipulation, is not associated with all or a portion of the polynucleotide with which it is associated in nature. The term "recombinant" as used with respect to a protein or polypeptide means a polypeptide produced by expression of a recombinant polynucleotide. In general, the gene of interest is cloned and then expressed in transformed organisms, as described further below. The host organism expresses the foreign gene to produce the protein under expression conditions.

[0143] The term "transformation" refers to the insertion of an exogenous polynucleotide into a host cell, irrespective of the method used for the insertion. For example, direct uptake, transduction or f-mating are included. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, may be integrated into the host genome.

[0144] Recombinant host cells," "host cells," "cells", "cell lines," "cell cultures," and other such terms denoting microorganisms or higher eukaryotic cell lines cultured as unicellularentities refer to cells which can be, or have been, used as recipients for recombinant vector or other transferred DNA, and include the original progeny of the original cell which has been transfected.

[0145] A "coding sequence" or a sequence which "encodes" a selected polypeptide, is a nucleic acid molecule which is transcribed (in the case of DNA) and translated (in the case of mRNA) into a polypeptide in vivo when placed under the control of appropriate regulatory sequences (or "control elements"). The boundaries of the coding sequence can be determined by a start codon at the 5' (amino) terminus and a translation stop codon at the 3' (carboxy) terminus. A coding sequence can include, but is not limited to, cDNA from viral, prokaryotic or eukaryotic mRNA, genomic DNA sequences from viral or prokaryotic DNA, and even synthetic DNA sequences. A transcription termination sequence may be located 3' to the coding sequence.

[0146] Typical "control elements," include, but are not limited to, transcription promoters, transcription enhancer elements, transcription termination signals, polyadenylation sequences (located 3' to the translation stop codon), sequences for optimization of initiation of translation (located 5’ to the coding sequence), and translation termination sequences.

[0147] "Operably linked" refers to an arrangement of elements wherein the components so described are configured so as to perform their usual function. Thus, a given promoter operably linked to a coding sequence is capable of effecting the expression of the coding sequence when the proper enzymes are present. The promoter need not be contiguous with the coding sequence, so long as it functions to direct the expression thereof. Thus, for example, intervening untranslated yet transcribed sequences can be present between the promoter sequence and the coding sequence and the promoter sequence can still be considered "operably linked" to the coding sequence.

[0148] "Expression cassette" or "expression construct" refers to an assembly which is capable of directing the expression of the sequence(s) or gene(s) of interest. An expression cassette generally includes control elements, as described above, such as a promoter which is operably linked to (so as to direct transcription of) the sequence(s) or gene(s) of interest, and often includes a polyadenylation sequence as well. Within certain embodiments of the invention, the expression cassette described herein may be contained within a plasmid construct. In addition to the components of the expression cassette, the plasmid construct may also include, one or more selectable markers, a signal which allows the plasmid construct to exist as single stranded DNA (e.g., a M13origin of replication), at least one multiple cloning site, and a "mammalian" origin of replication (e.g., a SV40 or adenovirus origin of replication).

[0149] "Purified polynucleotide" refers to a polynucleotide of interest or fragment thereof which is essentially free, e.g., contains less than about 50%, preferably less than about 70%, and more preferably less than about at least 90%, of the protein with which the polynucleotide is naturally associated. Techniques for purifying polynucleotides of interest are well-known in the art and include, for example, disruption of the cell containing the polynucleotide with a chaotropic agent and separation of the polynucleotide(s) and proteins by ion-exchange chromatography, affinity chromatography and sedimentation according to density.

[0150] The term "transfection" is used to refer to the uptake of foreign DNA by a cell. A cell has been "transfected" when exogenous DNA has been introduced inside the cell membrane. A number of transfection techniques are generally known in the art. See, e.g., Graham et al. (1973) Virology, 52:456, Sambrook et al. (2001 ) Molecular Cloning, a laboratory manual, 3rd edition, Cold Spring Harbor Laboratories, New York, Davis et al. (1995) Basic Methods in Molecular Biology, 2nd edition, McGraw-Hill, and Chu et al. (1981 ) Gene 13:197. Such techniques can be used to introduce one or more exogenous DNA moieties into suitable host cells. The term refers to both stable and transient uptake of the genetic material, and includes uptake of peptide- or antibody-linked DNAs.

[0151] A "vector" is capable of transferring nucleic acid sequences to target cells (e.g., viral vectors, non-viral vectors, particulate carriers, and liposomes). Typically, "vector construct," "expression vector," and "gene transfer vector," mean any nucleic acid construct capable of directing the expression of a nucleic acid of interest and which can transfer nucleic acid sequences to target cells. Thus, the term includes cloning and expression vehicles, as well as viral vectors.

[0152] The term “hybridization” refers to the specific binding of a nucleic acid to a complementary nucleic acid via Watson-Crick base pairing.

[0153] "Gene transfer" or "gene delivery" refers to methods or systems for reliably inserting DNA or RNA of interest into a host cell. Such methods can result in transient expression of non-integrated transferred DNA, extrachromosomal replication and expression of transferred replicons (e.g., episomes), or integration of transferred genetic material into the genomic DNA of host cells. Gene delivery expression vectors include, but are not limited to, vectors derived from bacterial plasmid vectors, viral vectors, non- viral vectors, adenoviruses, lentiviruses, alphaviruses, pox viruses, and vaccinia viruses.

[0154] A polynucleotide "derived from" a designated sequence refers to a polynucleotide sequence which comprises a contiguous sequence of approximately at least about 6 nucleotides, preferably at least about 8 nucleotides, more preferably at least about 10-12 nucleotides, and even more preferably at least about 15-20 nucleotides corresponding, i.e. , identical or complementary to, a region of the designated nucleotide sequence. The derived polynucleotide will not necessarily be derived physically from the nucleotide sequence of interest, but may be generated in any manner, including, but not limited to, chemical synthesis, replication, reverse transcription or transcription, which is based on the information provided by the sequence of bases in the region(s) from which the polynucleotide is derived. As such, it may represent either a sense or an antisense orientation of the original polynucleotide.

[0155] A “CRISPR system" refers collectively to transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated ("Cas") genes. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elements that promote the formation of a CRISPR complex at the site of a target sequence.

[0156] The term "Cas9" as used herein encompasses type II clustered regularly interspaced short palindromic repeats (CRISPR) system Cas9 endonucleases from any species, and also includes biologically active fragments, variants, analogs, and derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks).

[0157] A Cas9 endonuclease binds to and cleaves DNA at a site comprising a sequence complementary to its bound guide RNA (gRNA). For purposes of Cas9 targeting, a gRNA may comprise a sequence "complementary" to a target sequence (e.g., in an intron of a TCR gene), capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.

[0158] A Cas9 polynucleotide, nucleic acid, oligonucleotide, protein, polypeptide, or peptide refers to a molecule derived from any source. The molecule need not be physically derived from an organism, but may be synthetically or recombinantly produced.Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP 002989955, WP 038434062, WP_01 1528583); Campylobacter jejuni (WP_022552435, YP_002344900),Campylobacter coll (WP 060786116); Campylobacter fetus (WP 059434633); Corynebacterium ulcerans (NC_015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP 033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861 ); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus torquisl (NC_018721 ); Streptococcus thermophilus (YP_820832), Streptococcus mutans (WP_061046374, WP_024786433); Listeria innocua (NP 472073); Listeria monocytogenes (WP 061665472); Legionella pneumophila (WP 062726656); Staphylococcus aureus (WP 001573634); Francisella tularensis (WP_032729892, WP_014548420), Enterococcus faecalis (WP_033919308); Lactobacillus rhamnosus (WP_048482595, WP_032965177); and Neisseria meningitidis (WP 061704949, YP 002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein, wherein the variant retains biological activity, such as Cas9 site-directed endonuclease activity. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol. 198(5)797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385-397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10):6091 -6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[0159] By "selectively binds" with reference to a guide RNA is meant that the guide RNA binds preferentially to a target sequence of interest or binds with greater affinity to the target sequence than to other genomic sequences. For example, a gRNA will bind to a substantially complementary sequence and not to unrelated sequences. A gRNA that selectively binds to a particular target DNA sequence will selectively direct binding of Cas9 to a substantially complementary sequence at the target site and not to unrelated sequences.

[0160] The term "donor polynucleotide" refers to a polynucleotide that provides a sequence of an intended edit to be integrated into the genome at a target locus by homology directed repair (HDR).

[0161] A "target site" or "target sequence" is the nucleic acid sequence recognized (i.e., sufficiently complementary for hybridization) by a guide RNA (gRNA) or a homology arm of a donor polynucleotide. The target site may be in an exon or an intron or a specific allele.

[0162] By "homology arm" is meant a portion of a donor polynucleotide that is responsible for targeting the donor polynucleotide to the genomic sequence to be edited in a cell. The donor polynucleotide typically comprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence flanking a nucleotide sequence comprising the intended edit to the genomic DNA. The homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively. The nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR or recombineering at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.

[0163] As used herein, the terms "complementary" or "complementarity" refers to polynucleotides that are able to form base pairs with one another. Base pairs are typically formed by hydrogen bonds between nucleotide units in an anti-parallel orientation between polynucleotide strands. Complementary polynucleotide strands can base pair in a Watson-Crick manner (e.g., A to T, A to U, C to G), or in any other manner that allows for the formation of duplexes. As persons skilled in the art are aware, when using RNA as opposed to DNA, uracil (U) rather than thymine (T) is the base that is considered to be complementary to adenosine. However, when a uracil is denoted in the context of the present invention, the ability to substitute a thymine is implied, unless otherwise stated. "Complementarity" may exist between two RNA strands, two DNA strands, or between a RNA strand and a DNA strand. It is generally understood that two or more polynucleotides may be "complementary" and able to form a duplex despite having less than perfect or less than 100% complementarity. Two sequences are "perfectlycomplementary" or "100% complementary" if at least a contiguous portion of each polynucleotide sequence, comprising a region of complementarity, perfectly base pairs with the other polynucleotide without any mismatches or interruptions within such region. Two or more sequences are considered "perfectly complementary" or "100% complementary" even if either or both polynucleotides contain additional non- complementary sequences as long as the contiguous region of complementarity within each polynucleotide is able to perfectly hybridize with the other. "Less than perfect" complementarity refers to situations where less than all of the contiguous nucleotides within such region of complementarity are able to base pair with each other. Determining the percentage of complementarity between two polynucleotide sequences is a matter of ordinary skill in the art. For purposes of Gas nuclease (e.g., Cas9 or Cas12a) targeting, a gRNA may comprise a sequence "complementary" to a target sequence, capable of sufficient base-pairing to form a duplex (i.e., the gRNA hybridizes with the target sequence). Additionally, the gRNA may comprise a sequence complementary to a PAM sequence, wherein the gRNA also hybridizes with the PAM sequence in a target DNA.

[0164] "Administering" a nucleic acid, such as a multi-module gene editing construct, viral vector, or a CRISPR system (expressing, e.g., a donor polynucleotide, guide RNA, Gas protein (e.g., Cas9, dCas9, Cas12a (Cpf1 ), Cas12d, or Cas13)) to a cell comprises transducing, transfecting, electroporating, translocating, fusing, phagocytosing, shooting or ballistic methods, etc., i.e., any means by which a nucleic acid can be transported across a cell membrane.

[0165] The terms "microRNA," "miRNA," and MiR" are interchangeable and refer to endogenous or artificial non-coding RNAs that are capable of regulating gene expression. It is believed that miRNAs function via RNA interference. When used herein in the context of inactivation, the use of the term microRNAs is intended to include also long non-coding RNAs, piRNAs, siRNAs, and the like. Endogenous (e.g., naturally occurring) miRNAs are typically expressed from RNA polymerase II promoters and are generated from a larger transcript.

[0166] The terms "siRNA" and "short interfering RNA" are interchangeable and refer to single-stranded or double-stranded RNA molecules that are capable of inducing RNA interference. The siRNA molecules typically have a duplex region that is between 18 and 30 base pairs in length.

[0167] The terms "shRNA" and "small hairpin RNA" are interchangeable and refer to RNA molecules, typically about 80 base pairs in length, that form a hairpin structure. shRNAmolecules are processed within a cell to form siRNA which knock down gene expression through RNA interference.

[0168] The terms "piRNA" and "Piwi-interacting RNA" are interchangeable and refer to a class of small RNAs involved in gene silencing. The piRNA molecules typically are between 26 and 31 nucleotides in length.

[0169] The terms "snRNA" and "small nuclear RNA" are interchangeable and refer to a class of small RNAs involved in a variety of processes including RNA splicing and regulation of transcription factors. The subclass of small nucleolar RNAs (snoRNAs) is also included. The term is also intended to include artificial snRNAs, such as antisense derivatives of snRNAs comprising antisense sequences directed against the PTGES3 gene.

[0170] The term "antisense", as used herein, refers to any composition containing nucleotide sequences which are complementary to a specific DNA or RNA sequence. The term "antisense strand" is used in reference to a nucleic acid strand that is complementary to the "sense" strand. Antisense molecules include peptide nucleic acids and may be produced by any method including synthesis or transcription. Once introduced into a cell, the complementary nucleotides combine with natural sequences produced by the cell to form duplexes and block either transcription or translation. The designation "negative" is sometimes used in reference to the antisense strand, and "positive" is sometimes used in reference to the sense strand.

[0171] A "barcode" refers to one or more nucleotide sequences that are used to identify a nucleic acid (e.g., genome editing module in a multi-module gene editing construct), cell (e.g., cell tracing sequence to identify a donor or lineage, unique molecular identifier), or condition with which the barcode is associated. Barcodes can be 3-1000 or more nucleotides in length, preferably 10-250 nucleotides in length, and more preferably 10- 30 nucleotides in length, including any length within these ranges, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length. Barcodes may be used, for example, to identify a single cell, subpopulation of cells, colony, or sample from which a nucleic acid originated. Barcodes may also be used to identify the position (i.e., positional barcode) of a cell, colony, or sample from which a nucleic acid originated, such as the position of a colony in a cellular array, the position of a well in a multi-well plate, or the position of a tube, flask, or other container in a rack. In particular, a barcode may be used to identify a genetically modified cell from which anucleic acid originated. In some embodiments, a barcode is used to identify a donor cell from which a genetically modified cell was generated. Alternatively, a unique barcode may be used to identify each genome editing module in a multi-module gene editing construct and / or each guide-RNA and / or donor polynucleotide used in multiplexed or multi-step genome editing. Furthermore, multiple barcodes can be used in combination to identify different features of a nucleic acid or cell. For example, positional barcoding (e.g., to identify the position of a cell, colony, culture, or sample in an array, multi-well plate, or rack) can be combined with barcodes identifying a cell donor / lineage and / or barcodes identifying genome editing modules, guide-RNAs, and / or donor polynucleotides used in genome editing.

[0172] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to any compound comprising naturally occurring or synthetic amino acid polymers or amino acid-like molecules including but not limited to compounds comprising amino and / or imino molecules. No particular size is implied by use of the terms “polypeptide,” “peptide,” and “protein” and these terms are used interchangeably. The “terms include post-expression modifications of the polypeptide, peptide, or protein such as glycosylation, acetylation, phosphorylation, and the like. Further, polypeptides, peptides, or proteins, as described herein may include additional molecules such as labels (e.g., fluorescent, bioluminescent, or radioactive), tags (e.g., histidine tag, epitope tag), or other chemical moieties.

[0173] The terms “antibodies” and “immunoglobulin” include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to an antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, monoclonal antibodies, hybrid antibodies, chimeric antibodies, humanized antibodies, single-chain antibodies, single-domain antibodies, nanobodies, bispecific antibodies, tri-specific antibodies, and other multi-specific antibodies, and fusion proteins comprising an antigen-binding portion of an antibody and a non-antibody protein.

[0174] “Antibody fragments” comprise a portion of an intact antibody, for example, the antigen binding or variable region of the intact antibody. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments. Papain digestion of antibodies produces two identical antigen-binding fragments, called “Fab” fragments, each with a single antigen-binding site, and a residual “Fc” fragment, a designation reflecting the ability to crystallize readily. Pepsin treatment yields anF(ab')2 fragment that has two antigen-combining sites and is still capable of cross-linking antigen.

[0175] “Single-chain Fv” or “sFv” antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. In some embodiments, the Fv polypeptide further comprises a polypeptide linker between the Vn and VL domains, which enables the sFv to form the desired structure for antigen binding.

[0176] The terms “specific binding,” “specifically binds,” and the like, refer to non-covalent or covalent preferential binding to a molecule relative to other molecules or moieties in a solution or reaction. In some embodiments, the affinity of one molecule for another molecule to which it specifically binds is characterized by a KD (dissociation constant) of 10'5M or less (e.g., 1 O’6M or less, 10’7M or less, 10’8M or less, 10’9M or less, 10-1° M or less, 10-11M or less, 10-12M or less). "Affinity" refers to the strength of binding, increased binding affinity being correlated with a lower KD. In an embodiment, affinity is determined by surface plasmon resonance (SPR), e.g., as used by Biacore systems. The affinity of one molecule for another molecule is determined by measuring the binding kinetics of the interaction, e.g., at 25°C.

[0177] The term “antigen-binding fragment” as used herein refers to any antibody fragment that specifically binds to a target antigen including, but not limited to, a diabody, a Fab, a Fab', a F(ab')2, an Fv fragment, a disulfide stabilized Fv fragment (dsFv), a (dsFv)2, a bispecific dsFv (dsFv-dsFv'), a disulfide stabilized diabody (ds diabody), a single-chain antibody molecule (scFv), an scFv dimer (bivalent diabody), a multispecific antibody formed from a portion of an antibody including one or more complementarity determining regions (CDRs).

[0178] The term "variable" refers to the fact that certain portions of the variable domains differ extensively in sequence among antibodies and are used in the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions both in the light-chain and the heavy-chain variable domains. The more highly conserved portions of variable domains are called the framework (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely adopting a D-sheet configuration, connected by three CDRs, which form loops connecting, and in some cases forming part of, the D-sheet structure. The CDRs in each chain are heldtogether in close proximity by the FR regions and, with the CDRs from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991 )). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular toxicity. VL and VH sequences can be reformatted as fragments, as single chain binding domains, linked to chimeric antigen receptors, and the like.

[0179] The term “antigen binding domain (ABD)” refers to a domain that specifically binds to a target antigen. The antigen binding domain region of an antibody may comprise a heavy-chain variable domain (VH) and a light-chain variable domain (VL) in non-covalent association as a single polypeptide or as a dimer. The three complementarity-determining regions of the heavy chain variable domain (CDR H1 , H2, H3) and three complementarity-determining regions of the light chain variable domain (CDR L1 , L2, L3) interact to define an antigen-binding site on the surface of an antibody. Collectively, the six CDRs of the light chain and heavy chain variable domains confer antigen-binding specificity to an antibody. An antigen binding domain region of a CAR may comprise all six CDRs of an antibody or a single variable domain or half of an Fv fragment comprising only three CDRs specific for an antigen, which still retains the ability to recognize and bind the target antigen. In some embodiments, the antigen-binding domain binds to one or more target antigens expressed on the surface of a target cell (e.g., cell surface markers).

[0180] The term "T cell" includes all types of immune cells expressing CD3 including T- helper cells (CD4+cells), cytotoxic T-cells (CD8+cells), natural killer T cells, T-regulatory cells (Treg) and gamma-delta T cells. The term "T cell" also includes genetically modified T cells, including T cells engineered to express a chimeric antigen receptor (CAR) and T cells from which the gene encoding the endogenous T cell receptor has been inactivated or deleted (i.e., TCR gene knockout).

[0181] The terms “T cell receptor” and “TCR” are used interchangeably and generally refer to a receptor found on the surface of T cells or T lymphocytes that is responsible for recognizing antigenic peptides bound to major histocompatibility complex (MHC) molecules. The TCR is a membrane-anchored heterodimeric protein comprising two different protein chains. In the majority of human T cells, the TCR consists of an alpha (a) chain and a beta (b) chain (encoded by TRA and TRB genes, respectively). In about5% of human T cells, the TCR consists of gamma and delta (g / d) chains (encoded by TRG and TRD genes, respectively). T cells expressing a TCR comprising alpha and beta chains are referred to as ab T cells, and T cells expressing a TCR comprising gamma and delta chains are referred to as gd T cells The ratio of ab T cells to gd T cells differs between species and may be altered by disease (such as leukemia). The variable domains of the TCR a-chain and p-chain each have three hypervariable or complementarity-determining regions (CDRs). CDR 1 and CDR3 bind to the antigenic peptide. CDR2 recognizes the MHC. The constants domains of the TCR a-chain and p- chain each have a cysteine that forms a disulfide bond that links the two chains. The TCR receptor a and chains associate with six additional adaptor proteins, including a delta chain, a gamma chain, two epsilon chains, and two zeta chains to form an octameric complex. The adaptor proteins comprise signaling motifs involved in TCR signaling.

[0182] Chimeric antigen receptor (CAR). A CAR may have any suitable architecture, as known in the art, comprising an antigen binding domain, usually provided in an scFv format, linked to T cell receptor effector functions. The term refers to artificial multimodule molecules capable of triggering or inhibiting the activation of an immune cell. A CAR will generally comprise an antigen binding domain, linker, transmembrane domain and cytoplasmic signaling domain. In some instances, a CAR will include one or more co-stimulatory domains and / or one or more co-inhibitory domains.

[0183] The antigen-binding domain of the CAR may include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a target antigen of interest. In some embodiments, the binding region is an antigen-binding region, such as an antibody or functional binding domain or antigen-binding fragment thereof. The antigen-binding region of the CAR can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a single-chain antibody, and any antigen-binding fragment thereof. Thus, in some embodiments, the antigen binding domain portion includes a mammalian antibody or an antigen-binding fragment thereof. An antigen-binding domain may comprise an antigenbinding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a VH domain, a VL domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, or a diabody; or a functional antigen-binding fragment thereof. In someembodiments, the antigen-binding domain is derived from the same cell type or the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR may include a human antibody, a humanized antibody, or an antigen-binding fragment thereof.

[0184] In some embodiments, the antigen binding domain is derived from a single chain antibody that selectively binds to a target antigen. In some embodiments, the antigen binding domain is provided by a single chain variable fragment (scFv). A scFv is a recombinant molecule in which the variable regions of the light and heavy immunoglobulin chains are connected in a single fusion polypeptide. Generally, the VH and VL sequences are joined by a linker sequence. See, for example, Ahmad (2012) Clinical and Developmental Immunology Article ID 980250, herein specifically incorporated by reference. In principle, there are no particular limitations to the length and / or amino acid composition of the linker peptide joining the VH and VL sequences. In some embodiments, any arbitrary single-chain peptide including about 1 to 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acid residues) can be used as a peptide linker. In some embodiments, the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues.

[0185] The transmembrane domain may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membranebound or transmembrane protein. In some embodiments, the transmembrane domain comprises at least the stalk and / or transmembrane region(s) of CD8, Megf10, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, Integrin subunit |35, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, and / or CD86. In some embodiments, the CAR transmembrane domain is derived from a type I membrane protein, such as, but not limited to, CD3£, CD4, CD8, or CD28. In other embodiments, the transmembrane domain is synthetic, in which case it will include predominantlyhydrophobic residues such as leucine, isoleucine, valine, phenylalanine, tryptophan, and alanine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be inserted at each end of a synthetic transmembrane domain.

[0186] In some embodiments, the CAR further comprises one or more linkers / spacers. For example, an extracellular spacer region may link the antigen binding domain to the transmembrane domain and / or an intracellular spacer region may link an intracellular signaling domain to the transmembrane domain. A spacer (linker) region linking the antigen binding domain to the transmembrane domain should be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition.

[0187] Various types of linkers may be used in the CARs described herein. In some embodiments, the linker includes a peptide linker / spacer sequence. In some embodiments, the spacer comprises the hinge region from an immunoglobulin, e.g., the hinge from any one of lgG1 , lgG2a, lgG2b, lgG3, lgG4, particularly the human protein sequences. Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. For many scFv based constructs, an IgG hinge is effective.

[0188] In principle, there are no particular limitations to the length and / or amino acid composition of a linker peptide sequence. In some embodiments, a linker peptide sequence comprises about 1 to 100 amino acid residues, including any number of residues within this range such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues. In some embodiments, the linker peptide sequence may include up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. In some embodiments, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular engulfment signaling domain or extracellular antigen binding domain of the CAR. In some embodiments the linkercomprises the amino acid sequence (G4S)nwhere n is 1 , 2, 3, 4, 5, etc., and in some embodiments, n is 3.

[0189] A cytoplasmic signaling domain, such as those derived from the T cell receptor □- chain, is employed as part of the CAR in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Endodomains from co-stimulatory molecules may be included in the cytoplasmic signaling portion of the CAR.

[0190] The term “co-stimulatory domain”, refers to a stimulatory domain, typically an endodomain, of a CAR that provides a secondary non-specific activation mechanism through which a primary specific stimulation is propagated. Examples of co-stimulation include antigen nonspecific T cell co-stimulation following antigen specific signaling through the T cell receptor and antigen nonspecific B cell co-stimulation following signaling through the B cell receptor. Co-stimulation, e.g., T cell co-stimulation, and the factors involved have been described in Chen & Flies. Nat Rev Immunol (2013) 13(4):227-42, the disclosure of which are incorporated herein by reference in their entirety. Non-limiting examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-1 BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0191] The term “co-inhibitory domain” refers to an inhibitory domain, typically an endodomain, derived from a receptor that provides secondary inhibition of primary antigen-specific activation mechanisms which prevents co-stimulation. Co-inhibition, e.g., T cell co-inhibition, and the factors involved have been described in Chen & Flies. Nat Rev Immunol (2013) 13(4):227-42 and Thaventhiran et al. J Clin Cell Immunol (2012) S12. In some embodiments, co-inhibitory domains homodimerize. A co-inhibitory domain can be an intracellular portion of a transmembrane protein. Non-limiting examples of suitable co-inhibitory polypeptides include, but are not limited to, CTLA-4 and PD-1 .

[0192] A first-generation CAR transmits the signal from antigen binding through only a single signaling domain, for example a signaling domain derived from the high-affinity receptor for IgE FCERI D D or the CD3 chain. The domain contains one or three immunoreceptor tyrosine-based activating motif(s) [ITAM(s)] for antigen-dependent T- cell activation. The ITAM-based activating signal endows T-cells with the ability to lyse the target tumor cells and secret cytokines in response to antigen binding.

[0193] Second-generation CARs include a co-stimulatory signal in addition to the CD3D signal. Coincidental delivery of the delivered co-stimulatory signal enhances cytokinesecretion and antitumor activity induced by CAR-transduced T-cells. The co-stimulatory domain will usually be membrane proximal relative to the CD3D domain. Third- generation CARs include a tripartite signaling domain, comprising for example a CD28, CD3 , 0X40 or 4-1 BB signaling region. In fourth generation, or “armored car” CAR-T cells, CAR-T cells are further genetically modified to express or block molecules and / or receptors to enhance immune activity.

[0194] CAR variants include split CARs wherein the extracellular portion, the ABD and the cytoplasmic signaling domain of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application Nos. US2014 / 016527, US1996 / 017060, US2013 / 063083; Fedorov et al. Sci Trans! Med (2013) ;5(215) :215ra172; Glienke et al. Front Pharmacol (2015) 6:21 ; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151 -5; Riddell et al. Cancer J (2014) 20(2):141 -4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1 ):91 -106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388- 98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; the disclosures of which are incorporated herein by reference in their entirety.

[0195] CAR variants also include bispecific or tandem CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. Tandem CARs (TanCAR) mediate bispecific activation of T cells through the engagement of two chimeric receptors designed to deliver stimulatory or costimulatory signals in response to an independent engagement of two different tumor associated antigens. iCARs use the dual antigen targeting to shout down the activation of an active CAR through the engagement of a second suppressive receptor equipped with inhibitory signaling domains

[0196] The dual recognition of different epitopes by two CARs diversely designed to either deliver killing through -chain or costimulatory signals, e.g., through CD28 allows a more selective activation of the reprogrammed T cells by restricting Tandem CAR's activity to cancer cell expressing simultaneously two antigens rather than one. The potency of delivered signals in engineered T cells will remain below threshold of activation and thusineffective in absence of the engagement of costimulatory receptor. The combinatorial antigen recognition enhances selective tumor eradication and protects normal tissues expressing only one antigen from unwanted reactions.

[0197] Inhibitory CARs (iCARs) are designed to regulate CAR-T cells activity through inhibitory receptor signaling module activation. This approach combines the activity of two CARs, one of which generates dominant negative signals limiting the responses of CAR-T cells activated by the activating receptor. iCARs can switch off the response of the counteracting activator CAR when bound to a specific antigen expressed only by normal tissues. In this way, iCARs-T cells can distinguish cancer cells from healthy ones, and reversibly block functionalities of transduced T cells in an antigen-selective fashion. CTLA-4 or PD-1 intracellular domains in iCARs trigger inhibitory signals on T lymphocytes, leading to less cytokine production, less efficient target cell lysis, and altered lymphocyte motility.

[0198] An ABD can be provided as a “chimeric bispecific binding member”, i.e., a chimeric polypeptide having dual specificity to two different binding partners (e.g., two different antigens). Non-limiting examples of chimeric bispecific binding members include bispecific antibodies, bispecific conjugated monoclonal antibodies (mab)2, bispecific antibody fragments (e.g., F(ab)2, bispecific scFv, bispecific diabodies, single chain bispecific diabodies, etc.), bispecific T cell engagers (BiTE), bispecific conjugated single domain antibodies, micabodies and mutants thereof, and the like. Non-limiting examples of chimeric bispecific binding members also include those chimeric bispecific agents described in Kontermann. MAbs. (2012) 4(2): 182-197; Stamova et al. Antibodies 2012, 1 (2), 172-198; Farhadfar et al. Leuk Res. (2016) 49:13-21 ; Benjamin et al. Ther Adv Hematol. (2016) 7(3):142-56; Kiefer et al. Immunol Rev. (2016) 270(1 ):178-92; Fan et al. J Hematol Oncol. (2015) 8:130; May et al. Am J Health Syst Pharm. (2016) 73(1 ):e6-e13; the disclosures of which are incorporated herein by reference in their entirety.

[0199] In some instances, a chimeric bispecific binding member may be a bispecific T cell engager (BiTE). A BiTE is generally made by fusing a specific binding member (e.g., a scFv) that binds an antigen to a specific binding member (e.g., a scFv) with a second binding domain specific for a T cell molecule such as CD3.

[0200] In some instances, a chimeric bispecific binding member may be a CAR-T cell adapter. As used herein, by “CAR-T cell adapter” is meant an expressed bispecific polypeptide that binds the antigen recognition domain of a CAR and redirects the CAR to a second antigen. Generally, a CAR-T cell adapter will have two binding regions, onespecific for an epitope on the CAR to which it is directed and a second epitope directed to a binding partner which, when bound, transduces the binding signal activating the CAR. Useful CAR-T cell adapters include but are not limited to e.g., those described in Kim et al. J Am Chem Soc. (2015) 137(8):2832-5; Ma et al. Proc Natl Acad Sci U S A. (2016) 1 13(4):E450-8 and Cao et al. Angew Chem Int Ed Engl. (2016) 55(26)7520-4; the disclosures of which are incorporated herein by reference in their entirety.

[0201] Effector CAR-T cells include autologous or allogeneic immune cells having cytolytic activity against a target cell. In some embodiments, a T cell is engineered to express a CAR. The term “T cells” refers to mammalian immune effector cells that may be characterized by expression of CD3 and / or a T cell antigen receptor.

[0202] In some embodiments, the CAR-T cells are engineered from a complex mixture of immune cells, e.g., tumor infiltrating lymphocytes (TILs) isolated from an individual in need of treatment. See, for example, Yang and Rosenberg (2016) Adv Immunol. 130:279-94, “Adoptive T Cell Therapy for Cancer; Feldman et al (2015) Semin Oncol. 42(4):626-39 “Adoptive Cell Therapy-Tumor-Infiltrating Lymphocytes, T-Cell Receptors, and Chimeric Antigen Receptors”; Clinical Trial NCT01 174121 , “Immunotherapy Using Tumor Infiltrating Lymphocytes for Patients With Metastatic Cancer”; Tran et al. (2014) Science 344(6184)641 -645, “Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer”.

[0203] In other embodiments, the engineered T cell is allogeneic with respect to the individual that is treated, e.g. see clinical trials NCT03121625; NCT03016377; NCT02476734; NCT02746952; NCT02808442. See for review Graham et al. (2018) Cells. 7(10) E155. In some embodiments an allogeneic engineered T cell is fully HLA matched. However not all patients have a fully matched donor, and a cellular product suitable for all patients independent of HLA type provides an alternative.

[0204] Allogeneic T cells may be administered in combination with intensification of lymphodepletion to allow CAR-T cells to expand and clear malignant cells prior to host immune recovery, e.g., by administration of Alemtuzumab (monoclonal anti-CD52), purine analogs, etc. The allogeneic T cells may be modified for resistance to Alemtuzumab. Gene editing can be used to prevent expression of HLA class I molecules on CAR-T cells, e.g. by deletion of □2-microglobulin.

[0205] In addition to modifying T cells, induced pluripotent stem (iPS) cell-derived CAR- T cells can be used. For example, donor T cells can be transduced with reprogramming factors to restore pluripotency, and then re-differentiated into T effector cells.

[0206] T cells for engineering, as described above, collected from a subject or a donor, may be separated from a mixture of cells by techniques that enrich for desired cells, or may be engineered and cultured without separation. An appropriate solution may be used for dispersion or suspension. Such solution will generally be a balanced salt solution, e.g. normal saline, PBS, Hank’s balanced salt solution, etc., conveniently supplemented with fetal calf serum or other naturally occurring factors, in conjunction with an acceptable buffer at low concentration, generally from 5-25 mM. Convenient buffers include HEPES, phosphate buffers, lactate buffers, etc.

[0207] Techniques for affinity separation may include magnetic separation, using antibody-coated magnetic beads, affinity chromatography, cytotoxic agents joined to a monoclonal antibody or used in conjunction with a monoclonal antibody, e.g., complement and cytotoxins, and "panning" with antibody attached to a solid matrix, e.g., a plate, or other convenient technique. Techniques providing accurate separation include fluorescence activated cell sorters, which can have varying degrees of sophistication, such as multiple color channels, low angle and obtuse light scattering detecting channels, impedance channels, etc. The cells may be selected against dead cells by employing dyes associated with dead cells (e.g., propidium iodide). Any technique may be employed which is not unduly detrimental to the viability of the selected cells. The affinity reagents may be specific receptors or ligands for the cell surface molecules indicated above. In addition to antibody reagents, peptide-MHC antigen and T cell receptor pairs may be used; peptide ligands and receptor; effector and receptor molecules, and the like.

[0208] The separated cells may be collected in any appropriate medium that maintains the viability of the cells, usually having a cushion of serum at the bottom of the collection tube. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove's medium, etc., frequently supplemented with fetal calf serum (FCS).

[0209] The collected and optionally enriched cell population may be used immediately for genetic modification, or may be frozen at liquid nitrogen temperatures and stored, being thawed and capable of being reused. The cells will usually be stored in 10% DMSO, 50% FCS, 40% RPMI 1640 medium.

[0210] Engineered CAR-T cells may be infused into a subject in any physiologically acceptable medium by any convenient route of administration, normally intravascularly, though CAR-T cells may also be introduced by other routes, where the cells may find an appropriate site for growth. Usually, at least 1 x106cells / kg will be administered, at least1 x107cells / kg, at least 1 x108cell s / kg , at least 1 x109cells / kg, at least 1 x1010cells / kg, or more, usually being limited by the number of T cells that are obtained during collection.

[0211] By “genetically engineered” or “genetically modified”, it is intended to mean that the genome of a cell has been altered. In some cases, the genome of the cell has been manipulated to express an expression product that is not normally naturally expressed by the cell. Examples of cells that have been genetically engineered include chimeric antigen receptor (CAR)-T cells that are T-cells that have been genetically engineered to express a CAR. A coding sequence encoding a CAR may be introduced on an expression vector into a cell to be engineered. For example, a CAR coding sequence may be introduced into the genome at the site of an endogenous T cell receptor gene. In some cases, cells are further engineered to delete an endogenous T cell receptor (i.e., TCR knockout). In some cases, a CRISPR / Cas9 system is used to genetically modify a T cell. A CRISPR / Cas9 system can be introduced into cells by transfection with mRNA or a plasmid that encodes Cas9 and a gRNA or by viral delivery of CRISPR components, e.g., using lentiviral, retroviral vectors, or non-integrating viruses, such as adenovirus and adeno-associated virus (AAV).

[0212] By “binding-triggered transcriptional switch” or “BTSS”, it is intended to mean a synthetic modular polypeptide or system of interacting polypeptides having an extracellular domain that includes a second member of a specific binding pair that binds a first member of the specific binding pair (e.g., an antigen), a binding-transducer and an intracellular domain. Upon binding of the first member of the specific binding pair to the BTTS the binding signal is transduced to the intracellular domain such that the intracellular domain becomes activated and performs a function, e.g., transcription activation, within the cell that it does not perform in the absence of the binding signal.

[0213] Examples of BTSS include the synNotch system, the modular extracellular sensor architecture (MESA) system, the TANGO system, the A2 Notch system, and the synthetic intramembrane proteolysis receptor (SNIPR) system, etc. The synNotch receptor may be for example as described in U.S. Patent No. 9,670,281 and described in more detail below. The MESA system may be as described in WO 2018 / 081039 A1 and comprises a self-containing sensing and signal transduction system, such that binding of a ligand (first member of the specific binding pair) to the receptor (second member of the specific binding pair) induces signaling to regulate expression of a target gene. In the MESA system, binding of the ligand to the receptor induces dimerization that results in proteolytic trans-cleavage of the system to release a transcriptional activator previouslysequestered at the plasma membrane. The TANGO system may be as described in Barnea et al., 2008 Proc. Natl. Acad. Sci. U.S.A., 105(1 ): 64-9. Briefly, the TANGO system sequesters a transcription factor to the cell membrane by physically linking it to a membrane-bound receptor (e.g., GPCRs, receptor kinases, Notch, steroid hormone receptors, etc.). Activation of the receptor fusion results in the recruitment of a signaling protein fused to a protease that then cleaves and releases the transcription factor to activate genes in the cell. The A2 Notch system may be as described in WO 2019099689 A1. Briefly, the A2 Notch system incorporates a force sensor cleavage domain which, upon cleavage induced upon binding of a ligand to the receptor, releases the intracellular domain into the cell. The SNIPR system may be described as in Zhu et al. (2022) Cell 185(8):1431 -1443.e16; herein incorporated by reference. Briefly, the SNIPR system uses a synthetic RiP receptor comprising an ectodomain comprising an extracellular regulatory element that specifically binds a ligand, a transmembrane domain, a juxtamembrane domain, and a transcription factor that can be cleaved from the SNIPR by a protease in response to binding of a ligand to the extracellular regulatory element.

[0214] In certain embodiments, the second binding member may be present on the surface of a genetically engineered cell, such as, a cell expressing a BTTS and a CAR under the control of the BTTS. In certain embodiments, the second binding member may be present on the surface of a genetically engineered cell, such as, a cell expressing the BTTS and a CAR under control of the BTTS.

[0215] In certain cases, the first binding member may bind to a synNotch receptor as described in U.S. Patent No. 9,670,281 . For example, the synNotch receptor may include an extracellular domain that includes the second binding member, where the second binding member is a single-chain Fv (scFv) or a nanobody and the first binding member present on the particles is an antigen to which the single-chain Fv (scFv) or a nanobody binds. In certain cases, the second binding member may be an anti-CD19, anti- mesothelin, anti-GFP antibody, scFv, or a nanobody and the first binding member may be CD19, mesothelin, GFP, respectively.

[0216] In certain embodiments, the BTTS is a chimeric Notch polypeptide comprising, from N-terminus to C-terminus and in covalent linkage: a) an extracellular domain comprising the second member of the specific-binding pair that is not naturally present in a Notch receptor polypeptide and that specifically binds to the first member of the specific-binding pair; b) a Notch regulatory region comprising a Lin 12-Notch repeat, an S2 proteolytic cleavage site, and a transmembrane domain comprising an S3 proteolyticcleavage site; c) an intracellular domain comprising a transcriptional activator or a transcriptional repressor that is heterologous to the Notch regulatory region and replaces a naturally-occurring intracellular Notch domain, wherein binding of the first member of the specific-binding pair to the second member of the specific-binding pair induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain; and a transcriptional control element, responsive to the transcriptional activator, operably linked to a nucleotide sequence encoding a chimeric antigen receptor (CAR). In certain cases, the cell may be a T-cell, such as, those described in U.S. Patent No. 9,670,281 , which is herein incorporated by reference.Multi-Module Gene Editing Constructs

[0217] Compositions and methods are provided for assembling a multi-module gene editing construct. A schematic of an exemplary embodiment of a method for assembling a multi-module gene editing construct is shown in FIG. 2. The method comprises: (a) providing a circular DNA or plasmid comprising a first recombinant polynucleotide, wherein the first recombinant polynucleotide comprises a first genome editing module and a first barcode to identify the first genome editing module, wherein the first genome editing module and the first barcode are separated by a first excisable staffer sequence, wherein the first excisable staffer sequence is flanked by a first excision-mediating site and a second excision-mediating site; (b) contacting the circular DNA or plasmid with an excising agent that binds to the first excision-mediating site and the second excisionmediating site, wherein the first excisable staffer sequence is excised from the first recombinant polynucleotide to produce a cleaved circular DNA or plasmid having the first genome editing module at a first cleaved end and the first barcode at a second cleaved end; (c) providing a second recombinant polynucleotide, wherein the second recombinant polynucleotide comprises a second genome editing module and a second barcode to identify the second genome editing module, wherein the second genome editing module and the second barcode are separated by a second excisable stuffer sequence, wherein the second excisable stuffer sequence is flanked by a third excisionmediating site and a fourth excision-mediating site; (d) ligating the second recombinant polynucleotide to the first cleaved end and the second cleaved end to produce a first ligation product, wherein the first genome module is connected to the second genome module, and wherein the second barcode is connected to the first barcode; (e) contacting the first ligation product with an excising agent that binds to the third excision-mediatingsite and the fourth excision-mediating site, wherein the second excisable staffer sequence is excised from the first ligation product to produce a first cleaved ligation product having the first genome editing module connected to the second genome module at a first end of the first cleaved ligation product and the second barcode connected to the first barcode at a second end of the first cleaved ligation product; (f) providing a third recombinant polynucleotide, wherein the third recombinant polynucleotide comprises a third genome editing module and a third barcode to identify the third genome editing module, wherein the third genome editing module and the third barcode are separated by a third excisable staffer sequence, wherein the third excisable staffer sequence is flanked by a fifth excision-mediating site and a sixth excision-mediating site; (g) ligating the third recombinant polynucleotide to the first end of the first cleaved ligation product and the second end of the first cleaved ligation product to produce a second ligation product comprising the first genome module, the second genome module, the third genome module, the third excisable staffer sequence flanked by the fifth excisionmediating site and the sixth excision-mediating site, and a 3’-barcode array comprising the third barcode, followed by the second barcode, followed by the first barcode; and (h) repeating steps (e) - (g) until assembly of the multi-module gene editing construct is completed.

[0218] Any number of genome editing modules can be added to the multi-module gene editing construct using this method. In some embodiments, the final assembled multimodule gene editing construct has two or more genome editing modules, three or more genome editing modules, four or more genome editing modules, five or more genome editing modules, six or more genome editing modules, seven or more genome editing modules, eight or more genome editing modules, nine or more genome editing modules, or ten or more genome editing modules.

[0219] The subject methods enable any type of genetic modification to be tested in combination with other genetic modifications. For example, each genome editing module in the multi-module gene editing construct can be designed to produce a different sequence-specific genetic perturbation. Exemplary sequence-specific genetic perturbations include a gene knockin, a gene knockout, a gene knock-down, a gene upregulation, an insertion of a coding or non-coding domain, a deletion of an endogenous protein coding domain, an insertion of a regulatory element, a deletion of a regulatory element, an epigenetic perturbation, and an insertion of a unique molecular identifier orcell tracing sequence. The assembled multi-module gene editing construct can be designed to test any combination of such sequence-specific genetic perturbations.

[0220] A host cell can be genetically modified with a multi-module gene editing construct to provide any desired number of genetic modifications. Accordingly, cells genetically modified with a multi-module gene editing construct may have multiple gene knockins and / or gene knockouts, and / or insertions and / or deletions of coding and / or non-coding domains. See, e.g., FIGS. 1 1 -14 for schematics of exemplary embodiments. In certain embodiments, the multi-module gene editing construct comprises a plurality of genome editing modules to knockin a plurality of full-length genes or coding or non-coding domains, knockdown expression of a plurality of genes, upregulate expression of a plurality of genes, knockout a plurality of genes, or any combination thereof. Schematics of exemplary genome editing modules that can be used to assemble a multi-module gene editing construct are shown in FIGS. 3-1 1 .

[0221] In certain embodiments, the excising agent, used for excision of the excisable stuffer sequences in the genome editing modules is a restriction enzyme or a site-specific recombinase. In some embodiments, each excision-mediating site comprises a recognition site for the same restriction enzyme or site-specific recombinase.

[0222] In some embodiments, a type IIS restriction enzyme is used for excision of the excisable stuffer sequences in the genome editing modules, wherein the excision sites comprise recognition sites (i.e., restriction sites) for the type IIS restriction enzyme. Exemplary type IIS restriction enzymes include, without limitation, Bsal, Bbsl, Bsgl, BsmAI, BsmBI-v2, BsmFI, Bsml, BspCNI, BspMI, BspQI, BsrDI, Bsrl, BtgZI, BtsCI, Btsl- v2, BtsIMutl, CspCI, Earl, Ecil, Esp3l, Faul, Fokl, Hgal, Hphl, HpyAV, Mboll, Mlyl, Mmel, Mnll, NmeAIII, PaqCI, Piel, Sapl, BspQI, and SfaNI.

[0223] In some embodiments, a site-specific recombinase is used for excision of the excisable stuffer sequences in the genome editing modules, wherein the excision sites comprise recognition sites for the site-specific recombinase. Exemplary site-specific recombinases include, without limitation, a Ore recombinase, which catalyzes sitespecific recombination between two loxP sites, a flippase (FLP) recombinase, which catalyzes site-specific recombination between two flippase recognition target (FRT) sites, a phiC31 recombinase, which catalyze site-specific recombination between two attachment (att) sites referred to as attB and attP, a DreO recombinase, which catalyzes site-specific recombination between two rox sites, or a Tre recombinase, which catalyzessite-specific recombination between two loxP sites that are modified with HIV long terminal repeats (loxLTR).

[0224] The recombinant polynucleotides comprising the genome editing modules, used to assemble a multi-module gene editing construct, and / or the excisable stuffer sequences in the genome editing modules may further comprise constant 5’-adapters, constant 3’-adapters, and / or sequencing primer binding sites to facilitate amplification and / or sequencing of the multi-module gene editing construct or amplification and / or sequencing of a portion of the multi-module gene editing construct comprising the 3’- barcode array of the multi-module gene editing construct. Any suitable type of adapter may be used in the constructs.

[0225] In some embodiments, the adapters include sites that allow nucleic acids to attach to a solid support for amplification and / or sequencing (e.g., the surface of a flow-cell for the Illumina platform or beads for the Ion Torrent platform). The 3’-adapter may be hybridized to an extension primer, wherein amplification is performed by extending the primer. In certain embodiments, the constructs comprise a sequencing platform adapter, which includes one or more nucleic acid domains selected from: a domain (e.g., a "capture site" or "capture sequence") that specifically binds to a surface-attached sequencing platform oligonucleotide (e.g., the P5 or P7 oligonucleotides attached to the surface of a flow cell in an Illumina® sequencing system); a sequencing primer binding domain (e.g., a domain to which the Read 1 or Read 2 primers of the Illumina® platform may bind); a barcode domain (e.g., a domain that uniquely identifies the sample source of the nucleic acid being sequenced to enable sample multiplexing by marking every molecule from a given sample with a specific barcode or "tag"); a barcode sequencing primer binding domain (a domain to which a primer used for sequencing a barcode binds); or any combination of such domains.

[0226] The adapters chosen for the preparation of a sequencing library should be compatible with the sequencing system to be used. A sequencing platform adapter domain, when present in an adapter, may include one or more nucleic acid domains of any length and sequence suitable for the sequencing platform of interest. In some embodiments, the nucleic acid domains are from 4 to 200 nucleotides in length. For example, the nucleic acid domains may be from 4 to 100 nucleotides in length, such as from 6 to 75, from 8 to 50, or from 10 to 40 nucleotides in length. According to certain embodiments, the sequencing platform adapter construct includes a nucleic acid domain that is from 2 to 8 nucleotides in length, such as from 9 to 15, from 16 to 22, from 23 to29, or from 30 to 36 nucleotides in length. The nucleic acid domains may have a length and sequence that enables a polynucleotide (e.g., an oligonucleotide) employed by the sequencing platform of interest to specifically bind to the nucleic acid domain, e.g., for solid phase amplification and / or sequencing. Adapter sequences are typically provided by the manufacturer of the sequencing platform (e.g., in technical documents provided with the sequencing system and / or available on the manufacturer's website). Based on such information, the sequence of any sequencing platform adapter domains, amplification primers, and / or the like, may be designed to include all or a portion of one or more nucleic acid domains in a configuration that enables sequencing the nucleic acid insert (e.g., barcode region of a multi-module gene editing construct) on the platform of interest.

[0227] In certain embodiments, each excisable stuffer sequence in the genome editing modules of a multi-module gene editing construct comprises or consists of the same sequence. In some embodiments, the excisable stuffer sequence comprises: a first recognition site for binding a restriction enzyme, wherein the restriction enzyme cuts at the first excision site; a second recognition site for binding the restriction enzyme, wherein the restriction enzyme cuts at the second excision site; a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site positioned between the safety cut site and the second recognition site for binding the restriction enzyme; a constant 5’-adapter positioned at the 5’-end of the excisable stuffer sequence; and a constant 3’-adapter positioned at the 3’-end of the excisable stuffer sequence.

[0228] An exemplary embodiment of a recombinant polynucleotide comprising a genome editing module for insertion of a full-length gene into a genome is shown in FIG. 3. In this exemplary embodiment, the recombinant polynucleotide comprises: a constant 5'- adapter sequence at the 5’-end of the recombinant polynucleotide; a genome editing module comprising a sequence encoding a full-length gene; an intron spacer sequence, wherein the intron spacer sequence is positioned between the constant 5’-adapter sequence at the 5’-end of the recombinant polynucleotide and the sequence encoding the full-length gene; an excisable stuffer sequence flanked by a first excision site and a second excision site, wherein the excisable stuffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excisionsite, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a constant 5’-adapter positioned at the 5’-end of the excisable staffer sequence, and a constant 3’-adapter positioned at the 3’-end of the excisable staffer sequence; a 2A multicistronic element, wherein the 2A multicistronic element is positioned between the sequence encoding the full-length gene and the excisable staffer sequence to separate an open reading frame of the full-length gene from any other open reading frame; a barcode for identification of the genome editing module comprising the sequence encoding the full-length gene, wherein the barcode is separated from the genome editing module by the excisable staffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.

[0229] An exemplary embodiment of a recombinant polynucleotide comprising a genome editing module for insertion of a coding or non-coding domain into a genome is shown in FIG. 4. In this exemplary embodiment, the recombinant polynucleotide comprises: a genome editing module comprising a sequence encoding a coding domain or a noncoding domain; an intron spacer sequence positioned before the sequence encoding the coding domain or the non-coding domain, wherein the intron comprises a 5’-splice donor site, a constant 5’-adapter, and a 3’-splice acceptor site, wherein the constant 5’-adapter is positioned within the 5’-splice donor site or between the 5’-splice donor site and the 3'- splice acceptor site, and wherein the constant 5’-adapter is removed by messenger RNA splicing; an excisable staffer sequence flanked by a first excision site and a second excision site, wherein the excisable staffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a constant 5’-adapter positioned at the 5’-end of the excisable staffer sequence, and a constant 3’-adapter positioned at the 3’-end of the excisable staffer sequence; a barcode for identification of the genome editing module comprising the sequence encoding the coding domain or the non-coding domain, wherein the barcodeis separated from the genome editing module by the excisable staffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.

[0230] As shown in an exemplary embodiment depicted in FIG. 5, constructs can be designed with intron spacer sequences such that scar regions generated by assembly of a multi-module gene editing construct are contained within the intron spacer sequences. Ane intron spacer sequence containing a scar region can be removed by mRNA splicing. In some embodiments, an intron comprising a 5’-splice donor site, a constant 5’-adapter, and a 3’-splice acceptor site is used in a construct, wherein the constant 5’-adapter is positioned between the 5’-splice donor site and the 3’-splice acceptor site such that the constant 5’-adapter as well as any scar region, if present, is removed by RNA splicing from the transcribed mRNA.

[0231] An exemplary embodiment of a recombinant polynucleotide comprising a genome editing module for knockdown of expression of a target gene is shown in FIG. 7. In this exemplary embodiment, the recombinant polynucleotide comprises: a genome editing module comprising a sequence encoding a full-length gene or a protein coding domain; a stop codon operably linked to the sequence encoding the full-length gene or protein coding domain, wherein the stop codon terminates translation of the sequence encoding the full-length gene or protein coding domain; a 3’-untranslated region following the stop codon, wherein the 3’-untranslated region comprises: a mRNA stabilizing element, a sequence encoding a regulatory RNA, a first 5’-adapter positioned between the mRNA stabilizing element and the sequence encoding the regulatory RNA; an excisable staffer sequence flanked by a first excision site and a second excision site, wherein the excisable staffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a second constant 5’-adapter positioned at the 5’-end of the excisable staffer sequence, and a first constant 3’-adapter positioned at the 3’-end of the excisable staffer sequence; a barcode for identification of the genome editing module comprising the sequence encoding the regulatory RNA, wherein the barcode is separated from the sequence encoding the regulatory RNA by the excisable staffer sequence; and a constant 3’-adapter sequenceat the 3’-end of the recombinant polynucleotide. In some embodiments, the regulatory RNA comprises a short hairpin RNA (shRNA). In some embodiments, the shRNA is a microRNA (miR)-embedded shRNA, wherein endogenous microRNA processing of the miR-embedded shRNA within a cell transfected with the multi-module gene editing construct results in excision of the shRNA such that the shRNA can inhibit expression of a target gene. In some embodiments, the miR-embedded shRNA is a miR-30-embedded shRNA or a miR-E-embedded shRNA. In certain embodiments, the 3’-untranslated region further comprises one or more additional sequences encoding one or more additional regulatory RNAs. In some embodiments, the one or more additional regulatory RNAs are shRNAs or miR-embedded shRNAs. In some embodiments, the mRNA stabilizing element is a triplex stabilizer.

[0232] An exemplary embodiment of a recombinant polynucleotide comprising a genome editing module for knockout of a target gene is shown in FIG. 9. In this exemplary embodiment, the recombinant polynucleotide comprises: a genome editing module comprising a sequence encoding a full-length gene or a protein coding domain; a stop codon operably linked to the sequence encoding the full-length gene or protein coding domain, wherein the stop codon terminates translation of the sequence encoding the full- length gene or protein coding domain; a 3’-untranslated region following the stop codon, wherein the 3’-untranslated region comprises: a mRNA stabilizing element, a sequence encoding a guide RNA (gRNA) for an RNA-guided nuclease, a first 5’-adapter positioned between the mRNA stabilizing element and the sequence encoding the gRNA; an excisable stuffer sequence flanked by a first excision site and a second excision site, wherein the excisable stuffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a second constant 5’-adapter positioned at the 5’-end of the excisable stuffer sequence, and a first constant 3’-adapter positioned at the 3’-end of the excisable stuffer sequence; a barcode for identification of the genome editing module comprising the sequence encoding the gRNA, wherein the barcode is separated from the sequence encoding the gRNA by the excisable stuffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide. In some embodiments, the mRNA stabilizing element is a triplex stabilizer.

[0233] The final assembled multi-module gene editing construct comprises a genome editing region comprising a series of genome editing modules followed by a barcode region (i.e., 3’-barcode array) comprising a series of barcodes identifying the individual genome editing modules in the order in which they were added to the construct (see, e.g., FIG. 2). Each barcode comprises one or more nucleotide sequences that are used to identify each genome editing module in the multi-module gene editing construct. Barcodes can be 3-1000 or more nucleotides in length, preferably 10-250 nucleotides in length, and more preferably 10-30 nucleotides in length, including any length within these ranges, such as 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides in length.

[0234] One or more additional barcodes may be used, for example, to identify a single cell, subpopulation of cells, colony, donor, or sample from which a nucleic acid originated. Barcodes may also be used to identify the position (i.e., positional barcode) of a cell, colony, or sample from which a nucleic acid originated, such as the position of a colony in a cellular array, the position of a well in a multi-well plate, or the position of a tube, flask, or other container in a rack. In particular, a barcode may be used to identify a genetically modified cell from which a nucleic acid originated. In some embodiments, a barcode is used to identify a donor cell from which a genetically modified cell was generated (i.e., donor barcode). Furthermore, multiple barcodes can be used in combination to identify different features of a nucleic acid or cell. For example, positional barcoding (e.g., to identify the position of a cell, colony, culture, or sample in an array, multi-well plate, or rack) can be combined with barcodes identifying a cell donor, cell lineage, and / or barcodes identifying genome editing modules, guide-RNAs, and / or donor polynucleotides used in genome editing.

[0235] In certain embodiments, one or more additional barcodes are added to the 3'- barcode array of a multi-module gene editing construct by adding a barcode module. For example, a barcode module may be added to the circular DNA or plasmid comprising the multi-module gene editing construct by a method comprising: contacting the multi-module gene editing construct with an excising agent, wherein an excisable stuffer sequence is excised from the multi-module gene editing construct to produce a cleaved circular DNA or plasmid comprising the genome editing modules of the multi-module gene editingconstruct at a first cleaved end and the 3’-barcode array comprising the barcodes of the multi-module gene editing construct at a second cleaved end; and ligating a polynucleotide comprising the barcode module comprising the one or more additional barcodes to the first cleaved end and the second cleaved end, wherein the barcode module comprising the one or more additional barcodes is added to the 3’-barcode array comprising the barcodes of the genome editing modules of the multi-module gene editing construct. In some embodiments, the one or more additional barcodes comprise a cell tracing sequence or unique molecular identifier sequence. For example, the cell tracing sequence may identify a clone, cell lineage, donor, or condition. In certain embodiments, the method further comprises sequencing the one or more additional barcodes to identify the clone, the cell lineage, or the donor from which a genetically modified cell comprising the multi-module gene editing construct was derived. In certain embodiments, the method further comprises performing single-cell RNA sequencing on a genetically modified cell comprising the multi-module gene editing construct.

[0236] In certain embodiments, genetically modified cells are plated in an ordered array on media suitable for their growth to produce arrayed colonies of clones, wherein each colony of clones comprises a different multi-module gene editing construct. The barcode region of the multi-module gene editing construct of an individual clone can be sequenced to identify the genome editing modules present in that clone. A positional barcode can be added to a multi-module gene editing construct to identify the position in the array of a colony of clones having a particular multi-module gene editing construct to allow cells with a particular multi-module gene editing construct and a corresponding combination of genetic modifications to be selected on demand.

[0237] Various gene editing approaches can be used to introduce sequence-specific genetic perturbations, including, without limitation, genome editing systems comprising clustered regularly interspaced short palindromic repeats (CRISPR) / CRISPR-associated (Cas) nucleases, meganucleases, zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs). See, e.g., CRISPR Gene Editing: Methods and Protocols (edited by Luo, Humana, 2019), Genome Editing and Engineering: From TALENs, ZFNs and CRISPRs to Molecular Surgery (edited by Appasani and Church, Cambridge University Press, 2018); herein incorporated by reference in their entireties. These gene editing techniques involve using an RNA-guided nuclease to create a double-strand break (DSB) in the DNA at a target site of an intended edit to the genome. In some embodiments, the site-specific nuclease comprises a DNA-binding domain thatdirects the nuclease to the target site. In other embodiments, the site-specific nuclease is an RNA-guided nuclease, wherein the nuclease forms a complex with a guide RNA (gRNA) that directs the RNA-guided nuclease to the target site. The site-specific nuclease can be used to create a DSB at a target site in an exon or an intron of a gene, for example, to alter a coding sequence or a regulatory region of a gene. Gene knockout may be achieved through either the non-homologous end joining (NHEJ) or the microhomology-mediated end-joining (MMEJ) DNA repair pathways, which generate small nucleotide insertions or deletions (indels) at the site of the DSB. Gene knockout results if an indel shifts the reading frame or introduces a premature stop codon in a gene coding sequence. Alternatively, gene editing can be performed with a donor polynucleotide template using homologous recombination (HR) to replace a portion of the genomic sequence with a modified sequence containing any desired genome edit. A donor polynucleotide may be used, for example, to create a gene knockin (e.g., exogenous gene or synthetic gene), add a coding domain or a non-coding domain, alter a gene regulatory region of a gene (e.g., gene knockdown or overexpression), add, mutate, or delete selected sequences in the genome, or make epigenetic changes. The subject methods can be used to genetically modify the genome of a cell, organoid, or organism with a multi-module gene editing construct.

[0238] In some embodiments, each genome editing module is capable of generating a different intended edit to the genome, such that the assembled multi-module gene editing construct is capable of producing a plurality of different intended edits at one or more genomic target loci. An RNA-guided nuclease is introduced into cells that is capable of forming complexes with guide RNAs, which may be encoded by the multi-module gene editing construct modules or provided separately, wherein the guide RNAs direct the complexes to one or more genomic target loci in the cells, where the RNA-guided nuclease creates double-stranded breaks in the genomic DNA. In the absence of a donor polynucleotide, gene knockout may be achieved through NHEJ or MMEJ DNA repair at target genomic loci.

[0239] Integration of donor polynucleotides, provided by a multi-module gene editing construct, at genomic target loci by homology directed repair (HDR) can be used to produce a plurality of edits in cells. Genome modification can be performed, for example, using HDR with a donor polynucleotide comprising a sequence comprising an intended genome edit flanked by a pair of homology arms responsible for targeting the donor polynucleotide to the target locus to be edited in a cell. The donor polynucleotide typicallycomprises a 5' homology arm that hybridizes to a 5' genomic target sequence and a 3' homology arm that hybridizes to a 3' genomic target sequence. The homology arms are referred to herein as 5' and 3' (i.e., upstream and downstream) homology arms, which relates to the relative position of the homology arms to the nucleotide sequence comprising the intended edit within the donor polynucleotide. The 5' and 3' homology arms hybridize to regions within the target locus in the genomic DNA to be modified, which are referred to herein as the "5' target sequence" and "3' target sequence," respectively.

[0240] The homology arm must be sufficiently complementary for hybridization to the target sequence to mediate homologous recombination between the donor polynucleotide and genomic DNA at the target locus. For example, a homology arm may comprise a nucleotide sequence having at least about 80-100% sequence identity to the corresponding genomic target sequence, including any percent identity within this range, such as at least 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity thereto, wherein the nucleotide sequence comprising the intended edit is integrated into the genomic DNA by HDR at the genomic target locus recognized (i.e., sufficiently complementary for hybridization) by the 5' and 3' homology arms.

[0241] In certain embodiments, the corresponding homologous nucleotide sequences in the genomic target sequence (i.e., the "5' target sequence" and "3' target sequence") flank a specific site for cleavage and / or a specific site for introducing the intended edit. The distance between the specific cleavage site and the homologous nucleotide sequences (e.g., each homology arm) can be several hundred nucleotides. In some embodiments, the distance between a homology arm and the cleavage site is 200 nucleotides or less (e.g., 0, 10, 20, 30, 50, 75, 100, 125, 150, 175, and 200 nucleotides). In most cases, a smaller distance may give rise to a higher gene targeting rate. In a preferred embodiment, the donor polynucleotide is substantially identical to the target genomic sequence, across its entire length except for the sequence changes to be introduced to a portion of the genome that encompasses both the specific cleavage site and the portions of the genomic target sequence to be altered.

[0242] A homology arm can be of any length, e.g. 10 nucleotides or more, 50 nucleotides or more, 100 nucleotides or more, 250 nucleotides or more, 300 nucleotides or more, 350 nucleotides or more, 400 nucleotides or more, 450 nucleotides or more, 500 nucleotides or more, 1000 nucleotides (1 kb) or more, 5000 nucleotides (5 kb) or more,10000 nucleotides (10 kb) or more, etc. In some instances, the 5' and 3' homology arms are substantially equal in length to one another, e.g. one may be 30% shorter or less than the other homology arm, 20% shorter or less than the other homology arm, 10% shorter or less than the other homology arm, 5% shorter or less than the other homology arm, 2% shorter or less than the other homology arm, or only a few nucleotides less than the other homology arm. In other instances, the 5' and 3' homology arms are substantially different in length from one another, e.g. one may be 40% shorter or more, 50% shorter or more, sometimes 60% shorter or more, 70% shorter or more, 80% shorter or more, 90% shorter or more, or 95% shorter or more than the other homology arm.

[0243] A donor polynucleotide encoded by a genome editing module in a multi-module gene editing construct may be used in combination with an RNA-guided nuclease (encoded by the multi-module gene editing construct or provided separately), which is targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by a guide RNA (gRNA). A target-specific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site. For example, the gRNA can be designed with a sequence complementary to a target sequence in the target gene. In some embodiments, the gRNA is designed with a sequence complementary to a disease-specific gene mutation to target the nuclease-gRNA complex to the site of a mutation in a diseased cell. The mutation may comprise an insertion, a deletion, or a substitution. For example, the mutation may include a single nucleotide variation, gene fusion, translocation, inversion, duplication, frameshift, missense, nonsense, or other mutation. The targeted minor allele may be a common genetic variant or a rare genetic variant. In certain embodiments, the gRNA is designed to selectively bind to a minor allele with single base-pair discrimination, for example, to allow binding of the nuclease-gRNA complex to a single nucleotide polymorphism (SNP). In particular, the gRNA may be designed to target disease-relevant mutations of interest for the purpose of genome editing to delete or deactivate a gene in a cell.

[0244] An RNA-guided nuclease can be targeted to a particular genomic sequence (i.e., genomic target sequence to be modified) by altering its guide RNA sequence. A targetspecific guide RNA comprises a nucleotide sequence that is complementary to a genomic target sequence, and thereby mediates binding of the nuclease-gRNA complex by hybridization at the target site. For example, the gRNA can be designed with a sequencecomplementary to a sequence of the genomic target locus to target the nuclease-gRNA complex to a target site.

[0245] In certain embodiments, the RNA-guided nuclease used for genome modification is a clustered regularly interspersed short palindromic repeats (CRISPR) system Cas nuclease. Any RNA-guided Cas nuclease capable of catalyzing site-directed cleavage of DNA to allow integration of donor polynucleotides by the HDR mechanism can be used in genome editing, including CRISPR system type I, type II, or type III Cas nucleases. Examples of Cas proteins include Cas1 , Cas1 B, Cas2, Cas3, Cas4, Cas5, Cas5e (CasD), Cas6, Cas6e, Cas6f, Cas7, Cas8a1 , Cas8a2, Cas8b, Cas8c, Cas9 (Csn1 or Csx12), Casi o, Cas10d, CasF, CasG, CasH, Csy1 , Csy2, Csy3, Cse1 (CasA), Cse2 (CasB), Cse3 (CasE), Cse4 (CasC), Csc1 , Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1 , Cmr3, Cmr4, Cmr5, Cmr6, Csb1 , Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1 , Csx15, Csf1 , Csf2, Csf3, Csf4, and Cu1966, and homologs or modified versions thereof.

[0246] In certain embodiments, a type II CRISPR system Cas9 endonuclease is used. Cas9 nucleases from any species, or biologically active fragments, variants, analogs, or derivatives thereof that retain Cas9 endonuclease activity (i.e., catalyze site-directed cleavage of DNA to generate double-strand breaks) may be used to perform genome modification as described herein. The Cas9 need not be physically derived from an organism, but may be synthetically or recombinantly produced. Cas9 sequences from a number of bacterial species are well known in the art and listed in the National Center for Biotechnology Information (NCBI) database. See, for example, NCBI entries for Cas9 from: Streptococcus pyogenes (WP 002989955, WP 038434062, WP 01 1528583); Campylobacter jejuni (WP 022552435, YP_002344900), Campylobacter coll (WP 0607861 16); Campylobacter fetus (WP 059434633); Corynebacterium ulcerans (NC 015683, NC_017317); Corynebacterium diphtheria (NC_016782, NC_016786); Enterococcus faecalis (WP 033919308); Spiroplasma syrphidicola (NC_021284); Prevotella intermedia (NC_017861 ); Spiroplasma taiwanense (NC_021846); Streptococcus iniae (NC_021314); Belliella baltica (NC_018010); Psychroflexus torquisl (NC_018721 ); Streptococcus thermophilus (YP_820832), Streptococcus mutans (WP 061046374, WP_024786433); Listeria innocua (NP_472073); Listeria monocytogenes (WP 061665472); Legionella pneumophila (WP 062726656); Staphylococcus aureus (WP 001573634); Francisella tularensis (WP 032729892, WP 014548420), Enterococcus faecalis (WP 033919308); Lactobacillus rhamnosus(WP 048482595, WP_032965177); and Neisseria meningitidis (WP_061704949, YP_002342100); all of which sequences (as entered by the date of filing of this application) are herein incorporated by reference. Any of these sequences or a variant thereof comprising a sequence having at least about 70-100% sequence identity thereto, including any percent identity within this range, such as 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, or 99% sequence identity thereto, can be used for genome editing, as described herein. See also Fonfara et al. (2014) Nucleic Acids Res. 42(4):2577-90; Kapitonov et al. (2015) J. Bacteriol. 198(5):797-807, Shmakov et al. (2015) Mol. Cell. 60(3):385-397, and Chylinski et al. (2014) Nucleic Acids Res. 42(10):6091 -6105); for sequence comparisons and a discussion of genetic diversity and phylogenetic analysis of Cas9.

[0247] The CRISPR-Cas system naturally occurs in bacteria and archaea where it plays a role in RNA-mediated adaptive immunity against foreign DNA. The bacterial type II CRISPR system uses the endonuclease, Cas9, which forms a complex with a guide RNA (gRNA) that specifically hybridizes to a complementary genomic target sequence, where the Cas9 endonuclease catalyzes cleavage to produce a double-stranded break. Targeting of Cas9 typically further relies on the presence of a 5' protospacer-adjacent motif (PAM) in the DNA at or near the gRNA-binding site.

[0248] The genomic target site will typically comprise a nucleotide sequence that is complementary to the gRNA, and may further comprise a protospacer adjacent motif (PAM). In certain embodiments, the target site comprises 20-30 base pairs in addition to a 3 base pair PAM. Typically, the first nucleotide of a PAM can be any nucleotide, while the two other nucleotides will depend on the specific Cas9 protein that is chosen. Exemplary PAM sequences are known to those of skill in the art and include, without limitation, NNG, NGN, NAG, and NGG, wherein N represents any nucleotide. In certain embodiments, the intron sequence of the TCR gene targeted by a gRNA comprises a mutation that creates a PAM within the intron, wherein the PAM promotes binding of the Cas9-gRNA complex to the intron.

[0249] In certain embodiments, the gRNA is 5-50 nucleotides, 10-30 nucleotides, 15-25 nucleotides, 18-22 nucleotides, or 19-21 nucleotides in length, or any length between the stated ranges, including, for example, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, or 35 nucleotides in length. The guide RNA may be a single guide RNA comprising crRNA and tracrRNA sequences in a single RNAmolecule, or the guide RNA may comprise two RNA molecules with crRNA and tracrRNA sequences residing in separate RNA molecules.

[0250] In another embodiment, the CRISPR nuclease from Prevotella and Francisella 1 (Cpf1 ) also referred to as CRISPR associated protein 12a (Cas12a) may be used. Cas12a is another class II CRISPR / Cas system RNA-guided nuclease with similarities to Cas9 and may be used analogously. Unlike Cas9, Cas12a does not require a tracrRNA and only depends on a crRNA in its guide RNA, which provides the advantage that shorter guide RNAs can be used with Cas12a for targeting than Cas9. Cas12a is capable of cleaving either DNA or RNA. The PAM sites recognized by Cas12a have the sequences 5'-YTN-3' (where "Y" is a pyrimidine and "N" is any nucleobase) or 5'-TTN-3', in contrast to the G-rich PAM site recognized by Cas9. Cas12a cleavage of DNA produces double-stranded breaks with sticky-ends having a 4 or 5 nucleotide overhang. For a discussion of Cas12a, see, e.g., Ledford et al. (2015) Nature. 526 (7571 ):17-17, Zetsche et al. (2015) Cell. 163 (3):759-771 , Murovec et al. (2017) Plant Biotechnol. J. 15(8):917-926, Zhang et al. (2017) Front. Plant Sci. 8:177, Fernandes et al. (2016) Postepy Biochem. 62(3):315-326; herein incorporated by reference.

[0251] C2c1 is another class II CRISPR / Cas system RNA-guided nuclease that may be used. C2c1 , similarly to Cas9, depends on both a crRNA and tracrRNA for guidance to target sites. For a description of C2c1 , see, e.g., Shmakov et al. (2015) Mol Cell. 60(3):385-397, Zhang et al. (2017) Front Plant Sci. 8:177; herein incorporated by reference.

[0252] In yet another embodiment, an engineered RNA-guided Fokl nuclease may be used. RNA-guided Fokl nucleases comprise fusions of inactive Cas9 (dCas9) and the Fokl endonuclease (Fokl-dCas9), wherein the dCas9 portion confers guide RNA- dependent targeting on Fokl. For a description of engineered RNA-guided Fokl nucleases, see, e.g., Havlicek et al. (2017) Mol. Ther. 25(2):342-355, Pan et al. (2016) Sci Rep. 6:35794, Tsai et al. (2014) Nat Biotechnol. 32(6):569-576; herein incorporated by reference.

[0253] The RNA-guided nuclease can be provided in the form of a protein, such as the nuclease complexed with a gRNA, or provided by a nucleic acid encoding the RNA- guided nuclease, such as an RNA (e.g., messenger RNA) or DNA (expression vector such as a plasmid or viral vector or multi-module gene editing construct comprising a coding sequence encoding the RNA-guided nuclease). Codon usage may be optimized to improve production of an RNA-guided nuclease in a particular cell, organoid, ororganism. For example, a nucleic acid encoding an RNA-guided nuclease can be modified to substitute codons having a higher frequency of usage in a human cell or a non-human mammalian cell, such as a non-human primate cell, a rodent cell, a mouse cell, a rat cell, or any other host cell of interest, as compared to the naturally occurring polynucleotide sequence. When a nucleic acid encoding the gRNA and / or RNA-guided nuclease is introduced into cells, the gRNA and / or RNA-guided nuclease can be transiently, conditionally, or constitutively expressed in the cell. A nucleic acid encoding the RNA-guided nuclease and / or gRNA can be introduced into a cell using any suitable transfection technique such as, but not limited to electroporation, nucleofection, or lipofection. Alternatively, a ribonucleoprotein complex of the gRNA and the RNA-guided nuclease may be introduced into a cell by microinjection into the cytoplasm or nucleus.

[0254] A CRISPR-Cas system can be introduced into cells with a viral vector that encodes a Cas nuclease (e.g., Cas9 or Cas12a) and / or a guide RNA (gRNA). Viral delivery of CRISPR components has been demonstrated using lentiviral, retroviral, adenovirus, and adeno-associated virus (AAV) vectors. For a description of methods of introducing a CRISPR system into cells with various viral vectors, see, e.g., Shalem et al. (2014) Science 343:84-87, Williams et al. (2016) Sci Rep. 6:2561 1 , Ran et al. (2015) Nature 520:186-191 , Swiech et al. (2015) Nat Biotechnol. 33:102-106; herein incorporated by reference.

[0255] Alternatively, a gRNA and a messenger RNA encoding the RNA-guided nuclease can be introduced into cells, wherein the RNA-guided nuclease is produced by translation of the messenger RNA in the cytoplasm. The gRNA and RNA-guided nuclease then form a complex in the cytoplasm and enter the nucleus. RNA transfection of cells can be performed using electroporation, cationic-lipid-mediated transfection, or using liposomes or lipid nanoparticles (LNPs) encapsulating the gRNA and mRNA. See, e.g., Billingsley et al. (2022) Nano Lett 22(1 ):533-542, Tchou et al. (2017) Cancer Immunol Res. 5(12):1152-1 161 , Ye et al. (2022) ACS Biomater Sci Eng. 8(2):722-733, Guevara et al. (2020) Front. Chem. 8:589959; herein incorporated by reference.

[0256] Donor polynucleotides and gRNAs are readily synthesized by standard techniques, e.g., solid phase synthesis via phosphoramidite chemistry, as disclosed in U.S. Patent Nos. 4,458,066 and 4,415,732, incorporated herein by reference; Beaucage et al., Tetrahedron (1992) 48:2223-2311 ; and Applied Biosystems User Bulletin No. 13 (1 April 1987). Other chemical synthesis methods include, for example, the phosphotriester method described by Narang et al., Meth. Enzymol. (1979) 68:90 andthe phosphodiester method disclosed by Brown et al., Meth. Enzymol. (1979) 68:109. In view of the short lengths of gRNAs (typically about 20 nucleotides in length) and donor polynucleotides (typically about 100-150 nucleotides), gRNA-donor polynucleotide cassettes can be produced by standard oligonucleotide synthesis techniques and subsequently ligated into constructs or vectors comprising a gene editing module.Combinatorial Library

[0257] Methods of producing a combinatorial library comprising a plurality of multi-module gene editing constructs are also provided. A population of cells can be transfected with a plurality of vectors (e.g., plasmids or viral vectors), wherein each vector comprises a different multi-module gene editing construct. The vectors can be introduced into cells using any suitable transfection technique such as, but not limited to electroporation, nucleofection, lipofection, chemical transfection, laser-mediated transfection, biolistic particle delivery, or microinjection. The subject methods allow multiple cells within a cell population to be edited in a single round with multiple genetic changes.

[0258] In some embodiments, the combinatorial library comprises 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 9 or more, 10 or more, 20 or more, 40 or more, 60 or more, 80 or more, 100 or more, 500 or more, or 1000 or more different multi-module gene editing constructs. In some embodiments, the combinatorial library comprises 2-1000 multi-module gene editing constructs, 10-500 multi-module gene editing constructs, or 50-100 multi-module gene editing constructs, including any number of multi-module gene editing constructs within these ranges such as 2, 3, 4, 5, 6, 7, 8, 9 ,10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 constructs.

[0259] Methods are also provided for conducting screening using a combinatorial library comprising a plurality of multi-module gene editing constructs. For example, a pool of cells containing the combinatorial library can be tested to determine which combination of genetic modifications is most effective in conferring a particular desired phenotype. Individual cells can be isolated, and the barcodes of the multi-module gene editing constructs can be sequenced to identify the genome editing modules and the sequence-specific genetic perturbations that are effective in conferring the desired phenotype. The cells of the combinatorial library can be further genetically modified to remove detrimental mutations and consolidate beneficial mutations to improve various cellular processes.

[0260] In some embodiments, adapter sequences are added to the multi-module gene editing constructs to facilitate high-throughput amplification or sequencing. For example, a pair of adapter sequences can be added at the 5’ and 3’ ends of the constructs to allow amplification and / or sequencing of multiple constructs simultaneously by the same set of primers. Additionally, restriction sites can be incorporated into constructs to facilitate cloning of the multi-module gene editing constructs into vectors. For example, constructs can be designed with a common 5’ restriction site and a common 3’ restriction site to facilitate ligation into vectors. A restriction digest that selectively cleaves each multimodule gene editing construct at the common 5’ restriction site and the common 3’ restriction site is performed to produce restriction fragments that can be cloned into vectors (e.g., plasmids or viral vectors), followed by transformation of cells with the vectors comprising the multi-module gene editing constructs to produce the combinatorial library.

[0261] Amplification of multi-module gene editing constructs may be performed, for example, before ligation into vectors or before sequencing barcodes. Any method for amplifying nucleic acids may be used, including, but not limited to polymerase chain reaction (PCR) and isothermal amplification methods such as recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), nicking enzyme amplification reaction (NEAR), ligase chain reaction (LGR), nucleic acid sequence based amplification (NASBA), transcription-mediated amplification (TMA), Q- beta amplification, and the like. In some embodiments, the multi-module gene editing constructs comprise common 5’ and 3’ priming sites to allow amplification of different constructs in parallel with a set of universal primers. In another embodiment, a set of selective primers is used to selectively amplify a subset of the constructs from a pooled mixture.Multiplexed Screening

[0262] The subject methods allow cells with different combinations of genetic modifications to be screened simultaneously in multiplexed assays. Cells can be tested individually or in large pools. Multiple different perturbation types can be screened together combinatorially. For example, gene knockdowns can be screened simultaneously with synthetic gene knockins, or gene knockouts or knockins of variable domains of genes. Furthermore, different types of genetic manipulations can be screenedcombinatorially across categories (testing knockouts, knockins, knockdowns, overexpression, etc.) in multiple combinations simultaneously.

[0263] In certain embodiments, detecting the effects of different sequence-specific genetic perturbations in a plurality of genetically modified host cells comprises detecting a change in cell morphology, cell growth, cell proliferation, gene expression, biological activity of a protein, or any combination thereof in the plurality of genetically modified host cells compared to an unmodified host cell.

[0264] In certain embodiments, a plurality of genetically modified host cells are contacted with a test agent to determine the effects of the different sequence-specific genetic perturbations in the plurality of genetically modified host cells on activity of the test agent. Genetically modified cells comprising multi-module gene editing constructs can be subjected to a plurality of candidate agents or other therapeutic intervention. Candidate agents encompass numerous chemical classes, e.g., small organic compounds having a molecular weight of more than 50 daltons and less than about 10,000 daltons, less than about 5,000 daltons, or less than about 2,500 daltons. Test agents can comprise functional groups necessary for structural interaction with proteins, e.g., hydrogen bonding, and can include at least an amine, carbonyl, hydroxyl or carboxyl group, or at least two of the functional chemical groups. The test agents can comprise cyclical carbon or heterocyclic structures and / or aromatic or polyaromatic structures substituted with one or more of the above functional groups. Test agents are also found among biomolecules including peptides, peptide fragments, receptor fragments, co-receptor fragments, saccharides, fatty acids, steroids, purines, pyrimidines, derivatives, structural analogs or combinations thereof.

[0265] Test agents are obtained from a wide variety of sources including libraries of synthetic or natural compounds. For example, numerous means are available for random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides and oligopeptides. Alternatively, libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means, and may be used to produce combinatorial libraries. Known pharmacological agents may be subjected to directed or random chemical modifications, such as acylation, alkylation, esterification, amidification, etc. to produce structural analogs. Moreover, screening may be directed to known pharmacologically activecompounds and chemical analogs thereof, or to new agents with unknown properties such as those created through rational drug design.

[0266] In some embodiments, test agents are synthetic compounds. A number of techniques are available for the random and directed synthesis of a wide variety of organic compounds and biomolecules, including expression of randomized oligonucleotides. See for example WO 94 / 24314, hereby expressly incorporated by reference, which discusses methods for generating new compounds, including random chemistry methods as well as enzymatic methods.

[0267] In another embodiment, the test agents are provided as libraries of natural compounds in the form of bacterial, fungal, plant and animal extracts that are available or readily produced. Additionally, natural or synthetically produced libraries and compounds are readily modified through conventional chemical, physical and biochemical means. Known pharmacological agents may be subjected to directed or random chemical modifications, including enzymatic modifications, to produce structural analogs.

[0268] In some embodiments, the test agents are organic moieties. In this embodiment, test agents are synthesized from a series of substrates that can be chemically modified. “Chemically modified” herein includes traditional chemical reactions as well as enzymatic reactions. These substrates generally include, but are not limited to, alkyl groups (including alkanes, alkenes, alkynes and heteroalkyl), aryl groups (including arenes and heteroaryl), alcohols, ethers, amines, aldehydes, ketones, acids, esters, amides, cyclic compounds, heterocyclic compounds (including purines, pyrimidines, benzodiazepins, beta-lactams, tetracylines, cephalosporins, and carbohydrates), steroids (including estrogens, androgens, cortisone, ecodysone, etc.), alkaloids (including ergots, vinca, curare, pyrollizdine, and mitomycines), organometallic compounds, hetero-atom bearing compounds, amino acids, and nucleosides. Chemical (including enzymatic) reactions may be done on the moieties to form new substrates or candidate agents which can then be tested using the present invention.

[0269] In some embodiments a test agent is assessed for any cytotoxic activity it may exhibit toward a living eukaryotic cell, using well-known assays, such as trypan blue dye exclusion, an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2 H-tetrazolium bromide) assay, and the like. Agents that do not exhibit significant cytotoxic activity are considered candidate agents.

[0270] Genetically modified cells comprising multi-module gene editing constructs may be tested in culture with one or a panel of cellular environments, where the cellular environment includes one or more of: exposure to a candidate agent of interest, contact with other cells, electrical stimulation, alterations in ionicity, alterations in temperature, contact with pro-inflammatory or anti-inflammatory agents, contact with infectious agents, e.g. bacterial, viral, fungal, or parasitic infectious agents, and the like, and where cells may vary in types of genetic modifications, in prior exposure to an environment of interest, in the dose of an agent that is provided, etc. Usually at least one control is included, for example, a negative control and / or a positive control. Culture of genetically modified cells is typically performed in a sterile environment, for example, at 37°C. in an incubator containing a humidified 92-95% air / 5-8% CO2 atmosphere. Cell culture may be carried out in nutrient mixtures containing undefined biological fluids such as fetal calf serum, or media which is fully defined and serum free. The effect of the altering the environment may be assessed by monitoring multiple output parameters, including morphological, functional, and genetic changes.

[0271] The agents are conveniently added in solution, or readily soluble form, to the medium used in culturing the genetically modified cells. The agents may be added in a flow-through system, as a stream, intermittent or continuous, or alternatively, adding a bolus of the compound, singly or incrementally, to an otherwise static solution. In a flow- through system, two fluids are used, where one is a physiologically neutral solution, and the other is the same solution with the test compound added. The first fluid is passed over the cells, followed by the second. In a single solution method, a bolus of the test compound is added to the volume of medium surrounding the cells. The overall concentrations of the components of the culture medium should not change significantly with the addition of the bolus, or between the two solutions in a flow through method.

[0272] Preferred agent formulations do not include additional components, such as preservatives, that may have a significant effect on the overall formulation. Thus, preferred formulations consist essentially of a biologically active compound and a physiologically acceptable carrier, e.g., water, ethanol, DMSO, etc. However, if a compound is liquid without a solvent, the formulation may consist essentially of the compound itself.

[0273] A plurality of assays may be run in parallel with different agent concentrations to obtain a differential response to the various concentrations. As known in the art, determining the effective concentration of an agent typically uses a range ofconcentrations resulting from 1 :10, or other log scale, dilutions. The concentrations may be further refined with a second series of dilutions, if necessary. Typically, one of these concentrations serves as a negative control, i.e. , at zero concentration or below the level of detection of the agent or at or below the concentration of agent that does not give a detectable change in the phenotype.

[0274] Any suitable method known in the art may be used for analyzing cells. Microscopy techniques including, without limitation, fluorescence microscopy, confocal microscopy, two-photon microscopy, multi-photon microscopy, light-field microscopy, expansion microscopy, and light sheet microscopy may be used, for example, to detect morphological changes resulting from the combinations of genetic modifications generated by a multi-module gene editing construct. Additionally, immunofluorescence may be used to detect changes in localization of antigens and expression of surface markers on genetically modified cells. Patch-clamping can be used to detect electrophysiological changes (e.g., particularly in excitable cells such as neurons and muscle cells). Microarray analysis and / or proteomic profiling techniques may be used to detect changes in gene expression and / or the distribution of proteins in genetically modified cells. Biochemical assays may be used to assess changes in activities of particular proteins in genetically modified cells.

[0275] Various methods can be utilized for quantifying the presence of selected parameters, For measuring the amount of a target analyte that is present, a convenient method is to label a molecule with a detectable moiety, which may be fluorescent, luminescent, radioactive, enzymatically active, etc., particularly a molecule specific for binding to the target analyte with high affinity. Fluorescent moieties are readily available for labeling virtually any biomolecule, structure, or cell type. Immunofluorescent moieties can be directed to bind not only to specific proteins but also specific conformations, cleavage products, or site modifications like phosphorylation. Individual peptides and proteins can be engineered to fluoresce, e.g., by expressing them as green fluorescent protein chimeras inside cells (for a review see Jones et al. (1999) Trends Biotechnol. 17(12):477-81 ). Cells can be genetically modified to provide fusions of an antibody to a fluorescent or bioluminescent protein.

[0276] Depending upon the label chosen, parameters may be measured using immunoassay techniques such as a radioimmunoassay (RIA) or enzyme linked immunosorbance assay (ELISA), homogeneous enzyme immunoassays, and related non-enzymatic techniques. These techniques utilize specific antibodies as reportermolecules, which are particularly useful due to their high degree of specificity for attaching to a single molecular target. U.S. Pat. No. 4,568,649 describes ligand detection systems, which employ scintillation counting. These techniques are particularly useful for protein or modified protein parameters or epitopes, or carbohydrate determinants. Cell readouts for proteins and other cell determinants can be obtained using fluorescent or otherwise tagged reporter molecules. Cell based ELISA or related non-enzymatic or fluorescence-based methods enable measurement of cell surface parameters and secreted parameters. Capture ELISA and related non-enzymatic methods usually employ two specific antibodies or reporter molecules and are useful for measuring parameters in solution. Flow cytometry methods are useful for measuring cell surface and intracellular parameters, as well as shape change and granularity and for analyses of beads used as antibody- or probe-linked reagents. Readouts from such assays may be the mean fluorescence associated with individual fluorescent antibody-detected cell surface molecules or cytokines, or the average fluorescence intensity, the median fluorescence intensity, the variance in fluorescence intensity, or some relationship among these.

[0277] Quantitative readouts of parameters may include baseline measurements in the absence of agents or a pre-defined genetic control condition and test measurements in the presence of a single or multiple agents or a genetic test condition. Furthermore, quantitative readouts of parameters may include long-term recordings and may therefore be used as a function of time (change of parameter value). Readouts may be acquired either spontaneously or in response to stimulation or perturbation of the cells. The quantitative readouts of parameters may further include a single determined value, the mean or median values of parallel, subsequent or replicate measurements, the variance of the measurements, various normalizations, the cross-correlation between parallel measurements, etc. and every statistic used to a calculate a meaningful and informative factor.Sequencing

[0278] Any high-throughput technique for sequencing can be used to sequence the 3’- barcode array of a multi-module gene editing construct to identify the genome editing modules in the multi-module gene editing construct in an individual cell as well as any other barcodes added to the barcode region of the multi-module gene editing construct such as cell tracing sequences, unique molecular identifier sequences, and / or positionalbarcodes. For example, all barcodes in a construct may be sequenced, including barcodes identifying the types of genetic modifications conferred by the genome editing modules of a multi-module gene editing construct and additional barcodes identifying the clone, the cell lineage, or the donor from which a genetically modified cell comprising the multi-module gene editing construct was derived and / or the location of a colony of clones of the genetically modified cell comprising the multi-module gene editing construct. The inclusion of a common sequencing primer binding site and a constant 5'-adapter and constant 3’-adapter in multi-module gene editing constructs facilitates high-throughput amplification and sequencing of the barcode regions.

[0279] DNA sequencing techniques include dideoxy sequencing reactions (Sanger method) using labeled terminators or primers and gel separation in slab or capillary, sequencing by synthesis using reversibly terminated labeled nucleotides, pyrosequencing, 454 sequencing, sequencing by synthesis using allele specific hybridization to a library of labeled clones followed by ligation, real time monitoring of the incorporation of labeled nucleotides during a polymerization step, polony sequencing, SOLID sequencing, and the like.

[0280] Certain high-throughput methods of sequencing comprise a step in which individual molecules are spatially isolated on a solid surface where they are sequenced in parallel. Such solid surfaces may include nonporous surfaces (such as in Solexa sequencing, e.g. Bentley et al, Nature, 456: 53-59 (2008) or Complete Genomics sequencing, e.g. Drmanac et al, Science, 327: 78-81 (2010)), arrays of wells, which may include bead- or particle-bound templates (such as with 454, e.g. Margulies et al, Nature, 437: 376-380 (2005) or Ion Torrent sequencing, e.g., U.S. patent publication 2010 / 0137143 or 2010 / 0304982), micromachined membranes (such as with SMRT sequencing, e.g. Eid et al, Science, 323: 133-138 (2009)), or bead arrays (as with SOLiD sequencing or polony sequencing, e.g. Kim et al, Science, 316: 1481 -1414 (2007)). Such methods may comprise amplifying the isolated molecules either before or after they are spatially isolated on a solid surface. Prior amplification may comprise emulsion-based amplification, such as emulsion PCR or rolling circle amplification.

[0281] Of particular interest is sequencing on the Illumina MiSeq, NextSeq, and HiSeq platforms, which use reversible-terminator sequencing by synthesis technology (see, e.g., Shen et al. (2012) BMC Bioinformatics 13:160; Junemann et al. (2013) Nat. Biotechnol. 31 (4):294-296; Glenn (201 1 ) Mol. Ecol. Resour. 11 (5):759-769; Thudi et al. (2012) Brief Funct. Genomics 1 1 (1 ):3-11 ; herein incorporated by reference); the OxfordNanopore Technologies Inc. MinlON, GridlON, and PromethlON nanopore sequencing platforms, which can be used to determine the sequences of DNA or RNA by monitoring changes in electrical current as nucleic acids are passed through a protein nanopore (see, e.g., Lu et al. (2016) Genomics Proteomics Bioinformatics 14(5):265-279, Petersen et al. (2019) J. Clin. Microbiol. 58(1 ) :e01315-19, Kono et al. (2019) Dev Growth Differ. 61 (5):316-326, Deamer et al. (2016) Nat. Biotechnol. 34(5):518-24, Madoui et al. (2015) BMC Genomics 16:327, Szalay et al. (2015) Nat. Biotechnol 33, 1087-1091 ; herein incorporated by reference); the PacBIO Single Molecule, Real-Time (SMRT) sequencing platforms, including the Sequel, HiFi, and RS II sequencing platforms (see, e.g., Ardui et al. (2018) Nucleic Acids Res. 46(5):2159-2168, An et al. (2018) Genes (Basel) 9(1 ):43), Nakano et al. (2017) Hum Cell. 30(3):149-161 ; herein incorporated by reference), the Omniome sequencing by binding (SBB®) short-read sequencing platform using high fidelity plasmonic nanohole arrays (see, e.g., Cetin et al. (2018) ACS Sens. 3(3):561 -568; herein incorporated by reference), the Gynapsys compact DNA sequencer, which uses metal oxide semiconductor (CMOS) sequencing chips for electronic data detection and sequencing by synthesis (SBS) chemistry, the Singular Genomics G4 benchtop sequencing platform, which uses SBS chemistry, and the Element Biosciences AVITI™ benchtop sequencer, which uses a modified form of SBS chemistry that reduces reagent usage.

[0282] The subject methods are also compatible with single-cell RNA sequencing technologies (see, e.g., Slovin et al. (2021 ) Methods Mol Biol. 2284:343-365, Jovic et al. (2022) Clin. Transl. Med. 12(3):e694, Zhang et al. (2020) Nat. Commun. 1 1 (1 ):21 18, Hwang et al. (2018) Exp Mol Med. 50(8):1 -14, Chen et al. (2019) Front Genet. 10:317, and Hedlund et al. (2018) Mol Aspects Med. 59:36-46; herein incorporated by reference). Single-cell RNA sequencing typically involves isolation of single cells, cell lysis, mRNA capture, reverse transcription (conversion of mRNA into cDNA), cDNA amplification, and sequencing of cDNA amplicons. Barcodes in the multi-module gene editing constructs can be used to identify mRNA molecules transcribed from the constructs to determine what genome editing modules were present in an individual cell.Amplification of Multi-Module Gene Editing Constructs

[0283] Any primer-dependent amplification method known in the art may be used for amplification of a complete multi-module gene editing construct or a portion of a multimodule gene editing construct comprising its barcode region. Nucleic acid amplificationmethods include, without limitation, polymerase chain reaction (PCR), rolling circle amplification, and isothermal amplification methods such as recombinase polymerase amplification (RPA), loop-mediated isothermal amplification (LAMP), strand displacement amplification (SDA), helicase-dependent amplification (HDA), nicking enzyme amplification reaction (NEAR), ligase chain reaction (LGR), nucleic acid sequence based amplification (NASBA), transcription-mediated amplification (TMA), Q- beta amplification, and the like.

[0284] In some embodiments, polymerase chain reaction (PCR)-based techniques are used to amplify a construct or barcode region. PCR is a technique for amplifying a desired target nucleic acid sequence contained in a nucleic acid molecule or mixture of molecules. In PCR, a pair of primers is employed in excess to hybridize to the complementary strands of the target nucleic acid. The primers are each extended by a polymerase using the target nucleic acid as a template. The extension products become target sequences themselves after dissociation from the original target strand. New primers are then hybridized and extended by a polymerase, and the cycle is repeated to geometrically increase the number of target sequence molecules. The PCR method for amplifying target nucleic acid sequences in a sample is well known in the art and has been described in, e.g., Innis et al. (eds.) PCR Protocols (Academic Press, NY 1990); Taylor (1991 ) Polymerase chain reaction: basic principles and automation, in PCR: A Practical Approach, McPherson et al. (eds.) IRL Press, Oxford; Saiki et al. (1986) Nature 324:163; as well as in U.S. Pat. Nos. 4,683,195, 4,683,202 and 4,889,818, all incorporated herein by reference in their entireties.

[0285] In particular, PCR uses relatively short oligonucleotide primers which flank the target nucleotide sequence to be amplified, oriented such that their 3' ends face each other, each primer extending toward the other. The polynucleotide sample is extracted and denatured, preferably by heat, and hybridized with first and second primers that are present in molar excess. Polymerization is catalyzed in the presence of the four deoxyribonucleotide triphosphates (dNTPs — dATP, dGTP, dCTP and dTTP) using a primer- and template-dependent polynucleotide polymerizing agent, such as any enzyme capable of producing primer extension products, for example, E. coli DNA polymerase I, Klenow fragment of DNA polymerase I, T4 DNA polymerase, thermostable DNA polymerases isolated from Thermus aquaticus (Taq), available from a variety of sources (for example, Perkin Elmer), Thermus thermophilus polymerase (United States Biochemicals), Bacillus stereothermophilus polymerase (Bio-Rad), Thermococcus inlitoralis polymerase (“Vent” polymerase, New England Biolabs), Pyrococcus species GB- D polymerase (“Deep Vent” polymerase, New England Biolabs), Pyrococcus woesei polymerase (Pwo polymerase, Sigma-Aldrich) or Pyrococcus furiosus polymerase (Pfu polymerase from Promega Corporation). This results in two “long products” which contain the respective primers at their 5' ends covalently linked to the newly synthesized complements of the original strands. The reaction mixture is then returned to polymerizing conditions, e.g., by lowering the temperature, inactivating a denaturing agent, or adding more polymerase, and a second cycle is initiated. The second cycle provides the two original strands, the two long products from the first cycle, two new long products replicated from the original strands, and two “short products” replicated from the long products. The short products have the sequence of the target sequence with a primer at each end. On each additional cycle, an additional two long products are produced, and a number of short products equal to the number of long and short products remaining at the end of the previous cycle. Thus, the number of short products containing the target sequence grows exponentially with each cycle. Preferably, PGR is carried out with a commercially available thermal cycler, e.g., Perkin Elmer.

[0286] RNA transcripts of a multi-module gene editing construct may be amplified by reverse transcribing the RNA into cDNA, and then performing PGR (RT-PCR), as described above. Alternatively, a single enzyme may be used for both steps as described in U.S. Pat. No. 5,322,770, incorporated herein by reference in its entirety. RNA may also be reverse transcribed into cDNA, followed by asymmetric gap ligase chain reaction (RT- AGLCR) as described by Marshall et al. (1994) PGR Meth. App. 4:80-84.

[0287] PGR primers should be of sufficient length to provide for hybridization to complementary template DNA under annealing conditions. The primers will generally be at least 6 bp in length, including but not limited to e.g., at least 10 bp in length, at least 15 bp in length, at least 16 bp in length, at least 17 bp in length, at least 18 bp in length, at least 19 bp in length, at least 20 bp in length, at least 21 bp in length, at least 22 bp in length, at least 23 bp in length, at least 24 bp in length, at least 25 bp in length, at least 26 bp in length, at least 27 bp in length, at least 28 bp in length, at least 29 bp in length, at least 30 bp in length, and may be as long as 60 bp in length or longer, where the length of the primers will generally range from 18 to 50 bp in length, including but not limited to, e.g., from about 20 to 35 bp in length. In some instances, the template DNA may be contacted with a single primer or a set of two primers (forward and reverse primers), depending on whether primer extension, linear or exponential amplification of thetemplate DNA is desired. Methods of PCR that may be employed in the subject methods include but are not limited to those described in U.S. Pat. Nos.: 4,683,202; 4,683,195; 4,800,159; 4,965,188 and 5,512,462, the disclosures of which are herein incorporated by reference.

[0288] Alternatively, a polymerase that preferentially uses dUTP rather than dTTP can be used to perform PCR. Such polymerases include archaeal family B DNA polymerases such as Nanoarchaeum equitans B DNA polymerase, which can utilize deaminated bases such as uracil and hypoxanthine and performs PCR with higher fidelity than Thermus aquaticus (Taq) DNA polymerase (e.g., as described in Choi et al. (2008) AppL Environ. Microbiol. 74(21 ): 6563-6569; herein incorporated by reference). In addition, engineered polymerases such as Q5U Hot Start High-Fidelity DNA Polymerase from New England Biolabs (Ipswich, MA) and Phusion U DNA polymerase from Thermo Fisher Scientific (Waltham, MA), which contain a mutation in the nucleotide-binding pocket that enables these polymerases to amplify templates containing uracil and inosine bases, may be used to perform PCR with dUTP. The use of polymerases that utilize UTP is useful for preventing carryover contamination in different PCR runs. The uracil-containing amplicon products of such polymerases can be digested by a uracil-DNA glycosylase to remove residual products from previous PCR amplifications and suppress template contamination between runs.

[0289] In addition, one or more PCR additives or enhancing agents may be included to improve the yield of the amplification reaction, for example, by reducing secondary structure in a nucleic acid or mispriming events. Such additives or enhancing agents include, but are not limited to, dimethyl sulfoxide (DMSO), N,N,N-trimethylglycine (betaine), formamide, glycerol, nonionic detergents (e.g., Triton X-100, Tween 20, and Nonidet P-40 (NP-40)), 7-deaza-2'-deoxyguanosine, bovine serum albumin, T4 gene 32 protein, polyethylene glycol, 1 ,2-propanediol, and tetramethylammonium chloride.

[0290] A PCR reaction will generally be carried out by cycling the reaction mixture between appropriate temperatures for annealing, elongation / extension, and denaturation for specific times. Such temperature and times will vary and will depend on the particular components of the reaction including, e.g., the polymerase and the primers as well as the expected length of the resulting PCR product. In some instances, e.g., where nested or two-step PCR are employed the cycling-reaction may be carried out in stages, e.g., cycling according to a first stage having a particular cycling program or using particulartemperature(s) and subsequently cycling according to a second stage having a particular cycling program or using particular temperature(s).

[0291] Multistep PCR processes may or may not include that addition of one or more reagents following the initiation of amplification. For example, in some instances, amplification may be initiated by elongation with the use of a polymerase and, following an initial phase of the reaction, additional reagent(s) (e.g., one or more additional primers, additional enzymes, etc.) may be added to the reaction to facilitate a second phase of the reaction. In some instances, amplification may be initiated with a first primer or a first set of primers and, following an initial phase of the reaction, additional reagent(s) (e.g., one or more additional primers, additional enzymes, etc.) may be added to the reaction to facilitate a second phase of the reaction. In certain embodiments, the initial phase of amplification may be referred to as “preamplification”.

[0292] In particular, the subject methods are applicable to digital PCR techniques. For digital PCR, a sample containing nucleic acids is separated into a large number of partitions before performing PCR. Partitioning can be achieved in a variety of ways known in the art, for example, by use of micro well plates, capillaries, emulsions, arrays of miniaturized chambers or nucleic acid binding surfaces. Separation of the sample may involve distributing any suitable portion including up to the entire sample among the partitions. Each partition includes a fluid volume that is isolated from the fluid volumes of other partitions. The partitions may be isolated from one another by a fluid phase, such as a continuous phase of an emulsion, by a solid phase, such as at least one wall of a container, or a combination thereof. In certain embodiments, the partitions may comprise droplets disposed in a continuous phase, such that the droplets and the continuous phase collectively form an emulsion.

[0293] The partitions may be formed by any suitable procedure, in any suitable manner, and with any suitable properties. For example, the partitions may be formed with a fluid dispenser, such as a pipette, with a droplet generator, by agitation of the sample (e.g., shaking, stirring, sonication, etc.), and the like. Accordingly, the partitions may be formed serially, in parallel, or in batch. The partitions may have any suitable volume or volumes. The partitions may be of substantially uniform volume or may have different volumes. Exemplary partitions having substantially the same volume are monodisperse droplets. Exemplary volumes for the partitions include an average volume of less than about 100, 10 or 1 mL, less than about 100, 10, or 1 n L, or less than about 100, 10, or 1 pL, among others.

[0294] After separation of the sample, PCR is carried out in the partitions. The partitions, when formed, may be competent for performance of one or more reactions in the partitions. Alternatively, one or more reagents may be added to the partitions after they are formed to render them competent for reaction. The reagents may be added by any suitable mechanism, such as a fluid dispenser, fusion of droplets, or the like.

[0295] In some embodiments, nucleic acids are amplified by emulsion PCR to compartmentalize the amplification reactions of individual DNA molecules. An aqueous PCR mixture with forward and reverse primers is mixed with an oil to create the emulsion. Preferably, each droplet of water in the oil emulsion contains one bead and one molecule of template DNA, such that individual molecules are amplified in separate emulsion droplets. After amplification, the emulsion is broken, e.g., using isopropanol and detergent with vortexing. In some embodiments, the gene fragment library and the sequencing library are bound to magnetic beads or superparamagnetic beads prior to amplification, wherein amplification and breaking of the emulsion is followed by magnetic separation of the beads. For a description of emulsion PCR, see, e.g., Kanagal- Shamanna et al. (2016) Methods Mol Biol. 1392:33-42, Zhu et al. (2012) Anal Bioanal Chem. 403(8):2127-43, Zhang et al. (2020) Lab Chip 20(13):2328-2333, Siu et al. (2021 ) Taianta 221 :121593, Zheng et al. (201 1 ) Nat. Protoc. 6(9) :1367-1376, and Kojima et al. (2015) Methods Mol. Biol. 2015;1347:87-100; herein incorporated by reference.

[0296] After PCR amplification, nucleic acids can be quantified by counting the partitions that contain PCR amplicons. Partitioning of the sample allows quantification of the number of different molecules by assuming that the population of molecules follows a Poisson distribution. For a description of digital PCR methods, see, e.g., Hindson et al. (2011 ) Anal. Chem. 83(22):8604-8610; Pohl and Shih (2004) Expert Rev. Mol. Diagn. 4(1 ):41 -47; Pekin et al. (201 1 ) Lab Chip 11 (13): 2156-2166; Pinheiro et al. (2012) Anal. Chem. 84 (2): 1003-101 1 ; Day et al. (2013) Methods 59(1 ):101 -107; herein incorporated by reference in their entireties.

[0297] In some instances, amplification may be carried out under isothermal conditions, e.g., by means of isothermal amplification. Methods of isothermal amplification generally make use of enzymatic means of separating DNA strands to facilitate amplification at constant temperature, such as, e.g., strand-displacing polymerase or a helicase, thus negating the need for thermocycling to denature DNA. Any convenient and appropriate means of isothermal amplification may be employed in the subject methods including but are not limited to: recombinase polymerase amplification (RPA), loop-mediatedisothermal amplification (LAMP), strand displacement amplification (SDA), helicasedependent amplification (HDA), nicking enzyme amplification reaction (NEAR), ligase chain reaction (LGR), nucleic acid sequence based amplification (NASBA), transcription- mediated amplification (TMA), Q-beta amplification, and the like.

[0298] RPA combines isothermal recombinase-mediated primer targeting with stranddisplacement DNA synthesis (Piepenburg et al. (2006) PLOS Biology. 4 (7): e204; herein incorporated by reference). The technique uses two primers together with a recombinase, a single-stranded DNA-binding protein, and a strand-displacing polymerase for amplification. Unlike PCR, heat is not required for melting of the DNA strands. Instead, a recombinase-primer complex is used for localized strand exchange to place oligonucleotide primers at homologous sequences of the DNA template. The single-stranded DNA-binding protein binds to the displaced template strand to prevent the primers from being ejected by branch migration. Dissociation of the recombinase leaves the 3'-end of the primer accessible to the strand displacing DNA polymerase (e.g., the large fragment of Bacillus subtilis Pol I), which catalyzes primer extension. Cyclic repetition of this process results in exponential amplification.

[0299] LAMP generally utilizes a plurality of primers, e.g., 4-6 primers, which may recognize a plurality of distinct regions, e.g., 6-8 distinct regions, of target DNA. Synthesis is generally initiated by a strand-displacing DNA polymerase with two of the primers forming loop structures to facilitate subsequent rounds of amplification. LAMP is rapid and sensitive. In addition, the magnesium pyrophosphate produced during the LAMP amplification reaction may, in some instances, be visualized without the use of specialized equipment, e.g., by eye.

[0300] SDA generally involves the use of a strand-displacing DNA polymerase (e.g., Bst DNA polymerase, Large (Klenow) Fragment polymerase, Klenow Fragment (3'-5' exo-), and the like) to initiate at nicks created by a strand-limited restriction endonuclease or nicking enzyme at a site contained in a primer. In SDA, the nicking site is generally regenerated with each polymerase displacement step, resulting in exponential amplification.

[0301] HDA generally employs: a helicase which unwinds double-stranded DNA unwinding to separate strands; primers, e.g., two primers, that may anneal to the unwound DNA; and a strand-displacing DNA polymerase for extension.

[0302] NEAR generally involves a strand-displacing DNA polymerase that initiates elongation at nicks, e.g., created by a nicking enzyme. NEAR is rapid and sensitive, quickly producing many short nucleic acids from a target sequence.

[0303] Nucleic acid sequence-based amplification (NASBA) is an isothermal RNA- specific amplification method that does not require thermal cycling instrumentation. RNA is initially reverse transcribed such that the single-stranded RNA target is copied into a double-stranded DNA molecule that serves as a template for RNA transcription. Detection of the amplified RNA is typically accomplished either by electrochemiluminescence or in real-time, for example, with fluorescently labeled molecular beacon probes. See, e.g., Lau et al. (2006) Dev. Biol. (Basel) 126:7-15; and Deiman et al. (2002) Mol. Biotechnol. 20(2):163-179.

[0304] The Ligase Chain Reaction (LCR) is an alternate method for nucleic acid amplification. In LCR, probe pairs are used which include two primary (first and second) and two secondary (third and fourth) probes, all of which are employed in molar excess to the target. The first probe hybridizes to a first segment of the target strand, and the second probe hybridizes to a second segment of the target strand, the first and second segments being contiguous so that the primary probes abut one another in 5' phosphates' hydroxyl relationship, and so that a ligase can covalently fuse or ligate the two probes into a fused product. In addition, a third (secondary) probe can hybridize to a portion of the first probe and a fourth (secondary) probe can hybridize to a portion of the second probe in a similar abutting fashion. If the target is initially double stranded, the secondary probes also will hybridize to the target complement in the first instance. Once the ligated strand of primary probes is separated from the target strand, it will hybridize with the third and fourth probes which can be ligated to form a complementary, secondary ligated product. It is important to realize that the ligated products are functionally equivalent to either the target or its complement. By repeated cycles of hybridization and ligation, amplification of the target sequence is achieved. This technique is described more completely in ERA 320,308 to K. Backman published Jun. 16, 1989 and EPA 439,182 to K. Backman et aL, published Jul. 31 , 1991 , both of which are incorporated herein by reference.

[0305] Other known methods for amplification of nucleic acids include, but are not limited to, self-sustained sequence replication (3SR) described by Guatelli et aL, Proc. Natl. Acad. Sci. USA (1990) 87:1874-1878 and J. Compton, Nature (1991 ) 350:91 -92 (1991 ); Q-beta amplification; strand displacement amplification (as described in Walker etaL, Clin. Chem. (1996) 42:9-13 and EPA 684,315; target mediated amplification, as described in International Publication No. WO 93 / 22461 , and the TaqMan™ assay.

[0306] In some instances, entire amplification methods may be combined, or aspects of various amplification methods may be recombined to generate a hybrid amplification method. For example, in some instances, aspects of PGR may be used, e.g., to generate the initial template or amplicon or first round or rounds of amplification, and an isothermal amplification method may be subsequently employed for further amplification. In some instances, an isothermal amplification method or aspects of an isothermal amplification method may be employed, followed by PGR for further amplification of the product of the isothermal amplification reaction. In some instances, a sample may be preamplified using a first method of amplification and may be further processed, including e.g., further amplified or analyzed, using a second method of amplification. As a non-limiting example, a sample may be preamplified by PGR and further analyzed by qPCR. In some instances, the method further comprises monitoring the amplification of a target DNA molecule such as is performed in, e.g., real-time PGR, also referred to herein as quantitative PGR (qPCR).

[0307] The fluorogenic 5' nuclease assay is conveniently performed using, for example, AmpliTaq Gold™ DNA polymerase, which has endogenous 5' nuclease activity, to digest an internal oligonucleotide probe labeled with both a fluorescent reporter dye and a quencher (see, Holland et al., Proc. Natl. Acad. Sci. USA (1991 ) 88:7276-7280; and Lee et al., Nucl. Acids Res. (1993) 21 :3761 -3766). Assay results are detected by measuring changes in fluorescence that occur during the amplification cycle as the fluorescent probe is digested, uncoupling the dye and quencher labels and causing an increase in the fluorescent signal that is proportional to the amplification of target nucleic acid.

[0308] The amplification products can be detected in solution or using solid supports. In this method, the TaqMan™ probe is designed to hybridize to a target sequence within the desired PGR product. The 5' end of the TaqMan™ probe contains a fluorescent reporter dye. The 3' end of the probe is blocked to prevent probe extension and contains a dye that will quench the fluorescence of the 5' fluorophore. During subsequent amplification, the 5' fluorescent label is cleaved off if a polymerase with 5' exonuclease activity is present in the reaction. Excision of the 5' fluorophore results in an increase in fluorescence that can be detected. For a detailed description of the TaqMan™ assay, reagents and conditions for use therein, see, e.g., Holland et al., Proc. Natl. Acad. Sci,U.S.A. (1991 ) 88:7276-7280; U.S. Pat. Nos. 5,538,848, 5,723,591 , and 5,876,930, all incorporated herein by reference in their entireties.

[0309] TMA is an isothermal, autocatalytic nucleic acid target amplification system that can provide more than a billion RNA copies of a target sequence, and thus provides a method of identifying target nucleic acid sequences present in very small amounts in a sample. For a detailed description of TMA assay methods, see, e.g., Hill (2001 ) Expert Rev. Mol. Diagn. 1 :445-55; WO 89 / 1050; WO 88 / 10315; EPO Publication No. 408,295; EPO Application No. 881 1394-8.9; WO91 / 02818; U.S. Pat. Nos. 5,399,491 , 6,686,156, and 5,556,771 , all incorporated herein by reference in their entireties.

[0310] Suitable DNA polymerases include reverse transcriptases, such as avian myeloblastosis virus (AMV) reverse transcriptase (available from, e.g., Seikagaku America, Inc.) and Moloney murine leukemia virus (MMLV) reverse transcriptase (available from, e.g., Bethesda Research Laboratories).Therapeutic Cells

[0311] In some embodiments, a cell is genetically modified using a multi-module gene editing construct to produce a therapeutic cell with multiple genetic changes that may be used in cellular therapy. The cell may be any suitable type of cell for transplanting to an individual in need. For example, the cell may be a stem cell, progenitor cell, or mature cell. The cell may be autologous, allogeneic, or xenogeneic.

[0312] In some cases, therapeutic cells include cells whose activity is conditional, e.g., cells that modulate their function based on the physiological state of the host and / or the environment of the host tissue. The therapeutic cell may be a type of cell that specifically possesses the functional activity by virtue of its cell type (e.g., by differentiating or having differentiated into a cell type that exhibits the functional activity) or may be genetically modified to exhibit the functional activity that was not exhibited by the cell before being genetically modified.

[0313] In some embodiments, the cell secretes a biological agent, e.g., a signaling molecule, a hormone, a growth factor, a cytokine, a chemokine, a neuropeptide, an enzyme, an antibody, etc. In some cases, the therapeutic cells include cells (e.g., immune cells, such as cytotoxic T lymphocytes) that interact with targets at or in the vicinity in the host tissue in which cells are transplanted.

[0314] Exemplary therapeutic molecules that can be secreted by a therapeutic cell include, without limitation, insulin, human growth hormone, thyroxine, glucagon-likepeptide-1 (GLP-1 ), GLP-1 (7-37), GLP-1 (7-36), and like GLP-1 receptor agonist polypeptides, GLP-2, interleukins 1 to 33 (e.g., IL-1 , IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11 , IL-12, IL-13, IL-17, IL-18, IL-21 , IL-22, IL-27, IL-33), interferon (a, p, y), GM-CSF, G-CSF, M-CSF, SCF, FAS ligands, TRAIL, leptin, adiponectin, blood coagulation factor Vlll / blood coagulation factor IX, von Willebrand factor, glucocerebrosidase, lipoprotein lipase (LPL), lecithin-cholesterol acyltransferase (LCAT), erythropoietin, apoA-l, albumin, atrial natriuretic peptide (ANP), luteinizing hormone releasing hormone (LHRH), angiostatin / endostatin, endogenous opioid peptides (enkephalins, endorphins, etc.), calcitonin / bone morphogenetic protein (BMP), pancreatic secretory trypsin inhibitors, catalase, superoxide dismutase, anti-TNF-a antibody, soluble IL-6 receptor, IL-1 receptor antagonist, a2 antitrypsin, etc.

[0315] In some cases, the cell is a stem cell or stem cell-derived cell. Stem cells of interest include, without limitation, hematopoietic stem cells, embryonic stem cells, adult stem cells, mesenchymal stem cells, neural stem cells, epidermal stem cells, endothelial stem cells, gastrointestinal stem cells, liver stem cells, cord blood stem cells, amniotic fluid stem cells, skeletal muscle stem cells, smooth muscle stem cells (e.g., cardiac smooth muscle stem cells), pancreatic stem cells, olfactory stem cells, induced pluripotent stem cells; and the like; as well as differentiated cells that can be cultured in vitro and used in a therapeutic regimen, where such cells include, but are not limited to, keratinocytes, adipocytes, cardiomyocytes, neurons, osteoblasts, pancreatic islet cells, retinal cells, and the like. The cell that is used will depend in part on the nature of the disorder or condition to be treated.

[0316] Suitable human embryonic stem (ES) cells include, but are not limited to, any of a variety of available human ES lines, e.g., BG01 (hESBGN-01 ), BG02 (hESBGN-02), BG03 (hESBGN-03) (BresaGen, Inc.; Athens, Ga.); SA01 (Sahlgrenska 1 ), SA02 (Sahlgrenska 2) (Cellartis AB; Goeteborg, Sweden); ES01 (HES-1 ), ES01 (HES-2), ES03 (HES-3), ES04 (HES-4), ES05 (HES-5), ES06 (HES-6) (ES Cell International; Singapore); UC01 (HSF-1 ), UC06 (HSF-6) (University of California, San Francisco; San Francisco, Calif.); WA01 (H1 ), WA07 (H7), WA09 (H9), WA09 / Oct4D10 (H9-hOct4-pGZ), WA13 (H13), WA14 (H14) (Wisconsin Alumni Research Foundation; WARF; Madison, Wis.). Cell line designations are given as the National Institutes of Health (NIII) code, followed in parentheses by the provider code.

[0317] Hematopoietic stem cells (HSCs) are mesoderm-derived cells that can be isolated from bone marrow, blood, cord blood, fetal liver and yolk sac. HSCs are characterized asCD34+and CD3“. HSCs can repopulate the erythroid, neutrophil-macrophage, megakaryocyte and lymphoid hematopoietic cell lineages in vivo. In vitro, HSCs can be induced to undergo at least some self- renewing cell divisions and can be induced to differentiate to the same lineages as is seen in vivo. As such, HSCs can be induced to differentiate into one or more of erythroid cells, megakaryocytes, neutrophils, macrophages, and lymphoid cells.

[0318] Neural stem cells (NSCs) are capable of differentiating into neurons, and glia (including oligodendrocytes, and astrocytes). A neural stem cell is a multipotent stem cell which is capable of multiple divisions, and under specific conditions can produce daughter cells which are neural stem cells, or neural progenitor cells that can be neuroblasts or glioblasts, e.g., cells committed to become one or more types of neurons and glial cells respectively. Methods of obtaining NSCs are known in the art.

[0319] Mesenchymal stem cells (MSCs) can be obtained from connective tissue including, without limitation, bone marrow, placenta, umbilical cord blood, adipose tissue, muscle, corneal stroma, and dental pulp of deciduous baby teeth. MSCs can differentiate to form muscle, bone, cartilage, fat, marrow stroma, and tendon. Methods of isolating MSCs are known in the art; and any known method can be used to obtain MSCs.

[0320] An induced pluripotent stem (iPS) cell is a pluripotent stem cell induced from a somatic cell, e.g., a differentiated somatic cell. iPS cells are capable of self-renewal and differentiation into cell fate-committed stem cells, including neural stem cells, as well as various types of mature cells. iPS cells can be generated from somatic cells, including skin fibroblasts, using, e.g., known methods. iPS cells can be generated from somatic cells (e.g., skin fibroblasts) by genetically modifying the somatic cells with one or more expression constructs encoding Oct-3 / 4 and Sox2. In some embodiments, somatic cells are genetically modified with one or more expression constructs comprising nucleotide sequences encoding Oct-3 / 4, Sox2, c-myc, and K1 f4. In some embodiments, somatic cells are genetically modified with one or more expression constructs comprising nucleotide sequences encoding Oct-4, Sox2, Nanog, and LIN28. Methods of generating iPS are known in the art, and any such method can be used to generate iPS.

[0321] In some embodiments, the therapeutic cells include insulin-secreting cells. The insulin-secreting cells may be any suitable type of insulin-secreting cell. In some cases, the insulin-secreting cells are a type of cell that secretes insulin (e.g., pancreatic p islet cells, or p-like cells). In some cases, the insulin-secreting cells are primary islet cells (e.g., mature p islet cells isolated from a pancreas). In some cases, the insulin-secretingcells are p cells, or p-like cells that are derived in vitro from immature cells, precursor cells, progenitor cells, or stem cells. The insulin-secreting cells may be derived from (i.e., obtained by differentiating) stem and / or progenitor cells such as hepatocytes (e.g., transdifferentiated hepatocytes), acinar cells, pancreatic duct cells, stem cells, embryonic stem cells (ES), partially differentiated stem cells, non-pluripotent stem cells, pluripotent stem cells, induced pluripotent stem cells (iPS cells), etc. Suitable insulin-secreting cells and methods of generating the same are described in, e.g., US20030082810; US20120141436; and Raikwar et al. (PLoS One. 2015 Jan 28;10(1 ):e01 16582), each of which are incorporated herein by reference.

[0322] The insulin-secreting cells may produce (e.g., secrete) insulin at a rate independent of the ambient / extracellular glucose concentration (e.g., the concentration of glucose in the host tissue in which the tissue graft is implanted), or may produce (e.g., secrete) insulin at a rate that depends on the ambient / extracellular glucose concentration. In some cases, the insulin-secreting cells constitutively secrete insulin. In some embodiments, the insulin-secreting cells increase insulin secretion when the ambient / extracellular glucose concentration increases, and decreases insulin secretion when the ambient / extracellular glucose concentration decreases.

[0323] In some embodiments, the therapeutic cells are immune cells such as, T cells, B cells, natural killer cells, neutrophils, eosinophils, mast cells, basophils, monocytes, macrophages, or dendritic cells. In some cases, the therapeutic cells are lymphocytes, such as CD4+ and / or CD8+ T lymphocytes, or B lymphocytes. In some embodiments, the therapeutic cells are CD4+helper T cells, CD8+cytotoxic T cells, natural killer T cells, or gamma delta T cells.

[0324] In some embodiments, the multi-module gene editing construct provides a gene knockin resulting in expression of an exogenous polypeptide in the cell. The exogenous polypeptide may be any type of protein / peptide of interest, including, without limitation, an enzyme, an extracellular matrix protein, a receptor, a transporter, an ion channel, or other membrane protein, a hormone, a neuropeptide, a growth factor, a cytokine, an antibody, or a cytoskeletal protein; or a fragment thereof, or a biologically active domain of interest.

[0325] In some embodiments, the exogenous protein is a chimeric antigen receptor (CAR) that binds specifically to a target antigen. For example, an immune cell such as a T cell or macrophage can be genetically modified using a multi-module gene editing construct to express a CAR that targets the immune cell to a pathogenic cell or particlein need of eradication. For example, lymphocytes, e.g., cytotoxic T cells, may be engineered to express a CAR that specifically binds to an antigen on a pathogenic cell that is associated with a disease, e.g., cancer or tumor, fibrosis, or antibiotic-resistant infection that is to be treated with the genetically modified cell.

[0326] In certain embodiments, at least one genome editing module in the multi-module gene editing construct produces a CAR gene knockin in a T cell, and at least one genome editing module in the multi-module gene editing construct produces a T cell receptor (TCR) gene knockout in the T cell. In some embodiments, another gene knockout at another genomic target locus is used to reduce graft versus host disease and / or improve cell function and survival. In certain embodiments, the multi-module gene editing construct is used for knockout of a gene encoding an alloantigen in order to reduce graft versus host disease. Exemplary alloantigens include, but are not limited to, major histocompatibility complex (MHC) class I alloantigens, MNS blood group alloantigens, CD1 , CD2, CD3, CD4, CD7, CD8, Ly-6, Qa-2, RT6, CD19, CD22, CD56, CD58 (LFA-3), CD59, and CDw90 (Thy 1 ), any one of which or any combination of which may be knocked out in the genetically modified cell using the methods described herein. In certain embodiments, at least one genome editing module in the multi-module gene editing construct produces a knockout of a CD5, CD52, CD70, BATF, LCK, PD-1 , LAG- 3, CTLA-4, P2-B2M, PD-1 , HLA-I, Fas, TGFBR2, PDCD-1 , DGK, EZH2, PAX5, or LDLR gene.

[0327] In some embodiments, the cells are further engineered to express a fluorescent protein, for example, to allow cells derived from different donors to be distinguished in multiplexed screening. In some embodiments, cells from the same donor are engineered to express the same fluorescent protein, and cells from different donors are engineered to express different fluorescent proteins. Exemplary fluorescent proteins include, without limitation, green fluorescent protein (GFP), enhanced green fluorescent protein (EGFP), superfolder GFP, emerald, Azami Green, mWasabi, TagGFP, TurboGFP, red fluorescent protein, blue fluorescent protein (BFP), EBFP, EBFP2, mTagBFP, Azurite, cyan fluorescent protein (CFP), mECFP, Cerulean, mCerulean, mTurquoise, CyPet, AmCyanl , Midori-lshi Cyan, yellow fluorescent protein (YFP), EYFP, Topaz, Venus, YPet, mCitrine, mBanana, orange fluorescent protein (OFP), Kusabira Orange, Kusabira Orange2, mOrange, mOrange2, dTomato, TagRFP, DsRed, DsRed2, mTangerine, red fluorescent protein (RFP), mRuby, mRuby2, mApple, mStrawberry, mCherry,mRaspberry, AsRed2, mRFP1 , JRed, dKeima-Tandem, Dronpa, mPlum, E2-Crimson, and aequorin.

[0328] In some instances, a population of cells may be enriched for those comprising desired genetic modifications by separating the genetically modified cells of interest from the remaining population. Separation of genetically modified cells typically relies upon the expression of a selectable marker co-integrated with the intended edits at target loci. After integration of a multi-module gene editing construct into the genome of a cell, positive selection is performed to isolate cells from a population, e.g. to create an enriched population of cells comprising the desired genetic modifications.

[0329] Cell separation may be accomplished by any convenient separation technique appropriate for the selection marker used, including, but not limited to flow cytometry, fluorescence activated cell sorting (FACS), magnetic-activated cell sorting (MACS), elutriation, immunopurification, and affinity chromatography. For example, if a fluorescent marker is used, cells may be separated by fluorescence activated cell sorting (FACS), whereas if a cell surface marker is used, cells may be separated from the heterogeneous population by affinity separation techniques, e.g., MACS, affinity chromatography, "panning" with an affinity reagent attached to a solid matrix, immunopurification with an antibody specific for the cell surface marker, or other convenient technique.

[0330] In certain embodiments, positive and / or negative selection of genetically modified cells is performed using a binding agent that specifically binds to a selection marker on a cell (e.g., such as produced from integration of a donor polynucleotide at a target genomic locus). Examples of binding agents include, without limitation, antibodies, antibody fragments, antibody mimetics, aptamers, and ligands. In some embodiments, the binding agent binds to the selection marker with high affinity.

[0331] The binding agent may be immobilized on a solid support to facilitate removal of cells having a selection marker from a liquid sample. The binding agent may be associated with the solid support either directly or indirectly. Binding agents may be immobilized on the surface of a solid support, such as, but not limited to, a non-magnetic bead, magnetic bead, rod, particle, plate, slide, wafer, strand, disc, membrane, film, or the inner surface of a tube, channel, column, flow cell device, or microfluidic device. A solid support may comprise various materials, including, but not limited to glass, quartz, silicon, metal, ceramic, plastic, nylon, polyacrylamide, agarose, resin, porous polymer monoliths, hydrogels, and composites thereof. Additionally, a substrate may be added to the surface of a solid support to facilitate attachment of a binding agent.

[0332] In positive selection, cells carrying a selection marker are collected, whereas in negative selection, cells carrying a selection marker are removed from a cell population. For example, in positive selection, a binding agent specific for a surface marker can be immobilized on a solid support (e.g., column or magnetic bead) and used to collect cells of interest on the solid support. Cells that are not of interest do not bind to the solid support (e.g., flow through the column or do not attach to the magnetic beads). In negative selection, the binding agent is used to deplete a cell population of cells that are not of interest. The cells of interest are those that do not bind to the binding agent (e.g., flow through the column or remain after the magnetic beads are removed).

[0333] In certain embodiments, the binding agent comprises an antibody that specifically binds to the selection marker on a cell. Any type of antibody may be used, including polyclonal and monoclonal antibodies, hybrid antibodies, altered antibodies, chimeric antibodies and, humanized antibodies, as well as: hybrid (chimeric) antibody molecules (see, for example, Winter et al. (1991 ) Nature 349:293-299; and U.S. Pat. No. 4,816,567); F(ab')2 and F(ab) fragments; Fvmolecules (noncovalent heterodimers, see, for example, Inbar et al. (1972) Proc Natl Acad Sci USA 69:2659-2662; and Ehrlich et al. (1980) Biochem 19:4091 -4096); single-chain Fv molecules (sFv) (see, e.g., Huston et al. (1988) Proc Natl Acad Sci USA 85:5879-5883); nanobodies or single-domain antibodies (sdAb) (see, e.g., Wang et al. (2016) Int J Nanomedicine 11 :3287-3303, Vincke et al. (2012) Methods Mol Biol 91 1 :15-26; dimeric and trimeric antibody fragment constructs; minibodies (see, e.g., Pack et al. (1992) Biochem 31 :1579-1584; Cumber et al. (1992) J Immunology 149B:120-126); humanized antibody molecules (see, e.g., Riechmann et al. (1988) Nature 332:323-327; Verhoeyan et al. (1988) Science 239:1534-1536; and U.K. Patent Publication No. GB 2,276,169, published 21 Sep. 1994); and, any functional fragments obtained from such molecules, wherein such fragments retain specific-binding properties of the parent antibody molecule (i.e., specifically binds to a selection marker on a cell).

[0334] In other embodiments, the binding agent comprises an aptamer that specifically binds to the selection marker on a cell. Any type of aptamer may be used, including a DNA, RNA, xeno-nucleic acid (XNA), or peptide aptamer that specifically binds to the target antibody isotype. Such aptamers can be identified, for example, by screening a combinatorial library. Nucleic acid aptamers (e.g., DNA or RNA aptamers) that bind selectively to a target antibody isotype can be produced by carrying out repeated rounds of in vitro selection or systematic evolution of ligands by exponential enrichment(SELEX). Peptide aptamers that bind to a selection marker on a cell may be isolated from a combinatorial library and improved by directed mutation or repeated rounds of mutagenesis and selection. For a description of methods of producing aptamers, see, e.g., Aptamers: Tools for Nanotherapy and Molecular Imaging (R.N. Veedu ed., Pan Stanford, 2016), Nucleic Acid and Peptide Aptamers: Methods and Protocols (Methods in Molecular Biology, G. Mayer ed., Humana Press, 2009), Nucleic Acid Aptamers: Selection, Characterization, and Application (Methods in Molecular Biology, G. Mayer ed., Humana Press, 2016), Aptamers Selected by Cell-SELEX for Theranostics (W. Tan, X. Fang eds., Springer, 2015), Cox et al. (2001 ) Bioorg. Med. Chem. 9(10):2525-2531 ; Cox et al. (2002) Nucleic Acids Res. 30(20): e108, Kenan et al. (1999) Methods Mol Biol. 118:217-231 ; Platelia et al. (2016) Biochim. Biophys. Acta Nov 16 pii: S0304- 4165(16)30447-0, and Lyu et al. (2016) Theranostics 6(9):1440-1452; herein incorporated by reference in their entireties.

[0335] In yet other embodiments, the binding agent comprises an antibody mimetic. Any type of antibody mimetic may be used, including, but not limited to, affibody molecules (Nygren (2008) FEBS J. 275 (1 1 ):2668-2676), affilins (Ebersbach et al. (2007) J. Mol. Biol. 372 (1 ):172-185), affimers (Johnson et al. (2012) Anal. Chem. 84 (15):6553-6560), affitins (Krehenbrink et al. (2008) J. Mol. Biol. 383 (5):1058-1068), alphabodies (Desmet et al. (2014) Nature Communications 5:5237), anticalins (Skerra (2008) FEBS J. 275 (1 1 ):2677-2683), avimers (Silverman et al. (2005) Nat. Biotechnol. 23 (12):1556-1561 ), darpins (Stumpp et al. (2008) Drug Discov. Today 13 (15-16):695-701 ), fynomers (Grabulovski et al. (2007) J. Biol. Chem. 282 (5):3196-3204), and monobodies (Koide et al. (2007) Methods Mol. Biol. 352:95-109).

[0336] Dead cells may be selected against by employing dyes that preferentially stain dead cells (e.g. propidium iodide). Any technique may be employed which is not unduly detrimental to the viability of the genetically modified cells.

[0337] Compositions that are highly enriched for cells having a desired genetic modification can be produced in this manner. By "highly enriched" is meant that the genetically modified cells are 70% or more, 75% or more, 80% or more, 85% or more, 90% or more, or 95% or more, or 98% or more of the cell composition. In other words, the composition may be a substantially pure composition of genetically modified cells.

[0338] Genetically modified cells produced by the methods described herein may be used immediately. Alternatively, the cells may be frozen at liquid nitrogen temperatures and stored for long periods of time before being thawed and used. In such cases, cells maybe frozen in 10% DMSO, 50% serum, 40% buffered medium, or some other such solution as is commonly used in the art to preserve cells at such freezing temperatures, and thawed in a manner as commonly known in the art for thawing frozen cultured cells.Methods of Transplanting Genetically Modified Cells into an Individual

[0339] Also provided herein are methods of transplanting cells that are genetically modified with a multi-module gene editing construct, as described herein, into an individual, for example, to treat a disease. In some embodiments, the cells are encapsulated in a biocompatible carrier, matrix, or scaffold. Suitable matrices include a polymeric mesh or sponge or a polymeric hydrogel.

[0340] A hydrogel is defined as a substance formed when an organic polymer (natural or synthetic) is cross-linked via covalent, ionic, or hydrogen bonds to create a three- dimensional open-lattice structure, which entraps water molecules to form a gel. In general, these polymers are at least partially soluble in aqueous solutions, such as water, buffered salt solutions, or aqueous alcohol solutions that have charged side groups, or a monovalent ionic salt thereof. Any suitable hydrogel polymers can be used to form a hydrogel. Exemplary hydrogel polymers include, without limitation, natural polymers such as polysaccharides, including hyaluronic acid, chitosan, heparin, alginate, cellulose, dextran, and agarose, and proteins, including fibrin, fibrinogen, collagen, elastin, gelatin, silk, laminin, fibronectin, albumin, thrombin, and keratin; modified natural polymers, including hydroxymethylcellulose, hydroxyethylcellulose, gelatin methacrylate, polyanionic N-carboxymethyl chitosan, and polycationic N-trimethyl chitosan; and synthetic polymers, including polyvinyl alcohol, N-vinylpyrrolidone, polyethylene glycol, polyethylene glycol) diacrylate, polyacrylamide, poly(N-isopropylacrylamide), sodium polyacrylate, acrylate polymers and copolymers such as hydroxyethyl methacrylate, ethyl methacrylate, propylene glycol methacrylate, ethylene glycol di-methyl acrylate, methyl methacrylate, glycidyl methacrylate, and glycol methacrylate, poly(N- isopropylacrylamide-co-acrylic acid), polyesters, polyurethanes, nylon, synthetic polyamino acids, prolamines; and combinations thereof, and other such molecules, including recombinant versions of such polymers. In some embodiments, the hydrogel comprises polyacrylamide.

[0341] The method may include implanting (e.g., surgically implanting) a matrix or scaffold containing the therapeutic cells at an implantation site of a host individual. The host individual may be suffering from a condition, e.g., a disease, that may be treated byproviding the therapeutic cells to the individual. The therapeutic cells may be any suitable therapeutic cells, as described above, and the type of therapeutic cells may depend on the disease to be treated.

[0342] The implantation site may be any suitable location (e.g., surgically accessible location) in the individual. In some cases, the implantation site is in a kidney, liver, omentum, peritoneum, abdomen, or submuscular or subcutaneous tissue. In some cases, the implantation site is at or in the vicinity of a tissue that is affected by the disease (e.g., a tissue with a solid tumor, fibrotic tissue, infected tissue).

[0343] A medical practitioner may locate the site for transplantation of the therapeutic cells, for example, by medical imaging (e.g., ultrasound, radiography, or MRI). In some embodiments, a contrast agent is included in the composition comprising the therapeutic cells to allow confirmation of the location of the cells by medical imaging after transplantation. In some embodiments, the contrast agent is a microbubble (e.g., for use in ultrasound) or a radiopaque contrast agent (e.g., for use in radiography). The contrast agent may be contained in the same composition as the therapeutic cells or in a different composition and used prior to or after transplantation.CAR-T Cells

[0344] The approaches described herein can be applied to engineering T cells with multiple genetic modifications for immunotherapy, particularly for the generation of chimeric antigen receptor (CAR)-T cells. A multi-module gene editing construct may be designed with a genome editing module comprising a coding sequence encoding a chimeric antigen receptor that specifically binds to a target antigen. The chimeric antigen receptor localizes a T cell to sites where target cells are present that express the target antigen. Binding of a CAR-T cell to a target antigen on the surface of a cell activates the T cell resulting in secretion of cytokines, which regulate other immune cells, and killing of target cells. For example, CAR-T cells may be engineered to target an antigen that is expressed on the surface of tumors but not on healthy cells to selectively kill tumor cells. In another example, CAR-T cells may also be engineered to target an antigen that is expressed on the surface of activated fibroblasts or fibrotic tissue, which may be used to selectively eliminate fibrotic tissue. In another example, CAR-T cells may also be engineered to target an antigen that is expressed on the surface of a pathogen (e.g., bacterium, virus, fungus, or parasite) to eradicate a pathogen. In a further example, CAR- T cells may be engineered to target an antigen that is expressed on the surface of anautoreactive immune cell (e.g., autoreactive T cell or B cell) to eliminate autoreactive immune cells. Thus, CAR-T cells may be used for the treatment of various diseases, including cancer, fibrosis, infections such as bacterial infections (e.g., multidrug resistant bacteria), viral infections, fungal infections, and parasitic infections, and autoimmune diseases.

[0345] The T cell, from which the CAR-T cell is derived, may be autologous or allogeneic. In some embodiments, the CAR-T cell is an effector T cell (e.g., a helper CD4+T cell, a cytotoxic CD8+T cell, a natural killer T cell, or a gamma delta T cell) or a regulatory T cell (Treg) that has been genetically modified to express a CAR.

[0346] A CAR may have any suitable architecture, known in the art, wherein the CAR comprises an antigen binding domain linked to T cell receptor effector functions. The term “CAR” refers to an artificial multi-module molecule capable of triggering or inhibiting the activation of an immune cell. A CAR will generally comprise an antigen binding domain, linker, transmembrane domain and cytoplasmic signaling domain. In some instances, a CAR includes one or more co-stimulatory domains and / or one or more co- inhibitory domains.

[0347] The antigen-binding domain of a CAR may include any naturally occurring, synthetic, semi-synthetic, or recombinantly produced binding partner for a target antigen of interest. In some embodiments, the binding region is an antigen-binding region, such as an antibody or functional binding domain or antigen-binding fragment thereof. The antigen-binding region of the CAR can include any domain that binds to the antigen and may include, but is not limited to, a monoclonal antibody, a polyclonal antibody, a synthetic antibody, a human antibody, a humanized antibody, a non-human antibody, a single-chain antibody, and any antigen-binding fragment thereof. Thus, in some embodiments, the antigen binding domain portion includes a mammalian antibody or an antigen-binding fragment thereof. An antigen-binding domain may comprise an antigenbinding fragment (Fab), a single-chain variable fragment (scFv), a nanobody, a VH domain, a VL domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, or a diabody; or a functional antigen-binding fragment thereof. In some embodiments, the antigen-binding domain is derived from the same cell type or the same species in which the CAR will ultimately be used. For example, for use in humans, the antigen-binding domain of the CAR may include a human antibody, a humanized antibody, or an antigen-binding fragment thereof.

[0348] In some embodiments, the antigen binding domain is derived from a single chain antibody that selectively binds to a target antigen. In some embodiments, the antigen binding domain is provided by a single chain variable fragment (scFv). A scFv is a recombinant molecule in which the variable regions of the light and heavy immunoglobulin chains are connected in a single fusion polypeptide. Generally, the VH and VL sequences are joined by a linker sequence. See, for example, Ahmad (2012) Clinical and Developmental Immunology Article ID 980250, herein specifically incorporated by reference. In principle, there are no particular limitations to the length and / or amino acid composition of the linker peptide joining the VH and VL sequences. In some embodiments, any arbitrary single-chain peptide including about 1 to 100 amino acid residues (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, etc. amino acid residues) can be used as a peptide linker. In some embodiments, the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues.

[0349] The transmembrane domain may be derived either from a natural or a synthetic source. Where the source is natural, the domain may be derived from any membranebound or transmembrane protein. In some embodiments, the transmembrane domain comprises at least the stalk and / or transmembrane region(s) of CD8, Megf10, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, Integrin subunit |35, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, and / or CD86. In some embodiments, the CAR transmembrane domain is derived from a type I membrane protein, such as, but not limited to, CD3 , CD4, CD8, or CD28. In other embodiments, the transmembrane domain is synthetic, in which case it will include predominantly hydrophobic residues such as leucine, isoleucine, valine, phenylalanine, tryptophan, and alanine. In some embodiments, a triplet of phenylalanine, tryptophan and valine will be inserted at each end of a synthetic transmembrane domain.

[0350] In some embodiments, the CAR further comprises one or more linkers / spacers. For example, an extracellular spacer region may link the antigen binding domain to the transmembrane domain and / or an intracellular spacer region may link an intracellular signaling domain to the transmembrane domain. A spacer (linker) region linking the antigen binding domain to the transmembrane domain should be flexible enough to allow the antigen binding domain to orient in different directions to facilitate antigen recognition.

[0351] Various types of linkers may be used in the CARs described herein. In some embodiments, the linker includes a peptide linker / spacer sequence. In some embodiments, the spacer comprises the hinge region from an immunoglobulin, e.g., the hinge from any one of lgG1 , lgG2a, lgG2b, lgG3, lgG4, particularly the human protein sequences. Alternatives include the CH2CH3 region of immunoglobulin and portions of CD3. For many scFv based constructs, an IgG hinge is effective.

[0352] In principle, there are no particular limitations to the length and / or amino acid composition of a linker peptide sequence. In some embodiments, a linker peptide sequence comprises about 1 to 100 amino acid residues, including any number of residues within this range such as 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 30, 40, 50, 60, 70, 80, 90, or 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 5 to 50, about 10 to 60, about 20 to 70, about 30 to 80, about 40 to 90, about 50 to 100, about 60 to 80, about 70 to 100, about 30 to 60, about 20 to 80, about 30 to 90 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25, about 20 to 40, about 30 to 50, about 40 to 60, about 50 to 70 amino acid residues. In some embodiments, the linker peptide sequence includes about 40 to 70, about 50 to 80, about 60 to 80, about 70 to 90, or about 80 to 100 amino acid residues. In some embodiments, the linker peptide sequence includes about 1 to 10, about 5 to 15, about 10 to 20, about 15 to 25 amino acid residues. In some embodiments, the linker peptide sequence may include up to 300 amino acids, preferably 10 to 100 amino acids and most preferably 25 to 50 amino acids. In some embodiments, a short oligo- or polypeptide linker, preferably between 2 and 10 amino acids in length may form the linkage between the transmembrane domain and the intracellular engulfment signaling domain or extracellular antigen binding domain of the CAR. In some embodiments the linker comprises the amino acid sequence (G4S)nwhere n is 1 , 2, 3, 4, 5, etc., and in some embodiments, n is 3.

[0353] A cytoplasmic signaling domain, such as those derived from the T cell receptor □- chain, is employed as part of the CAR in order to produce stimulatory signals for T lymphocyte proliferation and effector function following engagement of the chimeric receptor with the target antigen. Endodomains from co-stimulatory molecules may be included in the cytoplasmic signaling portion of the CAR.

[0354] The term “co-stimulatory domain”, refers to a stimulatory domain, typically an endodomain, of a CAR that provides a secondary non-specific activation mechanism through which a primary specific stimulation is propagated. Examples of co-stimulation include antigen nonspecific T cell co-stimulation following antigen specific signaling through the T cell receptor and antigen nonspecific B cell co-stimulation following signaling through the B cell receptor. Co-stimulation, e.g., T cell co-stimulation, and the factors involved have been described in Chen & Flies, Nat Rev Immunol (2013) 13(4):227-42, the disclosure of which is incorporated herein by reference in its entirety. Non-limiting examples of suitable co-stimulatory polypeptides include, but are not limited to, 4-1 BB (CD137), CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, and HVEM.

[0355] The term “co-inhibitory domain” refers to an inhibitory domain, typically an endodomain, derived from a receptor that provides secondary inhibition of primary antigen-specific activation mechanisms which prevents co-stimulation. Co-inhibition, e.g., T cell co-inhibition, and the factors involved have been described in Chen & Flies. Nat Rev Immunol (2013) 13(4):227-42 and Thaventhiran et al. J Clin Cell Immunol (2012) S12. In some embodiments, co-inhibitory domains homodimerize. A co-inhibitory domain can be an intracellular portion of a transmembrane protein. Non-limiting examples of suitable co-inhibitory polypeptides include, but are not limited to, CTLA-4 and PD-1 .

[0356] A first-generation CAR transmits the signal from antigen binding through only a single signaling domain, for example a signaling domain derived from the high-affinity receptor for IgE FccRlD D or the CD3 chain. The domain contains one or three immunoreceptor tyrosine-based activating motif(s) [ITAM(s)] for antigen-dependent T- cell activation. The ITAM-based activating signal endows T-cells with the ability to lyse the target tumor cells and secret cytokines in response to antigen binding.

[0357] Second-generation CARs include a co-stimulatory signal in addition to the CD3D signal. Coincidental delivery of the delivered co-stimulatory signal enhances cytokine secretion and antitumor activity induced by CAR-transduced T-cells. The co-stimulatory domain will usually be membrane proximal relative to the CD3D domain. Third- generation CARs include a tripartite signaling domain, comprising for example a CD28,CD3 , 0X40 or 4-1 BB signaling region. In fourth generation, or “armored car” CAR-T cells, CAR-T cells are further genetically modified to express or block molecules and / or receptors to enhance immune activity.

[0358] CAR variants include split CARs wherein the extracellular portion, the ABD and the cytoplasmic signaling domain of a CAR are present on two separate molecules. CAR variants also include ON-switch CARs which are conditionally activatable CARs, e.g., comprising a split CAR wherein conditional hetero-dimerization of the two portions of the split CAR is pharmacologically controlled. CAR molecules and derivatives thereof (i.e., CAR variants) are described, e.g., in PCT Application Nos. US2014 / 016527, US1996 / 017060, US2013 / 063083; Fedorov et al. Sci Trans! Med (2013) 5(215):215ra172; Glienke et al. Front Pharmacol (2015) 6:21 ; Kakarla & Gottschalk 52 Cancer J (2014) 20(2):151 -5; Riddell et al. Cancer J (2014) 20(2):141 -4; Pegram et al. Cancer J (2014) 20(2):127-33; Cheadle et al. Immunol Rev (2014) 257(1 ):91 -106; Barrett et al. Annu Rev Med (2014) 65:333-47; Sadelain et al. Cancer Discov (2013) 3(4):388- 98; Cartellieri et al., J Biomed Biotechnol (2010) 956304; herein incorporated by reference in their entireties.

[0359] CAR variants also include bispecific or tandem CARs, which include a secondary CAR binding domain that can either amplify or inhibit the activity of a primary CAR. CAR variants also include inhibitory chimeric antigen receptors (iCARs) which may, e.g., be used as a component of a bispecific CAR system, where binding of a secondary CAR binding domain results in inhibition of primary CAR activation. Tandem CARs (TanCAR) mediate bispecific activation of T cells through the engagement of two chimeric receptors designed to deliver stimulatory or costimulatory signals in response to an independent engagement of two different tumor associated antigens. iCARs use the dual antigen targeting to shout down the activation of an active CAR through the engagement of a second suppressive receptor equipped with inhibitory signaling domains.

[0360] The dual recognition of different epitopes by two CARs diversely designed to either deliver killing through -chain or costimulatory signals, e.g., through CD28 allows a more selective activation of the reprogrammed T cells by restricting Tandem CAR's activity to cancer cell expressing simultaneously two antigens rather than one. The potency of delivered signals in engineered T cells will remain below threshold of activation and thus ineffective in absence of the engagement of costimulatory receptor. The combinatorial antigen recognition enhances selective tumor eradication and protects normal tissues expressing only one antigen from unwanted reactions.

[0361] Inhibitory CARs (iGARs) are designed to regulate CAR-T cell activity through inhibitory receptor signaling module activation. This approach combines the activity of two CARs, one of which generates dominant negative signals limiting the responses of CAR-T cells activated by the activating receptor. iCARs can switch off the response of the counteracting activator CAR when bound to a specific antigen expressed only by normal tissues. In this way, iCARs-T cells can distinguish cancer cells from healthy ones, and reversibly block functionalities of transduced T cells in an antigen-selective fashion. CTLA-4 or PD-1 intracellular domains in iCARs trigger inhibitory signals on T lymphocytes, leading to less cytokine production, less efficient target cell lysis, and altered lymphocyte motility.

[0362] An ABD can be provided as a “chimeric bispecific binding member”, i.e., a chimeric polypeptide having dual specificity to two different binding partners (e.g., two different antigens). Non-limiting examples of chimeric bispecific binding members include bispecific antibodies, bispecific conjugated monoclonal antibodies (mab)2, bispecific antibody fragments (e.g., F(ab)2, bispecific scFv, bispecific diabodies, single chain bispecific diabodies, etc.), bispecific T cell engagers (BiTE) , bispecific conjugated single domain antibodies, micabodies and mutants thereof, and the like. Non-limiting examples of chimeric bispecific binding members also include those chimeric bispecific agents described in Kontermann. MAbs. (2012) 4(2): 182-197; Stamova et al. Antibodies 2012, 1 (2), 172-198; Farhadfar et al. Leuk Res. (2016) 49:13-21 ; Benjamin et al. Ther Adv Hematol. (2016) 7(3):142-56; Kiefer et al. Immunol Rev. (2016) 270(1 ):178-92; Fan et al. J Hematol Oncol. (2015) 8:130; May et al. Am J Health Syst Pharm. (2016) 73(1 ):e6-e13; the disclosures of which are incorporated herein by reference in their entirety.

[0363] In some instances, a chimeric bispecific binding member may be a bispecific T cell engager (BiTE). A BiTE is generally made by fusing a specific binding member (e.g., a scFv) that binds an antigen to a specific binding member (e.g., a scFv) with a second binding domain specific for a T cell molecule such as CD3.

[0364] In some instances, a chimeric bispecific binding member may be a CAR-T cell adapter. As used herein, by “CAR-T cell adapter” is meant an expressed bispecific polypeptide that binds the antigen recognition domain of a CAR and redirects the CAR to a second antigen. Generally, a CAR-T cell adapter will have two binding regions, one specific for an epitope on the CAR to which it is directed and a second epitope directed to a binding partner which, when bound, transduces the binding signal activating the CAR. Useful CAR-T cell adapters include but are not limited to e.g., those described inKim et al. J Am Ghem Soc. (2015) 137(8):2832-5; Ma et al. Proc Natl Acad Sci U S A. (2016) 1 13(4):E450-8 and Cao et al. Angew Ghem Int Ed Engl. (2016) 55(26)7520-4; the disclosures of which are incorporated herein by reference in their entirety.

[0365] Effector CAR-T cells include autologous or allogeneic immune cells having cytolytic activity against a target cell. In some embodiments, a patient's own T cells or T cells from a donor are engineered to express a CAR. In some embodiments, the CAR-T cells are engineered from a complex mixture of immune cells, e.g., tumor infiltrating lymphocytes (TILs) isolated from an individual in need of treatment. See, e.g., Yang and Rosenberg (2016) Adv Immunol. 130:279-94, “Adoptive T Cell Therapy for Cancer; Feldman et al (2015) Semin Oncol. 42(4):626-39 “Adoptive Cell Therapy-Tumor- Infiltrating Lymphocytes, T-Cell Receptors, and Chimeric Antigen Receptors”; Clinical Trial NCT01 174121 , “Immunotherapy Using Tumor Infiltrating Lymphocytes for Patients With Metastatic Cancer”; Tran et al. (2014) Science 344(6184)641 -645, “Cancer immunotherapy based on mutation-specific CD4+ T cells in a patient with epithelial cancer”. In other embodiments, stem cells, differentiated into T cells, are engineered to express a CAR. In some embodiments, induced pluripotent stem cell (IPSC)-derived T cells are engineered to express a CAR. See, e.g., Zhou et al. (2022) Cancers (Basel) 14(9):2266, Nezhad et al. (2021 ) Pharm Res 38(6):931 -945; herein incorporated by reference in their entireties.

[0366] A biological sample comprising T cells, from which CAR-T cells are generated, may be collected from a subject or a donor. The biological sample may include, without limitation, blood, lymphoid tissue (e.g., bone marrow, spleen, tonsils, lymph nodes), mucosal tissue (e.g., lungs, small intestine, and large intestine), skin, or a tissue where T cells have infiltrated. The T cells may be separated from a mixture of cells prior to engineering the T cells to generate CAR-T cells. Alternatively, T cells may be engineered and cultured without separation from other cells.

[0367] T cells may be separated from other cells using any suitable cell separation technique such as, but not limited to, centrifugation-based cell separation, positive or negative selection against surface markers on cells (e.g., with antibody-coated beads), affinity chromatography, panning and immunopanning techniques, fluorescence activated cell sorting (FACS), or magnetic-activated cell sorting (MACS). Affinity reagents may be employed comprising specific receptors or ligands specific for cell surface molecules. The T cells may be separated from dead cells by employing viability dyes(e.g., propidium iodide). Any technique may be employed which is not unduly detrimental to the viability of the T cells.

[0368] The cells may be collected in any appropriate medium that maintains the viability of the cells. Various media are commercially available and may be used according to the nature of the cells, including dMEM, HBSS, dPBS, RPMI, Iscove’s medium, etc., which may be supplemented with fetal calf serum (FCS). The collected cells may be used immediately or frozen (e.g., at liquid nitrogen temperatures) prior to use.

[0369] In some embodiments, CAR-T cells are expanded in culture prior to screening, as described further below, or use in therapy. The CAR-T cells require activation for expansion in vitro or ex vivo, which can be accomplished by co-incubating T cells with natural antigen-presenting cells (e.g., dendritic cells) or artificial antigen-presenting cells or particles that present antigen and / or activating signals to the CAR-T cells. See, e.g., Rhodes et al. (2018) Mol Immunol. 98:13-18, Couture et al. (2019) Front Immunol. 10:1081 , Turtle (2010) Cancer J. 16(4):374-81 , Wang et al. (2017) Theranostics 7(14):3504-3516, Est-Witte et al. (2021 ) Semin Immunol. 56:101541 , Perica et al. (2014) Nanomedicine. 10 (1 ): 1 19-129, Latouche et al. (2000) Nature Biotechnology. 18 (4): 405-409; herein incorporated by reference.

[0370] In some embodiments, the multi-module gene editing construct is designed to produce one or more additional genetic modifications in the T cell, for example, to reduce graft versus host disease, T-cell exhaustion, and / or cytokine-related toxicities, improve T-cell effector function and survival, and / or facilitate multiplexed pooled screening. In some embodiments, the multi-module gene editing construct is designed with a module to produce a gene knockin of a CAR and a module to produce a gene knockout of an endogenous T cell receptor (TCR) gene. TCR knockout reduces graft versus host disease and allows T cells from different donors to be pooled for multiplexed screening, as discussed in more detail in co-owned Provisional Patent Application entitled "Massively Parallel Mixed Lymphocyte Reactions," filed even date herewith, the disclosure of which is hereby incorporated by reference herein in its entirety. TCRs are membrane-anchored heterodimeric proteins that require both protein chains of the TCR to be present in order to function. Accordingly, TCR receptors can be knocked out by deleting or disrupting expression of one or both of the protein chains that form the heterodimer. For TCR knockout in ab T cells, wherein the TCR consists of an alpha (a) chain (TRAC), encoded by a TRA gene, and a TCR beta (b) chain (TRBC), encoded by a TRB gene, expression of TRAC and / or TRBC can be disrupted. For TCR knockout ingd T cells, wherein the TCR consists of a gamma chain (TRGC), encoded by a TRG gene, and a delta chain (TRDC), encoded by a TRD gene, expression of TRGC and / or TRDC can be disrupted.

[0371] In some embodiments, the multi-module gene editing construct is designed with an additional module to produce a gene knockout of a gene encoding a cytokine to reduce cytokine-related toxicity resulting from CAR-T cell therapy. In some embodiments, the cytokine is GM-CSF or IL-6.

[0372] In some embodiments, the multi-module gene editing construct is designed with an additional module to knockout a gene encoding an alloantigen to reduce graft versus host disease. In some embodiments, the alloantigen is a major histocompatibility complex (MHC) class I alloantigen or an MNS blood group alloantigen. In some embodiments, the alloantigen is CD1 , CD2, CD3, CD4, CD7, CD8, Ly-6, Qa-2, RT6, CD19, CD22, CD56, CD58 (LFA-3), CD59, or CDw90 (Thy 1 ) that is knocked out.

[0373] In some embodiments, the multi-module gene editing construct is designed with an additional module to knockout a CD5, CD52, CD70, BATF, LCK, PD-1 , LAG-3, CTLA- 4, [32-B2M, PD-1 , HLA-I, Fas, TGFBR2, PDCD-1 , DGK, EZH2, PAX5, or LDLR gene.

[0374] In some embodiments, a CAR-T cell is further engineered to comprise a binding- triggered transcriptional switch (BTSS) that regulates expression of the chimeric antigen receptor or activity of the CAR-T cell. By BTSS, is intended to mean a synthetic modular polypeptide or system of interacting polypeptides having an extracellular domain that includes a second member of a specific binding pair that binds a first member of the specific binding pair (e.g., an antigen), a binding-transducer and an intracellular domain. Upon binding of the first member of the specific binding pair to the BTTS the binding signal is transduced to the intracellular domain such that the intracellular domain becomes activated and performs a function, e.g., transcription activation, within the cell that it does not perform in the absence of the binding signal. In certain embodiments, the second binding member may be present on the surface of a genetically engineered cell, such as, a cell expressing a BTTS and a CAR under the control of the BTTS.

[0375] Examples of binding-triggered transcriptional switches include the synNotch system, the modular extracellular sensor architecture (MESA) system, the TANGO system, the A2 Notch system, and the synthetic intramembrane proteolysis receptor (SNIPR) system, etc. The synNotch receptor may be for example as described in U.S. Patent No. 9,670,281 and described in more detail below. The MESA system may be as described in WO 2018 / 081039 A1 and comprises a self-containing sensing and signaltransduction system, such that binding of a ligand (first member of the specific binding pair) to the receptor (second member of the specific binding pair) induces signaling to regulate expression of a target gene. In the MESA system, binding of the ligand to the receptor induces dimerization that results in proteolytic trans-cleavage of the system to release a transcriptional activator previously sequestered at the plasma membrane. The TANGO system may be as described in Barnea et al., 2008 Proc. Natl. Acad. Sci. U.S.A., 105(1 ): 64-9. Briefly, the TANGO system sequesters a transcription factor to the cell membrane by physically linking it to a membrane-bound receptor (e.g., GPCRs, receptor kinases, Notch, steroid hormone receptors, etc.). Activation of the receptor fusion results in the recruitment of a signaling protein fused to a protease that then cleaves and releases the transcription factor to activate genes in the cell. The A2 Notch system may be as described in WO 2019099689 A1 . Briefly, the A2 Notch system incorporates a force sensor cleavage domain which, upon cleavage induced upon binding of a ligand to the receptor, releases the intracellular domain into the cell. The SNIPR system may be described as in Zhu et al. (2022) Cell 185(8):1431 -1443.e16; herein incorporated by reference. Briefly, the SNIPR system uses a synthetic RIP receptor comprising an ectodomain comprising an extracellular regulatory element that specifically binds a ligand, a transmembrane domain, a juxtamembrane domain, and a transcription factor that can be cleaved from the SNIPR by a protease in response to binding of a ligand to the extracellular regulatory element.

[0376] In certain cases, the first binding member may bind to a synNotch receptor as described in U.S. Patent No. 9,670,281 . For example, the synNotch receptor may include an extracellular domain that includes the second binding member, where the second binding member is a single-chain Fv (scFv) or a nanobody and the first binding member present on the particles is an antigen to which the single-chain Fv (scFv) or a nanobody binds. In certain cases, the second binding member may be an anti-CD19, anti- mesothelin, anti-GFP antibody, scFv, or a nanobody and the first binding member may be CD19, mesothelin, GFP, respectively.

[0377] In certain embodiments, the BTTS is a chimeric Notch polypeptide comprising, from N-terminus to C-terminus and in covalent linkage: a) an extracellular domain comprising the second member of the specific-binding pair that is not naturally present in a Notch receptor polypeptide and that specifically binds to the first member of the specific-binding pair; b) a Notch regulatory region comprising a Lin 12-Notch repeat, an S2 proteolytic cleavage site, and a transmembrane domain comprising an S3 proteolyticcleavage site; c) an intracellular domain comprising a transcriptional activator or a transcriptional repressor that is heterologous to the Notch regulatory region and replaces a naturally-occurring intracellular Notch domain, wherein binding of the first member of the specific-binding pair to the second member of the specific-binding pair induces cleavage at the S2 and S3 proteolytic cleavage sites, thereby releasing the intracellular domain; and a transcriptional control element, responsive to the transcriptional activator, operably linked to a nucleotide sequence encoding a chimeric antigen receptor (CAR). In certain cases, the cell may be a T-cell, such as, those described in U.S. Patent No. 9,670,281 , which is herein incorporated by reference.Multiplexed Screening of CAR-T Cells

[0378] The subject methods of genetically modifying cells with multi-module gene editing constructs enable CAR-T cells with multiple different combinations of genetic modifications to be tested simultaneously. In some embodiments, a multi-module gene editing construct is designed to produce a knockin of a CAR as well as one or more additional genetic modifications, for example, to reduce graft versus host disease, T-cell exhaustion, and / or cytokine-related toxicities, improve T-cell effector function and survival, and / or facilitate multiplexed pooled screening. In some embodiments, a multimodule gene editing construct is used to produce CAR-T cells from multiple donors wherein the T cells have a knockin of the CAR and a knockout of their endogenous TCRs. Such CAR-T cells with knockouts of their endogenous TCRs can be pooled and tested simultaneously in multiplexed assays. See, e.g., co-owned Provisional Patent Application, entitled "Massively Parallel Mixed Lymphocyte Reactions,” filed even date herewith, the disclosure of which is hereby incorporated by reference herein in its entirety.

[0379] Activation of CAR-T cells can be determined by measuring cell proliferation, expression of activation markers (e.g., detection of CD69, HLA-DR, IL2RA, and / or CD25), and production of effector cytokines (e.g., IFN-g, TNF-a, TNF-b, IL-1 , IL-2, IL-3, IL-4, IL-5, IL-9, IL-10, IL-12, IL-13, and IL-25). Multiplexed screening of CAR-T cell cytotoxic activity can be performed in vitro to validate activity against target cells before further testing individual CAR-T cell in vivo in animal models and human clinical trials.

[0380] Cytotoxicity of CD8+ CAR-T cells involves exocytosis of granules containing the pore-forming toxin, perforin, proapoptotic serine proteases, and granzymes that lyse target cells. Cytotoxicity of CD4+ CAR-T cells involves secretion of cytokines and apoptotic factors such as TNF-a, INF-g, and TRAIL that induce apoptosis of target cellsor activate macrophages to engulf tumor cells. Perforin, proapoptotic serine proteases, granzymes, cytokines, and apoptotic factors can be measured, for example, using a multiplexed enzyme-linked immunosorbent assay (ELISA). Cytolysis can be assayed in vitro based on the release of compounds containing radioactive isotopes such as51Cr from radiolabeled target cells. Alternatively a membrane-permeable live-cell labeling dye such as calcein acetoxymethyl ester of calcein (Calcein / AM) can be used to distinguish live cells from dead cells. In the Calcein / AM assay, intracellular esterases cleave the acetoxymethyl (AM) ester group to produce a membrane-impermeable calcein fluorescent dye that is retained in live cells. Apoptotic and dead cells without intact cell membranes do not retain the calcein fluorescent dye. A lactate dehydrogenase (LDH) assay can also be used to evaluate cytotoxicity. LDH is a cytoplasmic enzyme, which is released into the extracellular space when the plasma membrane is damaged. Cytotoxicity is monitored by detecting LDH release from cells. See, e.g., Lieberman (2003) Nat Rev Immunol 3(5):361 -370, Neri et al. (2001 ) Clin Diagn Lab Immunol 8(6) : 1131 -1135, Smith et al. (2011 ) PLoS One 6(1 1 ):e26908, Chan et al. (2013) Methods Mol Biol 979:65-70; herein incorporated by reference in their entireties.

[0381] Flow cytometry can also be used to assess cell proliferation, activation, and cytotoxicity. The percentage of target cells that are live, apoptotic, or dead can be determined by staining target cells with viability dyes such that the live and dead cell populations can be distinguished based on differences in fluorescence. For example, Annexin V-FITC can be used to label target cells that are at an early stage of apoptosis. Propidium iodide can be used to label target cells that are at a late stage of apoptosis or dead. Lipophilic dyes, such as PKH67 and PKH26 can be used to label the cell membranes of target cells for measuring proliferation of CAR-T cells by flow cytometry. In addition, T cell activation can also be detected by immunofluorescent labeling of activation markers such as CD69, HLA-DR, IL2RA, and CD25. See, e.g., Zaritskaya et al. (2010) Expert Rev Vaccines 9(6):601 -616, Fischer et al. (2002) J Immunol Methods 259(1 -2):159-169, Aubry et al. (1999) Cytometry 37(3):197-204, and Tario et al. (201 1 ) Methods Mol Biol 699:1 19-164; herein incorporated by reference in their entireties.

[0382] Cell proliferation can also be detected and quantified, for example, using a cell counter or staining of CAR-T cells with a fluorescent tracking dye, such as carboxyfluorescein succinimidyl ester (CFSE).

[0383] The CAR-T cells may be further tested for efficacy in treating a disease in vivo, e.g., in an animal. For example, CAR-T cells can be tested for cytotoxicity againstcancerous cells in an animal with solid tumors. In some embodiments, human xenograft tumors are implanted in animals, followed by administration of CAR-T cells, and evaluation of antitumor responses. An exemplary animal model of cancer is a NOD Scid Gamma (NSG) mouse transplanted with human tumors. NSG mice are completely deficient in adaptive immunity and severely deficient in innate immunity, which avoids transplant rejection of CAR-T cells and patient-derived xenografts.

[0384] Antitumor responses can be evaluated by various methods known in the art. The volume of a subcutaneous tumor can be measured by using a digital caliper. Internal tumors can be measured by x-ray imaging, computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography (PET), or singlephoton emission computed tomography (SPECT). In some cases, the CAR-T cells are further modified to express a bioluminescent protein such as luciferase to allow monitoring of tumors by bioluminescence imaging or a fluorescent protein such as green fluorescent protein to allow monitoring of tumors by fluorescence imaging.

[0385] In addition, tumors can be removed from the animals and measured after the treatment with CAR-T cells is completed. Immunohistochemistry of tumor specimens can be used to detect T cell infiltration into tumors and quantitate target antigen expression. Cytokine profiling of tumors treated with CAR-T cells can also be performed.

[0386] In another example, CAR-T cells can be tested for cytotoxicity against activated fibroblasts or fibrotic tissue in an animal with fibrosis. The extent of fibrosis can be monitored in an animal in vivo, for example, by x-ray imaging, computed tomography (CT), ultrasound (US), magnetic resonance imaging (MRI), positron emission tomography (PET), or single-photon emission computed tomography (SPECT). In addition, fibrotic tissue can be removed from the animals and measured after the treatment with CAR-T cells is completed. Immunohistochemistry of fibrotic tissue specimens can be used to detect T cell infiltration into fibrotic tissue and quantitate target antigen expression. Cytokine profiling of fibrotic tissue treated with CAR-T cells can also be performed.

[0387] An animal model can be used not only to determine efficacy but also the toxicity or side effects of treatment with a CAR-T cell. Furthermore, this disclosure pertains to uses of CAR-T cells, identified by the above-described screening assays for treatment of a disease such as, but not limited to, cancer, fibrosis, an infection, or an autoimmune disease. A CAR-T cell, identified by the above-described screening assays for treatment of a disease, may be expanded in culture in the presence of a natural antigen-presentingcell (e.g., dendritic cell) or an artificial antigen-presenting cell or particle under selective conditions prior to formulation into a pharmaceutical composition and administration.KitsAny of the compositions described herein may be provided in a kit for genetically modifying cells to introduce multiple sequence-specific genetic modifications. In some embodiments, the kit comprises a multi-module gene editing construct. In some embodiments, the kit comprises a plurality of recombinant polynucleotides comprising different genome editing modules for assembling a multi-module gene editing construct according to the methods described herein. The kit may further comprise other agents for performing the subject methods such as an RNA-guided nuclease for genome editing (e.g., a Cas nuclease), a restriction enzyme (e.g., type IIS restriction enzyme) for excising excisable stuffer sequences during assembly of the multi-module gene editing construct, and / or reagents for sequencing barcodes of the multi-module gene editing construct. A kit may also include a binding agent for performing negative or positive selection to separate successfully edited cells from unsuccessfully edited cells in a sample. In some cases, the binding agent comprises a magnetic bead comprising an antibody specific for a cellular marker that can be used for enrichment of a selected population of cells using magnetic separation. Additionally, the kit may include cells, transfection agents, buffers, media suitable for culturing cells, tissue culture plates, flasks, test tubes, vials, and the like, and optionally one or more other factors, such as cytokines (e.g., IL-2, IL-3, IL-6, IL- 7, IL-15, TNFZI, IFN-D , and GM-CSF), growth factors, antibiotics, or other media supplements, and the like.

[0388] Such kits generally will comprise, in suitable means, distinct containers for each individual reagent or solution. Suitable containers for the compositions include, for example, bottles, vials, syringes, and test tubes. Containers can be formed from a variety of materials, including glass or plastic. A container may have a sterile access port (for example, the container may be a vial having a stopper pierceable by a hypodermic injection needle).

[0389] The kit may also provide a delivery device for administration of CAR-T cells to a patient. For example, kits may comprise a container having a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The kit can further comprise a container comprising a pharmaceutically-acceptable buffer, such as phosphate-buffered saline, Ringer'ssolution, or dextrose solution. It can also contain other materials useful to the end-user, including other pharmaceutically acceptable formulating solutions such as buffers, diluents, filters, needles, and syringes or other delivery device.

[0390] In addition to the above components, the subject kits may further include (in certain embodiments) instructions for practicing the subject methods. These instructions may be present in the subject kits in a variety of forms, one or more of which may be present in the kit. One form in which these instructions may be present is as printed information on a suitable medium or substrate, e.g., a piece or pieces of paper on which the information is printed, in the packaging of the kit, in a package insert, and the like. Yet another form of these instructions is a computer readable medium, e.g., diskette, compact disk (CD), DVD, flash drive, and the like, on which the information has been recorded. Yet another form of these instructions that may be present is a website address which may be used via the internet to access the information at a removed site.Examples of Non-Limiting Aspects of the Disclosure

[0391] Aspects, including embodiments, of the present subject matter described above may be beneficial alone or in combination, with one or more other aspects or embodiments. Without limiting the foregoing description, certain non-limiting aspects of the disclosure numbered 1 -130 are provided below. As will be apparent to those of skill in the art upon reading this disclosure, each of the individually numbered aspects may be used or combined with any of the preceding or following individually numbered aspects. This is intended to provide support for all such combinations of aspects and is not limited to combinations of aspects explicitly provided below.1 . A method of assembling a multi-module gene editing construct, the method comprising:(a) providing a circular DNA or plasmid comprising a first recombinant polynucleotide, wherein the first recombinant polynucleotide comprises a first genome editing module and a first barcode to identify the first genome editing module, wherein the first genome editing module and the first barcode are separated by a first excisable stuffer sequence, wherein the first excisable stuffer sequence is flanked by a first excision-mediating site and a second excision-mediating site;(b) contacting the circular DNA or plasmid with an excising agent that binds to the first excision-mediating site and the second excision-mediating site, wherein the firstexcisable stutter sequence is excised from the first recombinant polynucleotide to produce a cleaved circular DNA or plasmid having the first genome editing module at a first cleaved end and the first barcode at a second cleaved end;(c) providing a second recombinant polynucleotide, wherein the second recombinant polynucleotide comprises a second genome editing module and a second barcode to identify the second genome editing module, wherein the second genome editing module and the second barcode are separated by a second excisable stuffer sequence, wherein the second excisable stuffer sequence is flanked by a third excisionmediating site and a fourth excision-mediating site;(d) ligating the second recombinant polynucleotide to the first cleaved end and the second cleaved end to produce a first ligation product, wherein the first genome module is connected to the second genome module, and wherein the second barcode is connected to the first barcode;(e) contacting the first ligation product with an excising agent that binds to the third excision-mediating site and the fourth excision-mediating site, wherein the second excisable stuffer sequence is excised from the first ligation product to produce a first cleaved ligation product having the first genome editing module connected to the second genome module at a first end of the first cleaved ligation product and the second barcode connected to the first barcode at a second end of the first cleaved ligation product;(f) providing a third recombinant polynucleotide, wherein the third recombinant polynucleotide comprises a third genome editing module and a third barcode to identify the third genome editing module, wherein the third genome editing module and the third barcode are separated by a third excisable stuffer sequence, wherein the third excisable stuffer sequence is flanked by a fifth excision-mediating site and a sixth excisionmediating site;(g) ligating the third recombinant polynucleotide to the first end of the first cleaved ligation product and the second end of the first cleaved ligation product to produce a second ligation product comprising the first genome module, the second genome module, the third genome module, the third excisable stuffer sequence flanked by the fifth excision-mediating site and the sixth excision-mediating site, and a 3’-barcode array comprising the third barcode, followed by the second barcode, followed by the first barcode; and(h) repeating steps (e) - (g) until assembly of the multi-module gene editing construct is completed.2. The method of aspect 1 , wherein each genome editing module in the multimodule gene editing construct can produce a different sequence-specific genetic perturbation.3. The method of aspect 1 or 2, wherein the multi-module gene editing construct can produce at least 3,...

Claims

What is claimed is:1 . A method of assembling a multi-module gene editing construct, the method comprising:(a) providing a circular DNA or plasmid comprising a first recombinant polynucleotide, wherein the first recombinant polynucleotide comprises a first genome editing module and a first barcode to identify the first genome editing module, wherein the first genome editing module and the first barcode are separated by a first excisable stuffer sequence, wherein the first excisable stuffer sequence is flanked by a first excision-mediating site and a second excision-mediating site;(b) contacting the circular DNA or plasmid with an excising agent that binds to the first excision-mediating site and the second excision-mediating site, wherein the first excisable stuffer sequence is excised from the first recombinant polynucleotide to produce a cleaved circular DNA or plasmid having the first genome editing module at a first cleaved end and the first barcode at a second cleaved end;(c) providing a second recombinant polynucleotide, wherein the second recombinant polynucleotide comprises a second genome editing module and a second barcode to identify the second genome editing module, wherein the second genome editing module and the second barcode are separated by a second excisable stuffer sequence, wherein the second excisable stuffer sequence is flanked by a third excisionmediating site and a fourth excision-mediating site;(d) ligating the second recombinant polynucleotide to the first cleaved end and the second cleaved end to produce a first ligation product, wherein the first genome module is connected to the second genome module, and wherein the second barcode is connected to the first barcode;(e) contacting the first ligation product with an excising agent that binds to the third excision-mediating site and the fourth excision-mediating site, wherein the second excisable stuffer sequence is excised from the first ligation product to produce a first cleaved ligation product having the first genome editing module connected to the second genome module at a first end of the first cleaved ligation product and the second barcode connected to the first barcode at a second end of the first cleaved ligation product;(f) providing a third recombinant polynucleotide, wherein the third recombinant polynucleotide comprises a third genome editing module and a third barcode to identify the third genome editing module, wherein the third genome editing module and the thirdbarcode are separated by a third excisable stutter sequence, wherein the third excisable stutter sequence is flanked by a fifth excision-mediating site and a sixth excisionmediating site;(g) ligating the third recombinant polynucleotide to the first end of the first cleaved ligation product and the second end of the first cleaved ligation product to produce a second ligation product comprising the first genome module, the second genome module, the third genome module, the third excisable staffer sequence flanked by the fifth excision-mediating site and the sixth excision-mediating site, and a 3’-barcode array comprising the third barcode, followed by the second barcode, followed by the first barcode; and(h) repeating steps (e) - (g) until assembly of the multi-module gene editing construct is completed.

2. The method of claim 1 , wherein each genome editing module in the multimodule gene editing construct can produce a different sequence-specific genetic perturbation.

3. The method of claim 1 or 2, wherein the multi-module gene editing construct can produce at least 3, at least 4, at least 5, or at least 6 sequence-specific genetic perturbations selected from a gene knockin, a gene knockout, a gene knockdown, a gene upregulation, an insertion of a synthetic gene, an insertion of a non-coding domain, an insertion of a protein coding domain, a deletion of an endogenous protein coding domain, an insertion of a regulatory element, a deletion of a regulatory element, an epigenetic perturbation, and an insertion of a unique molecular identifier or cell tracing sequence.

4. The method of claim 3, wherein the multi-module gene editing construct comprises a plurality of genome editing modules to knockin a plurality of full-length genes or coding or non-coding domains, knockdown expression of a plurality of genes, upregulate expression of a plurality of genes, knockout a plurality of genes, or a combination thereof.

5. The method of any one of claims 1 -4, wherein at least one recombinant polynucleotide further comprises a constant 5’-adapter at the 5’-end of the recombinant polynucleotide and a constant 3’-adapter at the 3’-end of the recombinant polynucleotide.

6. The method of any one of claims 1 -5, wherein at least one recombinant polynucleotide further comprises an intron comprising a 5’-splice donor site, a constant 5’-adapter, and a 3’-splice acceptor site, wherein the constant 5’-adapter is positioned between the 5’-splice donor site and the 3’-splice acceptor site, and wherein the constant 5’-adapter is removed by messenger RNA splicing.

7. The method of any one of claims 1 -6, wherein each excisable stuffer sequence further comprises a constant 5’-adapter at the 5’-end of the excisable stuffer sequence and a constant 3’-adapter at the 3’-end of the excisable stuffer sequence.

8. The method of any one of claims 1 -7, wherein each excisable stuffer sequence comprises or consists of the same sequence.

9. The method of any one of claims 1 -8, wherein each excisable stuffer sequence further comprises a safety cut site.

10. The method of any one of claims 1 -9, wherein each excisable stuffer sequence further comprises a sequencing primer binding site.1 1. The method of claim 10, further comprising sequencing the 3’-barcode array using a sequencing primer that binds to the sequencing primer binding site.

12. The method of any one of claims 1 -1 1 , wherein the excising agent is a restriction enzyme or a site-specific recombinase.

13. The method of claim 12, wherein each excision-mediating site comprises a recognition site for the same restriction enzyme or site-specific recombinase.

14. The method of claim 12 or 13, wherein the restriction enzyme is a type IIS restriction enzyme.

15. The method of claim 14, wherein the type IIS restriction enzyme is Bsal, Bbsl, Bsgl, BsmAI, BsmBI-v2, BsmFI, Bsml, BspCNI, BspMI, BspQI, BsrDI, Bsrl, BtgZI, BtsCI, Btsl-v2, BtsIMutl, CspCI, Earl, Ecil, Esp3l, Faul, Fokl, Hgal, Hphl, HpyAV, Mboll, Mlyl, Mmel, Mnll, NmeAIII, PaqCI, Piel, Sapl, BspQI, or SfaNI.

16. The method of any one of claims 12-15, wherein the excisable staffer sequence comprises: a first recognition site for binding the restriction enzyme, wherein the restriction enzyme cuts at the first excision site; a second recognition site for binding the restriction enzyme, wherein the restriction enzyme cuts at the second excision site; a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site positioned between the safety cut site and the second recognition site for binding the restriction enzyme; a constant 5’-adapter positioned at the 5’-end of the excisable staffer sequence; and a constant 3’-adapter positioned at the 3’-end of the excisable staffer sequence.

17. The method of any one of claims 1 -16, wherein at least one recombinant polynucleotide comprises a genome editing module comprising a sequence encoding a full-length gene, a coding domain, a non-coding domain, a regulatory RNA, or a guide RNA (gRNA).

18. The method of claim 17, wherein the regulatory RNA comprises a short hairpin RNA (shRNA).

19. The method of claim 18, wherein the shRNA is a microRNA (miR)- embedded shRNA, wherein endogenous microRNA processing of the miR-embedded shRNA within a cell transfected with the multi-module gene editing construct results in excision of the shRNA such that the shRNA can inhibit expression of a target gene.

20. The method of claim 19, wherein the miR-embedded shRNA is a miR-30- embedded shRNA or a miR-E-embedded shRNA.

21. The method of any one of claims 17-20, wherein said at least one recombinant polynucleotide further comprises a multicistronic element, wherein the multicistronic element is positioned between the sequence encoding the full-length gene or the coding domain and the excisable stuffer sequence.

22. The method of claim 21 , wherein the multicistronic element is a 2A multicistronic element.

23. The method of any one of claims 17-22, wherein said at least one recombinant polynucleotide further comprises an intron spacer sequence positioned between the constant 5’-adapter sequence at the 5’-end of the recombinant polynucleotide and the sequence encoding the full-length gene or the coding domain.

24. The method of claim 17, wherein said at least one recombinant polynucleotide comprises: a constant 5’-adapter sequence at the 5’-end of the recombinant polynucleotide; a genome editing module comprising a sequence encoding a full-length gene; an intron spacer sequence, wherein the intron spacer sequence is positioned between the constant 5’-adapter sequence at the 5’-end of the recombinant polynucleotide and the sequence encoding the full-length gene; an excisable stuffer sequence flanked by a first excision site and a second excision site, wherein the excisable stuffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme,a constant 5’-adapter positioned at the 5’-end of the excisable staffer sequence, and a constant 3’-adapter positioned at the 3’-end of the excisable staffer sequence; a 2A multicistronic element, wherein the 2A multicistronic element is positioned between the sequence encoding the full-length gene and the excisable staffer sequence to separate an open reading frame of the full-length gene from any other open reading frame; a barcode for identification of the genome editing module comprising the sequence encoding the full-length gene, wherein the barcode is separated from the genome editing module by the excisable staffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.

25. The method of claim 17, wherein said at least one recombinant polynucleotide comprises: a genome editing module comprising a sequence encoding a coding domain or a non-coding domain; an intron spacer sequence positioned before the sequence encoding the coding domain or the non-coding domain, wherein the intron comprises a 5’-splice donor site, a constant 5’-adapter, and a 3’-splice acceptor site, wherein the constant 5’-adapter is positioned within the 5’-splice donor site or between the 5’-splice donor site and the 3’- splice acceptor site, and wherein the constant 5’-adapter is removed by messenger RNA splicing; an excisable staffer sequence flanked by a first excision site and a second excision site, wherein the excisable staffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme,a constant 5’-adapter positioned at the 5’-end of the excisable staffer sequence, and a constant 3’-adapter positioned at the 3’-end of the excisable staffer sequence; a barcode for identification of the genome editing module comprising the sequence encoding the coding domain or the non-coding domain, wherein the barcode is separated from the genome editing module by the excisable staffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.

26. The method of claim 25, wherein a scar region generated by assembly of the multi-module gene editing construct is contained within the intron spacer sequence, and wherein the intron spacer sequence containing the scar region is removed by messenger RNA splicing.

27. The method of claim 17, wherein said at least one recombinant polynucleotide comprises: a genome editing module comprising a sequence encoding a full-length gene or a protein coding domain; a stop codon operably linked to the sequence encoding the full-length gene or protein coding domain, wherein the stop codon terminates translation of the sequence encoding the full-length gene or protein coding domain; a 3’-untranslated region following the stop codon, wherein the 3’-untranslated region comprises: a mRNA stabilizing element, a sequence encoding a regulatory RNA, a first 5’-adapter positioned between the mRNA stabilizing element and the sequence encoding the regulatory RNA; an excisable staffer sequence flanked by a first excision site and a second excision site, wherein the excisable staffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site,a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a second constant 5’-adapter positioned at the 5’-end of the excisable staffer sequence, and a first constant 3’-adapter positioned at the 3’-end of the excisable staffer sequence; a barcode for identification of the genome editing module comprising the sequence encoding the regulatory RNA, wherein the barcode is separated from the sequence encoding the regulatory RNA by the excisable staffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.

28. The method of claim 27, wherein the regulatory RNA comprises a short hairpin RNA (shRNA).

29. The method of claim 28, wherein the shRNA is a microRNA (miR)- embedded shRNA, wherein endogenous microRNA processing of the miR-embedded shRNA within a cell transfected with the multi-module gene editing construct results in excision of the shRNA such that the shRNA can inhibit expression of a target gene.

30. The method of claim 29, wherein the miR-embedded shRNA is a miR-30- embedded shRNA or a miR-E-embedded shRNA.

31. The method of claim 30, wherein the 3’-untranslated region further comprises one or more additional sequences encoding one or more additional regulatory RNAs.

32. The method of claim 31 , wherein the one or more additional regulatoryRNAs are shRNAs or miR-embedded shRNAs.

33. The method of claim 17, wherein said at least one recombinant polynucleotide comprises: a genome editing module comprising a sequence encoding a full-length gene or a protein coding domain; a stop codon operably linked to the sequence encoding the full-length gene or protein coding domain, wherein the stop codon terminates translation of the sequence encoding the full-length gene or protein coding domain; a 3’-untranslated region following the stop codon, wherein the 3’-untranslated region comprises: a mRNA stabilizing element, a sequence encoding a guide RNA (gRNA) for an RNA-guided nuclease, a first 5’-adapter positioned between the mRNA stabilizing element and the sequence encoding the gRNA; an excisable stuffer sequence flanked by a first excision site and a second excision site, wherein the excisable stuffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a second constant 5’-adapter positioned at the 5’-end of the excisable stuffer sequence, and a first constant 3’-adapter positioned at the 3’-end of the excisable stuffer sequence; a barcode for identification of the genome editing module comprising the sequence encoding the gRNA, wherein the barcode is separated from the sequence encoding the gRNA by the excisable stuffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.

34. The method of any one of claims 27-33, wherein the mRNA stabilizing element is a triplex stabilizer.

35. The method of claim 34, wherein the RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nuclease.

36. The method of claim 35, wherein the Cas nuclease is Cas9, Cas12a, Cas12d, Cas13a, Cas13b, Cas13d, or a dead Cas9 (dCas9).

37. The method of any one of claims 33-36, wherein the gRNA forms a complex with the RNA-guided nuclease such that the gRNA directs the RNA-guided nuclease to a genomic target locus in a cell, wherein the RNA-guided nuclease creates a doublestranded break in the genomic DNA at the genomic target locus, wherein DNA repair of the double-stranded break by non-homologous end joining creates an insertion or deletion (indel) resulting in gene knockout at the genomic target locus.

38. The method of any one of claims 33-36, wherein the multi-module gene editing construct further comprises a genome editing module comprising a sequence encoding a donor polynucleotide, wherein the gRNA can form a complex with the RNA- guided nuclease such that the gRNA directs the RNA-guided nuclease to a genomic target locus in a cell, wherein the RNA-guided nuclease creates a double-stranded break in the genomic DNA at the genomic target locus, wherein a donor polynucleotide is integrated at the genomic target locus by homology directed repair (HDR).

39. The method of any one of claims 33-38, wherein the multi-module gene editing construct further comprises a genome editing module comprising a sequence encoding the RNA-guided nuclease.

40. The method of any one of claims 1 -39, further comprising sequencing the barcodes in the multi-module gene editing construct to identify the genome editing modules in the multi-module gene editing construct.41 . The method of any one of claims 1 -40, further comprising adding a barcode module comprising one or more additional barcodes to the circular DNA or plasmid comprising the multi-module gene editing construct.

42. The method of claim 41 , wherein said adding the barcode module comprising the one or more additional barcodes comprises: contacting the multi-module gene editing construct with an excising agent, wherein an excisable stuffer sequence is excised from the multi-module gene editing construct to produce a cleaved circular DNA or plasmid comprising the genome editing modules of the multi-module gene editing construct at a first cleaved end and the 3’-barcode array comprising the barcodes of the multi-module gene editing construct at a second cleaved end; and ligating a polynucleotide comprising the barcode module comprising the one or more additional barcodes to the first cleaved end and the second cleaved end, wherein the barcode module comprising the one or more additional barcodes is added to the 3'- barcode array comprising the barcodes of the genome editing modules of the multimodule gene editing construct.

43. The method of claim 41 or 42, wherein the one or more additional barcodes comprise a cell tracing sequence or unique molecular identifier sequence.

44. The method of claim 43, wherein the cell tracing sequence identifies a clone, cell lineage, donor, or condition.

45. The method of claim 44, further comprising sequencing the one or more additional barcodes to identify the clone, the cell lineage, or the donor from which a genetically modified cell comprising the multi-module gene editing construct was derived.

46. The method of any one of claims 1 -45, further comprising performing single-cell RNA sequencing on a genetically modified cell comprising the multi-module gene editing construct.

47. The method of any one of claims 1 -46, further comprising integrating the multi-module gene editing construct into a target locus in the genome of a host cell.

48. The method of claim 47, wherein said integrating comprises using a CRISPR system.

49. The method of any one of claims 1 -48, further comprising cloning the multimodule gene editing construct into a vector.

50. The method of claim 49, wherein the vector is a plasmid or a viral vector.51 . The method of claim 50, wherein the viral vector is a lentivirus vector, retrovirus vector, or adeno-associated virus vector.

52. The method of any one of claims 1 -51 , further comprising transfecting a host cell with the vector or the multi-module gene editing construct.

53. The method of claim 52, wherein the host cell is transfected transiently or stably.

54. The method of claim 52 or 53, wherein said transfecting comprises performing electroporation, nucleofection, or lipofection.

55. The method of any one of claims 1 -54, wherein at least one genome editing module in the multi-module gene editing construct produces a chimeric antigen receptor (GAR) gene knockin in a T cell.

56. The method of claim 55, wherein at least one genome editing module in the multi-module gene editing construct produces a T cell receptor (TCR) gene knockout in the T cell.

57. The method of claim 55 or 56, wherein at least one genome editing module in the multi-module gene editing construct produces a knockout of a gene encoding an alloantigen.

58. The method of claim 57, wherein the alloantigen is a major histocompatibility complex (MHC) class I alloantigen or an MNS blood group alloantigen.

59. The method of claim 58, wherein the alloantigen is CD1 , CD2, CD3, CD4, CD7, CD8, Ly-6, Qa-2, RT6, CD19, CD22, CD56, CD58 (LFA-3), CD59, or CDw90 (Thy 1)-60. The method of any one of claims 1 -59, wherein at least one genome editing module in the multi-module gene editing construct produces a knockout of a CD5, CD52, CD70, BATF, LCK, PD-1 , LAG-3, CTLA-4, 2-B2M, PD-1 , HLA-I, Fas, TGFBR2, PDCD- 1 , DGK, EZH2, PAX5, or LDLR gene.

61. The method of any one of claims 55-60, wherein at least one genome editing module further engineers the T cell to further comprise a binding-triggered transcriptional switch that regulates expression of the chimeric antigen receptor or activation of the T cell.

62. The method of claim 61 , wherein the binding-triggered transcriptional switch comprises a synthetic notch receptor, a modular extracellular sensor architecture (MESA), or a synthetic intramembrane proteolysis receptor (SNIPR).

63. The method of claim 62, wherein the synthetic notch receptor comprises i) an extracellular ligand-binding domain that specifically binds to a second target antigen on the target cell, and ii) an intracellular domain, wherein binding of the extracellular ligand-binding domain to the second target antigen results in cleavage of the intracellular domain to release a transcription factor from the intracellular domain, wherein the transcription factor that is released from the intracellular domain induces expression of the chimeric antigen receptor on the CAR-T cell.

64. A multi-module gene editing construct produced by the method of any one of claims 1 -63.

65. A combinatorial library comprising a plurality of different multi-module gene editing constructs produced by the method of any one of claims 1 -63.

66. A vector comprising the multi-module gene editing construct of claim 64.

67. The vector of claim 66, wherein the vector is a plasmid or a viral vector.

68. A host cell comprising the multi-module gene editing construct of claim 64.

69. The host cell of claim 68, wherein the host cell is a mammalian host cell.

70. The host cell of claim 69, wherein the mammalian host cell is a human host cell.71 . The host cell of claim 69 or 70, wherein the mammalian host cell is an immune cell.

72. The host cell of claim 71 , wherein the immune cell is a T cell, a B cell, a natural killer cell, a neutrophil, an eosinophil, a mast cell, a basophil, a monocyte, a macrophage, or a dendritic cell.

73. The method of claim 72, wherein the T cell is a helper CD4+T cell, a cytotoxic CD8+T cell, a natural killer T cell, or a gamma delta T cell.

74. A method of producing a genetically modified host cell, the method comprising introducing the multi-module gene editing construct of claim 64 into a host cell, wherein the multi-module gene editing construct genetically modifies the genome of the host cell to generate a genetically modified host cell.

75. The method of claim 74, further comprising introducing an RNA-guided nuclease into the host cell.

76. The method of claim 75, wherein the RNA-guided nuclease is provided by a vector, or as an mRNA sequence encoding the RNA-guided nuclease along with codelivery of a guide RNA or plasmid expressing a guide RNA, or as a ribonucleoprotein complex of the RNA-guided nuclease with a guide RNA.

77. The method of claim 76, wherein the RNA-guided nuclease is provided by the multi-module gene editing construct.

78. A method of simultaneously screening the effects of a plurality of sequencespecific genetic perturbations, the method comprising: introducing the multi-module gene editing construct of claim 64 into a host cell, wherein the multi-module gene editing construct genetically modifies the genome of the host cell to generate a genetically modified host cell comprising the plurality of sequencespecific genetic perturbations; and detecting an effect of the plurality of sequence-specific genetic perturbations on the genetically modified host cell.

79. The method of claim 78, wherein said detecting comprises detecting a change in cell morphology, cell growth, cell proliferation, gene expression, biological activity of a protein, or any combination thereof in the genetically modified host cell compared to an unmodified host cell.

80. The method of claim 78 or 79, further comprising: contacting the genetically modified host cell with a test agent; and detecting an effect of the plurality of sequence-specific genetic perturbations on activity of the test agent.81 . The method of any one of claims 78-80, further comprising introducing an RNA-guided nuclease into the host cell.

82. The method of claim 81 , wherein the RNA-guided nuclease is provided by a vector, or as an mRNA sequence encoding the RNA-guided nuclease along with codelivery of a guide RNA or plasmid expressing a guide RNA, or as a ribonucleoprotein complex of the RNA-guided nuclease with a guide RNA.

83. The method of claim 81 , wherein the RNA-guided nuclease is provided by the multi-module gene editing construct.

84. A method of simultaneously screening the effects of a plurality of sequencespecific genetic perturbations, the method comprising: providing a combinatorial library comprising a plurality of genetically modified host cells, wherein each genetically modified host cell comprises a different multi-module gene editing construct produced according to the method of any one of claims 1 -63, wherein the different multi-module gene editing constructs generate different sequence-specific genetic perturbations in each genetically modified host cell; and detecting effects of the different sequence-specific genetic perturbations in the plurality of genetically modified host cells; and sequencing the 3’-barcode arrays of the multi-module gene editing constructs in one or more genetically modified host cells of interest out of the plurality of genetically modified host cells to identify the genome editing modules present in the multi-module gene editing constructs in the one or more genetically modified host cells of interest.

85. The method of claim 84, wherein said detecting comprises detecting a change in cell morphology, cell growth, cell proliferation, gene expression, biological activity of a protein, or any combination thereof in the plurality of genetically modified host cells compared to an unmodified host cell.

86. The method of claim 84 or 85, further comprising: contacting the plurality of genetically modified host cells with a test agent; and detecting effects of the different sequence-specific genetic perturbations in the plurality of genetically modified host cells on activity of the test agent.

87. A composition comprising the genetically modified host cell of any one of claims 68-72 and a pharmaceutically acceptable excipient or carrier.

88. A method of performing cellular therapy, the method comprising administering a therapeutically effective amount of the composition of claim 87 to a subject.

89. The method of claim 88, wherein the genetically modified host cell is autologous or allogeneic.

90. The method of claim 88 or 89, wherein the genetically modified host cell is an immune cell.

91. The method of claim 90, wherein the immune cell is a T cell, a B cell, a natural killer cell, a neutrophil, an eosinophil, a mast cell, a basophil, a monocyte, a macrophage, or a dendritic cell.

92. The method of claim 91 , wherein the T cell is a helper CD4+T cell, a cytotoxic CD8+T cell, a natural killer T cell, or a gamma delta T cell.

93. The method of claim 91 or 92, wherein the T cell comprises a multi-module gene editing construct that comprises a genome editing module that produces a gene knockin of a gene encoding a chimeric antigen receptor (CAR) that specifically binds to a target antigen.

94. The method of claim 93, wherein the multi-module gene editing construct further comprises a genome editing module that produces a gene knockout of a gene encoding a T cell receptor (TCR) protein chain.

95. The method of claim 94, wherein the TCR protein chain is a T cell receptor alpha chain, a T cell receptor beta chain, a T cell receptor delta chain, or a T cell receptor gamma chain.

96. The method of any one of claims 93-95, wherein the CAR comprises a transmembrane domain linked to an extracellular antigen binding domain and an intracellular signaling domain, wherein the extracellular antigen-binding domain specifically binds to the target antigen.

97. The method of claim 96, wherein the extracellular antigen binding domain comprises a single chain variable fragment (scFv), an antigen-binding fragment (Fab), a nanobody, a heavy chain variable (VH) domain, a light chain variable (VL) domain, a single domain antibody (sdAb), a shark variable domain of a new antigen receptor (VNAR), a single variable domain on a heavy chain (VHH), a bispecific antibody, a diabody, or a functional fragment thereof that binds specifically to the antigen.

98. The method of claim 96 or 97, wherein the intracellular signaling domain is a CD3-zeta intracellular signaling domain or a ZAP-70 intracellular signaling domain.

99. The method of claim 96 or 97, wherein the intracellular signaling domain comprises an immunoreceptor tyrosine-based activation motif (ITAM).

100. The method of any one of claims 93-99, wherein the CAR further comprises a costimulatory domain.

101. The method of claim 100, wherein the costimulatory domain is a 4-1 BB, CD28, ICOS, OX-40, BTLA, CD27, CD30, GITR, or HVEM costimulatory domain.

102. The method of any one of claims 96-101 , wherein the transmembrane domain is a CD8, Megfl O, FcRy, Bail , MerTK, TIM4, Stabilin-1 , Stabilin-2, RAGE, CD300f, integrin subunit av, integrin subunit |35, CD36, LRP1 , SCARF1 , C1 Qa, Axl, CD45, or CD86 transmembrane domain.

103. The method of any one of claims 93-102, wherein the target antigen is on a cancer cell, a tumor cell, an activated fibroblast, an autoreactive immune cell, a pathogen, or a diseased cell.

104. The method of claim 103, wherein the target antigen is a tumor antigen or a tumor-associated antigen.

105. The method of claim 103, wherein the pathogen is a virus, a bacterium, a fungus, or a parasite.

106. The method of claim 105, wherein the target antigen is a viral antigen, a bacterial antigen, a fungal antigen or a parasite antigen.

107. The method of claim 103, wherein the autoreactive immune cell is an autoreactive T cell or B cell.

108. The method of claim 107, wherein the target antigen is an antigen on the autoreactive T cell or B cell.

109. The method of any one of claims 93-108, wherein the multi-module gene editing construct further comprises a genome editing module that engineers the T cell to further comprise a binding-triggered transcriptional switch that regulates expression of the chimeric antigen receptor or activation of the T cell.

110. The method of claim 109, wherein the binding-triggered transcriptional switch comprises a synthetic notch receptor, a modular extracellular sensor architecture (MESA), or a synthetic intramembrane proteolysis receptor (SNIPR).11 1. The method of claim 110, wherein the synthetic notch receptor comprises i) an extracellular ligand-binding domain that specifically binds to a second target antigen on the target cell, and ii) an intracellular domain, wherein binding of the extracellular ligand-binding domain to the second target antigen results in cleavage of the intracellular domain to release a transcription factor from the intracellular domain, wherein the transcription factor that is released from the intracellular domain induces expression of the chimeric antigen receptor on the CAR-T cell.

112. A recombinant polynucleotide comprising: a constant 5’-adapter sequence at the 5’-end of the recombinant polynucleotide; a genome editing module comprising a sequence encoding a genetic perturbation; an excisable stuffer sequence flanked by a first excision-mediating site and a second excision-mediating site, wherein the excisable stuffer comprises: a first recognition site for binding a restriction enzyme, wherein the restriction enzyme cuts at the first excision site, a second recognition site for binding the restriction enzyme, wherein the restriction enzyme cuts at the second excision site, and a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the first recognition site for binding the restriction enzyme and the second recognition site for binding the restriction enzyme; a barcode associated with the genetic perturbation, wherein the barcode is separated from the genome editing module by the excisable stuffer sequence; anda constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.

113. The recombinant polynucleotide of claim 1 12, wherein the genetic perturbation is a gene knockin, a gene knockout, a gene knock-down, a gene upregulation, an insertion of a synthetic gene, an insertion of a non-coding domain, an insertion of a domain, a deletion of a domain, an insertion of a regulatory element, a deletion of a regulatory element, an epigenetic perturbation, or an insertion of a unique molecular identifier or cell tracing sequence.1 14. The recombinant polynucleotide of claim 1 12 or 1 13, wherein the genome editing module comprises a sequence encoding a full-length gene, a coding domain, a non-coding domain, or a regulatory RNA.

115. The recombinant polynucleotide of any one of claims 1 12-1 14, further comprising an intron comprising a 5’-splice donor site, a constant 5’-adapter, and a 3'- splice acceptor site, wherein the constant 5’-adapter is positioned between the 5’-splice donor site and the 3’-splice acceptor site, and wherein the constant 5’-adapter is removed by messenger RNA splicing.

116. A recombinant polynucleotide comprising: a constant 5’-adapter sequence at the 5’-end of the recombinant polynucleotide; a genome editing module comprising a sequence encoding a full-length gene; an intron spacer sequence, wherein the intron spacer sequence is positioned between the constant 5’-adapter sequence at the 5’-end of the recombinant polynucleotide and the sequence encoding the full-length gene; an excisable stuffer sequence flanked by a first excision site and a second excision site, wherein the excisable stuffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site,a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a constant 5’-adapter positioned at the 5’-end of the excisable stuffer sequence, and a constant 3’-adapter positioned at the 3’-end of the excisable stuffer sequence; a 2A multicistronic element, wherein the 2A multicistronic element is positioned between the sequence encoding the full-length gene and the excisable stuffer sequence to separate an open reading frame of the full-length gene from any other open reading frame; a barcode for identification of the genome editing module comprising the sequence encoding the full-length gene, wherein the barcode is separated from the genome editing module by the excisable stuffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.1 17. A recombinant polynucleotide comprising: a genome editing module comprising a sequence encoding a coding domain or a non-coding domain; an intron spacer sequence positioned before the sequence encoding the coding domain or the non-coding domain, wherein the intron comprises a 5’-splice donor site, a constant 5’-adapter, and a 3’-splice acceptor site, wherein the constant 5’-adapter is positioned within the 5’-splice donor site or between the 5’-splice donor site and the 3’- splice acceptor site, and wherein the constant 5’-adapter is removed by messenger RNA splicing; an excisable stuffer sequence flanked by a first excision site and a second excision site, wherein the excisable stuffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site,a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a constant 5’-adapter positioned at the 5’-end of the excisable stuffer sequence, and a constant 3’-adapter positioned at the 3’-end of the excisable stuffer sequence; a barcode for identification of the genome editing module comprising the sequence encoding the coding domain or the non-coding domain, wherein the barcode is separated from the genome editing module by the excisable stuffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.

118. A recombinant polynucleotide comprising: a genome editing module comprising a sequence encoding a full-length gene or a protein coding domain; a stop codon operably linked to the sequence encoding the full-length gene or protein coding domain, wherein the stop codon terminates translation of the sequence encoding the full-length gene or protein coding domain; a 3’-untranslated region following the stop codon, wherein the 3’-untranslated region comprises: a mRNA stabilizing element, a sequence encoding a regulatory RNA, a first 5’-adapter positioned between the mRNA stabilizing element and the sequence encoding the regulatory RNA; an excisable stuffer sequence flanked by a first excision site and a second excision site, wherein the excisable stuffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site,a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a second constant 5’-adapter positioned at the 5’-end of the excisable staffer sequence, and a first constant 3’-adapter positioned at the 3’-end of the excisable stuffer sequence; a barcode for identification of the genome editing module comprising the sequence encoding the regulatory RNA, wherein the barcode is separated from the sequence encoding the regulatory RNA by the excisable stuffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.

119. The recombinant polynucleotide of claim 1 18, wherein the regulatory RNA comprises a short hairpin RNA (shRNA).

120. The recombinant polynucleotide of claim 119, wherein the shRNA is a microRNA (miR)-embedded shRNA, wherein endogenous microRNA processing of the miR-embedded shRNA within a cell transfected with the recombinant polynucleotide results in excision of the shRNA such that the shRNA can inhibit expression of a target gene.

121. The recombinant polynucleotide of claim 120, wherein the miR-embedded shRNA is a miR-30-embedded shRNA or a miR-E-embedded shRNA.

122. The recombinant polynucleotide of any one of claims 118-121 , wherein the 3’-untranslated region further comprises one or more additional sequences encoding one or more additional regulatory RNAs.

123. The recombinant polynucleotide of claim 122, wherein the one or more additional regulatory RNAs are shRNAs or miR-embedded shRNAs.

124. A recombinant polynucleotide comprising: a genome editing module comprising a sequence encoding a full-length gene or a protein coding domain; a stop codon operably linked to the sequence encoding the full-length gene or protein coding domain, wherein the stop codon terminates translation of the sequence encoding the full-length gene or protein coding domain; a 3’-untranslated region following the stop codon, wherein the 3’-untranslated region comprises: a mRNA stabilizing element, a sequence encoding a guide RNA (gRNA) for an RNA-guided nuclease, a first 5’-adapter positioned between the mRNA stabilizing element and the sequence encoding the gRNA; an excisable stuffer sequence flanked by a first excision site and a second excision site, wherein the excisable stuffer sequence comprises: a first recognition site for binding a Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the first excision site, a second recognition site for binding the Type IIS restriction enzyme, wherein the Type IIS restriction enzyme cuts at the second excision site, a safety cut site positioned between the first excision site and the second excision site, a sequencing primer binding site, wherein the sequencing primer binding site is positioned between the safety cut site and the second recognition site for binding the Type IIS restriction enzyme, a second constant 5’-adapter positioned at the 5’-end of the excisable stuffer sequence, and a first constant 3’-adapter positioned at the 3’-end of the excisable stuffer sequence; a barcode for identification of the genome editing module comprising the sequence encoding the gRNA, wherein the barcode is separated from the sequence encoding the gRNA by the excisable stuffer sequence; and a constant 3’-adapter sequence at the 3’-end of the recombinant polynucleotide.

125. The recombinant polynucleotide of claim 124, wherein the RNA-guided nuclease is a clustered regularly interspaced short palindromic repeats (CRISPR)- associated (Cas) nuclease.

126. The recombinant polynucleotide of claim 125, wherein the Cas nuclease is Cas9, Cas12a, Cas12d, Cas13a, Cas13b, Cas13d, or a dead Cas9 (dCas9).

127. The recombinant polynucleotide of any one of claims 124-126, wherein the gRNA forms a complex with the RNA-guided nuclease such that the gRNA directs the RNA-guided nuclease to a genomic target locus in a cell, wherein the RNA-guided nuclease creates a double-stranded break in the genomic DNA at the genomic target locus, wherein DNA repair of the double-stranded break by non-homologous end joining creates an insertion or deletion (indel) resulting in gene knockout at the genomic target locus.

128. The recombinant polynucleotide of any one of claims 124-127, wherein the 3’-untranslated region further comprises a sequence encoding a donor polynucleotide, wherein the gRNA can form a complex with the RNA-guided nuclease such that the gRNA directs the RNA-guided nuclease to a genomic target locus in a cell, wherein the RNA-guided nuclease creates a double-stranded break in the genomic DNA at the genomic target locus, wherein a donor polynucleotide is integrated at the genomic target locus by homology directed repair (HDR).

129. The recombinant polynucleotide of any one of claims 124-128, wherein the type IIS restriction enzyme is Bsal, Bbsl, Bsgl, BsmAI, BsmBI-v2, BsmFI, Bsml, BspCNI, BspMI, BspQI, BsrDI, Bsrl, BtgZI, BtsCI, Btsl-v2, BtsIMutl, CspCI, Earl, Ecil, Esp3l, Faul, Fokl, Hgal, Hphl, HpyAV, Mboll, Mlyl, Mmel, Mnll, NmeAIII, PaqCI, Piel, Sapl, BspQI, or SfaNI.

130. The recombinant polynucleotide of any one of claims 118-129, wherein the mRNA stabilizing element is a triplex stabilizer.

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