Compositions and methods for genetically modifying transforming growth factor beta receptor type 2 (TGFΒr2)

Abstract

Compositions and methods for editing, e.g., altering a DNA sequence, within a TGFβR2 are provided. Compositions and methods for reducing or eliminating TGFβR2 protein expression in a cell are provided. Compositions and methods for immunotherapy are provided.

Description

COMPOSITIONS AND METHODS FOR GENETICALLY MODIFYING TRANSFORMING GROWTH FACTOR BETA RECEPTOR TYPE 2 (TGF^R2) I. CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit under 35 USC 119(e) of US Provisional Application No.63 / 519,487, filed August 14, 2023, and US Provisional Application No. 63 / 519,733, filed August 15, 2023, the content of each of which is herein incorporated by reference in its entirety. II. REFERENCE TO ELECTRONIC SEQUENCE LISTING

[0002] This application contains a sequence listing, which has been submitted electronically in XML file format and is hereby incorporated by reference in its entirety. Said XML file, created on August 12, 2024, is named “01155-0057-00PCT.xml” and is 2,244,990 bytes in size. III. INTRODUCTION AND SUMMARY

[0003] The present disclosure particularly relates to genetic modification of the Transforming Growth Factor Beta Receptor Type 2 (TGF^R2) gene. The present disclosure also relates to CRISPR / Cas9 genome editing systems.

[0004] Transforming Growth Factor Beta (TGF-^) is a secreted cytokine with pleiotropic effects on processes ranging from development to carcinogenesis to immune signaling. Within the context of the immune system, TGF-^ signaling maintains homeostasis by promoting self-tolerance and suppressing inflammation. It accomplishes this through a broad repertoire of mechanisms, including subverting the antigen presentation capability of dendritic cells; mitigating the cytotoxicity of natural killer cells; suppressing the acquisition of a pro-inflammatory phenotype in macrophages; and inhibiting T cell activation, differentiation, and proliferation (Batlle and Massague 2019).

[0005] The interplay between TGF-^ signaling and T cell biology has garnered considerable interest, largely on account of its role in cancer pathophysiology and implications for the development of new therapeutic approaches. Although TGF-^ normally functions as a tumor suppressor, suppressing proliferation and inducing apoptosis in pre-malignant cells, many cancers harbor mutations that inactivate the TGF-^ pathway, thereby allowing them to escape the anti-tumorigenic effects of TGF-^ signaling. Paradoxically, in the context of TGF-^resistance, high levels of TGF-^ may contribute to tumorigenesis by protecting cancer cells from the immune system. For example, high levels of TGF-^ within a tumor microenvironment may promote the exclusion of T cells from the tumor (Tauriello et al. 2018). Moreover, TGF-^ signaling may prevent naïve T cells from differentiating into helper T cells, thereby reducing immune surveillance within the tumor microenvironment (Sad and Mosmann 1994). As a final example, TGF-^ may also inhibit T cell proliferation in response to the tumor (Donkor et al.2011).

[0006] Thus, there exists a need for improved methods and compositions for modifying cells to overcome the problem of TGF-^-mediated immunosuppression. In particular, there is an unmet need for methods that mitigate the inhibitory effect of TGF-^ signaling on T cell activation, differentiation, and expansion.

[0007] Provided herein are compositions and methods for genetically modifying a TGF^R2 sequence, and the cells with genetic modifications in the TGF^R2 sequence and their use in various methods, e.g., to promote an immune response, e.g., in immunooncology and infectious disease. Also provided are methods of promoting an immune response and treating cancer or infectious disease using the provided compositions.

[0008] The present disclosure relates to populations of cells including cells with a genetic modification in the TGF^R2 sequence as provided herein. The cells may be used in adoptive T cell transfer therapies.

[0009] The present disclosure relates to compositions and uses of the cells with genetic modification of the TGF^R2 sequence for use in therapy, e.g., cancer therapy and immunotherapy. The present disclosure relates to and provides gRNA molecules, CRISPR systems, cells, and methods useful for genome editing of cells.

[0010] Provided herein is an engineered cell comprising a genetic modification in a TGF^R2 sequence, within the genomic coordinates chr3:30606864-30691614.

[0011] In some embodiments, the disclosure provides engineered cells with reduced or eliminated surface expression of TGF^R2 protein as a result of a genetic modification in the TGF^R2 gene. The engineered cell compositions produced by the methods disclosed herein have desirable properties, including e.g., reduced or eliminated expression of TGFBR2 protein, reduced chronic TGF^R2-mediated aberrant immune responses such as T-cell exhaustion, thereby enhanced immune responses.

[0012] Also disclosed is the use of a composition or formulation of a cell of any of the foregoing embodiments for the preparation of a medicament for treating a subject. Thesubject may be a human or animal (e.g., human or non-human animal, e.g., cynomolgus monkey). Preferably, the subject is human.

[0013] Also disclosed are any of the foregoing compositions or formulations for use in producing a genetic modification (e.g., an insertion, a substitution, or a deletion) a TGF^R2 gene sequence. In certain embodiments, the genetic modification within the sequence results in a change in the nucleic acid sequence that prevents translation of a full-length protein prior to genetic modification of the genomic locus, e.g., by forming a frameshift or nonsense mutation, such that translation is terminated prematurely. The genetic modification can include insertion, substitution, or deletion at a splice site, i.e., a splice acceptor site or a splice donor site, such that the abnormal splicing results in a frameshift mutation, nonsense mutation, or truncated mRNA, such that translation is terminated prematurely. Genetic modifications can also disrupt translation or folding of the encoded protein resulting in premature translation termination.

[0014] In another aspect, the present disclosure provides a method of treating a subject that includes administering cells (e.g., a population of cells) prepared by a method of preparing cells described herein, for example, a method of any of the aforementioned aspects and embodiments of methods of preparing cells.

[0015] Also disclosed are any of the foregoing compositions or formulations for use in producing a genetic modification (e.g., an insertion, a substitution, or a deletion) in a TGF^R2 sequence. In certain embodiments, the genetic modification within the sequence results in a change in the nucleic acid sequence that prevents translation of a full-length protein prior to genetic modification of the genomic locus, e.g., by forming a frameshift or nonsense mutation, such that translation is terminated prematurely. The genetic modification can include insertion, substitution, or deletion at a splice site, i.e., a splice acceptor site or a splice donor site, such that the abnormal splicing results in a frameshift mutation, nonsense mutation, or truncated mRNA, such that translation is terminated prematurely. Genetic modifications can also disrupt translation or folding of the encoded protein resulting in premature translation termination.

[0016] In another aspect, the present disclosure provides a method of providing an enhanced immunotherapy to a subject, the method including administering to the subject an effective amount of cells as described herein, for example, a cell of any of the aforementioned cell aspects and embodiments.

[0017] In some embodiments, the engineered cell comprises a genetic modification within any one of the genomic coordinates listed in Table 2. In some embodiments, the geneticmodification is within the genomic coordinates targeted by a guide RNA comprising a guide sequence of any one of SEQ ID NOs: 1-117.

[0018] Further embodiments are provided throughout and described in the claims and Figures. IV. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG.1 shows mean percent indel and mean pSMAD2 / 3 negative T cells following TGFBR2 editing with SpyCas9.

[0020] FIG.2A shows mean percent indel in T cells following TGFBR2 editing with SpyCas9 using various doses.

[0021] FIG.2B shows mean pSMAD2 / 3 negative T cells following TGFBR2 editing with SpyCas9 using various doses.

[0022] FIGs.3A-3C show mean percent indel following TGFBR2 editing with SpyCas9 using various doses in T cells from three distinct apheresis donors.

[0023] FIG.4 shows mean percent indel and mean TGFBR2 negative T cells using pSMAD2 / 3 as a cell marker. following TGFBR2 editing with SpyCas9 base editor.

[0024] FIGs.5A-5D show the impact of single (SKO) IEE (immune enhancing edit) knockouts with either construct 5719, construct 5718, or a construct 4645 against a 786-O tumor cell line measured by the percent of viable tumor cells remaining. Unedited cells were used as a control. Constructs 5719 and 5718 were tested alone or with TGF^R2 SKO. FIG. 5A shows the percent tumor cell viability for construct 5719 without the presence of TGF^ and FIG.5B shows the results for construct 5719 in the presence of TGF^. FIG.5C shows the percent tumor cell viability for construct 5718 without the presence of TGF^ and FIG. 5D shows the results for construct 5718 in the presence of TGF^.

[0025] FIGs.6A-6D show the in-vitro rechallenge of four anti-CD70 CAR constructs alone or with a SKO IEE edit against a 768-O tumor cell line, measured by tumor cell area (mm2). The constructs were compared to benchmark construct 4645 and TRAC KO alone. FIG.6A shows the results for construct 5719, FIG.6B shows the results for construct 5281, FIG.6C shows the results for construct 5715, and FIG.6D shows the results for construct 6115.

[0026] FIGs.7A-7D show the in-vitro rechallenge of four anti-CD70 CAR constructs alone, with a SKO IEE edit against a ACHN tumor cell line, measured by tumor cell area (mm2). The constructs were compared to benchmark construct 4645 and TRAC KO alone. FIG.7A shows the results for construct 5719, FIG.7B shows the results for construct 5281, FIG.7C shows the results for construct 5715, and FIG.7D shows the results for construct 6115.

[0027] FIGs.8A-8C show the efficacy of three anti-CD70 CAR constructs alone, with a SKO IEE edit in a 786-O mouse tumor cell model against benchmark construct 4645, measured by tumor volume (mm3). FIG.8A shows the results for construct 5719, FIG.8B shows the results for construct 5715, and FIG.8C shows the results for construct 5281.

[0028] FIG.9A-9C show the rechallenge results measured by tumor volume (mm3) for the anti-CD70 CAR constructs with a SKO IEE edit that fully controlled tumor growth in FIGs. 8A-C. Constructs were compared to mice with tumor only. FIG.9A shows the rechallenge results for construct 5719 + TGF^R2 KO. FIG.9B shows the rechallenge results for construct 5281 + TGF^R2 KO. FIG.9C shows the rechallenge results for construct 5715 + TGF^R2 KO.

[0029] FIG.10 shows the mean OVCAR3 tumor size in NOG IL15 mice treated with engineered T cells with and without TGFBR2 KO. V. DETAILED DESCRIPTION

[0030] Reference will now be made in detail to certain embodiments of the disclosure, examples of which are illustrated in the accompanying drawings. While the present teachings are described in conjunction with various embodiments, it is not intended to limit the present teachings to those embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art.

[0031] The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any material incorporated by reference contradicts any term defined in this specification or any other express content of this specification, this specification controls.

[0032] Provided herein are the following numbered embodiments:

[0033] Embodiment 1 is an engineered cell, comprising a genetic modification within genomic coordinates chr3:30606864-30691614.

[0034] Embodiment 2 is an engineered cell, which has reduced or eliminated surface expression of TGF R2 protein relative to an unmodified cell, comprising a genetic modification within genomic coordinates chr3:30606864-30691614.

[0035] Embodiment 3 is the engineered cell of embodiment 1 or 2, wherein the genetic modification is within the genomic coordinates targeted by a guide RNA comprising a guide sequence of any one of SEQ ID NOs: 1-117.

[0036] Embodiment 4 is the engineered cell of any one of embodiments 1-3, which has reduced or eliminated surface expression of TGF R2 relative to an unmodified cell,comprising a genetic modification within any one of the genomic coordinates listed in Table 2.

[0037] Embodiment 5 is the engineered cell of any one of embodiments 1-4, wherein the genetic modification is within genomic coordinates chosen from: (a) chr3:30672267- 30672287; chr3:30644743-30644763; chr3:30671764-30671784; chr3:30672177-30672197; chr3:30606958-30606978; chr3:30606870-30606890; chr3:30606961-30606981; chr3:30606911-30606931; chr3:30606909-30606929; chr3:30606954-30606974; chr3:30606865-30606885; chr3:30606947-30606967; chr3:30606864-30606884; chr3:30606916-30606936; chr3:30606871-30606891; chr3:30606930-30606950; chr3:30606957-30606977; chr3:30623239-30623259; chr3:30623238-30623258; chr3:30623240-30623260; chr3:30623263-30623283; chr3:30644843-30644863; chr3:30644757-30644777; chr3:30650418-30650438; chr3:30650323-30650343; chr3:30650327-30650347; chr3:30650317-30650337; chr3:30650326-30650346; chr3:30650318-30650338; chr3:30650319-30650339; chr3:30650393-30650413; chr3:30672152-30672172; chr3:30672410-30672430; chr3:30672296-30672316; chr3:30671871-30671891; chr3:30672193-30672213; chr3:30671791-30671811; chr3:30672024-30672044; chr3:30671784-30671804; chr3:30672322-30672342; chr3:30672192-30672212; chr3:30672270-30672290; chr3:30671941-30671961; chr3:30671752-30671772; chr3:30672387-30672407; chr3:30671929-30671949; chr3:30672266-30672286; chr3:30672295-30672315; chr3:30672021-30672041; chr3:30671932-30671952; chr3:30671835-30671855; chr3:30672023-30672043; chr3:30671908-30671928; chr3:30672212-30672232; chr3:30671854-30671874; chr3:30671701-30671721; chr3:30672294-30672314; chr3:30672193-30672213; chr3:30671821-30671841; chr3:30672268-30672288; chr3:30672250-30672270; chr3:30672421-30672441; chr3:30672400-30672420; chr3:30672249-30672269; chr3:30672196-30672216; chr3:30672340-30672360; chr3:30671842-30671862; chr3:30671739-30671759; chr3:30674221-30674241; chr3:30674178-30674198; chr3:30674085-30674105; chr3:30674136-30674156; chr3:30674220-30674240; chr3:30674184-30674204; chr3:30688451-30688471; chr3:30688403-30688423; chr3:30688434-30688454; chr3:30688432-30688452; chr3:30688402-30688422; chr3:30688388-30688408; chr3:30688429-30688449; chr3:30688510-30688530; chr3:30688416-30688436; chr3:30691489-30691509; chr3:30691522-30691542; chr3:30691427-30691447; chr3:30691519-30691539; chr3:30691435-30691455; chr3:30691594-30691614; chr3:30691409-30691429; chr3:30691463-30691483; andchr3:30691475-30691495; and (b) chr3:30671764-30671784; chr3:30672177-30672197; chr3:30606958-30606978; chr3:30606961-30606981; chr3:30606916-30606936; chr3:30606957-30606977; chr3:30650418-30650438; chr3:30672193-30672213; chr3:30672024-30672044; chr3:30672023-30672043; chr3:30672421-30672441; chr3:30674178-30674198; chr3:30688510-30688530; chr3:30691409-30691429; chr3:30606962-30606982; chr3:30606974-30606994; chr3:30606975-30606995; chr3:30644885-30644905; chr3:30644893-30644913; chr3:30650250-30650270; chr3:30671618-30671638; chr3:30671763-30671783; chr3:30671983-30672003; chr3:30672088-30672108; chr3:30672094-30672114; chr3:30672099-30672119; chr3:30674083-30674103; chr3:30674198-30674218; chr3:30688476-30688496; chr3:30688481-30688501; chr3:30688490-30688510; chr3:30688491-30688511; chr3:30688507-30688527; chr3:30691396-30691416; chr3:30691397-30691417; chr3:30691412-30691432; chr3:30691413-30691433; chr3:30691582-30691602; and chr3:30691583-30691603.

[0038] Embodiment 6 is the engineered cell of any one of embodiments 1-5, wherein the genetic modification is within the genomic coordinates chosen from: chr3:30671941- 30671961 and chr3:30671739-30671759.

[0039] Embodiment 7 is the engineered cell of any one of embodiments 1-6, wherein the genetic modification is within genomic coordinates targeted by a guide RNA comprising a guide sequence of SEQ ID NO: 43 or 68.

[0040] Embodiment 8 is the engineered cell of any one of embodiments 1-5, wherein the genetic modification is within the genomic coordinates chosen from: chr3:30644885- 30644905; chr3:30671618-30671638; chr3:30671983-30672003; chr3:30672094-30672114; chr3:30672177-30672197; and chr3:30674198-30674218.

[0041] Embodiment 9 is the engineered cell of any one of embodiments 1-5 and 8, wherein the genetic modification is within genomic coordinates targeted by a guide RNA comprising a guide sequence of any one of SEQ ID NOs: 4, 95, 98, 100, 102, and 105.

[0042] Embodiment 10 is a composition comprising a guide RNA and optionally an RNA- guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the guide RNA comprises: a. a guide sequence selected from SEQ ID NOs: 1-117; b. a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-117; c. a guide sequence that is at least 85%, 90%, or 95% identical to a sequence selected from SEQ ID NOs: 1-117; d. a sequence that comprises 10 contiguous nucleotides ±10 nucleotides of a genomic coordinate listed in Table 2; e. at least17, 18, 19, or 20 contiguous nucleotides of a sequence from (d); or f. a guide sequence that is at least 85%, 90%, or 95% identical to a sequence selected from (d).

[0043] Embodiment 11 is the composition of embodiment 10, for use in altering a DNA sequence within the TGF R2 gene in a cell.

[0044] Embodiment 12 is a pharmaceutical composition comprising, or use of, the composition of embodiments 10 for inducing a double stranded break or a single stranded break within a TGF R2 gene in a cell, modifying the nucleic acid sequence of a TGF R2 gene in a cell, or reducing expression of a TGF R2 gene in a cell.

[0045] Embodiment 13 is a method of making an engineered human cell, which has reduced or eliminated surface expression of TGF R2 protein relative to an unmodified cell, comprising contacting a cell with the composition of embodiment 10.

[0046] Embodiment 14 is a method of reducing surface expression of TGF R2 protein in a cell relative to an unmodified cell, comprising contacting a cell with a composition comprising a guide RNA and optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the guide RNA comprises: a. a guide sequence selected from SEQ ID NOs: 1-117; b. a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence of selected from SEQ ID NOs: 1-117; c. a guide sequence that is at least 85%, 90%, or 95% identical to a sequence of any one of SEQ ID NOs: 1-117; d. a sequence that comprises 10 contiguous nucleotides ±10 nucleotides of a genomic coordinate listed in Table 2; e. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence from (d); or f. a guide sequence that is at least 85%, 90%, or 95% identical to a sequence selected from (d).

[0047] Embodiment 15 is the composition, use, or method of any one of embodiments 10-14, wherein the RNA-guided DNA binding agent is a cleavase and the guide RNA comprises a guide sequence of SEQ ID NO: 43 or 68.

[0048] Embodiment 16 is the composition, use, or method of any one of embodiments 10-14, wherein the RNA-guided DNA binding agent is a base editor and wherein the guide RNA comprises a guide sequence of any one of SEQ ID NO: 4, 95, 98, 100, 102 and 105.

[0049] Embodiment 17 is a population of cells comprising the engineered cell produced by use of the composition of embodiment 10-12, 15, and 16, or the method of any one of embodiments 13-16.

[0050] Embodiment 18 is a pharmaceutical composition comprising (a) the engineered cell produced by the composition or method of any one of embodiments 10-16; or (b) the population of cells of embodiment 17.

[0051] Embodiment 19 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-18, wherein the genetic modification comprises at least 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates.

[0052] Embodiment 20 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-19, wherein the genetic modification comprises at least 5, 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates.

[0053] Embodiment 21 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-20, wherein the genetic modification comprises an insertion, a deletion, or a substitution.

[0054] Embodiment 22 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-21, wherein the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within the genomic coordinates.

[0055] Embodiment 23 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-22, wherein the genetic modification comprises an indel.

[0056] Embodiment 24 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-23, wherein the genetic modification comprises an insertion of a heterologous coding sequence.

[0057] Embodiment 25 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-24, wherein the genetic modification comprises a substitution.

[0058] Embodiment 26 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-25, wherein the genetic modification comprises an A to G substitution.

[0059] Embodiment 27 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-25, wherein the genetic modification comprises a C to T substitution.

[0060] Embodiment 28 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-27, wherein the cells are engineered with a genomic editing system.

[0061] Embodiment 29 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 28, wherein the genomic editing system comprises an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.

[0062] Embodiment 30 is the composition of embodiment 29, wherein the nucleic acid encoding the RNA-guided DNA binding agent comprises an mRNA comprising an open reading frame (ORF) encoding the RNA-guided DNA binding agent.

[0063] Embodiment 31 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 29 or 30, wherein the RNA-guided DNA-binding agent or the RNA-guided DNA-binding agent encoded by the nucleic acid comprises a Cas9 nuclease.

[0064] Embodiment 32 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiment 29-31, wherein the RNA-guided DNA binding agent is a nuclease.

[0065] Embodiment 33 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 29-32, wherein the RNA-guided DNA binding agent is a Cas9 nuclease.

[0066] Embodiment 34 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 33, wherein the Cas9 is S. pyogenes Cas9.

[0067] Embodiment 35 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 34, wherein the S. pyogenes Cas9 comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 853, or an ORF encoding a S. pyogenes Cas9 having at least 90% identity to the sequence of SEQ ID NO: 853.

[0068] Embodiment 36 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 35, wherein the ORF encoding the amino acid sequence has at least 85% identity to any one of SEQ ID NOs: 813, 814, and 816-819.

[0069] Embodiment 37 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 31-36, wherein the nuclease has double stranded endonuclease activity.

[0070] Embodiment 38 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 31-36, wherein the nuclease has nickase activity.

[0071] Embodiment 39 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 31-36, wherein the nuclease is catalytically inactive.

[0072] Embodiment 40 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 31-36, wherein the nuclease further comprises a heterologous functional domain.

[0073] Embodiment 41 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 40, wherein the nuclease is a nickase and the heterologous functional domain is a deaminase.

[0074] Embodiment 42 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 41, wherein the deaminase is a cytidine deaminase or an adenine deaminase.

[0075] Embodiment 43 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 42, wherein the deaminase is a cytidine deaminase.

[0076] Embodiment 44 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 43, wherein the deaminase is an apolipoprotein B mRNA editing enzyme (APOBEC) deaminase.

[0077] Embodiment 45 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 41-44, wherein the nuclease and the deaminase comprise an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 851, 852, and 858 or an ORF encoding an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 851, 852, and 858.

[0078] Embodiment 46 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 45, wherein the ORF encoding the amino acid sequence has at least 85% identity to SEQ ID NO: 811, 812, or 815.

[0079] Embodiment 47 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 43-46, further comprising a uracil glycosylase inhibitor (UGI) or nucleic acid encoding a UGI, wherein the nuclease does not comprise a UGI or the nucleic acid encoding the nuclease does not encode a UGI.

[0080] Embodiment 48 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 47, wherein the UGI comprises an amino acidsequence having at least 90% identity to SEQ ID NO: 859, or an ORF encoding an amino acid sequence having at least 90% identity to the sequence of SEQ ID NO: 859.

[0081] Embodiment 49 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 48, wherein the ORF encoding the amino acid sequence has at least 85% identity to any one of SEQ ID NOs: 823-826, optionally SEQ ID NO: 823.

[0082] Embodiment 50 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 45-49, wherein the ORF is a modified ORF.

[0083] Embodiment 51 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 45-50, wherein the nuclease has nickase activity.

[0084] Embodiment 52 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 45-51, wherein the Cas9 nuclease comprises S. pyogenes (Spy) Cas9.

[0085] Embodiment 53 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 52, wherein the nucleic acid encoding the Cas9 nuclease is an mRNA comprising a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 811-822, 827, or 828.

[0086] Embodiment 54 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 45-52, wherein the nucleic acid encoding the base editor comprises a nucleotide sequence that is at least 80%, 85%, 90%, 95%, 98%, or 100% identical to SEQ ID NO: 811.

[0087] Embodiment 55 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 45-52, wherein the nucleic acid encoding the base editor comprises the nucleotide sequence of SEQ ID NO: 811.

[0088] Embodiment 56 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 3-55, wherein the guide RNA is a dual guide RNA (dgRNA).

[0089] Embodiment 57 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 3-55, wherein the guide RNA is a single guide RNA (sgRNA).

[0090] Embodiment 58 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 57, wherein the sgRNA is a Spy sgRNA.

[0091] Embodiment 59 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 58, wherein the Spy sgRNA further comprises one or more of: A. a shortened hairpin 1 region, or a substituted and optionally shortened hairpin 1 region, wherein 1. at least one of the following pairs of nucleotides are substituted in hairpin 1 with Watson-Crick pairing nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1- 10, or H1-4 and H1-9, and the hairpin 1 region optionally lacks a. any one or two of H1-5 through H1-8, b. one, two, or three of the following pairs of nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, and H1-4 and H1-9, or c. 1-8 nucleotides of hairpin 1 region; or 2. the shortened hairpin 1 region lacks 4-8 nucleotides, preferably 4-6 nucleotides; and a. one or more of positions H1-1, H1-2, or H1-3 is deleted or substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601) or b. one or more of positions H1-6 through H1-10 is substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601); or 3. the shortened hairpin 1 region lacks 5-10 nucleotides, preferably 5-6 nucleotides, and one or more of positions N18, H1-12, or n is substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601); or B. a shortened upper stem region, wherein the shortened upper stem region lacks 1-6 nucleotides and wherein the 6, 7, 8, 9, 10, or 11 nucleotides of the shortened upper stem region include less than or equal to 4 substitutions relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601); or C. a substitution relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601) at any one or more of LS6, LS7, US3, US10, B3, N7, N15, N17, H2-2 and H2-14, wherein the substituent nucleotide is neither a pyrimidine that is followed by an adenine, nor an adenine that is preceded by a pyrimidine; or D. an Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601) with an upper stem region, wherein the upper stem modification comprises a modification to any one or more of US1-US12 in the upper stem region.

[0092] Embodiment 60 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 59, wherein the guide RNA lacks 6 or 8 nucleotides in shortened hairpin 1.

[0093] Embodiment 61 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 59 or 60, wherein H-1 and H-3 are deleted.

[0094] Embodiment 62 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 59-61, wherein the guide RNA further comprises a 3’ tail, wherein the 3’ tail is 1-4 nucleotides in length, optionally 1 nucleotide in length.

[0095] Embodiment 63 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 59, comprising a sequence or modification pattern asset forth in Tables 6-7, wherein the N’s are collectively the guide sequence, N, A, C, G, and U are ribonucleotides (2’-OH), “m” indicates a 2’-O-Me modification, “f” indicates a 2’- fluoro modification, and a “*” indicates a phosphorothioate linkage between nucleotides.

[0096] Embodiment 64 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 3-63, wherein the guide RNA comprises at least one end modification.

[0097] Embodiment 65 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 64, wherein the modification comprises a 5’ end modification.

[0098] Embodiment 66 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 64 or 65, wherein the modification comprises a 3’ end modification.

[0099] Embodiment 67 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 64-66, wherein the guide RNA comprises a modification in a hairpin region.

[0100] Embodiment 68 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 67, wherein the modification in a hairpin region is also an end modification.

[0101] Embodiment 69 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 64-68, wherein the modification comprises a 2’-O-methyl (2’-O-Me) modified nucleotide.

[0102] Embodiment 70 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 64-69, wherein the modification comprises a phosphorothioate (PS) bond between nucleotides.

[0103] Embodiment 71 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 64-70, wherein the modification comprises a 2’-O-methyl (2’-O-Me) modified nucleotide with a phosphorothioate (PS) bond to a 3’ adjacent nucleotide.

[0104] Embodiment 72 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 64-71, wherein the modification comprises a 2’-fluor (2’F) modified nucleotide.

[0105] Embodiment 73 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiment 65-72, wherein the 5’ end modificationcomprises a 2’-O-methyl (2’-O-Me) modified nucleotide with a phosphorothioate (PS) bond to a 3’ adjacent nucleotide at nucleotides 1-3 of the 5’ end of the guide sequence.

[0106] Embodiment 74 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 3-73, wherein the guide RNA is associated with a lipid nanoparticle (LNP).

[0107] Embodiment 75 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 74, wherein the LNP comprises a cationic lipid, a helper lipid, a neutral lipid, a stealth lipid, or a combination of two or more thereof.

[0108] Embodiment 76 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 75, wherein the cationic lipid is (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate.

[0109] Embodiment 77 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 75 or 76, wherein the helper lipid is cholesterol.

[0110] Embodiment 78 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 75-77, wherein the neutral lipid is 1,2- distearoyl-sn-glycero-3-phosphocholine (DSPC).

[0111] Embodiment 79 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 75-78, wherein the stealth lipid is 1,2- dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (PEG2k-DMG).

[0112] Embodiment 80 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 75-79, wherein the LNP comprises (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate, DSPC, cholesterol, and PEG2k-DMG.

[0113] Embodiment 81 is a pharmaceutical composition comprising the engineered cell of any one of embodiments 1-80.

[0114] Embodiment 82 is a population of cells comprising the engineered cell of any one of embodiments 1-80.

[0115] Embodiment 83 is a pharmaceutical composition comprising a population of cells, wherein the population of cells comprises a plurality of the engineered cell of any one of embodiments 1-80.

[0116] Embodiment 84 is the pharmaceutical composition of embodiment 83, further comprising a pharmaceutical excipient.

[0117] Embodiment 85 is a method of administering the engineered cell, population of cells, or pharmaceutical composition of any one of embodiments 1-84 to a subject in need thereof.

[0118] Embodiment 86 is a method of administering the engineered cell, population of cells, or pharmaceutical composition of any one of embodiments 1-84 to a subject as an adoptive cell transfer (ACT) therapy.

[0119] Embodiment 87 is a method of administering the engineered cell, population of cells, or pharmaceutical composition of any one of embodiments 1-84 to a subject as an immunotherapy.

[0120] Embodiment 88 is an engineered cell, population of cells, or pharmaceutical composition of any one of embodiments 1-84, for use as an ACT therapy.

[0121] Embodiment 89 is a method of treating a disease or disorder comprising administering the engineered cell, population of cells, or pharmaceutical composition of any one of embodiments 1-84 to a subject in need thereof.

[0122] Embodiment 90 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 3-89, wherein the guide RNA is provided to the cell in a vector.

[0123] Embodiment 91 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 10-90, wherein the nucleic acid encoding the RNA-guided DNA binding agent is provided to the cell in the same vector as the guide RNA.

[0124] Embodiment 92 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-91, wherein an exogenous nucleic acid is provided to the cell, optionally in a vector.

[0125] Embodiment 93 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 90-92, wherein the vector is a viral vector.

[0126] Embodiment 94 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 93, wherein the vector is an AAV.

[0127] Embodiment 95 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-94, wherein the genetic modification inhibits expression of the gene in which the genetic modification is present.

[0128] Embodiment 96 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-95, wherein the genetic modification inhibits expression of the TGF R2 gene.

[0129] Embodiment 97 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-96, wherein the engineered cell has reduced surface expression of TGF R2 protein relative to an unmodified cell.

[0130] Embodiment 98 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 97, wherein cell surface expression of TGF R2 protein is below the level of detection.

[0131] Embodiment 99 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-98, wherein the cell comprises an exogenous nucleic acid encoding a targeting receptor that is expressed on the surface of the engineered cell.

[0132] Embodiment 100 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 99, wherein the targeting receptor is a T cell receptor (TCR).

[0133] Embodiment 101 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 100, wherein the targeting receptor is a WT1 TCR, optionally wherein the WT1 TCR comprises the amino acid sequence of SEQ ID NO: 1003, and / or optionally wherein the exogenous nucleic acid encoding the targeting receptor comprises the nucleic acid sequence of SEQ ID NO: 1002.

[0134] Embodiment 102 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 101, wherein the cell comprises an exogenous nucleic acid encoding a CD8 coreceptor that is expressed on the surface of the engineered cell, optionally wherein the CD8 coreceptor comprises the amino acid sequence of SEQ ID NO: 1005 and / or 1007, and / or optionally wherein the exogenous nucleic acid encoding the CD8 coreceptor comprises the nucleic acid sequence of SEQ ID NO: 1004 and / or 1006.

[0135] Embodiment 103 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 99, wherein the targeting receptor is a chimeric antigen receptor (CAR).

[0136] Embodiment 104 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-103, wherein the engineered cellfurther comprises a genetic modification in one or more of CIITA, HLA-A, HLA-B, TRBC, or TRAC gene.

[0137] Embodiment 105 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-104, wherein the engineered cell further has reduced surface expression of one or more of MHC class II protein, HLA-A, HLA-B, TRBC, or TRAC relative to an unmodified cell.

[0138] Embodiment 106 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 104 or 105, wherein the engineered cell comprises: i. a genetic modification in the HLA-A gene within the genomic coordinates chr6:29942891- 29942915 or chr6:29942609-29942633; ii. a genetic modification in the HLA-B gene within the genomic coordinates chr6:31355222-31355246, chr6:31355221-31355245, or chr6:31355205-31355229; iii. a genetic modification in the TRBC gene within the genomic coordinates chr7:142792047-142792067; iv. a genetic modification in the TRAC gene within the genomic coordinates chr14:22547524-22547544, chr14:22550574-22550598, or chr14:22550544-22550568; v. a genetic modification in the CIITA gene within the genomic coordinates chr16:10907504-10907528 or chr16:10906643-10906667; or vi. a combination of two or more of (i)-(v).

[0139] Embodiment 107 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 104-106, wherein the engineered cell comprises at least one genetic modification (i) within the genomic coordinates targeted by a HLA-A guide RNA comprising a guide sequence of SEQ ID NO: 403 or 404; (ii) within the genomic coordinates targeted by a HLA-B guide RNA comprising a guide sequence of SEQ ID NO: 406, 405 or 407; (iii) within the genomic coordinates targeted by a TRBC guide RNA comprising a guide sequence of SEQ ID NO: 414; (iv) within the genomic coordinates targeted by a TRAC guide RNA comprising a guide sequence of SEQ ID NO: 408, 409, or 413; (v) within the genomic coordinates targeted by a CIITA guide RNA comprising a guide sequence of SEQ ID NO: 401 or 402; or (vi) a combination of two or more of (i)-(v).

[0140] Embodiment 108 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-107, wherein the engineered cell is an immune cell.

[0141] Embodiment 109 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 108, wherein the engineered cell is a monocyte, macrophage, mast cell, dendritic cell, or granulocyte.

[0142] Embodiment 110 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 108, wherein the engineered cell is a lymphocyte.

[0143] Embodiment 111 is the engineered cell, population of cells, pharmaceutical composition, or method of embodiment 110, wherein the engineered cell is a T cell.

[0144] Embodiment 112 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-111, wherein the cell is a CD4+ T cell or a CD8+T cell.

[0145] Embodiment 113 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-111, wherein the cell is a memory T cell.

[0146] Embodiment 114 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-113, wherein the cell is a primary cell.

[0147] Embodiment 115 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-114, wherein the cell is a tissue- specific primary cell.

[0148] Embodiment 116 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-115, wherein the cell is an activated cell.

[0149] Embodiment 117 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-115, wherein the cell is a non-activated cell.

[0150] Embodiment 118 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-117, wherein the cell is an allogeneic cell.

[0151] Embodiment 119 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-107, wherein the cell is a stem cell.

[0152] Embodiment 120 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-119, for use in administering to a subject as an adoptive cell transfer (ACT) therapy.

[0153] Embodiment 121 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-119, for use in treating a subject with cancer.

[0154] Embodiment 122 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-119, for use in treating a subject with an infectious disease.

[0155] Embodiment 123 is the engineered cell, population of cells, pharmaceutical composition, or method of any one of embodiments 1-119, for use in treating a subject with an autoimmune disease.

[0156] Embodiment 124 is the population or the pharmaceutical composition of any one of embodiments 17-123, wherein the population of cells is at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% TGF R2 negative as measured by flow cytometry.

[0157] Embodiment 125 is the population or pharmaceutical composition of any one of embodiments 17-124, wherein at least 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the population of cells comprises the genetic modification in the TGF R2 gene, as measured by next-generation sequencing (NGS). A. Definitions

[0158] Unless stated otherwise, the following terms and phrases as used herein are intended to have the following meanings:

[0159] The term “or combinations thereof” as used herein refers to all permutations and combinations of the listed terms preceding the term. For example, “A, B, C, or combinations thereof” is intended to include at least one of: A, B, C, AB, AC, BC, or ABC, and if order is important in a particular context, also BA, CA, CB, ACB, CBA, BCA, BAC, or CAB. Continuing with this example, expressly included are combinations that contain repeats of one or more item or term, such as BB, AAA, AAB, BBC, CBBA, CABA, and so forth. The skilled artisan will understand that typically there is no limit on the number of items or terms in any combination, unless otherwise apparent from the context.

[0160] As used herein, the term “kit” refers to a packaged set of related components, such as one or more polynucleotides or compositions and one or more related materials such as delivery devices (e.g., syringes), solvents, solutions, buffers, instructions, or desiccants.

[0161] An “allogeneic” cell, as used herein, refers to a cell originating from a donor subject of the same species as a recipient subject, wherein the donor subject and recipient subject have genetic dissimilarity, e.g., genes at one or more loci that are not identical. Thus, e.g., a cell is allogeneic with respect to the subject to be administered the cell. As used herein, a cellthat is removed or isolated from a donor, that will not be re-introduced into the original donor, is considered an allogeneic cell.

[0162] An “autologous” cell, as used herein, refers to a cell derived from the same subject to whom the material will later be re-introduced. Thus, e.g., a cell is considered autologous if it is removed from a subject and it will then be re-introduced into the same subject.

[0163] The term “TGF^R2” or “TGFBR2” as used herein in the context of protein, refers to a transmembrane protein that has a protein kinase domain, forms a heterodimeric complex with Transforming Growth Factor Beta (TGF-^) receptor type-1, and binds TGF-beta. The term “TGF^R2” or “TGFBR2” as used herein in the context of nucleic acids refers to the gene encoding the Transforming Growth Factor Beta (TGF-^) receptor type-2 protein molecule. The human gene has accession number NC_000003.12 (30606356..30694142).

[0164] As used herein, the term “within the genomic coordinates” includes the boundaries of the genomic coordinate range given. For example, if chr6:29942854- chr6:29942913 is given, the coordinates chr6:29942854-chr6:29942913 are encompassed. Throughout this application, the referenced genomic coordinates are based on genomic annotations in the GRCh38 (also referred to as hg38) assembly of the human genome from the Genome Reference Consortium, available at the National Center for Biotechnology Information website. Tools and methods for converting genomic coordinates between one assembly and another are known in the art and can be used to convert the genomic coordinates provided herein to the corresponding coordinates in another assembly of the human genome, including conversion to an earlier assembly generated by the same institution or using the same algorithm (e.g., from GRCh38 to GRCh37), and conversion of an assembly generated by a different institution or algorithm (e.g., from GRCh38 to NCBI33, generated by the International Human Genome Sequencing Consortium). Available methods and tools known in the art include, but are not limited to, NCBI Genome Remapping Service, available at the National Center for Biotechnology Information website, UCSC LiftOver, available at the UCSC Genome Brower website, and Assembly Converter, available at the Ensembl.org website.

[0165] “Polynucleotide” and “nucleic acid” are used herein to refer to a multimeric compound comprising nucleosides or nucleoside analogs which have nitrogenous heterocyclic bases or base analogs linked together along a backbone, including conventional RNA, DNA, mixed RNA-DNA, and polymers that are analogs thereof. A nucleic acid “backbone” can be made up of a variety of linkages, including one or more of sugar- phosphodiester linkages, peptide-nucleic acid bonds (“peptide nucleic acids” or PNA; PCTNo. WO 95 / 32305), phosphorothioate linkages, methylphosphonate linkages, or combinations thereof. Sugar moieties of a nucleic acid can be ribose, deoxyribose, or similar compounds with substitutions, e.g., 2’ methoxy, 2’ halide, or a 2’-O-(2-methoxyethyl) (2’-O- moe) substitutions. An RNA may comprise one or more deoxyribose nucleotides, e.g. as modifications, and similarly a DNA may comprise one or more ribonucleotides. Nitrogenous bases can be conventional bases (A, G, C, T, U), analogs thereof (e.g., modified uridines such as 5-methoxyuridine, pseudouridine, or N1-methylpseudouridine, or others); inosine; derivatives of purines or pyrimidines (e.g., N4-methyl deoxyguanosine, deaza- or aza-purines, deaza- or aza-pyrimidines, pyrimidine bases with substituent groups at the 5 or 6 position (e.g., 5-methylcytosine), purine bases with a substituent at the 2, 6, or 8 positions, 2-amino-6- methylaminopurine, O6-methylguanine, 4-thio-pyrimidines, 4-amino-pyrimidines, 4- dimethylhydrazine-pyrimidines, and O4-alkyl-pyrimidines; US Pat. No.5,378,825 and PCT No. WO 93 / 13121). For general discussion see The Biochemistry of the Nucleic Acids 5-36, Adams et al., ed., 11thed., 1992). Nucleic acids can include one or more “abasic” residues where the backbone includes no nitrogenous base for position(s) of the polymer (US Pat. No. 5,585,481). A nucleic acid can comprise only conventional RNA or DNA sugars, bases and linkages, or can include both conventional components and substitutions (e.g., conventional nucleosides with 2’ methoxy substituents, or polymers containing both conventional nucleosides and one or more nucleoside analogs). Nucleic acid includes “locked nucleic acid” (LNA), an analogue containing one or more LNA nucleotide monomers with a bicyclic furanose unit locked in an RNA mimicking sugar conformation, which enhance hybridization affinity toward complementary RNA and DNA sequences (Vester and Wengel, 2004, Biochemistry 43(42):13233-41). Nucleic acid includes “unlocked nucleic acid” enables the modulation of the thermodynamic stability and also provides nuclease stability. RNA and DNA have different sugar moieties and can differ by the presence of uracil or analogs thereof in RNA and thymine or analogs thereof in DNA.

[0166] “Polypeptide” as used herein refers to a multimeric compound comprising amino acid residues that can adopt a three-dimensional conformation. Polypeptides include but are not limited to enzymes, enzyme precursor proteins, regulatory proteins, structural proteins, receptors, nucleic acid binding proteins, antibodies, etc. Polypeptides may, but do not necessarily, comprise post-translational modifications, non-natural amino acids, prosthetic groups, and the like.

[0167] “Guide RNA,” “gRNA,” and simply “guide” are used herein interchangeably to refer to, for example, either a single guide RNA or the combination of a crRNA and a trRNA (alsoknown as tracrRNA). The crRNA and trRNA may be associated as a single RNA strand (as a single guide RNA, sgRNA) or, for example, in two separate RNA strands (dual guide RNA, dgRNA). “Guide RNA” or “gRNA” refers to each type. The trRNA may be a naturally- occurring sequence, or a trRNA sequence with modifications or variations.

[0168] As used herein, a “guide sequence” refers to a sequence within a guide RNA that is complementary to a target sequence and functions to direct a guide RNA to a target sequence for binding or modification (e.g., cleavage) by an RNA-guided DNA binding agent.

[0169] For example, the Spy Cas9 guide sequence can be 16-, 17-, preferably 18-, 19-, or 20-nucleotides in length, such that, in some embodiments, the Spy Cas9 guide sequence comprises 16, 17, 18, 19, or 20 contiguous nucleotides of a guide sequence provided in Table 2, or a reverse complement thereof. In some embodiments, the guide sequence is complementary to the guide sequence. In some embodiments, the degree of complementarity or identity between a guide sequence and its corresponding target sequence in a genome is at least 80%, 85%, preferably 90%, or 95%, or is 100%. For example, in some embodiments, the guide sequence comprises a sequence that is at least 80%, 85%, preferably 90%, or 95%, or is 100% identical or complementary to 20 contiguous nucleotides of its corresponding target sequence. In other embodiments, the guide sequence and its corresponding target sequence may contain at least one mismatch, i.e., one nucleotide that is not identical or not complementary, depending on the reference sequence. For example, the guide sequence and the target sequence may contain 1, 2, 3, or 4 mismatches within the duplex formed between the guide and the target sequence, where the total length of the target sequence is 16, 17, 18, 19, 20 nucleotides, or more. In some embodiments, the guide sequence and the target sequence may contain 1-4 mismatches where the guide sequence comprises at least 20 nucleotides. In some embodiments, the guide sequence and the target region may contain 1, 2, 3, or 4 mismatches where the guide sequence comprises 20 nucleotides. That is, the guide sequence and the target region may form a duplex region having 16, 17, 18, 19, 20 base pairs, or more. In certain embodiments, the duplex region may include 1, 2, 3, or 4 mismatches such that guide strand and target sequence are not fully complementary. For example, a guide strand and target sequence may be complementary over a 20 nucleotide region, including 2 mismatches, such that the guide sequence and target sequence are 90% complementary providing a duplex region of 18 base pairs out of 20. More tolerated mismatch positions are known in the art, for example, PAM distal mismatches tend to be better tolerated than PAM proximal matches. Mismatch tolerances at other positions are known in the art (see, e.g., Sternberg et al., 2015, Nature:527:110-113).

[0170] Target sequences for RNA-guided DNA binding agents, as defined by the guide sequence of a guide RNA, may be present on either the positive or negative strand. Tables and other disclosures provided herein may recite genomic coordinates or position within a nucleotide sequence as a target sequence. It is understood that the guide can be complementary to either the positive or negative strand of the DNA as defined by the genomic coordinates or position within a nucleotide sequence. The sequence to which the guide is complementary depends on the presence of an appropriate PAM for the RNA guided DNA binding protein on the opposite strand. Thus, in some embodiments, when the guide sequence binds the reverse complement of a target sequence, i.e., the guide sequence is identical to certain nucleotides of the sense (positive) strand of the target sequence, when the PAM is present in the sense strand, except for the substitution of U for T in the guide sequence.

[0171] As used herein, an “RNA-guided DNA binding agent” means a polypeptide or complex of polypeptides having RNA and DNA binding activity, or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence-specific and depends on the presence of a PAM and the sequence of the guide RNA. Exemplary RNA-guided DNA binding agents include Cas cleavases / nickases and inactivated forms thereof (“dCas DNA binding agents”). “Cas nuclease” or “Cas9 protein”, as used herein, encompasses Cas cleavases, Cas nickases, and dCas DNA binding agents. The dCas DNA binding agent may be a dead nuclease comprising non-functional nuclease domains (RuvC or HNH domain). In some embodiments the Cas cleavase or Cas nickase encompasses a dCas DNA binding agent modified to permit DNA cleavage, e.g. via fusion with a FokI domain. Cas cleavases / nickases and dCas DNA binding agents include a Csm or Cmr complex of a type III CRISPR system, the Cas10, Csm1, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases.

[0172] As used herein, a “Class 2 Cas nuclease” is a single-chain polypeptide with RNA- guided DNA binding activity. Class 2 Cas nucleases include Class 2 Cas cleavases / nickases (e.g., H840A or D10A variants of Spy Cas9 and D16A and H588A of Nme Cas9, e.g., Nme2Cas9), which further have RNA-guided DNA cleavases or nickase activity, and Class 2 dCas DNA binding agents, in which cleavase / nickase activity is inactivated. Class 2 Cas nucleases include, for example, Cas9, Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g., K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. Cpf1 protein,Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9, and contains a RuvC-like nuclease domain. Cpf1 sequences of Zetsche are incorporated by reference in their entirety. See, e.g., Zetsche, Tables S1 and S3. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0173] Exemplary nucleotide and polypeptide sequences of Cas9 molecules are provided below. Methods for identifying alternate nucleotide sequences encoding Cas9 polypeptide sequences, including alternate naturally occurring variants, are known in the art. Sequences with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of the Cas9 nucleic acid sequences, or nucleic acid sequences encoding the amino acid sequences provided herein are also contemplated. In certain embodiments, the nucleotide sequence encoding the Cas9 amino acid sequence is not a naturally occurring Cas9 nucleotide sequence. Sequences with at least 95%, 96%, 97%, 98%, or 99% identity to any of the Cas9 amino acid sequences provided herein are also contemplated. In certain embodiments, the Cas9 amino acid sequence is not a naturally occurring Cas9 sequence.

[0174] Exemplary open reading frames and amino acid sequences for Cas9 (SEQ ID NO: 813, 814, 816-819, 853-857) and uracil glycosylase inhibitors (SEQ ID NO: 823-826, 859, 860) are provided in Table 10.

[0175] As used herein, the term “editor” refers to an agent comprising a polypeptide that is capable of making a modification within a DNA sequence. In some embodiments, the editor is a cleavase, such as a Cas9 cleavase. In some embodiments, the editor is capable of deaminating a base within a DNA molecule, and it may be called a base editor. In some embodiments, the editor is capable of deaminating a cytosine (C) in DNA. In some embodiments, the editor is a fusion protein comprising an RNA-guided nickase fused to a cytidine deaminase. In some embodiments, the editor is a fusion protein comprising an RNA- guided nickase fused to an APOBEC3A deaminase (A3A). In some embodiments, the editor comprises a Cas9 nickase fused to an APOBEC3A deaminase (A3A). In some embodiments, the editor is a fusion protein comprising an RNA-guided nickase fused to a cytidine deaminase and a UGI. In some embodiments, the editor lacks a UGI. Exemplary editors used herein may be described in WO2022125968 published June 16, 2022, the contents of which are incorporated by reference. Exemplary editors may be a single polypeptide comprising a H. sapiens APOBEC3A linked to S. pyogenes-D10A Cas9 nickase by an XTEN linker. An mRNA encoding the same is provided herein (e.g., SEQ ID NO: 811).

[0176] As used herein, a “cytidine deaminase” means a polypeptide or complex of polypeptides that is capable of cytidine deaminase activity, that is catalyzing the hydrolyticdeamination of cytidine or deoxycytidine, typically resulting in uridine or deoxyuridine. Cytidine deaminases encompass enzymes in the cytidine deaminase superfamily, and in particular, enzymes of the APOBEC family (APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups of enzymes), activation-induced cytidine deaminase (AID or AICDA) and CMP deaminases (see, e.g., Conticello et al., Mol. Biol. Evol.22:367-77, 2005; Conticello, Genome Biol.9:229, 2008; Muramatsu et al., J. Biol. Chem.274: 18470-6, 1999); Carrington et al., Cells 9:1690 (2020)).

[0177] As used herein, the term “APOBEC3” refers to a APOBEC3 protein, such as an APOBEC3 protein expressed by any of the seven genes (A3A-A3H) of the human APOBEC3 locus. The APOBEC3 may have catalytic DNA or RNA editing activity. An amino acid sequence of APOBEC3A has been described (UniPROT accession ID: p31941) and is included herein as SEQ ID NO: 850. In some embodiments, the APOBEC3 protein is a human APOBEC3 protein or a wild-type protein. Variants include proteins having a sequence that differs from wild-type APOBEC3 protein by one or several mutations (i.e. substitutions, deletions, insertions), such as one or several single point substitutions. For instance, a shortened APOBEC3 sequence could be used, e.g. by deleting several N-term or C-term amino acids, preferably one to four amino acids at the C-terminus of the sequence. As used herein, the term “variant” refers to allelic variants, splicing variants, and natural or artificial mutants, which are homologous to an APOBEC3 reference sequence. The variant is “functional” in that it shows a catalytic activity of DNA or RNA editing. In some embodiments, an APOBEC3 (such as a human APOBEC3A) has a wild-type amino acid position 57 (as numbered in the wild-type sequence). In some embodiments, an APOBEC3 (such as a human APOBEC3A) has an asparagine at amino acid position 57 (as numbered in the wild-type sequence).

[0178] As used herein, a “nickase” is an enzyme that creates a single-strand break (also known as a “nick”) in double strand DNA, i.e., cuts one strand but not the other of the DNA double helix. As used herein, an “RNA-guided DNA nickase” means a polypeptide or complex of polypeptides having DNA nickase activity, wherein the DNA nickase activity is sequence-specific and depends on the sequence of the RNA. Exemplary RNA-guided DNA nickases include Cas nickases. Cas nickases include nickase forms of a Csm or Cmr complex of a type III CRISPR system, the Cas10, Csm1, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the Cas3 subunit thereof, and Class 2 Cas nucleases. Class 2 Cas nickases include variants in which only one of the two catalytic domains is inactivated, which have RNA-guided DNA nickase activity. Class 2 Cas nickases include polypeptides in whicheither the HNH or RuvC catalytic domain is inactivated, for example, Cas9 for example, Cas9 (e.g., H840A, D10A, or N863A variants of SpyCas9 or D16A variant of NmeCas9). Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain or RuvC or RuvC-like domains for N. meningitidis include Nme2Cas9 D16A (HNH nickase) and Nme2Cas9 H588A (RuvC nickase), Cpf1, C2c1, C2c2, C2c3, HF Cas9 (e.g., N497A, R661A, Q695A, Q926A variants), HypaCas9 (e.g., N692A, M694A, Q695A, H698A variants), eSPCas9(1.0) (e.g, K810A, K1003A, R1060A variants), and eSPCas9(1.1) (e.g., K848A, K1003A, R1060A variants) proteins and modifications thereof. Cpf1 protein, Zetsche et al., Cell, 163: 1-13 (2015), is homologous to Cas9, and contains a RuvC-like protein domain. Cpf1 sequences of Zetsche are incorporated by reference in their entirety. See, e.g., Zetsche, Tables S1 and S3. “Cas9” encompasses S. pyogenes (Spy) Cas9, the variants of Cas9 listed herein, and equivalents thereof. See, e.g., Makarova et al., Nat Rev Microbiol, 13(11): 722-36 (2015); Shmakov et al., Molecular Cell, 60:385-397 (2015).

[0179] As used herein, the term “fusion protein” refers to a hybrid polypeptide which comprises polypeptides from at least two different proteins or sources. One polypeptide may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxy- terminal (C- terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxy-terminal fusion protein,” respectively. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

[0180] The term “linker,” as used herein, refers to a chemical group or a molecule linking two adjacent molecules or moieties. Typically, the linker is positioned between, or flanked by, two groups, molecules, or other moieties and connected to each one via a covalent bond. In some embodiments, the linker is an amino acid or a plurality of amino acids (e.g., a peptide or protein) such as a 16-amino acid residue “XTEN” linker, or a variant thereof (See, e.g., the Examples; and Schellenberger et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol.27, 1186-1190 (2009)). In some embodiments, the XTEN linker comprises the sequence SGSETPGTSESATPES (SEQ ID NO: 901), SGSETPGTSESA (SEQ ID NO: 902), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 903).

[0181] As used herein, the term “uracil glycosylase inhibitor”, “uracil-DNA glycosylase inhibitor”, or “UGI” refers to a protein that is capable of inhibiting a uracil-DNA glycosylase (UDG) base-excision repair enzyme (e.g., UniPROT ID: P14739; SEQ ID NO: 859).

[0182] As used herein, “open reading frame” or “ORF” of a gene refers to a sequence consisting of a series of codons that specify the amino acid sequence of the protein that the gene codes for. The ORF begins with a start codon (e.g., ATG in DNA or AUG in RNA) and ends with a stop codon, e.g., TAA, TAG or TGA in DNA or UAA, UAG, or UGA in RNA.

[0183] As used herein, “ribonucleoprotein” (RNP) or “RNP complex” refers to a guide RNA together with an RNA-guided DNA binding agent, such as a Cas nuclease, e.g., a Cas cleavase, Cas nickase, or dCas DNA binding agent (e.g., Cas9). In some embodiments, the guide RNA guides the RNA-guided DNA binding agent such as Cas9 to a target sequence, and the guide RNA hybridizes with and the agent binds to the target sequence; in cases where the agent is a cleavase or nickase, binding can be followed by cleaving or nicking.

[0184] As used herein, a first sequence is considered to “comprise a sequence with at least X% identity to” a second sequence if an alignment of the first sequence to the second sequence shows that X% or more of the positions of the second sequence in its entirety are matched by the first sequence. For example, the sequence AAGA comprises a sequence with 100% identity to the sequence AAG because an alignment would give 100% identity in that there are matches to all three positions of the second sequence. The differences between RNA and DNA (generally the exchange of uridine for thymidine or vice versa) and the presence of nucleoside analogs such as modified uridines do not contribute to differences in identity or complementarity among polynucleotides as long as the relevant nucleotides (such as thymidine, uridine, or modified uridine) have the same complement (e.g., adenosine for all of thymidine, uridine, or modified uridine; another example is cytosine and 5-methylcytosine, both of which have guanosine or modified guanosine as a complement). Thus, for example, the sequence 5’-AXG where X is any modified uridine, such as pseudouridine, N1-methyl pseudouridine, or 5-methoxyuridine, is considered 100% identical to AUG in that both are perfectly complementary to the same sequence (5’-CAU). Exemplary alignment algorithms are the Smith-Waterman and Needleman-Wunsch algorithms, which are well-known in the art. One skilled in the art will understand what choice of algorithm and parameter settings are appropriate for a given pair of sequences to be aligned; for sequences of generally similar length and expected identity >50% for amino acids or >75% for nucleotides, the Needleman- Wunsch algorithm with default settings of the Needleman-Wunsch algorithm interface provided by the EBI at the www.ebi.ac.uk web server is generally appropriate.

[0185] “mRNA” is used herein to refer to a polynucleotide and comprises an open reading frame that can be translated into a polypeptide (i.e., can serve as a substrate for translation by a ribosome and amino-acylated tRNAs). mRNA can comprise a phosphate-sugar backbone including ribose residues or analogs thereof, e.g., 2’-methoxy ribose residues. In some embodiments, the sugars of an mRNA phosphate-sugar backbone consist essentially of ribose residues, 2’-methoxy ribose residues, or a combination thereof.

[0186] As used herein, “indel” refers to an insertion or deletion mutation consisting of a number of nucleotides that are either inserted, deleted, or inserted and deleted, e.g. at the site of double-stranded breaks (DSBs), in a target nucleic acid. As used herein, when indel formation results in an insertion, the insertion is a random insertion at the site of a DSB and is not generally directed by or based on a template sequence.

[0187] As used herein, “reduced or eliminated” expression of a protein on a cell refers to a partial or complete loss of expression of the protein relative to an unmodified cell. In some embodiments, the surface expression of a protein on a cell is measured by flow cytometry and has “reduced” or “eliminated” surface expression relative to an unmodified cell as evidenced by a reduction in fluorescence signal upon staining with the same antibody against the protein. A cell that has “reduced” or “eliminated” surface expression of a protein by flow cytometry relative to an unmodified cell may be referred to as “negative” for expression of that protein as evidenced by a fluorescence signal similar to a cell stained with an isotype control antibody. The “reduction” or “elimination” of protein expression can be measured by other known techniques in the field with appropriate controls known to those skilled in the art.

[0188] As used herein, “knockdown” refers to a decrease in expression of a particular gene product (e.g., protein, mRNA, or both), e.g., as compared to expression of an unedited target sequence. Knockdown of a protein can be measured by detecting total cellular amount of the protein from a sample, such as a tissue, fluid, or cell population of interest. It can also be measured by measuring a surrogate, marker, or activity for the protein. Methods for measuring knockdown of mRNA are known and include analyzing mRNA isolated from a sample of interest. In some embodiments, “knockdown” may refer to some loss of expression of a particular gene product, for example a decrease in the amount of mRNA transcribed or a decrease in the amount of protein expressed by a cell or population of cells (including in vivo populations such as those found in tissues).

[0189] As used herein, “knockout” or “KO” refers to a loss of expression from a particular gene or of a particular protein in a cell. Knockout can result in a decrease in expression belowthe level of detection of the assay. Knockout can be measured either by detecting total cellular amount of a protein in a cell, a tissue or a population of cells.

[0190] As used herein, a “target sequence” or “genomic target sequence” refers to a sequence of nucleic acid in a target gene that has complementarity to the guide sequence of the gRNA. The interaction of the target sequence and the guide sequence directs an RNA-guided DNA binding agent to bind, and potentially nick or cleave (depending on the activity of the agent), within the target sequence.

[0191] As used herein, the term “subject” is intended to include living organisms in which an immune response can be elicited, including e.g., mammals, primates, humans.

[0192] As used herein, “treatment” refers to any administration or application of a therapeutic for disease or disorder in a subject, and includes inhibiting the disease, arresting its development, relieving one or more symptoms of the disease, curing the disease, or preventing one or more symptoms of the disease, including recurrence of the symptom.

[0193] Reference will now be made in detail to certain embodiments of the invention, examples of which are illustrated in the accompanying drawings. While the invention is described in conjunction with the illustrated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications, and equivalents, which may be included within the invention as defined by the appended claims and included embodiments.

[0194] Before describing the present teachings in detail, it is to be understood that the disclosure is not limited to specific compositions or process steps, as such may vary. It should be noted that, as used in this specification and the appended claims, the singular form “a”, “an” and “the” include plural references unless the context clearly dictates otherwise. Thus, for example, reference to “a conjugate” includes a plurality of conjugates and reference to “a cell” includes a plurality of cells (e.g., a population of cells) and the like.

[0195] Numeric ranges are inclusive of the numbers defining the range. Measured and measurable values are understood to be approximate, taking into account significant digits and the error associated with the measurement.

[0196] Also, the use of “comprise”, “comprises”, “comprising”, “contain”, “contains”, “containing”, “include”, “includes”, and “including” are not intended to be limiting. It is to be understood that both the foregoing general description and detailed description are exemplary and explanatory only and are not restrictive of the teachings. Unless specifically noted in the specification, embodiments in the specification that recite “comprising” various components are also contemplated as “consisting of” or “consisting essentially of” the recitedcomponents; embodiments in the specification that recite “consisting of” various components are also contemplated as “comprising” or “consisting essentially of” the recited components; and embodiments in the specification that recite “consisting essentially of” various components are also contemplated as “consisting of” or “comprising” the recited components (this interchangeability does not apply to the use of these terms in the claims).

[0197] The term “or” is used in an inclusive sense, i.e., equivalent to “and / or,” unless the context clearly indicates otherwise.

[0198] The term “about”, when used before a list, modifies each member of the list. The term “about” is understood to encompass tolerated variation or error within the art, e.g., 2 standard deviations from the mean, or the sensitivity of the method used to take a measurement. When “about” is present before the first value of a series, it can be understood to modify each value in the series.

[0199] Ranges are understood to include the numbers at the end of the range and all logical values therebetween. For example, 5-10 nucleotides is understood as 5, 6, 7, 8, 9, or 10 nucleotides, whereas 5-10% is understood to contain 5% and all possible values through 10%.

[0200] At least 17 nucleotides of a 20 nucleotide sequence is understood to include 17, 18, 19, or 20 nucleotides of the sequence provided, thereby providing an upper limit even if one is not specifically provided as it would be clearly understood. Similarly, up to 3 nucleotides would be understood to encompass 0, 1, 2, or 3 nucleotides, providing a lower limit even if one is not specifically provided. When “at least”, “up to”, or other similar language modifies a number, it can be understood to modify each number in the series.

[0201] As used herein, “no more than” or “less than” is understood as the value adjacent to the phrase and logical lower values or integers, as logical from context, to zero. For example, a duplex region of “no more than 2 nucleotide base pairs” has a 2, 1, or 0 nucleotide base pairs. When “no more than” or “less than” is present before a series of numbers or a range, it is understood that each of the numbers in the series or range is modified.

[0202] As used herein, ranges include both the upper and lower limit.

[0203] In the event of a conflict between a sequence in the application and an indicated accession number or position in an accession number, the sequence in the application predominates.

[0204] As used herein, it is understood that when the maximum amount of a value is represented by 100% (e.g., 100% inhibition or 100% encapsulation) that the value is limited by the method of detection. For example, 100% inhibition is understood as inhibition to alevel below the level of detection of the assay, and 100% encapsulation is understood as no material intended for encapsulation can be detected outside the vesicles.

[0205] The section headings used herein are for organizational purposes only and are not to be construed as limiting the desired subject matter in any way. In the event that any material incorporated by reference contradicts any term defined in this specification or any other express content of this specification, this specification controls. While the present teachings are described in conjunction with various embodiments, it is not intended that the present teachings be limited to such embodiments. On the contrary, the present teachings encompass various alternatives, modifications, and equivalents, as will be appreciated by those of skill in the art. B. Genetically Modified Cells 1. Engineered Cell Compositions

[0206] The present disclosure provides engineered cell compositions which have reduced or eliminated surface expression of TGF^R2 protein relative to an unmodified cell, comprising a genetic modification in the TGF^R2 gene.

[0207] In some embodiments, an engineered cell is provided which has reduced or eliminated surface expression of TGF^R2 protein relative to an unmodified cell, comprising a genetic modification in the TGF^R2 gene, wherein the genetic modification is within the genomic coordinates chr3:30606864-30691614. In some embodiments, an engineered cell is provided which has reduced or eliminated surface expression of TGF^R2 protein relative to an unmodified cell, comprising a genetic modification in the TGF^R2 gene, wherein the genetic modification comprises at least one nucleotide within the genomic coordinates chr3:30606864-30691614. In some embodiments, an engineered cell is provided which has reduced or eliminated surface expression of TGF^R2 protein relative to an unmodified cell, comprising a genetic modification in the TGFBR2 gene, wherein the genetic modification is within the genomic coordinates chr3:30606864-30691614.

[0208] In some embodiments, for each given range of genomic coordinates, a range may encompass + / - 10 nucleotides on either end of the specified coordinates. For example, if chr3:30606864-30606884 is given, in some embodiments the genomic target sequence or genetic modification may fall within chr3:30606864-30606884. In some embodiments, for each given range of genomic coordinates, the range may encompass + / - 5 nucleotides on either end of the range.

[0209] In some embodiments, a given range of genomic coordinates may comprise a target sequence on both strands of the DNA (i.e., the plus (+) strand and the minus (-) strand).

[0210] Genetic modifications in the TGF^R2 gene are described further herein. In some embodiments, a genetic modification in the TGF^R2 gene comprises any one or more of an insertion, deletion, substitution, or deamination of at least one nucleotide in a target sequence.

[0211] The engineered cells described herein may comprise a genetic modification in any TGF^R2 allele of the TGF^R2 gene. The TGF^R2 gene is located in chromosome 3.

[0212] In some embodiments, an engineered cell is provided which has reduced or eliminated surface expression of TGF^R2 protein relative to an unmodified cell, comprising a genetic modification in a TGF^R2 gene, wherein the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within any one of the genomic coordinates listed in Table 2.

[0213] In some embodiments, an engineered cell is provided which has reduced or eliminated surface expression of TGF^R2 protein relative to an unmodified cell, comprising a genetic modification in a TGF^R2 gene, wherein the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within any one of the genomic coordinates listed in Table 2, wherein the genetic modification comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides within the genomic coordinates.

[0214] In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 1 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 2 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 3 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 4 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 5 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 6 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 7 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 8 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 9 contiguous nucleotides within the genomic coordinates. In some embodiments, the genetic modification comprises at least 10 contiguous nucleotides within the genomic coordinates.

[0215] In some embodiments, an engineered cell is provided which has reduced or eliminated surface expression of TGF^R2 protein relative to an unmodified cell, comprising a genetic modification in a TGF^R2 gene, wherein the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within any one of the genomic coordinates listed in Table 2, wherein the genetic modification comprises at least one C to T substitution or at least one A to G substitution within the genomic coordinates.

[0216] In some embodiments, an engineered cell is provided wherein the TGF^R2 expression is reduced or eliminated by a gene editing system that binds to a TGF^R2 genomic target sequence comprising at least 5 contiguous nucleotides within any one of the genomic coordinates listed in Table 2. In some embodiments, the TGF^R2 genomic target sequence comprises at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the TGF^R2 genomic target sequence comprises at least 15 contiguous nucleotides within the genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent, such as an S. pyogenes Cas9, or a base editor that comprises an S. pyogenes.

[0217] In some embodiments, an engineered cell is provided which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell, comprising a genetic modification in a TGFBR2 gene, wherein the genetic modification is within the genomic coordinates chosen from chr3:30672267-30672287; chr3:30644743-30644763; chr3:30671764-30671784; chr3:30672177-30672197; chr3:30606958-30606978; chr3:30606870-30606890; chr3:30606961-30606981; chr3:30606911-30606931; chr3:30606909-30606929; chr3:30606954-30606974; chr3:30606865-30606885; chr3:30606947-30606967; chr3:30606864-30606884; chr3:30606916-30606936; chr3:30606871-30606891; chr3:30606930-30606950; chr3:30606957-30606977; chr3:30623239-30623259; chr3:30623238-30623258; chr3:30623240-30623260; chr3:30623263-30623283; chr3:30644843-30644863; chr3:30644757-30644777; chr3:30650418-30650438; chr3:30650323-30650343; chr3:30650327-30650347; chr3:30650317-30650337; chr3:30650326-30650346; chr3:30650318-30650338; chr3:30650319-30650339; chr3:30650393-30650413; chr3:30672152-30672172; chr3:30672410-30672430; chr3:30672296-30672316; chr3:30671871-30671891; chr3:30672193-30672213; chr3:30671791-30671811; chr3:30672024-30672044; chr3:30671784-30671804; chr3:30672322-30672342; chr3:30672192-30672212; chr3:30672270-30672290; chr3:30671941-30671961; chr3:30671752-30671772; chr3:30672387-30672407; chr3:30671929-30671949; chr3:30672266-30672286;chr3:30672295-30672315; chr3:30672021-30672041; chr3:30671932-30671952; chr3:30671835-30671855; chr3:30672023-30672043; chr3:30671908-30671928; chr3:30672212-30672232; chr3:30671854-30671874; chr3:30671701-30671721; chr3:30672294-30672314; chr3:30672193-30672213; chr3:30671821-30671841; chr3:30672268-30672288; chr3:30672250-30672270; chr3:30672421-30672441; chr3:30672400-30672420; chr3:30672249-30672269; chr3:30672196-30672216; chr3:30672340-30672360; chr3:30671842-30671862; chr3:30671739-30671759; chr3:30674221-30674241; chr3:30674178-30674198; chr3:30674085-30674105; chr3:30674136-30674156; chr3:30674220-30674240; chr3:30674184-30674204; chr3:30688451-30688471; chr3:30688403-30688423; chr3:30688434-30688454; chr3:30688432-30688452; chr3:30688402-30688422; chr3:30688388-30688408; chr3:30688429-30688449; chr3:30688510-30688530; chr3:30688416-30688436; chr3:30691489-30691509; chr3:30691522-30691542; chr3:30691427-30691447; chr3:30691519-30691539; chr3:30691435-30691455; chr3:30691594-30691614; chr3:30691409-30691429; chr3:30691463-30691483; and chr3:30691475-30691495. In some embodiments, an engineered cell is provided wherein the TGFBR2 expression is reduced or eliminated by a gene editing system that binds to a TGFBR2 genomic target sequence comprising at least 5 contiguous nucleotides within the genomic coordinates chosen from: chr3:30672267-30672287; chr3:30644743-30644763; chr3:30671764-30671784; chr3:30672177-30672197; chr3:30606958-30606978; chr3:30606870-30606890; chr3:30606961-30606981; chr3:30606911-30606931; chr3:30606909-30606929; chr3:30606954-30606974; chr3:30606865-30606885; chr3:30606947-30606967; chr3:30606864-30606884; chr3:30606916-30606936; chr3:30606871-30606891; chr3:30606930-30606950; chr3:30606957-30606977; chr3:30623239-30623259; chr3:30623238-30623258; chr3:30623240-30623260; chr3:30623263-30623283; chr3:30644843-30644863; chr3:30644757-30644777; chr3:30650418-30650438; chr3:30650323-30650343; chr3:30650327-30650347; chr3:30650317-30650337; chr3:30650326-30650346; chr3:30650318-30650338; chr3:30650319-30650339; chr3:30650393-30650413; chr3:30672152-30672172; chr3:30672410-30672430; chr3:30672296-30672316; chr3:30671871-30671891; chr3:30672193-30672213; chr3:30671791-30671811; chr3:30672024-30672044; chr3:30671784-30671804; chr3:30672322-30672342; chr3:30672192-30672212; chr3:30672270-30672290; chr3:30671941-30671961; chr3:30671752-30671772; chr3:30672387-30672407; chr3:30671929-30671949; chr3:30672266-30672286; chr3:30672295-30672315;chr3:30672021-30672041; chr3:30671932-30671952; chr3:30671835-30671855; chr3:30672023-30672043; chr3:30671908-30671928; chr3:30672212-30672232; chr3:30671854-30671874; chr3:30671701-30671721; chr3:30672294-30672314; chr3:30672193-30672213; chr3:30671821-30671841; chr3:30672268-30672288; chr3:30672250-30672270; chr3:30672421-30672441; chr3:30672400-30672420; chr3:30672249-30672269; chr3:30672196-30672216; chr3:30672340-30672360; chr3:30671842-30671862; chr3:30671739-30671759; chr3:30674221-30674241; chr3:30674178-30674198; chr3:30674085-30674105; chr3:30674136-30674156; chr3:30674220-30674240; chr3:30674184-30674204; chr3:30688451-30688471; chr3:30688403-30688423; chr3:30688434-30688454; chr3:30688432-30688452; chr3:30688402-30688422; chr3:30688388-30688408; chr3:30688429-30688449; chr3:30688510-30688530; chr3:30688416-30688436; chr3:30691489-30691509; chr3:30691522-30691542; chr3:30691427-30691447; chr3:30691519-30691539; chr3:30691435-30691455; chr3:30691594-30691614; chr3:30691409-30691429; chr3:30691463-30691483; and chr3:30691475-30691495. In some embodiments, the TGFBR2 genomic target sequence comprises at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the TGFBR2 genomic target sequence comprises at least 15 contiguous nucleotides within the genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent, such as an S. pyogenes Cas9.

[0218] In some embodiments, an engineered cell is provided which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell, comprising a genetic modification in a TGFBR2 gene, wherein the genetic modification is within the genomic coordinates chosen from chr3:30671764-30671784; chr3:30672177-30672197; chr3:30606958-30606978; chr3:30606961-30606981; chr3:30606916-30606936; chr3:30606957-30606977; chr3:30650418-30650438; chr3:30672193-30672213; chr3:30672024-30672044; chr3:30672023-30672043; chr3:30672421-30672441; chr3:30674178-30674198; chr3:30688510-30688530; chr3:30691409-30691429; chr3:30606962-30606982; chr3:30606974-30606994; chr3:30606975-30606995; chr3:30644885-30644905; chr3:30644893-30644913; chr3:30650250-30650270; chr3:30671618-30671638; chr3:30671763-30671783; chr3:30671983-30672003; chr3:30672088-30672108; chr3:30672094-30672114; chr3:30672099-30672119; chr3:30674083-30674103; chr3:30674198-30674218; chr3:30688476-30688496; chr3:30688481-30688501; chr3:30688490-30688510; chr3:30688491-30688511;chr3:30688507-30688527; chr3:30691396-30691416; chr3:30691397-30691417; chr3:30691412-30691432; chr3:30691413-30691433; chr3:30691582-30691602; and chr3:30691583-30691603. In some embodiments, an engineered cell is provided wherein the TGFBR2 expression is reduced or eliminated by a gene editing system that binds to a TGFBR2 genomic target sequence comprising at least 5 contiguous nucleotides within the genomic coordinates chosen from: chr3:30671764-30671784; chr3:30672177-30672197; chr3:30606958-30606978; chr3:30606961-30606981; chr3:30606916-30606936; chr3:30606957-30606977; chr3:30650418-30650438; chr3:30672193-30672213; chr3:30672024-30672044; chr3:30672023-30672043; chr3:30672421-30672441; chr3:30674178-30674198; chr3:30688510-30688530; chr3:30691409-30691429; chr3:30606962-30606982; chr3:30606974-30606994; chr3:30606975-30606995; chr3:30644885-30644905; chr3:30644893-30644913; chr3:30650250-30650270; chr3:30671618-30671638; chr3:30671763-30671783; chr3:30671983-30672003; chr3:30672088-30672108; chr3:30672094-30672114; chr3:30672099-30672119; chr3:30674083-30674103; chr3:30674198-30674218; chr3:30688476-30688496; chr3:30688481-30688501; chr3:30688490-30688510; chr3:30688491-30688511; chr3:30688507-30688527; chr3:30691396-30691416; chr3:30691397-30691417; chr3:30691412-30691432; chr3:30691413-30691433; chr3:30691582-30691602; and chr3:30691583-30691603. In some embodiments, the TGFBR2 genomic target sequence comprises at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the TGFBR2 genomic target sequence comprises at least 15 contiguous nucleotides within the genomic coordinates. In some embodiments, the gene editing system comprises an RNA-guided DNA binding agent, such as a base editor comprising a cytidine deaminase and an S. pyogenes Cas9 nickase.

[0219] In some embodiments, an engineered cell is provided which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell, comprising a genetic modification in a TGFBR2 gene, wherein the genetic modification is within the genomic coordinates chosen from chr3:30650418-30650438; chr3:30671941-30671961; chr3:30672212-30672232; chr3:30672193-30672213; chr3:30671739-30671759; and chr3:30691475-30691495. In some embodiments, an engineered cell is provided wherein the TGFBR2 expression is reduced or eliminated by a gene editing system that binds to a TGFBR2 genomic target sequence comprising at least 5 contiguous nucleotides within the genomic coordinates chosen from chr3:30650418-30650438; chr3:30671941-30671961;chr3:30672212-30672232; chr3:30672193-30672213; chr3:30671739-30671759; and chr3:30691475-30691495.

[0220] In some embodiments, an engineered cell is provided which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell, comprising a genetic modification in a TGFBR2 gene, wherein the genetic modification is within the genomic coordinates chosen from chr3:30671941-30671961 and chr3:30671739-30671759. In some embodiments, an engineered cell is provided wherein the TGFBR2 expression is reduced or eliminated by a gene editing system that binds to a TGFBR2 genomic target sequence comprising at least 5 contiguous nucleotides within the genomic coordinates chosen from chr3:30671941-30671961 and chr3:30671739-30671759.

[0221] In some embodiments, an engineered cell is provided which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell, comprising a genetic modification in a TGFBR2 gene, wherein the genetic modification is within the genomic coordinates chosen from chr3:30671764-30671784; chr3:30672177-30672197; chr3:30606958-30606978; chr3:30606961-30606981; chr3:30606916-30606936; chr3:30606957-30606977; chr3:30650418-30650438; chr3:30672193-30672213; chr3:30672024-30672044; chr3:30672023-30672043; chr3:30672421-30672441; chr3:30674178-30674198; chr3:30688510-30688530; and chr3:30691409-30691429. In some embodiments, an engineered cell is provided wherein the TGFBR2 expression is reduced or eliminated by a gene editing system that binds to a TGFBR2 genomic target sequence comprising at least 5 contiguous nucleotides within the genomic coordinates chosen from chr3:30671764-30671784; chr3:30672177-30672197; chr3:30606958-30606978; chr3:30606961-30606981; chr3:30606916-30606936; chr3:30606957-30606977; chr3:30650418-30650438; chr3:30672193-30672213; chr3:30672024-30672044; chr3:30672023-30672043; chr3:30672421-30672441; chr3:30674178-30674198; chr3:30688510-30688530; and chr3:30691409-30691429.

[0222] In some embodiments, an engineered cell is provided which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell, comprising a genetic modification in a TGFBR2 gene, wherein the genetic modification is within the genomic coordinates chosen from: chr3:30644885-30644905; chr3:30671618-30671638; chr3:30671983-30672003; chr3:30672094-30672114; chr3:30672177-30672197; and chr3:30674198-30674218. In some embodiments, an engineered cell is provided wherein the TGFBR2 expression is reduced or eliminated by a gene editing system that binds to a TGFBR2 genomic target sequence comprising at least 5 contiguous nucleotides within thegenomic coordinates chosen from chr3:30644885-30644905; chr3:30671618-30671638; chr3:30671983-30672003; chr3:30672094-30672114; chr3:30672177-30672197; and chr3:30674198-30674218.

[0223] In some embodiments, the TGFBR2 genomic target sequence comprises at least 10 contiguous nucleotides within the genomic coordinates. In some embodiments, the TGFBR2 genomic target sequence comprises at least 15 contiguous nucleotides within the genomic coordinates.

[0224] In some embodiments, the TGFBR2 genomic target sequence comprises at least 17, 18, 19, or 20, contiguous nucleotides within the genomic coordinates.

[0225] In some embodiments, the gene editing system comprises a transcription activator- like effector nuclease (TALEN). In some embodiments, the gene editing system comprises a zinc finger nuclease. In some embodiments, the gene editing system comprises a CRISPR / Cas system, such as a class 2 system. In some embodiments, the gene editing system comprises an RNA-guided DNA-binding agent or a nucleic acid encoding an RNA- guided DNA binding agent.

[0226] Exemplary RNA-guided DNA binding agents are shown in Table 1 below. Table 1. Exemplary RNA-guided DNA binding agents.*Exemplary base editor based on deaminase-SpyCas9 nickase or deaminase-NmeCas9 nickase. As is apparent, the base editor specificity, including PAM, will vary with its nickase.

[0227] In some embodiments, the RNA-guided DNA-binding agent or nucleic acid encoding the RNA-guided DNA binding agent comprises a Cas9 protein. In some embodiments, the RNA-guided DNA binding agent is selected from one of: S. pyogenes Cas9, Neisseria meningitidis Cas9, e.g. an Nme2Cas9, S. thermophilus Cas9, S. aureus Cas9, Francisella novicida Cpf1, Acidaminococcus sp. Cpf1, Lachnospiraceae bacterium Cpf1, C- to-T base editor, A-to-G base editor, Cas12a, Mad7 nuclease, ARCUS nucleases, and CasX. In some embodiments, the RNA-guided DNA binding agent comprises a polypeptide selected from one of: S. pyogenes Cas9, Neisseria meningitidis Cas9, e.g. an Nme2Cas9, S. thermophilus Cas9, S. aureus Cas9, Francisella novicida Cpf1, Acidaminococcus sp. Cpf1, Lachnospiraceae bacterium Cpf1, C-to-T base editor, A-to-G base editor, Cas12a, and CasX.

[0228] In some embodiments, the RNA-guided DNA-binding agent or nucleic acid encoding the RNA-guided DNA binding agent is S. pyogenes Cas9.

[0229] In some embodiments, the RNA-guided DNA-binding agent or nucleic acid encoding the RNA-guided DNA binding agent is S. thermophilus Cas9. In some embodiments, the RNA-guided DNA-binding agent or nucleic acid encoding the RNA-guided DNA binding agent is S. aureus Cas9. In some embodiments, the RNA-guided DNA-binding agent or nucleic acid encoding the RNA-guided DNA binding agent is Cpf1 from F. novicida. In some embodiments, the RNA-guided DNA-binding agent or nucleic acid encoding the RNA- guided DNA binding agent is Cpf1 from Acidaminococcus sp. In some embodiments, the RNA-guided DNA-binding agent or nucleic acid encoding the RNA-guided DNA binding agent is Cpf1 from Lachnospiraceae bacterium ND2006. In some embodiments, the RNA- guided DNA-binding agent or nucleic acid encoding the RNA-guided DNA binding agent isCas12a. In some embodiments, the RNA-guided DNA-binding agent or nucleic acid encoding the RNA-guided DNA binding agent is CasX.

[0230] In some embodiments, the RNA-guided DNA-binding agent or nucleic acid encoding the RNA-guided DNA binding agent is a C to T base editor. In some embodiments, the RNA- guided DNA-binding agent or nucleic acid encoding the RNA-guided DNA binding agent is a A to G base editor. In some embodiments, the base editor comprises a deaminase and an RNA-guided nickase. In some embodiments, the RNA-guided DNA-binding agent or nucleic acid encoding the RNA-guided DNA binding agent comprises a APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the RNA-guided nickase is a SpyCas9 nickase.

[0231] In any of the above embodiments, the genome editing system comprises an RNA- guided DNA binding agent, or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9. In some embodiments, the RNA-guided DNA binding agent is an S. pyogenes Cas9. In some embodiments, the RNA-guided DNA binding agent is a base editor. In some embodiments the base editor comprises a C to T deaminase and an RNA-guided nickase such as an S. pyogenes Cas9 nickase. In some embodiments the base editor comprises a A to G deaminase and an RNA-guided nickase such as a D10A SpyCas9 nickase.

[0232] In some embodiments, the gene editing system further comprises a uracil glycosylase inhibitor (UGI), and the UGI and the base editor are comprised in a single polypeptide. In some embodiments, the gene editing system comprises a UGI, and the UGI and the base editor are comprised in different polypeptides. In some embodiments, the base editor comprises a cytidine deaminase and an RNA-guided nickase. In some embodiments, the cytidine deaminase, the RNA-guided nickase, and the UGI are comprised in a single polypeptide. In some embodiments, the cytidine deaminase, the RNA-guided nickase, and the UGI are comprised in different polypeptides. In some embodiments, the cytidine deaminase and the RNA-guided nickase are comprised in a single polypeptide, and wherein the UGI is comprised in a different polypeptide.

[0233] The engineered cell may be any of the exemplary cell types disclosed herein.

[0234] In some embodiments, the disclosure provides a pharmaceutical composition comprising any one of the engineered cells disclosed herein. In some embodiments, the pharmaceutical composition comprises a population of any one of the engineered cells disclosed herein. In some embodiments, the population of engineered cells is at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%,95%, 96%, 97%, 98%, or 99% TGFBR2 negative as measured by flow cytometry. In some embodiments, at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the population of cells comprises the genetic modification in the TGFBR2 gene, as measured by next-generation sequencing (NGS). C. Methods and Compositions for Reducing or Eliminating Surface Expression of TGFBR2

[0235] The present disclosure provides methods and compositions for reducing or eliminating surface expression of TGFBR2 protein relative to an unmodified cell by genetically modifying the TGFBR2 gene. The resultant genetically modified cell may also be referred to herein as an engineered cell. In some embodiments, an already-genetically modified (or engineered) cell may be the starting cell for further genetic modification using the methods or compositions provided herein. In some embodiments, the cell is an allogeneic cell. In some embodiments, a cell with reduced or eliminated surface expression of TGFBR2 protein is useful for immunotherapy. In some embodiments, a cell with reduced or eliminated surface expression of TGFBR2 protein is useful for adoptive cell transfer therapies. In some embodiments, editing of the TGFBR2 gene is combined with additional genetic modifications to yield a cell that is desirable for allogeneic transplant purposes.

[0236] In some embodiments, the methods comprise reducing surface expression of TGFBR2 protein in a cell relative to an unmodified cell, comprising contacting a cell with composition comprising (a) a guide RNA comprising (i) a guide sequence selected from SEQ ID NOs: 1- 117; or (ii) at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-117; or (iii) a guide sequence at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 1-117; or (iv) a guide sequence that binds a target site comprising a genomic region listed in Table 2; or (v) a guide sequence that is complementary to at least 17, 18, 19, or 20 contiguous nucleotides of a genomic region listed in Table 2; or (vi) a guide sequence that is at least 95%, 90%, 85%, identical to a sequence selected from (v); and optionally (b) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.

[0237] In some embodiments, the methods further comprise contacting the cell with an RNA- guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA binding agent comprises a Cas9 protein.

[0238] In some embodiments, the RNA-guided DNA binding agent is S. pyogenes Cas9. In some embodiments, the guide RNA is a S. pyogenes Cas9 guide RNA.

[0239] In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain.

[0240] In some embodiments the RNA-guided DNA binding agent is a C to T base editor. In some embodiments the RNA-guided DNA binding agent is a A to G base editor. In some embodiments, the base editor comprises a deaminase and an RNA-guided nickase. In some embodiments the RNA-guided DNA binding agent comprises a APOBEC3A deaminase (A3A) and an RNA-guided nickase. In some embodiments, the RNA-guided nickase is a SpyCas9 nickase.

[0241] In some embodiments, the surface expression of TGFBR2 protein (i.e., engineered cell) is thereby reduced or eliminated.

[0242] In some embodiments, the methods comprise making an engineered human cell, which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell, comprising contacting a cell with composition comprising (a) a guide RNA comprising (i) a guide sequence selected from SEQ ID NOs: 1-117; or (ii) at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-117; or (iii) a guide sequence at least 95%, 90%, or 85% identical to a sequence selected from SEQ ID NOs: 1-117; or (iv) a guide sequence that binds a target site comprising a genomic region listed in Table 2; or (v) a guide sequence that is complementary to at least 17, 18, 19, or 20 contiguous nucleotides of a genomic region listed in Table 2; or (vi) a guide sequence that is at least 95%, 90%, 85%, identical to a sequence selected from (v); and optionally (b) an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the methods further comprise contacting the cell with an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.. In some embodiments, the RNA-guided DNA binding agent is SpyCas9. In some embodiments, the guide RNA is a Spy guide RNA. In some embodiments, the RNA-guided DNA binding agent comprises a deaminase domain. In some embodiments the RNA-guided DNA binding agent comprises an APOBEC3A deaminase (A3A) and an RNA-guided nickase.

[0243] In some embodiments, the composition further comprises a uracil glycosylase inhibitor (UGI). In some embodiments, the composition comprises an RNA-guided DNA binding agent that the RNA-guided DNA binding agent generates a cytosine (C) to thymine (T) conversion with the TGFBR2 genomic target sequence. In some embodiments, the composition comprises an RNA-guided DNA binding agent that generates an adenosine (A) to guanine (G) conversion with the TGFBR2 genomic target sequence.

[0244] In some embodiments, the surface expression of TGFBR2 protein (i.e., engineered cell) is thereby reduced or eliminated.

[0245] In some embodiments, an engineered cell produced by the methods described herein is provided. In some embodiments, the compositions disclosed herein further comprise a pharmaceutically acceptable carrier. In some embodiments, a cell produced by the compositions disclosed herein comprising a pharmaceutically acceptable carrier is provided. In some embodiments, compositions comprising the cells disclosed herein are provided. 1. TGFBR2 guide RNAs

[0246] The methods and compositions provided herein disclose guide RNAs useful for reducing or eliminating the surface expression of TGFBR2 protein. In some embodiments, such guide RNAs direct an RNA-guided DNA binding agent to a TGFBR2 genomic target sequence and may be referred to herein as “TGFBR2 guide RNA.” In some embodiments, the TGFBR2 guide RNA directs an RNA-guided DNA binding agent to a human TGFBR2 genomic target sequence. In some embodiments, the TGFBR2 guide RNA comprises a guide sequence selected from SEQ ID NOs: 1-117.

[0247] In some embodiments, a composition is provided comprising a guide RNA described herein and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.

[0248] In some embodiments, a composition is provided comprising a single-guide RNA (sgRNA) comprising a guide sequence selected from SEQ ID NOs: 1-117. In some embodiments, a composition is provided comprising TGFBR2 sgRNA described herein and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.

[0249] In some embodiments, a composition is provided comprising an TGFBR2 dual-guide RNA (dgRNA) comprising a guide sequence selected from SEQ ID NOs: 1-117. In some embodiments, a composition is provided comprising an TGFBR2 dgRNA described herein and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.

[0250] In some embodiments, the TGFBR2 gRNA comprises a guide sequence selected from any one of SEQ ID NOs: 1-117. Exemplary TGFBR2 target and guide sequences are shown below in Table 2 (SEQ ID NOs: 1-117). The guide sequence disclosed in this Table may be unmodified, modified with the exemplary modification pattern shown in Tables 3-7, or modified with a different modification pattern disclosed herein or available in the art.Table 2. Exemplary TGFBR2 guide RNA genomic coordinates and guide sequencesTable 3. Exemplary full and modified guide RNAs

[0251] In some embodiments, the TGFBR2 gRNA comprises a guide sequence selected from any one of SEQ ID NOs: 1-117. In some embodiments, the TGFBR2 guide RNA comprises a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-117. In some embodiments, the TGFBR2 guide RNA comprises a guide sequence that is at least 95%, 90%, or 85%identical to a sequence selected from SEQ ID NOs: 1-117. In some embodiments, the TGFBR2 guide RNA comprises a guide sequence that is at least 95% identical to a sequence selected from SEQ ID NOs: 1-117. In some embodiments, the TGFBR2 guide RNA comprises a sequence of any one of the guide RNA sequences as shown in Table 2.

[0252] In some embodiments, the TGFBR2 guide RNA comprises a guide sequence that comprises at least 10 contiguous nucleotides ± 10 nucleotides of a genomic coordinate listed in Table 2. As used herein, at least 10 contiguous nucleotides ± 10 nucleotides of a genomic coordinate means, for example, at least 10 contiguous nucleotides within the genomic coordinates wherein the genomic coordinates include 10 nucleotides in the 5’ direction and 10 nucleotides in the 3’ direction from the ranges listed in Table 2. For example, a TGFBR2 guide RNA may comprise 10 contiguous nucleotides within the genomic coordinates: chr3:30650418-30650438; chr3:30671941-30671961; chr3:30672212-30672232; chr3:30672193-30672213; chr3:30671739-30671759; or chr3:30691475-30691495, including the boundary nucleotides of these ranges. In some embodiments, the TGFBR2 guide RNA comprises a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence that comprises 10 contiguous nucleotides ± 10 nucleotides of a genomic coordinate listed in Table 2. In some embodiments, the TGFBR2 guide RNA comprises a guide sequence that is at least 95%, 90%, or 85% identical to a sequence selected from a sequence that is 17, 18, 19, or 20 contiguous nucleotides of a sequence that comprises 10 contiguous nucleotides ± 10 nucleotides of a genomic coordinate listed in Table 2.

[0253] In some embodiments, the Table 2 guide RNA comprises a guide sequence that comprises at least 15 contiguous nucleotides ± 10 nucleotides of a genomic coordinate listed in Table 2. In some embodiments, the TGFBR2 guide RNA comprises a guide sequence that comprises at least 20 contiguous nucleotides ± 10 nucleotides of a genomic coordinate listed in Table 2.

[0254] In some embodiments, a composition is provided comprising a S. pyogenes Cas9 and a TGFBR2 gRNA that comprises (i) a guide sequence selected from any one of SEQ ID NOs: 1-91 and 117; (ii) a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides ofany one of SEQ ID NOs: 1-91 and 117; or (iii) a guide sequence that is at least 95%, 90%, or 85% identical to any one of SEQ ID NOs: 1-91 and 117.

[0255] In some embodiments, a composition is provided comprising a base editor described herein, comprising a deaminase and an RNA-guided nickase being a SpyCas9 nickase; and a TGFBR2 gRNA that comprises (i) a guide sequence selected from any one of SEQ ID NOs: 3, 4, 5, 7, 14, 16, 24, 36, 38, 52, 62, 70, 82, 90, and 92-116; (ii) a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of any one of SEQ ID NOs: 3, 4, 5, 7, 14, 16, 24, 36, 38, 52, 62, 70, 82, 90, and 92-116; or (iii) a guide sequence that is at least 95%, 90%, or 85% identical to any one of SEQ ID NOs: 3, 4, 5, 7, 14, 16, 24, 36, 38, 52, 62, 70, 82, 90, and 92-116.

[0256] Additional embodiments of TGFBR2 guide RNAs are provided herein, including e.g., exemplary modifications to the guide RNA. 2. Genetic modifications to TGFBR2

[0257] In some embodiments, the methods and compositions disclosed herein genetically modify at least one nucleotide in the TGFBR2 gene in a cell. Genetic modifications encompass the population of modifications that results from contact with a gene editing system (e.g., the population of edits that result from Cas9 and a TGFBR2 guide RNA, or the population of edits that result from BC22 and an TGFBR2 guide RNA).

[0258] In some embodiments, the genetic modification is within the genomic coordinates chr3:30606864-30691614. In some embodiments, the genetic modification comprises at least one nucleotide within the genomic coordinates chr3:30606864-30691614.

[0259] In some embodiments, the genetic modification is within any one of the genomic coordinates listed in Table 2. In some embodiments, the genetic modification comprises at least 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 contiguous nucleotides within any one of the genomic coordinates listed in Table 2.

[0260] In some embodiments, the genetic modification comprises at least one nucleotide within the genomic coordinates chosen from: chr3:30672267-30672287; chr3:30644743- 30644763; chr3:30671764-30671784; chr3:30672177-30672197; chr3:30606958-30606978; chr3:30606870-30606890; chr3:30606961-30606981; chr3:30606911-30606931; chr3:30606909-30606929; chr3:30606954-30606974; chr3:30606865-30606885; chr3:30606947-30606967; chr3:30606864-30606884; chr3:30606916-30606936; chr3:30606871-30606891; chr3:30606930-30606950; chr3:30606957-30606977; chr3:30623239-30623259; chr3:30623238-30623258; chr3:30623240-30623260;chr3:30623263-30623283; chr3:30644843-30644863; chr3:30644757-30644777; chr3:30650418-30650438; chr3:30650323-30650343; chr3:30650327-30650347; chr3:30650317-30650337; chr3:30650326-30650346; chr3:30650318-30650338; chr3:30650319-30650339; chr3:30650393-30650413; chr3:30672152-30672172; chr3:30672410-30672430; chr3:30672296-30672316; chr3:30671871-30671891; chr3:30672193-30672213; chr3:30671791-30671811; chr3:30672024-30672044; chr3:30671784-30671804; chr3:30672322-30672342; chr3:30672192-30672212; chr3:30672270-30672290; chr3:30671941-30671961; chr3:30671752-30671772; chr3:30672387-30672407; chr3:30671929-30671949; chr3:30672266-30672286; chr3:30672295-30672315; chr3:30672021-30672041; chr3:30671932-30671952; chr3:30671835-30671855; chr3:30672023-30672043; chr3:30671908-30671928; chr3:30672212-30672232; chr3:30671854-30671874; chr3:30671701-30671721; chr3:30672294-30672314; chr3:30672193-30672213; chr3:30671821-30671841; chr3:30672268-30672288; chr3:30672250-30672270; chr3:30672421-30672441; chr3:30672400-30672420; chr3:30672249-30672269; chr3:30672196-30672216; chr3:30672340-30672360; chr3:30671842-30671862; chr3:30671739-30671759; chr3:30674221-30674241; chr3:30674178-30674198; chr3:30674085-30674105; chr3:30674136-30674156; chr3:30674220-30674240; chr3:30674184-30674204; chr3:30688451-30688471; chr3:30688403-30688423; chr3:30688434-30688454; chr3:30688432-30688452; chr3:30688402-30688422; chr3:30688388-30688408; chr3:30688429-30688449; chr3:30688510-30688530; chr3:30688416-30688436; chr3:30691489-30691509; chr3:30691522-30691542; chr3:30691427-30691447; chr3:30691519-30691539; chr3:30691435-30691455; chr3:30691594-30691614; chr3:30691409-30691429; chr3:30691463-30691483; and chr3:30691475-30691495.

[0261] In some embodiments, the genetic modification comprises at least one nucleotide within the genomic coordinates chosen from: (b) chr3:30671764-30671784; chr3:30672177- 30672197; chr3:30606958-30606978; chr3:30606961-30606981; chr3:30606916-30606936; chr3:30606957-30606977; chr3:30650418-30650438; chr3:30672193-30672213; chr3:30672024-30672044; chr3:30672023-30672043; chr3:30672421-30672441; chr3:30674178-30674198; chr3:30688510-30688530; chr3:30691409-30691429; chr3:30606962-30606982; chr3:30606974-30606994; chr3:30606975-30606995; chr3:30644885-30644905; chr3:30644893-30644913; chr3:30650250-30650270; chr3:30671618-30671638; chr3:30671763-30671783; chr3:30671983-30672003; chr3:30672088-30672108; chr3:30672094-30672114; chr3:30672099-30672119;chr3:30674083-30674103; chr3:30674198-30674218; chr3:30688476-30688496; chr3:30688481-30688501; chr3:30688490-30688510; chr3:30688491-30688511; chr3:30688507-30688527; chr3:30691396-30691416; chr3:30691397-30691417; chr3:30691412-30691432; chr3:30691413-30691433; chr3:30691582-30691602; chr3:30691583-30691603; and chr3:30691475-30691495.

[0262] In some embodiments, the genetic modification comprises at least one nucleotide within the genomic coordinates chosen from: chr3:30650418-30650438; chr3:30671941- 30671961; chr3:30672212-30672232; chr3:30672193-30672213; chr3:30671739-30671759; and chr3:30691475-30691495.

[0263] In some embodiments, the genetic modification comprises at least one nucleotide within the genomic coordinates chosen from: chr3:30671764-30671784; chr3:30672177- 30672197; chr3:30606958-30606978; chr3:30606961-30606981; chr3:30606916-30606936; chr3:30606957-30606977; chr3:30650418-30650438; chr3:30672193-30672213; chr3:30672024-30672044; chr3:30672023-30672043; chr3:30672421-30672441; chr3:30674178-30674198; chr3:30688510-30688530; and chr3:30691409-30691429.

[0264] In some embodiments, the genetic modification comprises at least one nucleotide within the genomic coordinates chosen from: chr3:30671941-30671961 and chr3:30671739- 30671759. In some embodiments, the genetic modification comprises at least one nucleotide within the genomic coordinates chosen from: chr3:30644885-30644905; chr3:30671618- 30671638; chr3:30671983-30672003; chr3:30672094-30672114; chr3:30672177-30672197; and chr3:30674198-30674218.

[0265] In some embodiments, the modification to TGFBR2 comprises any one or more of an insertion, deletion, substitution, or deamination of at least one nucleotide in a target sequence. In some embodiments, the modification to TGFBR2 comprises an insertion of 1, 2, 3, 4 or 5 or more nucleotides in a target sequence. In some embodiments, the modification to TGFBR2 comprises a deletion of 1, 2, 3, 4 or 5 or more nucleotides in a target sequence. In other embodiments, the modification to TGFBR2 comprises an insertion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more nucleotides in a target sequence. In other embodiments, the modification to TGFBR2 comprises a deletion of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25 or more nucleotides in a target sequence. In some embodiments, the modification to TGFBR2 comprises an indel, which is generally defined in the art as an insertion or deletion of less than 1000 base pairs (bp). In some embodiments, the modification to TGFBR2 comprises an indel which results in a frameshift mutation in a target sequence. In some embodiments, the modification to TGFBR2 comprises a substitution of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20 or 25or more nucleotides in a target sequence. In some embodiments, the modification to TGFBR2 comprises one or more of an insertion, deletion, or substitution of nucleotides resulting from the incorporation of a template nucleic acid. In some embodiments, the modification to TGFBR2 comprises an insertion of a donor nucleic acid in a target sequence. In some embodiments, the modification to TGFBR2 is not transient.

[0266] In some embodiments, the genetic modification results in a change in the nucleic acid sequence that prevents translation of a full-length protein having an amino acid sequence of the full-length protein prior to genetic modification.

[0267] In some embodiments, the genetic modification results in a change in the nucleic acid sequence that results in a premature stop codon in a coding sequence of the full-length protein. In some embodiments, the genetic modification results in a change in the nucleic acid sequence that results in a change in splicing of a pre-mRNA from the genomic locus. In some embodiments, the inhibition results in reduced cell surface expression of a protein from the gene comprising a genetic modification. 3. Efficacy of guide RNAs

[0268] The efficacy of a TGFBR2 guide RNA may be determined by techniques available in the art that assess the editing efficiency of a guide RNA, and the surface expression of TGFBR2 protein. In some embodiments, the reduction or elimination of surface expression of TGFBR2 protein may be determined by comparison to an unmodified cell (or “relative to an unmodified cell”). An engineered cell or cell population may also be compared to a population of unmodified cells.

[0269] An “unmodified cell” (or “unmodified cells”) refers to a control cell (or cells) of the same type of cell in an experiment or test, wherein the “unmodified” control cell has not been contacted with a TGFBR2 guide. Therefore, an unmodified cell (or cells) may be a cell that has not been contacted with a guide RNA, or a cell that has been contacted with a guide RNA that does not target TGFBR2.

[0270] In some embodiments, the efficacy of a TGFBR2 guide RNA is determined by measuring levels of surface expression of TGFBR2 protein. In some embodiments, TGFBR2 protein levels are measured by flow cytometry (e.g., with an antibody against TGFBR2). Surface expression of TGFBR2 protein may be measured by flow cytometry as commonly known in the art. One skilled in the art will be familiar with techniques for measuring surface expression of protein such as TGFBR2 protein, by flow cytometry. In some embodiments, TGFBR2 protein levels are indirectly measured by phosphoSMAD (pSMAD) signalmeasurement. The level of intracellular phosphoSMAD signal correlates to the level of TGFBR2 protein levels. One skilled in the art will be familiar with techniques for assaying TGFBR2 protein levels by measuring phosphoSMAD signal. An exemplary measurement of levels of surface expression of TGFBR2 protein by flow cytometry is discussed in Examples 1-5. In some embodiments, the population of cells is enriched (e.g., by FACS or MACS) and is at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, or 94% TGFBR2 negative as measured by flow cytometry relative to a population of unmodified cells. In some embodiments, the population of cells is not enriched (e.g., by FACS or MACS) and is at least 65%, 70%, 80%, 90%, 91%, 92%, 93%, or 94% TGFBR2 negative as measured by flow cytometry relative to a population of unmodified cells. In some embodiments, the population of cells is at least 65% TGFBR2 negative as measured by flow cytometry relative to a population of unmodified cells. In some embodiments, the population of cells is at least 70% TGFBR2 negative as measured by flow cytometry relative to a population of unmodified cells. In some embodiments, the population of cells is at least 80% TGFBR2 negative as measured by flow cytometry relative to a population of unmodified cells. In some embodiments, the population of cells is at least 90% TGFBR2 negative as measured by flow cytometry relative to a population of unmodified cells. In some embodiments, the population of cells is at least 100% TGFBR2 negative as measured by flow cytometry relative to a population of unmodified cells. D. Methods and Compositions for Additional Genetic Modifications

[0271] In some embodiments, multiplex gene editing may be performed in a cell. In some embodiments, the methods comprise reducing or eliminating surface expression of TGFBR2 protein comprising genetically modifying the TGFBR2 gene comprising contacting the cell with a composition comprising a TGFBR2 guide RNA disclosed herein; and optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, the method further comprising contacting with one or more compositions selected from: (a) a guide RNA that directs an RNA-guided DNA binding agent to the TGFBR2 gene; (b) a guide RNA that directs an RNA-guided DNA binding agent to a locus in the genome of the cell other than TGFBR2; and (c) a donor nucleic acid for insertion in the genome of the cell.

[0272] In some embodiments, an engineered cell which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell is provided, that further has reduced or eliminated surface expression of one or more of MHC class II protein, MHC-Iprotein, TRAC, or TRBC relative to an unmodified cell. In some embodiments, an engineered cell which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell is provided, that further has reduced or eliminated surface expression of HLA-A relative to an unmodified cell. In some embodiments, an engineered cell which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell is provided, further has reduced or eliminated surface expression of HLA-A relative to an unmodified cell and one or more of MHC class II protein, TRAC, or TRBC relative to an unmodified cell. Such methods and compositions for reduced or eliminated surface expression of one or more of MHC class II protein, MHC-I protein, TRAC, or TRBC are further described in e.g., International Publication Nos. WO 2020 / 081613, WO 2022 / 125982, WO 2022 / 140586, and WO 2022 / 140587, and International Application Nos. PCT / US2023 / 068498 and PCT / US2023 / 068499, the contents of each of which are hereby incorporated in their entireties. For example, further detailed description of the guide RNAs for reducing or eliminating the expression of TRBC and / or TRAC proteins and for genetic modifications of TRBC and / or TRAC are provided in International Publication No. WO 2020 / 081613, the entire contents of which are incorporated herein by reference. For example, further detailed description of the guide RNAs for reducing or eliminating the expression of HLA-A and / or CIITA proteins and for genetic modifications of HLA-A and / or CIITA are provided in International Publication No. WO 2022 / 125982, the entire contents of which are incorporated herein by reference. For example, further detailed description of the guide RNAs for reducing or eliminating the expression of HLA-A protein and for genetic modifications of HLA-A are provided in International Publication No. WO 2022 / 140586, the entire contents of which are incorporated herein by reference. For example, further detailed description of the guide RNAs for reducing or eliminating the expression of HLA-A and / or CIITA proteins and for genetic modifications of HLA-A and / or CIITA are provided in International Publication No. WO 2022 / 140587, the entire contents of which are incorporated herein by reference. For example, further detailed description of the guide RNAs for reducing or eliminating the expression of HLA-A and / or HLA-B proteins and for genetic modifications of HLA-A and / or HLA-B are provided in International Application No. PCT / US2023 / 068498, the entire contents of which are incorporated herein by reference. For example, further detailed description of the guide RNAs for reducing or eliminating the expression of HLA-A, TRAC, TRBC, and / or CIITA proteins and for genetic modifications of HLA-A, TRAC, TRBC, and / or CIITA are provided in International Application No. PCT / US2023 / 068499, the entire contents of which are incorporated herein by reference.

[0273] In some embodiments, an engineered cell is provided which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell, comprising a genetic modification in the TGFBR2 gene, wherein the genetic modification comprises at least one nucleotide within any one of the genomic coordinates shown in Tables 2 and 3, and wherein the engineered cell further comprises a genetic modification in one or more of CIITA, HLA- A, HLA-B, TRAC, or TRBC gene.

[0274] In some embodiments, the methods and compositions comprise reducing or eliminating surface expression of TGFBR2 protein by genetically modifying TGFBR2 with a gene editing system, and inserting an exogenous nucleic acid encoding a targeting receptor, or other polypeptide (expressed on the cell surface or secreted) into the cell by genetic modification.

[0275] In some embodiments, an engineered cell is provided which has reduced or eliminated surface expression TGFBR2 protein relative to an unmodified cell, comprising a genetic modification in the TGFBR2 gene, wherein the genetic modification comprises at least one nucleotide within any one of the genomic coordinates shown in Table 2, and wherein the engineered cell further comprises an exogenous nucleic acid. In some embodiments, the engineered cell comprises an exogenous nucleic acid encoding a targeting receptor that is expressed on the surface of the engineered cell. In some embodiments, the targeting receptor is a CAR or a universal CAR. In some embodiments, the targeting receptor is a TCR. In some embodiments, the targeting receptor is a WT1 TCR. In some embodiments, the targeting receptor is a ligand for the receptor. In some embodiments, the targeting receptor is a hybrid CAR / TCR. In some embodiments, the targeting receptor comprises an antigen recognition domain (e.g., a cancer antigen recognition domain) and a subunit of a TCR. In some embodiments, the targeting receptor is a cytokine receptor. In some embodiments, the targeting receptor is a chemokine receptor. In some embodiments, the targeting receptor is a B cell receptor (BCR). In some embodiments, the engineered cell further comprises an exogenous nucleic acid encoding a CD8 coreceptor that is expressed on the surface of the engineered cell.

[0276] In some embodiments, the engineered cell further comprises an exogenous nucleic acid encoding a polypeptide that is secreted by the engineered cell (i.e., a soluble polypeptide). In some embodiments, the exogenous nucleic acid encodes a therapeutic polypeptide. In some embodiments, the secreted polypeptide is an antibody. In some embodiments, the secreted polypeptide is an enzyme. In some embodiments, the exogenous nucleic acid encodes an antibody encodes a cytokine. In some embodiments, the exogenousnucleic acid encodes a chemokine. In some embodiments, the exogenous nucleic acid encodes a fusion protein.

[0277] In some embodiments, the engineered cell is an allogeneic cell therapy. In some embodiments, the engineered cell is transferred to a recipient that has the same HLA-A allele as the engineered human cell. In some embodiments, the engineered cell is transferred to a recipient that has the same HLA-C allele as the engineered human cell. In some embodiments, the engineered cell is transferred to a recipient that has the same HLA-A and HLA-C alleles as the engineered human cell. Thus, the engineered cells disclosed herein provide a partial HLA match to a recipient, thereby reducing the risk of an adverse immune response. In some embodiments, an engineered cell which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell is provided, that further has reduced or eliminated surface expression of MHC class II protein. In some embodiments, the engineered cell has a genetic modification in a gene that reduces or eliminates surface expression of MHC class II protein.

[0278] In some embodiments, methods for reducing or eliminating surface expression of TGFBR2 by genetically modifying TGFBR2 as disclosed herein are provided, wherein the methods and compositions further provide for reducing or eliminating surface expression of MHC class II protein relative to an unmodified cell. In some embodiments, MHC class II protein expression is reduced or eliminated by contacting the cell with a CIITA guide RNA.

[0279] MHC class II expression is impacted by a variety of proteins. In some embodiments, MHC class II protein expression is reduced or eliminated by genetically modifying a gene selected from: CIITA, HLA-DR, HLA-DQ, HLA-DP, RFX5, RFXB / ANK, RFXAP, CREB, NF-YA, NF-YB, and NF-YC. In some embodiments, MHC class II protein expression is reduced or eliminated by genetically modifying the CIITA gene.

[0280] In some embodiments, the engineered cell has a genetic modification in the CIITA gene. In some embodiments, the engineered cell has a genetic modification in the HLA-DR gene. In some embodiments, the engineered cell has a genetic modification in the HLA-DQ gene. In some embodiments, the engineered cell has a genetic modification in the HLA-DP gene. In some embodiments, the engineered cell has a genetic modification in the RFX gene. In some embodiments, the engineered cell has a genetic modification in the CREB gene. In some embodiments, the engineered cell has a genetic modification in the Nuclear Factor (NF)-gamma gene.

[0281] In some embodiments, methods are provided for making an engineered cell which has reduced or eliminated expression of TGFBR2 protein relative to an unmodified cell, furthercomprising reducing or eliminating the surface expression of MHC class II protein in the cell relative to an unmodified cell. In some embodiments, the methods comprise contacting the cell with a CIITA guide RNA.

[0282] In some embodiments, an engineered cell which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell is provided, that further has reduced or eliminated surface expression of TRAC protein. In some embodiments, an engineered cell which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell is provided, that further has reduced or eliminated surface expression of TRBC protein.

[0283] In some embodiments, an engineered cell which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell is provided, that further has reduced or eliminated surface expression of HLA-A protein. In some embodiments, an engineered cell which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell is provided, that further has reduced or eliminated surface expression of HLA-B protein. In some embodiments, an engineered cell which has reduced or eliminated surface expression of TGFBR2 protein relative to an unmodified cell is provided, that further has reduced or eliminated surface expression of HLA-A protein and HLA-B protein.

[0284] In some embodiments, the engineered cells comprise a genetic modification in one or more of the HLA-A, HLA-B, TRAC, TRBC, or CIITA genes. In some embodiments, the genetic modification in the HLA-A gene is within the HLA-A target genomic coordinates shown in Tables 10A-10B (such as chr6:29942891-29942915, chr6:29942609-29942633, or chr6:29942864-29942884). In some embodiments, the genetic modification in the HLA-B gene is within the HLA-B target genomic coordinates shown in Tables 10A-10B (such as chr6:31355222-31355246, chr6:31355221-31355245, or chr6:31355205-31355229). In some embodiments, the genetic modification in the TRAC gene is within the TRAC target genomic coordinates shown in Tables 10A-10B (such as chr14:22550574-22550598, chr14:22550544- 22550568, or chr14:22547524- 22547544). In some embodiments, the genetic modification in the CIITA gene is within the CIITA target genomic coordinates shown in Tables 10A-10B (such as chr16:10907504-10907528, chr16:10906643-10906667, or chr16:10906853- 10906873). In some embodiments, the genetic modification in the TRBC gene is within the TRBC target genomic coordinates shown in Tables 10A-10B (such as chr7:142792690- 142792714 or chr7:142792047-142792067).

[0285] In some embodiments, in any of the methods and compositions disclosed herein, the HLA-A guide RNA is an HLA-A guide RNA that comprises a guide sequence disclosed in Tables 10A-10B, such as a guide sequence selected from SEQ ID NOs: 403, 404, and 412. In some embodiments, in any of the methods and compositions disclosed herein, the HLA-B guide RNA is an HLA-B guide RNA that that comprises a guide sequence disclosed in Tables 10A-10B, such as a guide sequence selected from SEQ ID NOs: 405-407. In some embodiments, in any of the methods and compositions disclosed herein, the TRAC guide RNA is a TRAC guide RNA that comprises a guide sequence disclosed in Tables 10A-10B, such as a guide sequence selected from SEQ ID NOs: 408, 409, and 413. In some embodiments, in any of the methods and compositions disclosed herein, the CIITA guide RNA is a CIITA guide RNA that that comprises a guide sequence disclosed in Tables 10A- 10B, such as a guide sequence selected from SEQ ID NOs: 401, 402, and 411. In some embodiments, in any of the methods and compositions disclosed herein, the TRBC guide RNA is a TRBC guide RNA that that comprises a guide sequence disclosed in Tables 10A- 10B, such as a guide sequence selected from SEQ ID NOs: 410 or 414.

[0286] In some embodiments, in any of the methods and compositions disclosed herein, the HLA-A guide RNA disclosed herein is a single guide RNA comprising the sequence disclosed in Tables 10A-10B. In some embodiments, in any of the methods and compositions disclosed herein, the HLA-B guide RNA disclosed herein is a single guide RNA comprising the sequence disclosed in Tables 10A-10B. In some embodiments, in any of the methods and compositions disclosed herein, the CIITA guide RNA disclosed herein is a single guide RNA comprising the sequence disclosed in Tables 10A-10B. In some embodiments, in any of the methods and compositions disclosed herein, the TRAC guide RNA disclosed herein is a single guide RNA comprising the sequence disclosed in Tables 10A-10B. In some embodiments, in any of the methods and compositions disclosed herein, the TRBC guide RNA disclosed herein is a single guide RNA comprising the sequence disclosed in Tables 10A-10B. E. Exogenous nucleic acids knock in

[0287] In some embodiments, the present disclosure provides methods and compositions for reducing or eliminating surface expression of TGFBR2 protein by genetically modifying TGFBR2 as disclosed herein, wherein the methods and compositions further provide for expression of a protein encoded by an exogenous nucleic acid (e.g., an antibody, chimeric antigen receptor (CAR), T cell receptor (TCR), cytokine or cytokine receptor, chemokine orchemokine receptor, enzyme, fusion protein, or other type of cell surface bound or soluble polypeptide). In some embodiments, the exogenous nucleic acid encodes a protein that is expressed on the cell surface. For example, in some embodiments, the exogenous nucleic acid encodes a targeting receptor expressed on the cell surface (described further herein). In some embodiments, the genetically modified cell may function as a “cell factory” for the expression of a secreted polypeptide encoded by an exogenous nucleic acid, including e.g., as a source for continuous production of a polypeptide in vivo (as described further herein). In some embodiments, the cell is an allogeneic cell.

[0288] In some embodiments, the methods comprise reducing surface expression of TGFBR2 protein comprising genetically modifying the TGFBR2 gene comprising contacting the cell with a composition comprising a TGFBR2 guide RNA disclosed herein, the method further comprising contacting the cell with an exogenous nucleic acid.

[0289] In some embodiments, the methods comprise reducing or eliminating surface expression of TGFBR2 protein, comprising genetically modifying the cell with one or more compositions comprising a TGFBR2 guide RNA as disclosed herein, an exogenous nucleic acid encoding a polypeptide (e.g., a targeting receptor), and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.

[0290] In some embodiments, the methods comprise reducing or eliminating surface expression of TGFBR2 protein and MHC class II protein, comprising genetically modifying the cell with one or more compositions comprising a TGFBR2 guide RNA as disclosed herein, a CIITA guide RNA, an exogenous nucleic acid encoding a polypeptide (e.g., a targeting receptor), and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.

[0291] In some embodiments, the methods comprise reducing or eliminating surface expression of TGFBR2 protein, one or more of HLA-A, HLA-B, CIITA, or TRAC protein, comprising genetically modifying the cell with one or more compositions comprising a TGFBR2 guide RNA as disclosed herein, at least one guide RNA that targets one of HLA-A, HLA-B, CIITA, or TRAC protein, an exogenous nucleic acid encoding a polypeptide (e.g., a targeting receptor), and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.

[0292] In some embodiments, the exogenous nucleic acid encodes a polypeptide that is expressed on the surface of the cell. In some embodiments, the exogenous nucleic acid encodes a soluble polypeptide. As used herein, “soluble” polypeptide refers to a polypeptide that is secreted by the cell. In some embodiments, the soluble polypeptide is a therapeuticpolypeptide. In some embodiments, the soluble polypeptide is an antibody. In some embodiments, the soluble polypeptide is an enzyme. In some embodiments, the soluble polypeptide is a cytokine. In some embodiments, the soluble polypeptide is a chemokine. In some embodiments, the soluble polypeptide is a fusion protein.

[0293] In some embodiments, the exogenous nucleic acid encodes an antibody. In some embodiments, the exogenous nucleic acid encodes an antibody fragment (e.g., Fab, Fab2). In some embodiments, the exogenous nucleic acid encodes is a full-length antibody. In some embodiments, the exogenous nucleic acid encodes is a single-chain antibody (e.g., scFv). In some embodiments, the antibody is an IgG, IgM, IgD, IgA, or IgE. In some embodiments, the antibody is an IgG antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the heavy chain constant region contains mutations known to reduce effector functions. In some embodiments, the heavy chain constant region contains mutations known to enhance effector functions. In some embodiments, the antibody is a bispecific antibody. In some embodiments, the antibody is a single-domain antibody (e.g., VH domain-only antibody).

[0294] In some embodiments, the exogenous nucleic acid encodes a neutralizing antibody. A neutralizing antibody neutralizes the activity of its target antigen. In some embodiments, the antibody is a neutralizing antibody against a virus antigen. In some embodiments, the antibody neutralizes a target viral antigen, blocking the ability of the virus to infect a cell. In some embodiments, a cell-based neutralization assay may be used to measure the neutralizing activity of an antibody. The particular cells and readout will depend on the target antigen of the neutralizing antibody. The half maximal effective concentration (EC50) of the antibody can be measured in a cell-based neutralization assay, wherein a lower EC50 is indicative of more potent neutralizing antibody.

[0295] In some embodiments, the exogenous nucleic acid encodes an antibody that binds to an antigen associated with a disease or disorder (see e.g., diseases and disorders described in Section IV).

[0296] In some embodiments, the exogenous nucleic acid encodes a polypeptide that is expressed on the surface of the cell (i.e., a cell surface bound protein). In some embodiments, the exogenous nucleic acid encodes a targeting receptor. A “targeting receptor” is a receptor present on the surface of a cell, e.g., a T cell, to permit binding of the cell to a target site, e.g., a specific cell or tissue in an organism. In some embodiments, the targeting receptor is a CAR. In some embodiments, the targeting receptor is a universal CAR (UniCAR). In some embodiments, the targeting receptor is a proliferation-inducing ligand (APRIL). In someembodiments, the targeting receptor is a TCR. In some embodiments, the targeting receptor is a TCR, and the cell further comprises and / or is engineered with an exogenous nucleic acid encoding a CD8 coreceptor that is expressed on the surface of the cell. In some embodiments, the CD8 coreceptor comprises the amino acid sequence of SEQ ID NO: 1005. In some embodiments, the CD8 coreceptor comprises the amino acid sequence of SEQ ID NO: 1007. In some embodiments, the CD8 coreceptor comprises the amino acid sequences of SEQ ID NOs: 1005 and 1007. In some embodiments, the exogenous nucleic acid encoding the CD8 coreceptor comprises the nucleic acid sequence of SEQ ID NO: 1004. In some embodiments, the exogenous nucleic acid encoding the CD8 coreceptor comprises the nucleic acid sequence of SEQ ID NO: 1006. In some embodiments, the exogenous nucleic acid encoding the CD8 coreceptor comprises the nucleic acid sequences of SEQ ID NOs: 1004 and 1006. In some embodiments, the targeting receptor is a TRuC. In some embodiments, the targeting receptor is a B cell receptor (BCR) (e.g., expressed on a B cell). In some embodiments, the targeting receptor is chemokine receptor. In some embodiments, the targeting receptor is a cytokine receptor.

[0297] In some embodiments, targeting receptors include a chimeric antigen receptor (CAR), a T-cell receptor (TCR), and a receptor for a cell surface molecule operably linked through at least a transmembrane domain in an internal signaling domain capable of activating a T cell upon binding of the extracellular receptor portion. In some embodiments, a CAR refers to an extracellular antigen recognition domain, e.g., an scFv, VHH, nanobody; operably linked to an intracellular signaling domain, which activates the T cell when an antigen is bound. CARs are composed of four regions: an antigen recognition domain, an extracellular hinge region, a transmembrane domain, and an intracellular T-cell signaling domain. Such receptors are well known in the art (see, e.g., WO2020092057, WO2019191114, WO2019147805, WO2018208837). A universal CAR (UniCAR) for recognizing various antigens (see, e.g., EP 2990416 A1) and a reversed universal CAR (RevCAR) that promotes binding of an immune cell to a target cell through an adaptor molecule (see, e.g., WO2019238722) are also contemplated. CARs can be targeted to any antigen to which an antibody can be developed and are typically directed to molecules displayed on the surface of a cell or tissue to be targeted. In some embodiments, the targeting receptor comprises an antigen recognition domain (e.g., a cancer antigen recognition domain and a subunit of a TCR (e.g., a TRuC). (See Baeuerle et al. Nature Communications 2087 (2019).)

[0298] In some embodiments, the exogenous nucleic acid encodes a TCR. In some embodiments, the exogenous nucleic acid encodes a genetically modified TCR. In someembodiments, the exogenous nucleic acid encodes is a genetically modified TCR with specificity for a polypeptide expressed by cancer cells. In some embodiments, the exogenous nucleic acid encodes a targeting receptor specific for Wilms’ tumor gene (WT1) antigen. In some embodiments, the exogenous nucleic acid encodes the WT1-specific TCR (see e.g., WO2020 / 081613A1). In some embodiments, the WT1-specific TCR comprises the amino acid sequence of SEQ ID NO: 1003. In some embodiments, the exogenous nucleic acid encoding the WT1-specific TCR comprises the nucleic acid sequence of SEQ ID NO: 1002.

[0299] In some embodiments, an exogenous nucleic acid is inserted into the genome of the target cell. In some embodiments, the exogenous nucleic acid is integrated into the genome of the target cell. In some embodiments, the exogenous nucleic acid is integrated into the genome of the target cell by homologous recombination (HR). In some embodiments, the exogenous nucleic acid is integrated into the genome of the target cell by blunt end insertion. In some embodiments, the exogenous nucleic acid is integrated into the genome of the target cell by non-homologous end joining. In some embodiments, the exogenous nucleic acid is integrated into a safe harbor locus in the genome of the cell. In some embodiments, the exogenous nucleic acid is integrated into one of the TRAC locus, B2M locus, AAVS1 locus, or CIITA locus. In some embodiments, the lipid nucleic acid assembly composition is a lipid nanoparticle (LNP).

[0300] In some embodiments, the methods produce a composition comprising an engineered cell having reduced or eliminated surface expression of TGFBR2 protein and comprising an exogenous nucleic acid. In some embodiments, the methods produce a composition comprising an engineered cell having reduced or eliminated surface expression of TGFBR2 protein and that secretes or expresses a polypeptide encoded by an exogenous nucleic acid integrated into the genome of the cell. In some embodiments, the methods produce a composition comprising an engineered cell having reduced or eliminated surface expression of TGFBR2 protein, or reduced or eliminated TGFBR2 levels in the cell nucleus, and having reduced or eliminated surface expression of MHC class II protein expression, and secreting or expressing a polypeptide encoded by an exogenous nucleic acid integrated into the genome of the cell. In some embodiments, the engineered cell elicits a reduced response from CD4+ T cells, or CD8+ T cells.

[0301] In some embodiments, an allogeneic cell is provided wherein the cell has reduced or eliminated surface expression of MHC class II and TGFBR2 protein, wherein the cell comprises a modification in the TGFBR2 gene as disclosed herein, wherein the cellcomprises a modification in the CIITA gene, and wherein the cell further comprises an exogenous nucleic acid encoding a polypeptide (e.g., a targeting receptor).

[0302] In some embodiments, the present disclosure provides methods for reducing or eliminating surface expression of TGFBR2 protein by genetically modifying TGFBR2 as disclosed herein, wherein the methods further provide for reducing expression of one or more additional target genes (e.g., HLA-A, HLA-B, CIITA, TRAC, TRBC). In some embodiments, the additional genetic modifications provide further advantages for use of the genetically modified cells for adoptive cell transfer applications.

[0303] In some embodiments, the methods comprise reducing or eliminating surface expression of TGFBR2 protein, comprising genetically modifying the cell with one or more compositions comprising a TGFBR2 guide RNA as disclosed herein, a CIITA guide RNA, an exogenous nucleic acid encoding polypeptide (e.g., a targeting receptor), a guide RNA that directs an RNA-guided DNA binding agent to a target sequence located in an another gene, thereby reducing or eliminating expression of the other gene, and an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent. In some embodiments, the additional target gene is TRAC. In some embodiments, the additional target gene is TRBC.

[0304] In some embodiments, the method disclosed herein further comprises contacting the cell with a DNA-dependent protein kinase inhibitor (DNAPKi), optionally wherein the DNAPKi is Compound 1 or “DNAPKI Compound 1”: 9-(4,4-difluorocyclohexyl)-7-methyl- 2-((7-methyl-[1,2,4]triazolo[1,5-a]pyridin-6-yl)amino)-7,9-dihydro-8H-purin-8-one, also. F. Exemplary Genome Editing Systems

[0305] Various suitable gene editing systems may be used to make the engineered cells disclosed herein, including but not limited to the CRISPR / Cas system; zinc finger nuclease (ZFN) system; and the transcription activator-like effector nuclease (TALEN)system. Generally, the gene editing systems involve the use of engineered cleavage systems to induce a double strand break (DSB) or a nick (e.g., a single strand break, or SSB) in a target DNA sequence. Cleavage or nicking can occur through the use of specific nucleases such as engineered ZFN, TALENs, or using the CRISPR / Cas system with an engineered guide RNA to guide specific cleavage or nicking of a target DNA sequence. Further, targeted nucleases are being developed based on the Argonaute system (e.g., from T. thermophilus, known as ‘TtAgo’, see Swarts et al (2014) Nature 507(7491): 258-261), which also may have the potential for uses in gene editing and gene therapy.

[0306] In some embodiments, the gene editing system is a TALEN system. Transcription activator-like effector nucleases (TALEN) are restriction enzymes that can be engineered to cut specific sequences of DNA. They are made by fusing a TAL effector DNA-binding domain to a DNA cleavage domain (a nuclease which cuts DNA strands). Transcription activator-like effectors (TALEs) can be engineered to bind to a desired DNA sequence, to promote DNA cleavage at specific locations (see, e.g., Boch, 2011, Nature Biotech). The restriction enzymes can be introduced into cells, for use in gene editing or for gene editing in situ, a technique known as gene editing with engineered nucleases. Such methods and compositions for use therein are known in the art. See, e.g., WO2019147805, WO2014040370, WO2018073393, the contents of which are hereby incorporated in their entireties.

[0307] In some embodiments, the gene editing system is a zinc-finger system. Zinc-finger nucleases (ZFNs) are artificial restriction enzymes generated by fusing a zinc finger DNA- binding domain to a DNA-cleavage domain. Zinc finger domains can be engineered to target specific desired DNA sequences to enables zinc-finger nucleases to target unique sequences within complex genomes. The non-specific cleavage domain from the type IIs restriction endonuclease FokI is typically used as the cleavage domain in ZFNs. Cleavage is repaired by endogenous DNA repair machinery, allowing ZFN to precisely alter the genomes of higher organisms. Such methods and compositions for use therein are known in the art. See, e.g., WO2011091324, the contents of which are hereby incorporated in their entireties.

[0308] In some embodiments, the gene editing system is a CRISPR / Cas system, including e.g., a CRISPR guide RNA comprising a guide sequence and a RNA-guided DNA binding agent, and described further herein. In some embodiments the gene editing system comprises a base editor comprising a deaminase and an RNA-guided nickase. In some embodiments the gene editing system comprises a base editor comprising a cytidine deaminase and an RNA- guided nickase. In some embodiments, the gene editing system comprises a DNApolymerase. Further description of the gene editing system methods and compositions for use therein are known in the art. See e.g., WO2019 / 067910, WO2021 / 188840A1, WO2019 / 051097, and PCT / US2021 / 062922 filed December 10, 2021, and US Provisional Application No.63 / 275,425 filed November 3, 2021, the contents of each of which are hereby incorporated in their entireties. Exemplary nucleotide and polypeptide sequences for the genome editing system disclosed herein are provided below in Table 10. Methods for identifying alternate nucleotide sequences encoding polypeptide sequences provided herein, including alternate naturally occurring variants, are known in the art. Sequences with at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% identity to any of the nucleic acid sequences, or nucleic acid sequences encoding the amino acid sequences provided herein are also contemplated. G. CRISPR Guide RNA

[0309] Provided herein are guide sequences useful for modifying a target sequence, e.g., using a guide RNA comprising a disclosed guide sequence with an RNA-guided DNA binding agent (e.g., a CRISPR / Cas system).

[0310] In some aspects, provided herein is a guide RNA comprising: A. a guide sequence comprising a sequence at least 80%, 85%, preferably 90%, or 95% identical to or complementary to at least 20 contiguous nucleotides of any one of the guide sequences of Table 2.

[0311] In some embodiments, the guide RNAs provided herein further comprise one or more of: A. a shortened hairpin 1 region, or a substituted and optionally shortened hairpin 1 region, wherein 1. at least one of the following pairs of nucleotides are substituted in hairpin 1 with Watson-Crick pairing nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, or H1-4 and H1-9, and the hairpin 1 region optionally lacks a. any one or two of H1-5 through H1-8, b. one, two, or three of the following pairs of nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, and H1-4 and H1-9, or c.1-8 nucleotides of hairpin 1 region; or 2. the shortened hairpin 1 region lacks 4-8 nucleotides, preferably 4-6 nucleotides; and a. one or more of positions H1-1, H1-2, or H1-3 is deleted or substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601) or b. one or more of positions H1-6 through H1-10 is substituted relative to Exemplary SpyCas9 sgRNA-1(SEQ ID NO: 601); or 3. the shortened hairpin 1 region lacks 5-10 nucleotides, preferably 5-6 nucleotides, and one or more of positions N18, H1-12, or n is substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601); or B. a shortened upper stem region, wherein the shortened upper stem regionlacks 1-6 nucleotides and wherein the 6, 7, 8, 9, 10, or 11 nucleotides of the shortened upper stem region include less than or equal to 4 substitutions relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601); or C. a substitution relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601) at any one or more of LS6, LS7, US3, US10, B3, N7, N15, N17, H2-2 and H2-14, wherein the substituent nucleotide is neither a pyrimidine that is followed by an adenine, nor an adenine that is preceded by a pyrimidine; or D. an Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601) with an upper stem region, wherein the upper stem modification comprises a modification to any one or more of US1-US12 in the upper stem region.

[0312] In some embodiments, the guide RNA lacks 6 nucleotides in shortened hairpin 1.

[0313] In some embodiments, the guide RNA lacks 8 nucleotides in shortened hairpin 1.

[0314] In some embodiments, H-1 and H-3 are deleted.

[0315] In some embodiments, the guide RNA further comprises a 3’ tail.

[0316] In some embodiments, the 3’ tail is 1-4 nucleotides in length, optionally 1 nucleotide in length.

[0317] In some embodiments, the guide RNA comprises an upper stem region comprising a modification to any one or more of US1-US12 in the upper stem region.

[0318] In some embodiments, the guide RNAs described herein comprise a nucleotide sequence selected from the sequences in Table 3.

[0319] In some embodiments, the guide RNA comprises a modified nucleotide sequence selected from the modified Spy guide scaffold sequences in Table 6, wherein the modified nucleotide sequence is 3’ of the guide sequence.

[0320] In some embodiments, the guide RNAs described herein are modified according to the pattern of a nucleotide sequence selected from the modified Spy guide RNA sequences in Tables 6-7.

[0321] In some embodiments, the guide comprises a nucleotide sequence selected from the unmodified Spy guide RNA Sequences in Table 5, wherein the N20’s are collectively a guide sequence described herein.

[0322] In some embodiments, each nucleotide of the unmodified Spy guide RNA Sequences in Tables 4-5 is any natural or non-natural nucleotide.

[0323] In some embodiments, the guide RNA is modified according to a pattern selected from the modification patterns in Tables 6-7, wherein the (mN*)3N17 refers to the guide sequence described herein in which the first three nucleotides comprises a 2’-O-Me modification and a phosphorothioate linkage.

[0324] In some embodiments, the guide RNAs described herein comprise a sequence or modification pattern set forth in Tables 6-7.

[0325] Guide sequences targeted to sites adjacent to an appropriate PAM, e.g., a Spy Cas9 PAM, e.g., may further comprise additional nucleotides, which can be referred to as a scaffold sequence or a conserved portion, to form a crRNA or a crRNA joined to a trRNA to form a sgRNA e.g., with the following exemplary nucleotide sequence following the guide sequence at its 3’ end (Tables 4 and 6). The #mer refers to the length of the crRNA or the sgRNA when a 20 nucleotide guide sequence is included 5’ to the scaffold sequence provided in Table 4 and 6.

[0326] In some aspects, provided herein is a composition comprising a guide RNA described herein. Table 4: Exemplary Unmodified Spy Scaffold Sequences

[0327] In some embodiments, the guide RNA comprises a nucleotide sequence selected from the unmodified Spy guide RNA Sequences in Table 5, wherein the N20’s are collectively any one of the guide sequences of Tables 2-3. In some embodiments, each nucleotide of the unmodified Spy guide RNA Sequences in Table 5 is any natural or non-natural nucleotide. Table 5: Exemplary Unmodified Spy Guide RNA SequencesWherein the Ns collectively are a guide sequence provided herein.

[0328] In the case of a sgRNA, the guide sequences may be integrated into the following modified guide scaffold motifs (Table 6). The #mer refers to the length of the sgRNA when a 20 nucleotide guide sequence, either a modified or unmodified sequence, is included 5’ to the scaffold sequence provided in Table 6: Table 6: Exemplary Modified Spy Guide Scaffold Sequenceswherein “m” indicates a 2’-O-Me modification, “f” indicates a 2’-fluoro modification, a “*” indicates a phosphorothioate linkage between nucleotides, and no modification in the context of a modified sequence indicates an RNA (2’-OH) and phosphodiesterase linkage.

[0329] A guide sequence is present on the 5’ end of the conserved portion of the guide RNA. In certain embodiments, the guide sequence is 20-25, preferably 22-24 nucleotides in length. In certain embodiments, the guide sequence comprises one or more chemical modifications, for example modifications at one or more of nucleotides 1, 2, and 3, optionally all of nucleotides 1, 2, and 3 at the 5’ end of the guide RNA. In certain embodiments, the modification comprises a 2’-O-Me modification.

[0330] In certain embodiments, the guide sequence is a chemically modified sequence. In certain embodiments, the chemically modified guide sequence is (mN*)3(N)13-17. In certain embodiments, the guide sequence is (mN*)3(N)17, i.e., mN*mN*mN*NNNNNNNNNNNNNNNNN. In certain embodiments, each N of the (N)13-17 or the (N)17 is unmodified. In certain embodiments, the each N in the (N)13-17 or the (N)17 is independently modified, e.g., independently modified with a 2’-O-methyl modification.

[0331] In some embodiments, the sgRNA comprises any of the modification patterns shown herein, where N is any natural or non-natural nucleotide, and wherein the totality of the N’s comprise any one of the guide sequences disclosed in Tables 2-3. In some embodiments, the modified sgRNA comprises a sequence shown in Table 7. Table 7: Exemplary Modified Spy Guide RNA Sequenceswherein “m” indicates a 2’-O-Me modification, “f” indicates a 2’-fluoro modification, and a “*” indicates a phosphorothioate linkage between nucleotides, and no modification in the context of a modified sequence indicates an RNA (2’-OH), wherein the totality of N’s comprise a guide sequence comprising a sequence at least 85%, preferably 90% or 95% identical to or complementary to at least 17, 18, 19, or 20 contiguous nucleotides of any of the guide sequences disclosed herein in Tables 2-3, where the N’s are replaced with any of the guide sequences disclosed herein in Tables 2-3. In certain embodiments, when the totality of N’s comprise a guide sequence, within N17, each N of the N17 may be independently modified, e.g., modified with a 2’-OMe modification.

[0332] In some embodiments, the sgRNA comprises a nucleotide sequence selected from the sequences in Tables 4-7.

[0333] In some embodiments, the guide RNA comprises a modified nucleotide sequence selected from the modified Spy guide scaffold sequences in Table 6, wherein the modified nucleotide sequence is 3’ of the guide sequence or , wherein the guide RNA is modified according to a pattern selected from the modification patterns in Table 7, wherein the (mN*)3N17 refers to the guide sequence 2 in which the first three nucleotides comprises a 2’- O-Me modification and a phosphorothioate linkage..

[0334] In the case of a sgRNA, the guide sequences may further comprise a SpyCas9 sgRNA scaffold sequence. An example of a SpyCas9 sgRNA scaffold sequence is shown in the Table 8 below (SEQ ID NO: 601: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAU AAGGCUAGUC CGUUAUCAACUUGAAAAAGU GGCACCGAGUCGGUGC – “Exemplary SpyCas9 sgRNA-1”), included at the 3’ end of the guide sequence, and provided with the domains as shown in the table below. LS is lower stem. B is bulge. US is upperstem. H1 and H2 are hairpin 1 and hairpin 2, respectively. Collectively H1 and H2 are referred to as the hairpin region. A model of the structure containing both a guide sequence and a scaffold sequence is provided in Figure 10A of WO2019237069, which is incorporated herein by reference. ^

[0335] The nucleotide sequence of Exemplary SpyCas9 sgRNA-1 may serve as a template sequence for specific chemical modifications, sequence substitutions and truncations.

[0336] In certain embodiments, the gRNA is an sgRNA or a dgRNA, for example, and it optionally comprises a chemical modification. In some embodiments, the modified sgRNA comprises a guide sequence and a SpyCas9 sgRNA sequence, e.g., Exemplary SpyCas9 sgRNA-1. A gRNA, such as an sgRNA, may include modifications on the 5’ end of the guide sequence or on the 3’ end of the SpyCas9 sgRNA sequence, such as, e.g., Exemplary SpyCas9 sgRNA-1 at one or more of the terminal nucleotides, e.g., at 1, 2, 3, or 4 of the nucleotides at the 3’ end or at the 5’ end. In certain embodiments, the modified nucleotide is selected from a 2’-O-methyl (2’-OMe) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O- moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, and an inverted abasic modified nucleotide; or is a combination thereof. In certain embodiments, the modified nucleotide includes a 2’-OMe modified nucleotide. In certain embodiments, the modified nucleotide includes a PS linkage. In certain embodiments, the modified nucleotide includes a 2’-OMe modified nucleotide and a PS linkage.

[0337] In certain embodiments, using SEQ ID NO: 601 GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC UUGAAAAAGUGGCACCGAGUCGGUGC “Exemplary SpyCas9 sgRNA-1,” see WO2019237069, the contents of which are incorporated herein by reference). The portions of the Exemplary SpyCas9 sgRNA-1 and position numbering scheme are set forth in Table 8 below.

[0338] As an example, the Exemplary SpyCas9 sgRNA-1 further includes one or more of: A. a shortened hairpin 1 region, or a substituted and optionally shortened hairpin 1 region, wherein 1. at least one of the following pairs of nucleotides are substituted in hairpin 1 with Watson-Crick pairing nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, or H1-4 and H1-9, and the hairpin 1 region optionally lacks a. any one or two of H1-5 through H1-8,b. one, two, or three of the following pairs of nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, and H1-4 and H1-9, or c. 1-8 nucleotides of hairpin 1 region; or 2. the shortened hairpin 1 region lacks 4-8 nucleotides, preferably 4-6 nucleotides; and a. one or more of positions H1-1, H1-2, or H1-3 is deleted or substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601) or b. one or more of positions H1-6 through H1-10 is substituted relative to Exemplary SpyCas9 sgRNA-1(SEQ ID NO: 601); or 3. the shortened hairpin 1 region lacks 5-10 nucleotides, preferably 5-6 nucleotides, and one or more of positions N18, H1-12, or n is substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601); or B. a shortened upper stem region, wherein the shortened upper stem region lacks 1-6 nucleotides and wherein the 6, 7, 8, 9, 10, or 11 nucleotides of the shortened upper stem region include less than or equal to 4 substitutions relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601); or C. a substitution relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601) at any one or more of LS6, LS7, US3, US10, B3, N7, N15, N17, H2-2 and H2-14, wherein the substituent nucleotide is neither a pyrimidine that is followed by an adenine, nor an adenine that is preceded by a pyrimidine; or D. an Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601) with an upper stem region, wherein the upper stem modification comprises a modification to any one or more of US1-US12 in the upper stem region, wherein 1. the modified nucleotide is optionally selected from a 2’-O-methyl (2’- OMe) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, and an inverted abasic modified nucleotide, or is optionally a combination thereof; or 2. the modified nucleotide optionally includes a 2’-OMe modified nucleotide.

[0339] In certain embodiments, Exemplary SpyCas9 sgRNA-1, or an sgRNA, such as an sgRNA comprising an Exemplary SpyCas9 sgRNA-1, further includes a 3’ tail, e.g., a 3’ tailof 1, 2, 3, 4, or more nucleotides. In certain embodiments, the tail includes one or more modified nucleotides. In certain embodiments, the modified nucleotide is selected from a 2’- O-methyl (2’-OMe) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a 2’ deoxy (2’H-) modified nucleotide, an abasic nucleotide, a locked nucleic acid (LNA) nucleotide, an unlocked nucleic acid (UNA) nucleotide, a phosphorothioate (PS) linkage between nucleotides, and a terminal inverted abasic modified nucleotide; or is a combination thereof. In certain embodiments, the modified nucleotide includes a 2’-OMe modified nucleotide. In certain embodiments, the modified nucleotide includes a PS linkage between nucleotides. In certain embodiments, the modified nucleotide includes a 2’-OMe modified nucleotide and a PS linkage between nucleotides.

[0340] In certain embodiments, the hairpin region includes one or more modified nucleotides. In certain embodiments, the modified nucleotide is selected from a 2’-O-methyl (2’-OMe) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, and an inverted abasic modified nucleotide; or is a combination thereof. In certain embodiments, the modified nucleotide includes a 2’-OMe modified nucleotide.

[0341] In certain embodiments, the upper stem region includes one or more modified nucleotides. In certain embodiments, the modified nucleotide is selected from a 2’-O-methyl (2’-OMe) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, and an inverted abasic modified nucleotide; or is a combination thereof. In certain embodiments, the modified nucleotide includes a 2’-OMe modified nucleotide.

[0342] In certain embodiments, the Exemplary SpyCas9 sgRNA-1 comprises one or more YA dinucleotides, wherein Y is a pyrimidine, wherein the YA dinucleotide includes a modified nucleotide. In certain embodiments, the modified nucleotide selected from a 2’-O- methyl (2’-OMe) modified nucleotide, a 2’-O-(2-methoxyethyl) (2’-O-moe) modified nucleotide, a 2’-fluoro (2’-F) modified nucleotide, a phosphorothioate (PS) linkage between nucleotides, and an inverted abasic modified nucleotide, or is a combination thereof. In certain embodiments, the modified nucleotide includes a 2’-OMe modified nucleotide.

[0343] In certain embodiments, the Exemplary SpyCas9 sgRNA-1 comprises one or more YA dinucleotides, wherein Y is a pyrimidine, wherein the YA dinucleotide includes a sequence substituted nucleotide, wherein the pyrimidine is substituted for a purine. In certain embodiments, when the pyrimidine forms a Watson-Crick base pair in the single guide, theWatson-Crick based nucleotide of the sequence substituted pyrimidine nucleotide is substituted to maintain Watson-Crick base pairing.Linker containing gRNAs

[0344] In certain embodiments, the gRNA comprises one or more internal linkers. As used herein, “internal linker” describes a non-nucleotide segment joining two nucleotides within a guide RNA. If the gRNA contains a spacer region, the internal linker is located outside of the spacer region (e.g., in the scaffold or conserved region of the gRNA). For Type V guides, it is understood that the last hairpin is the only hairpin in the structure, i.e., the repeat-anti-repeat region. The length of an internal linker may be dependent on, for example, the number of nucleotides replaced by the linker and the position of the linker in the gRNA. Internal linkers and their use in the context of gRNA are provided in WO2022261292.

[0345] gRNAs disclosed herein may comprise an internal linker. In general, any internal linker compatible with the function of the gRNA may be used. It may be desirable for the linker to have a degree of flexibility. In some embodiments, the internal linker comprises at least two, three, four, five, six, or more on-pathway single bonds. A bond is on-pathway if it is part of the shortest path of bonds between the two nucleotides whose 5’ and 3’ positions are connected to the linker.

[0346] As used herein the length of the internal linker can be defined by its bridging length. The “bridging length” of an internal linker as used herein refers to the distance or number of atoms in the shortest chain of atoms on the pathway from the first atom of the linker (bound to a 3’ substituent, such as an oxygen or phosphate, of the preceding nucleotide to the last atom of the linker (bound to a 5’ substituent, such as an oxygen or phosphate) of the following nucleotide) (e.g., from ~ to # in the structure of Formula (I) described below). Approximate predicted bridging lengths for various linkers are provided in a table below.

[0347] Exemplary predicted linker lengths by number of atoms, number of ethylene glycol units, approximate linker length in Angstroms on the assumption that an ethylene glycol monomer is about 3.7 Angstroms, and suitable location for substitution of at least the entire loop portion of a hairpin structure are provided in the table 8 below. Substitution of two nucleotides requires a linker length of at least about 11 Angstroms. Substitution of at least 3 nucleotides requires a linker length of at least about 16 Angstroms.Table 9A

[0348] In some embodiments, the internal linker comprises a structure of formula (I): ~-L0-L1-L2-# (I) wherein: ~ indicates a bond to a 3’ substituent of the preceding nucleotide; # indicates a bond to a 5’ substituent of the following nucleotide; L0 is null or C1-3 aliphatic; L1 is –[E1-(R1)]m-, where each R1is independently a C1-5aliphatic group, optionally substituted with 1 or 2 E2, each E1and E2are independently a hydrogen bond acceptor, or are each independently chosen from cyclic hydrocarbons, and heterocyclic hydrocarbons, and each m is 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10; and L2 is null, C1-3aliphatic, or is a hydrogen bond acceptor.

[0349] In some embodiments, L1 comprises one or more -CH2CH2O-, -CH2OCH2-, or -OCH2CH2- units (“ethylene glycol subunits”). In some embodiments, thenumber of -CH2CH2O-, -CH2OCH2-, or -OCH2CH2- units is in the range of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.

[0350] In some embodiments, m is 1, 2, 3, 4 or 5. In some embodiments, m is 1, 2, or 3. In some embodiments, m is 6, 7, 8, 9, or 10.

[0351] In some embodiments, L0 is null. In some embodiments, L0 is -CH2- or -CH2CH2-.

[0352] In some embodiments, L2 is null. In some embodiments, L2 is -O-, -S-, or C1-3aliphatic. In some embodiments, L2 is -O-. In some embodiments, L2 is -S-. In some embodiments, L2 is -CH2- or -CH2CH2-.

[0353] In the tables herein, L1 and L2, are optionally, C9 and C18, respectively as follows:

[0354] In certain embodiments, the internal linker has a bridging length of about 3-30 atoms, optionally 12-21 atoms, and the linker substitutes for at least 2 nucleotides of the gRNA. In certain embodiments, the internal linker has a bridging length of about 6-18 atoms, optionally about 6-12 atoms, and the linker substitutes for at least 2 nucleotides of the gRNA. In certain embodiments, the internal linker substitutes for 2-12 nucleotides.

[0355] In some embodiments, the guide RNA comprises a nucleic acid sequence of SEQ ID NO: 601, including modifications disclosed elsewhere herein. Table 9B shows various embodiments of the gRNA structures and species with possible number of internal linkers and positions..

[0356] In certain embodiments, the internal linker is in a repeat-anti-repeat region of the gRNA. In certain embodiments, the internal linker substitutes for at least 4 nucleotides of the repeat-anti-repeat region of the gRNA. In certain embodiments, the internal linker substitutes for the loop in the repeat-anti-repeat region of a Spy Cas9 gRNA, corresponding to nucleotides 13-16 in SEQ ID NO: 601.

[0357] In certain embodiments, the internal linker substitutes for 2, 3, or 4 nucleotides of the nexus region of the gRNA. In certain embodiments, the internal linker substitutes for the loop in the nexus region of a Spy Cas9 gRNA corresponding to nucleotides 33-36 of SEQ ID NO: 601.

[0358] In certain embodiments, the internal linker is in a hairpin region of the gRNA. In certain embodiments, the internal linker substitutes for at least 4 nucleotides of the hairpin region of the gRNA. In certain embodiments, the internal linker substitutes for the loop in the hairpin 1 region of a Spy Cas9 gRNA, corresponding to nucleotides 53-56 in SEQ ID NO: 601. Table 9C. Exemplary SpyCas9 guide RNAs comprising linkersNucleotide modifications in modified sequences are indicated in Table 9C as follows: wherein “m” indicates a 2’-O-Me modification, a “*” indicates a phosphorothioate linkage between nucleotides, and within the individually indicated nucleotides, no modification indicates an RNA (2’-OH) with a phosphodiesterase backbone, a “(dS)” indicates an abasic site having 1’,2’-dideoxyribose modification (e.g., dSpacer from IDT).

[0359] In some embodiments, a composition comprising one or more guide RNAs comprising a guide sequence of any one in Tables 2-3 is provided. In some embodiments, a composition comprising one or more guide RNAs comprising a guide sequence of any one in Tables 2-3 is provided, wherein the nucleotides of SEQ ID: 617 follow the guide sequence at its 3’ end. In some embodiments, the one or more guide RNAs comprising a guide sequence of any one in Tables 2-3, wherein the nucleotides of SEQ ID NO: 617 follow the guide sequence at its 3’ end, is modified according to the modification pattern of any one of the sequences shown in Table 6 (e.g., SEQ ID NO: 641). In some embodiments, the one or more guide RNAs comprising a guide sequence of any one in Tables 2-3, wherein the nucleotidesof SEQ ID NO: 601 follow the guide sequence at its 3’ end, is modified according to the modification pattern of any one of the sequences shown in Table 7 (e.g., SEQ ID NO: 658).

[0360] In some embodiments, an sgRNA comprising the guide sequence of any one listed in Tables 2-3 and any conserved portion of an sgRNA shown in Tables 4-7, optionally having a modification pattern of any of an sgRNA shown in Tables 6-7, optionally wherein the sgRNA comprises a 5’ and 3’ end modification (if not already shown in the construct of Table 7) is provided.

[0361] In some embodiments, the sgRNA comprises any of the modification patterns shown below in Table 7, where N is any natural or non-natural nucleotide, and wherein the totality of the N’s comprise a guide sequence as described herein in Table 2. Table 7 does not depict the guide sequence portion of the sgRNA. The modifications remain as shown in Table 7 despite the substitution of N’s for the nucleotides of a guide sequence. That is, although the nucleotides of the guide replace the “N’s”, the nucleotides are modified as shown in Table 7.

[0362] In some embodiments, a composition comprising one or more guide RNAs comprising a guide sequence of any one in Tables 2-3 is provided. In one aspect, a composition comprising one or more gRNAs is provided, comprising a guide sequence that is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the nucleic acids of any one in Tables 2-3.

[0363] In other embodiments, a composition is provided that comprises at least one, e.g., at least two gRNA’s comprising guide sequences selected from any two or more of the guide sequences shown in any one in Tables 2-3. In some embodiments, the composition comprises at least two gRNA’s that each comprise a guide sequence at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, or 90% identical to any of the guide sequences shown in any one in Tables 2-3.

[0364] In some embodiments, the guide RNA compositions of the present disclosure are designed to recognize (e.g., hybridize to) a target sequence. For example, the target sequence may be recognized and cleaved by a provided Cas cleavase comprising a guide RNA. In some embodiments, an RNA-guided DNA binding agent, such as a Cas cleavase, may be directed by a guide RNA to a target sequence, where the guide sequence of the guide RNA hybridizes with the target sequence and the RNA-guided DNA binding agent, such as a Cas cleavase, cleaves the target sequence.

[0365] In some embodiments, the selection of the one or more guide RNAs is determined based on target sequences within the target gene. In some embodiments, the compositions comprising one or more guide sequences comprise a guide sequence that is complementary tothe corresponding genomic region shown in Table 2, according to coordinates from human reference genome hg38. Guide sequences of further embodiments may be complementary to sequences in the close vicinity of any one of the genomic coordinate listed in Table 2 within the target gene. For example, guide sequences of further embodiments may be complementary to sequences that comprise 10 contiguous nucleotides ± 10 nucleotides of a genomic coordinate listed in Table 2. Without being bound by any particular theory, modifications (e.g., frameshift mutations resulting from indels occurring as a result of a nuclease-mediated DSB) in certain regions of the target gene may be less tolerable than mutations in other regions, thus the location of a DSB is an important factor in the amount or type of protein knockdown that may result. In some embodiments, a gRNA complementary or having complementarity to a target sequence within the target gene used to direct an RNA- guided DNA binding agent to a particular location in the target gene.

[0366] In some embodiments, the guide sequence is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, or 80% identical to a target sequence present in the target gene. In some embodiments, the guide sequence is at least 99%, 98%, 97%, 96%, 95%, 94%, 93%, 92%, 91%, 90%, 85%, or 80% identical to a target sequence present in the human target gene.

[0367] In some embodiments, the target sequence may be complementary to the guide sequence of the guide RNA. In some embodiments, the degree of complementarity or identity between a guide sequence of a guide RNA and its corresponding target sequence may be at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the target sequence and the guide sequence of the gRNA may be 100% complementary or identical. In other embodiments, the target sequence and the guide sequence of the gRNA may contain at least one mismatch. For example, the target sequence and the guide sequence of the gRNA may contain 1, 2, 3, or 4 mismatches, where the total length of the guide sequence is 20. In some embodiments, the target sequence and the guide sequence of the gRNA may contain 1-4 mismatches where the guide sequence is 20 nucleotides. VI. RNA-guided DNA binding agent

[0368] In some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease as described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease, is provided, used, or administered.

[0369] In some embodiments, the composition further comprises an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent.

[0370] In some embodiments, the nucleic acid encoding the RNA-guided DNA binding agent comprises an mRNA comprising an open reading frame (ORF) encoding the RNA-guided DNA binding agent.

[0371] In some embodiments, the RNA-guided DNA binding agent is a nuclease .

[0372] In some embodiments, the RNA-guided DNA binding agent is a Cas9 nuclease.

[0373] In some embodiments, the Cas9 is S. pyogenes Cas9.

[0374] In some embodiments, the S. pyogenes Cas9 comprises an amino acid sequence having at least 90% identity to a sequence selected from SEQ ID NOs: 853-857 or an ORF encoding a S. pyogenes Cas9 having at least 90% identity to a sequence selected from SEQ ID NOs: 853-857. In some embodiments, the S. pyogenes Cas9 comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 853 or an ORF encoding a S. pyogenes Cas9 having at least 90% identity to SEQ ID NO: 853.

[0375] In some embodiments, the ORF encoding the amino acid sequence has at least 85% identity to any one of SEQ ID NOs: 813, 814, 816-819, 827, and 828. In some embodiments, the ORF encoding the amino acid sequence has at least 85% identity to SEQ ID NO: 813.

[0376] In some embodiments, the nuclease has double stranded endonuclease activity.

[0377] In some embodiments, the nuclease has nickase activity.

[0378] In some embodiments, the nuclease is catalytically inactive.

[0379] In some embodiments, the nuclease further comprises a heterologous functional domain.

[0380] In some embodiments, the nuclease is a nickase and the heterologous functional domain is a deaminase.

[0381] In some embodiments, the deaminase is a cytidine deaminase or an adenine deaminase.

[0382] In some embodiments, the deaminase is a cytidine deaminase.

[0383] In some embodiments, the deaminase is an apolipoprotein B mRNA editing enzyme (APOBEC) deaminase.

[0384] In some embodiments, the nuclease and the deaminase comprise an amino acid sequence having at least 90% identity to a sequence to SEQ ID NO: 851, 852, or 858 or an ORF encoding an amino acid sequence having at least 90% identity to SEQ ID NO: 851, 852, or 858. In some embodiments, the ORF encoding the amino acid sequence has at least 85% identity to SEQ ID NOs: 811, 812, or 815.

[0385] In some embodiments, the composition described herein further comprises a uracil glycosylase inhibitor (UGI) or nucleic acid encoding a UGI, wherein the nuclease polypeptide does not comprise a UGI or the nucleic acid encoding the polypeptide does not encode a UGI.

[0386] In some embodiments, the UGI comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 859 or 860 or an ORF encoding an amino acid sequence having at least 90% identity to SEQ ID NO: 859 or 860.

[0387] In some embodiments, the ORF encoding the amino acid sequence has at least 85% identity to a sequence selected from SEQ ID NOs: 823-826, optionally SEQ ID NO: 823.

[0388] In some embodiments, the ORF is a modified ORF.

[0389] RNA-guided DNA binding agents described herein encompass SpyCas9 and modified and variants thereof.

[0390] Modified versions having one catalytic domain, either RuvC or HNH, that is inactive are termed “nickases.” Nickases cut only one strand on the target DNA, thus creating a single-strand break. A single-strand break may also be known as a “nick.” In some embodiments, the compositions and methods comprise nickases. In some embodiments, the compositions and methods comprise a nickase RNA-guided DNA binding agent, such as a nickase Cas, e.g., a nickase Cas9, that induces a nick rather than a double strand break in the target DNA.

[0391] In some embodiments, the nuclease is modified to induce a point mutation or base change, e.g., through deamination.

[0392] In some embodiments, the Cas protein comprises a fusion protein comprising a Cas nuclease (e.g., SpyCas9), which is a nickase or is catalytically inactive, linked to a heterologous functional domain. In some embodiments, the Cas protein comprises a fusion protein comprising a catalytically inactive Cas nuclease (e.g., SpyCas9) linked to a heterologous functional domain (see, e.g., WO2014152432). In some embodiments, the catalytically inactive Cas9 is from the SpyCas9. In some embodiments, the catalytically inactive Cas comprises mutations that inactivate the Cas.

[0393] In some embodiments, the heterologous functional domain is a domain that modifies gene expression, histones, or DNA. In some embodiments, the heterologous functional domain is a transcriptional activation domain or a transcriptional repressor domain. In some embodiments, the nuclease is a catalytically inactive Cas nuclease, such as dCas9.

[0394] In some embodiments, the heterologous functional domain is a deaminase, such as a cytidine deaminase or an adenine deaminase. In certain embodiments, the heterologousfunctional domain is a C to T base converter (cytidine deaminase), such as an apolipoprotein B mRNA editing enzyme (APOBEC) deaminase. A heterologous functional domain such as a deaminase may be part of a fusion protein with a Cas nuclease having nickase activity or a Cas nuclease that is catalytically inactive discussed further below.

[0395] The RNA-guided DNA binding agent disclosed herein may further comprise a base- editing domain, such as a deaminase domain, that introduces a specific modification into a target nucleic acid.

[0396] In some embodiments, a nucleic acid is provided that comprises an open reading frame encoding a polypeptide comprising a cytidine deaminase (e.g., A3A), a C-terminal Cas9 nickase, and a first nuclear localization signal (NLS), wherein the polypeptide does not comprise a uracil glycosylase inhibitor (UGI).

[0397] In some embodiments, a second NLS is N-terminal to the Cas9 nickase. In some embodiments, the deaminase is N-terminal to an NLS (i.e., the first NLS or the second NLS). In some embodiments, the deaminase is N-terminal to all NLS in the polypeptide. In some embodiments, the deaminase is N-terminal to all NLS in the polypeptide, wherein the polypeptide does not comprise a uracil glycosylase inhibitor (UGI).

[0398] In some embodiments, the polynucleotide is DNA or RNA. In some embodiments, the polynucleotide is mRNA. In some embodiments, a polypeptide encoded by the mRNA is provided.

[0399] In some embodiments, the polypeptide comprising A3A and an RNA-guided nickase does not comprise a uracil glycosylase inhibitor (UGI).

[0400] In some embodiments, a composition is provided comprising a first polypeptide, or an mRNA encoding a first polypeptide, comprising a cytidine deaminase, which is optionally an APOBEC3A deaminase (A3A); a C-terminal Cas9 nickase; a first nuclear localization signal (NLS); and, optionally, a second NLS; wherein the first NLS and, when present, the second NLS are located to N-terminal to the sequence encoding the Cas9 nickase, wherein the first polypeptide does not comprise a uracil glycosylase inhibitor (UGI); and a second polypeptide, or an mRNA encoding a second polypeptide, comprising a uracil glycosylase inhibitor (UGI), wherein the second polypeptide is different from the first polypeptide.

[0401] In some embodiments, methods of modifying a target gene are provided comprising administering the compositions described herein. In some embodiments, the method comprises delivering to a cell a first nucleic acid comprising a first open reading frame encoding a first polypeptide comprising a cytidine deaminase, which is optionally an APOBEC3A deaminase (A3A); a C-terminal Cas9 nickase; a first nuclear localization signal(NLS); and, optionally, a second NLS; wherein the first NLS and, when present, the second NLS are located to N-terminal to the sequence encoding the Cas9 nickase, wherein the first polypeptide does not comprise a uracil glycosylase inhibitor (UGI), and a second nucleic acid comprising a second open reading frame encoding a uracil glycosylase inhibitor (UGI), wherein the second nucleic acid is different from the first nucleic acid.

[0402] In some embodiments, the methods comprise delivering to a cell a polypeptide comprising a deaminase, which is optionally an APOBEC3A deaminase (A3A); a C-terminal Cas9 nickase; a first nuclear localization signal (NLS); and a second NLS; wherein the first NLS and the second NLS are located to N-terminal to the sequence encoding the Cas9 nickase, wherein the first polypeptide does not comprise a uracil glycosylase inhibitor (UGI), or a nucleic acid encoding the polypeptide, and delivering to the cell a uracil glycosylase inhibitor (UGI), or a nucleic acid encoding the UGI.

[0403] In some embodiments, a molar ratio of the mRNA encoding UGI to the mRNA encoding the APOBEC3A deaminase (A3A) and an RNA-guided nickase is from about 1:35 to from about 30:1. In some embodiments, the molar ratio of the mRNA encoding UGI to the mRNA encoding the APOBEC3A deaminase (A3A) and an RNA-guided nickase is not about 1:1.

[0404] Similarly, in some embodiments, the molar ratio discussed above for the mRNA encoding the UGI protein to the mRNA encoding the APOBEC3A deaminase (A3A) and an RNA-guided nickase are similar if delivering protein.

[0405] In some embodiments, the composition described herein further comprises at least one gRNA. In some embodiments, the composition described herein further comprises two gRNAs. In some embodiments, a composition is provided that comprises an mRNA described herein and at least one gRNA, e.g., two gRNAs. In some embodiments, the gRNA is a single guide RNA (sgRNA). In some embodiments, the gRNA is a dual guide RNA (dgRNA).

[0406] In some embodiments, the composition is capable of effecting genome editing upon administration to the subject. Cytidine deaminase; APOBEC3A Deaminase

[0407] Cytidine deaminases encompass enzymes in the cytidine deaminase superfamily, and in particular, enzymes of the APOBEC family (APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups of enzymes), activation-induced cytidine deaminase (AID or AICDA) and CMP deaminases (see, e.g., Conticello et al., Mol. Biol. Evol.22:367-77, 2005;Conticello, Genome Biol.9:229, 2008; Muramatsu et al., J. Biol. Chem.274: 18470-6, 1999); and Carrington et al., Cells 9:1690 (2020)).

[0408] In some embodiments, the cytidine deaminase disclosed herein is an enzyme of APOBEC family. In some embodiments, the cytidine deaminase disclosed herein is an enzyme of APOBEC1, APOBEC2, APOBEC4, and APOBEC3 subgroups. In some embodiments, the cytidine deaminase disclosed herein is an enzyme of APOBEC3 subgroup. In some embodiments, the cytidine deaminase disclosed herein is an APOBEC3A deaminase (A3A).In some embodiments, the deaminase comprises an APOBEC3A deaminase.

[0409] In some embodiments, an APOBEC3A deaminase (A3A) disclosed herein is a human A3A. In some embodiments, an APOBEC3A deaminase (A3A) disclosed herein is a human A3A. In some embodiments, the A3A is a wild-type A3A.

[0410] In some embodiment, the A3A is an A3A variant. A3A variants share homology to wild-type A3A, or a fragment thereof. In some embodiments, a A3A variant has at least about 80% identity, at least about 85% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, or at least about 99% identity to a wild type A3A. In some embodiments, the A3A variant may have 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 21, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50 or more amino acid changes compared to a wild type A3A. In some embodiments, the A3A variant comprises a fragment of an A3A, such that the fragment has at least about 80% identity, at least about 90% identity, at least about 95% identity, at least about 96% identity, at least about 97% identity, at least about 98% identity, at least about 99% identity, at least about 99.5% identity, or at least about 99.9% identity to the corresponding fragment of a wild-type A3A.

[0411] In some embodiments, an A3A variant is a protein having a sequence that differs from a wild-type A3A protein by one or several mutations, such as substitutions, deletions, insertions, one or several single point substitutions. In some embodiments, a shortened A3A sequence could be used, e.g. by deleting N-terminal, C-terminal, or internal amino acids. In some embodiments, a shortened A3A sequence is used where one to four amino acids at the C-terminus of the sequence is deleted. In some embodiments, an APOBEC3A (such as a human APOBEC3A) has a wild-type amino acid position 57 (as numbered in the wild-type sequence). In some embodiments, an APOBEC3A (such as a human APOBEC3A) has an asparagine at amino acid position 57 (as numbered in the wild-type sequence).

[0412] In some embodiments, the wild-type A3A is a human A3A (UniPROT accession ID: p31941, SEQ ID NO: 850).

[0413] In some embodiments, the A3A disclosed herein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 850. In some embodiments, the level of identity is at least 85%, at least 87%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, the A3A comprises an amino acid sequence having at least 87% identity to SEQ ID NO: 850. In some embodiments, the A3A comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 850. In some embodiments, the A3A comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 850. In some embodiments, the A3A comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 850. In some embodiments, the A3A comprises an amino acid sequence with at least 99% identity to A3A ID NO: 850. In some embodiments, the A3A comprises the amino acid sequence of SEQ ID NO: 850.

[0414] In some embodiments, the cytidine deaminase disclosed herein comprises an amino acid sequence having at least 80% identity to SEQ ID NO: 850.

[0415] In some embodiments, any of the foregoing levels of identity is at least 90%, at least 95%, at least 98%, at least 99%, or 100%. In some embodiments, the UGI comprises an amino acid sequence with at least 90% identity to SEQ ID NO: 859 or 860. In some embodiments, the UGI comprises an amino acid sequence with at least 95% identity to SEQ ID NO: 859 or 860. In some embodiments, the UGI comprises an amino acid sequence with at least 98% identity to SEQ ID NO: 859 or 860. In some embodiments, the UGI comprises an amino acid sequence with at least 99% identity to SEQ ID NO: 859 or 860. In some embodiments, the UGI comprises the amino acid sequence of SEQ ID NO: 859 or 860.

[0416] Linkers

[0417] In some embodiments, the polypeptide comprising the deaminase and the RNA- guided nickase described herein further comprises a linker that connects the deaminase and the RNA-guided nickase. In some embodiments, the linker is a peptide linker. In some embodiments, the nucleic acid encoding the polypeptide comprising the deaminase and the RNA-guided nickase further comprises a sequence encoding the peptide linker. In some embodiments, mRNAs encoding the deaminase-linker-RNA-guided nickase fusion protein are provided.

[0418] In some embodiments, the peptide linker is any stretch of amino acids having at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 15, at least 20, at least 25, at least 30, at least 40, at least 50, or more amino acids.

[0419] In some embodiments, the peptide linker is the 16 residue “XTEN” linker, or a variant thereof (See, e.g., the Examples; and Schellenberger et al. A recombinant polypeptide extends the in vivo half-life of peptides and proteins in a tunable manner. Nat. Biotechnol. 27, 1186-1190 (2009)). In some embodiments, the XTEN linker comprises the sequence SGSETPGTSESATPES (SEQ ID NO: 901), SGSETPGTSESA (SEQ ID NO: 902), or SGSETPGTSESATPEGGSGGS (SEQ ID NO: 903).

[0420] In some embodiments, the peptide linker comprises a (GGGGS)n (SEQ ID NO: 931), a (G)n, an (EAAAK)n(SEQ ID NO: 932), a (GGS)n, an SGSETPGTSESATPES (SEQ ID NO: 901) motif (see, e.g., Guilinger J P, Thompson D B, Liu D R. Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification. Nat. Biotechnol.2014; 32(6): 577-82; the entire contents are incorporated herein by reference), or an (XP)n motif, or a combination of any of these, wherein n is independently an integer between 1 and 30. See, WO2015089406, e.g., paragraph

[0012] , the entire content of which is incorporated herein by reference.

[0421] In some embodiments, the peptide linker comprises one or more sequences selected from SEQ ID NOs: 901-991. VII. Modified gRNAs and mRNAs

[0422] In some embodiments, the gRNA is chemically modified. A gRNA comprising one or more modified nucleosides or nucleotides is called a “modified” gRNA or “chemically modified” gRNA, to describe the presence of one or more non-naturally or naturally occurring components or configurations that are used instead of or in addition to the canonical A, G, C, and U residues. In some embodiments, a modified gRNA is synthesized with a non-canonical nucleoside or nucleotide, is here called “modified.” Modified nucleosides and nucleotides can include one or more of: (i) alteration, e.g., replacement, of one or both of the non-linking phosphate oxygens or of one or more of the linking phosphate oxygens in the phosphodiester backbone linkage (an exemplary backbone modification); (ii) alteration, e.g., replacement, of a constituent of the ribose sugar, e.g., of the 2' hydroxyl on the ribose sugar (an exemplary sugar modification); (iii) modification or replacement of a naturally occurring nucleobase, including with a non-canonical nucleobase (an exemplary base modification); and (iv) modification of the 3' end or 5' end of the oligonucleotide to provide exonuclease stability, e.g., with 2’ O-me, 2’ halide, or 2’ deoxy substituted ribose; or inverted abasic terminal nucleotide, or replacement of phosphodiester with phosphorothioate.

[0423] Chemical modifications such as those listed above can be combined to provide modified gRNAs or mRNAs comprising nucleosides and nucleotides (collectively “residues”) that can have two, three, four, or more modifications. For example, a modified residue can have a modified sugar and a modified nucleobase. In certain embodiments, up to 15% of the phosphate groups of a gRNA molecule are replaced with phosphorothioate groups. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 5' end of the RNA. In some embodiments, modified gRNAs comprise at least one modified residue at or near the 3' end of the RNA. In some embodiments, the gRNA comprises one, two, three or more modified residues. In some embodiments, at least 5% (e.g., at least 5%, 10%, 15%, preferably at least 20%, 25%, 30%, 35%, 40%, 45%, or 50%) of the positions in a modified gRNA are modified nucleosides or nucleotides. In some embodiments, at least 5% of the positions in the modified guide RNA are modified nucleotides or nucleosides. In some embodiments, at least 10% of the positions in the modified guide RNA are modified nucleotides or nucleosides. In some embodiments at least 15% of the positions in the modified gRNA are modified nucleotides or nucleosides. In some embodiments preferably at least 20% of the positions in the modified gRNA are modified nucleotides or nucleosides. In some embodiments, no more than 65% of the positions in the modified gRNA are modified nucleotides. In some embodiments, no more than 55% of the positions in the modified gRNA are modified nucleotides. In some embodiments, no more than 50% of the positions in the modified gRNA are modified nucleotides. In some embodiments, 10-70% of the positions in the modified gRNA are modified nucleotides. In some embodiments, 20-70% of the positions in the modified gRNA are modified nucleotides. In some embodiments, 20-50% of the positions in the modified gRNA are modified nucleotides and the nuclease is a SpyCas9 nuclease. In some embodiments, 30-70% of the positions in the modified gRNA are modified nucleotides and the nuclease is a SpyCas9 nuclease.

[0424] Unmodified nucleic acids can be prone to degradation by, e.g., intracellular nucleases or those found in serum. For example, nucleases can hydrolyze nucleic acid phosphodiester bonds. Accordingly, in one aspect the gRNAs described herein can contain one or more modified nucleosides or nucleotides, e.g., to introduce stability toward intracellular or serum- based nucleases. In some embodiments, the modified gRNA molecules described herein can exhibit a reduced innate immune response when introduced into a population of cells, both in vivo and ex vivo. The term “innate immune response” includes a cellular response toexogenous nucleic acids, including single stranded nucleic acids, which involves the induction of cytokine expression and release, particularly the interferons, and cell death.

[0425] In some embodiments of a backbone modification, the phosphate group of a modified residue can be modified by replacing one or more of the oxygens with a different substituent. Further, the modified residue, e.g., modified residue present in a modified nucleic acid, can include the replacement of an unmodified phosphate moiety with a modified phosphate group as described herein. In some embodiments, the backbone modification of the phosphate backbone can include alterations that result in either an uncharged linker or a charged linker with unsymmetrical charge distribution.

[0426] Examples of modified phosphate groups include, phosphorothioate, borano phosphate esters, methyl phosphonates, phosphoroamidates, phosphodithioate, alkyl or aryl phosphonates and phosphotriesters. The phosphorous atom in an unmodified phosphate group is achiral. However, replacement of one of the non-bridging oxygens with one of the above atoms or groups of atoms can render the phosphorous atom chiral. The stereogenic phosphorous atom can possess either the “R” configuration (herein Rp) or the “S” configuration (herein Sp). The backbone can also be modified by replacement of a bridging oxygen, (i.e., the oxygen that links the phosphate to the nucleoside), with nitrogen (bridged phosphoroamidates), sulfur (bridged phosphorothioates) and carbon (bridged methylenephosphonates). The replacement can occur at either linking oxygen or at both of the linking oxygens.

[0427] The phosphate group can be replaced by non-phosphorus containing connectors in certain backbone modifications, e.g., an amide linkage. In some embodiments, the charged phosphate group can be replaced by a neutral moiety. Examples of moieties which can replace the phosphate group can include, without limitation, e.g., methyl phosphonate, carboxymethyl, carbamate, amide, thioether. Further examples of moieties which can replace the phosphate group can include, without limitation, e.g., ethylene oxide linker, sulfonate, sulfonamide, thioformacetal, formacetal, methyleneimino, methylenemethylimino, methylenehydrazo, methylenedimethylhydrazo and methyleneoxymethylimino.

[0428] Scaffolds that can mimic nucleic acids can also be constructed wherein the phosphate linker and ribose sugar are replaced by nuclease resistant nucleoside or nucleotide surrogates. Such modifications may comprise backbone and sugar modifications. In some embodiments, the nucleobases can be tethered by a surrogate backbone. Examples can include, without limitation, the morpholino, cyclobutyl, pyrrolidine and peptide nucleic acid (PNA) nucleoside surrogates.

[0429] The modified nucleosides and modified nucleotides can include one or more modifications to the sugar group, i.e. at sugar modification. For example, the 2' hydroxyl group (OH) can be modified, e.g. replaced with a number of different “oxy” or “deoxy” substituents. In some embodiments, modifications to the 2' hydroxyl group can enhance the stability of the nucleic acid since the hydroxyl can no longer be deprotonated to form a 2'- alkoxide ion.

[0430] Examples of 2' hydroxyl group modifications can include alkoxy or aryloxy (OR, wherein “R” can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or a sugar); polyethyleneglycols (PEG), O(CH2CH2O)nCH2CH2OR wherein R can be, e.g., H or optionally substituted alkyl, and n can be an integer from 0 to 20 (e.g., from 0 to 4, from 0 to 8, from 0 to 10, from 0 to 16, from 1 to 4, from 1 to 8, from 1 to 10, from 1 to 16, from 1 to 20, from 2 to 4, from 2 to 8, from 2 to 10, from 2 to 16, from 2 to 20, from 4 to 8, from 4 to 10, from 4 to 16, and from 4 to 20). In some embodiments, the 2' hydroxyl group modification can be 2'-O-Me. In some embodiments, the 2' hydroxyl group modification can be a 2'-fluoro modification, which replaces the 2' hydroxyl group with a fluoride. In some embodiments, the 2' hydroxyl group modification can include “locked” nucleic acids (LNA) in which the 2' hydroxyl can be connected, e.g., by a C1-6 alkylene or C1-6 heteroalkylene bridge, to the 4' carbon of the same ribose sugar, where exemplary bridges can include methylene, propylene, ether, or amino bridges; O-amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino) and aminoalkoxy, O(CH2)n-amino, (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, or diheteroarylamino, ethylenediamine, or polyamino). In some embodiments, the 2' hydroxyl group modification can include "unlocked" nucleic acids (UNA) in which the ribose ring lacks the C2'-C3' bond. In some embodiments, the 2' hydroxyl group modification can include the methoxyethyl group (MOE), (OCH2CH2OCH3, e.g., a PEG derivative).2' modifications can include hydrogen (i.e. deoxyribose sugars); halo (e.g., bromo, chloro, fluoro, or iodo); amino (wherein amino can be, e.g., NH2; alkylamino, dialkylamino, heterocyclyl, arylamino, diarylamino, heteroarylamino, diheteroarylamino, or amino acid); NH(CH2CH2NH)nCH2CH2- amino (wherein amino can be, e.g., as described herein), -NHC(O)R (wherein R can be, e.g., alkyl, cycloalkyl, aryl, aralkyl, heteroaryl or sugar), cyano; mercapto; alkyl-thio-alkyl; thioalkoxy; and alkyl, cycloalkyl, aryl, alkenyl and alkynyl, which may be optionally substituted with e.g., an amino as described herein.

[0431] The sugar modification can comprise a sugar group which may also contain one or more carbons that possess the opposite stereochemical configuration than that of the corresponding carbon in ribose. Thus, a modified nucleic acid can include nucleotides containing e.g., arabinose, as the sugar. The modified nucleic acids can also include abasic sugars. These abasic sugars can also be further modified at one or more of the constituent sugar atoms. The modified nucleic acids can also include one or more sugars that are in the L form, e.g. L- nucleosides. As used herein, a single abasic sugar is not understood to result in a discontinuity of a duplex.

[0432] In certain embodiments, 2’ modifications, include, for example, modifications include 2’-OMe, 2’-F, 2’-H, optionally 2’-O-Me.

[0433] The modified nucleosides and modified nucleotides described herein, which can be incorporated into a modified nucleic acid, can include a modified base, also called a nucleobase. Examples of nucleobases include, but are not limited to, adenine (A), guanine (G), cytosine (C), and uracil (U). These nucleobases can be modified or wholly replaced to provide modified residues that can be incorporated into modified nucleic acids. The nucleobase of the nucleotide can be independently selected from a purine, a pyrimidine, a purine analog, and a pyrimidine analog. In some embodiments, the nucleobase can include, for example, naturally-occurring and synthetic derivatives of a base. In embodiments employing a dual guide RNA, each of the crRNA and the tracr RNA can contain modifications. Such modifications may be at one or both ends of the crRNA or tracr RNA. In embodiments comprising an sgRNA, one or more residues at one or both ends of the sgRNA may be chemically modified, or internal nucleosides may be modified, or the sgRNA may be chemically modified throughout. Certain embodiments comprise a 5' end modification. Certain embodiments comprise a 3' end modification. Certain embodiments comprise a 5’ end modification and a 3’ end modification.

[0434] In some embodiments, the guide RNAs disclosed herein comprise one of the structures / modification patterns disclosed in US20170114334, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNAs disclosed herein comprise one of the structures / modification patterns disclosed in WO2017 / 136794, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNAs disclosed herein comprise one of the modification patterns disclosed in WO2018 / 107028, the contents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNAs disclosed herein comprise one of the structures / modification patterns disclosed in WO2019 / 237069, thecontents of which are hereby incorporated by reference in their entirety. In some embodiments, the guide RNAs disclosed herein comprise one of the structures / modification patterns disclosed in WO2021 / 119275, the contents of which are hereby incorporated by reference in their entirety.

[0435] In some embodiments, the guide RNAs disclosed herein comprise one of the structures / modification patterns disclosed in WO2022261292, the contents of which are hereby incorporated by reference in their entirety.

[0436] The terms “mA,” “mC,” “mU,” or “mG” may be used to denote a nucleotide that has been modified with 2’-O-Me. The terms “fA,” “fC,” “fU,” or “fG” may be used to denote a nucleotide that has been substituted with 2’-F. A “*” may be used to depict a PS modification.

[0437] The terms A*, C*, U*, or G* may be used to denote a nucleotide that is linked to the next (e.g., 3’) nucleotide with a PS bond.

[0438] The terms “mA*,” “mC*,” “mU*,” or “mG*” may be used to denote a nucleotide that has been substituted with 2’-O-Me and that is linked to the next (e.g., 3’) nucleotide with a PS bond.

[0439] Any of the modifications described below may be present in the gRNAs and mRNAs described herein.

[0440] In the context of chemically modified sequences, “A,” “C,” “G,” “N,” and “U” denote an RNA nucleotide, i.e., 2’-OH with a phosphodiesterase linkage to the 3’ nucleotide.

[0441] The terms “mA,” “mC,” “mU,” or “mG” are used to denote an adenine, cytosine, uridine, or guanidine nucleotide, respectively, that has been modified with 2’-O-Me.

[0442] Modification with 2’-O-methyl can be depicted as follows:

[0443] Another chemical modification that has been shown to influence nucleotide sugar rings is halogen substitution. For example, 2’-fluoro (2’-F) substitution on nucleotide sugar rings can increase oligonucleotide binding affinity and nuclease stability.

[0444] In this application, the terms “fA,” “fC,” “ ,” or “fG” are used to denote a nucleotide that has been substituted with 2’-F.

[0445] Substitution of 2’-F can be depicted as follows:

[0446] Phosphorothioate (PS) linkage or bond refers to a bond where a sulfur is substituted for one non-bridging phosphate oxygen in a phosphodiester linkage, for example in the bonds between nucleotides bases. When phosphorothioates are used to generate oligonucleotides, the modified oligonucleotides may also be referred to as S-oligos.

[0447] A “*” is used to denote a PS modification. In this application, the terms A*, C*, U*, or G* may be used to denote a nucleotide that is linked to the next (e.g., 3’) nucleotide with a PS bond.

[0448] In this application, the terms “mA*,” “mC*,” “mU*,” or “mG*” are used to denote a nucleotide that has been substituted with 2’-O-Me and that is linked to the next (e.g., 3’) nucleotide with a PS bond.

[0449] The diagram below shows the substitution of S- into a non-bridging phosphate oxygen, generating a PS bond in lieu of a phosphodiester bond:

[0450] Abasic nucleotides refer to those which lack nitrogenous bases. The diagram below depicts an oligonucleotide with an abasic (also known as apurinic) site that lacks a base. As used herein, the presence of a single abasic site is not considered to disrupt a duplex, e.g., a duplex formed between the guide sequence of a guide RNA and a target site in the genome:

[0451] Inverted bases refer to those with linkages that are inverted from the normal 5’ to 3’ linkage (i.e., either a 5’ to 5’ linkage or a 3’ to 3’ linkage). Such inverted bases can only be present as a terminal nucleotide. In chemical synthesis methods performed 3’ to 5’, inverted bases do not have 5’ hydroxy available to grow the chain. For example:

[0452] An abasic nucleotide can be attached with an inverted linkage. For example, an abasic nucleotide may be attached to the terminal 5’ nucleotide via a 5’ to 5’ linkage, or an abasic nucleotide may be attached to the terminal 3’ nucleotide via a 3’ to 3’ linkage. An inverted abasic nucleotide at either the terminal 5’ or 3’ nucleotide may also be called an inverted abasic end cap.

[0453] In some embodiments, one or more of the first three, four, or five nucleotides at the 5' terminus, and one or more of the last three, four, or five nucleotides at the 3' terminus are modified. In some embodiments, the modification is a 2’-O-Me, 2’-F, inverted abasic nucleotide, PS bond, or other nucleotide modification well known in the art to increase stability or performance.

[0454] In some embodiments, the first four nucleotides at the 5' terminus, and the last four nucleotides at the 3' terminus are linked with phosphorothioate (PS) bonds.

[0455] In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise a 2'-O-methyl (2'-O-Me) modified nucleotide. In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise a 2'-fluoro (2'-F) modified nucleotide. In some embodiments, the first three nucleotides at the 5' terminus, and the last three nucleotides at the 3' terminus comprise an inverted abasic nucleotide.

[0456] In some embodiments, the Spy guide RNA comprises a modified sgRNA. In some embodiments, the sgRNA comprises the modification pattern shown in Tables 6-7, for example mN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAmU mAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCACGAAAGGGCACCGAGUCG GmU*mG*mC*mU (SEQ ID NO: 669); ormN*mN*mN*NNNNNNNNNNNNNNNNNGUUUUAGAmGmCmUmAmGmAmAmAmU mAmGmCAAGUUAAAAUAAGGCUAGUCCGUUAUCAmAmCmUmUmGmAmAmAm AmAmGmUmGmGmCmAmCmCmGmAmGmUmCmGmGmUmGmCmU*mU*mU*mU (SEQ ID NO: 658) where each A, C, G, U, and N is an RNA nucleotide, 2’-OH and phosphodiester linkage to the 3’ nucleotide, m indicates a 2'-O-methyl (2'-O-Me) modified nucleotide, and * indicates a phosphorothioate linkage between nucleotides , and where the totality of the N’s comprise a guide sequence that directs a nuclease to a target sequence, e.g., the target sequence that is complementary to a guide sequence. In certain embodiments, the guide sequence comprises a guide sequence as shown in Tables 2-3.

[0457] As noted above, in some embodiments, a composition or formulation disclosed herein comprises an mRNA comprising an open reading frame (ORF) encoding an RNA-guided DNA binding agent, such as a Cas nuclease, e.g. Cas9 nuclease, as described herein. In some embodiments, an mRNA comprising an ORF encoding an RNA-guided DNA binding agent, such as a Cas nuclease, e.g. Cas9 nuclease, is provided, used, or administered. In some embodiments, the ORF encoding an RNA-guided DNA nuclease is a “modified RNA-guided DNA binding agent ORF” or simply a “modified ORF,” which is used as shorthand to indicate that the ORF is modified.

[0458] In some embodiments, the mRNA or modified ORF may comprise a modified uridine at least at one, a plurality of, or all uridine positions. In some embodiments, the modified uridine is a uridine modified at the 5’ position, e.g., with a halogen, methyl, or ethyl. In some embodiments, the modified uridine is a pseudouridine modified at the 1 position, e.g., with a halogen, methyl, or ethyl. The modified uridine can be, for example, pseudouridine, N1- methyl-pseudouridine, 5-methoxyuridine, 5-iodouridine, or a combination thereof. In some embodiments, the modified uridine is 5-methoxyuridine. In some embodiments, the modified uridine is 5-iodouridine. In some embodiments, the modified uridine is pseudouridine. In some embodiments, the modified uridine is N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5- methoxyuridine. In some embodiments, the modified uridine is a combination of N1-methyl pseudouridine and 5-methoxyuridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and N1-methyl-pseudouridine. In some embodiments, the modified uridine is a combination of pseudouridine and 5-iodouridine. In some embodiments, the modified uridine is a combination of 5-iodouridine and 5-methoxyuridine.

[0459] In some embodiments, an mRNA disclosed herein comprises a 5’ cap, such as a Cap0, Cap1, or Cap2. A 5’ cap is generally a 7-methylguanine ribonucleotide (which may be further modified, as discussed below e.g. with respect to ARCA) linked through a 5’-triphosphate to the 5’ position of the first nucleotide of the 5’-to-3’ chain of the mRNA, i.e., the first cap- proximal nucleotide. In Cap0, the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2’-hydroxyl. In Cap1, the riboses of the first and second transcribed nucleotides of the mRNA comprise a 2’-methoxy and a 2’-hydroxyl, respectively. In Cap2, the riboses of the first and second cap-proximal nucleotides of the mRNA both comprise a 2’-methoxy. See, e.g., Katibah et al. (2014) Proc Natl Acad Sci USA 111(33):12025-30; Abbas et al. (2017) Proc Natl Acad Sci USA 114(11):E2106-E2115. Most endogenous higher eukaryotic mRNAs, including mammalian mRNAs such as human mRNAs, comprise Cap1 or Cap2. Cap0 and other cap structures differing from Cap1 and Cap2 may be immunogenic in mammals, such as humans, due to recognition as “non-self” by components of the innate immune system such as IFIT-1 and IFIT-5, which can result in elevated cytokine levels including type I interferon. Components of the innate immune system such as IFIT-1 and IFIT-5 may also compete with eIF4E for binding of an mRNA with a cap other than Cap1 or Cap2, potentially inhibiting translation of the mRNA.

[0460] A cap can be included co-transcriptionally. For example, ARCA (anti-reverse cap analog; Thermo Fisher Scientific Cat. No. AM8045) is a cap analog comprising a 7- methylguanine 3’-methoxy-5’-triphosphate linked to the 5’ position of a guanine ribonucleotide which can be incorporated in vitro into a transcript at initiation. ARCA results in a Cap0 cap in which the 2’ position of the first cap-proximal nucleotide is hydroxyl. See, e.g., Stepinski et al., (2001) “Synthesis and properties of mRNAs containing the novel ‘anti- reverse’ cap analogs 7-methyl(3'-O-methyl)GpppG and 7-methyl(3'deoxy)GpppG,” RNA 7: 1486–1495. The ARCA structure is shown below.

[0461] CleanCapTMAG (m7G(5')ppp(5')(2'OMeA)pG; TriLink Biotechnologies Cat. No. N- 7113) or CleanCapTMGG (m7G(5')ppp(5')(2'OMeG)pG; TriLink Biotechnologies Cat. No. N-7133) can be used to provide a Cap1 structure co-transcriptionally.3’-O-methylated versions of CleanCapTMAG and CleanCapTMGG are also available from TriLinkBiotechnologies as Cat. Nos. N-7413 and N-7433, respectively, or CleanCap AU: TriLink Biotechnologies as Cat. Nos. N-7114. The CleanCapTMAG structure is shown below.

[0462] Alternatively, a cap can be added to an RNA post-transcriptionally. For example, Vaccinia capping enzyme is commercially available (New England Biolabs Cat. No. M2080S) and has RNA triphosphatase and guanylyltransferase activities, provided by its D1 subunit, and guanine methyltransferase, provided by its D12 subunit. As such, it can add a 7- methylguanine to an RNA, so as to give Cap0, in the presence of S-adenosyl methionine and GTP. See, e.g., Guo, P. and Moss, B. (1990) Proc. Natl. Acad. Sci. USA 87, 4023-4027; Mao, X. and Shuman, S. (1994) J. Biol. Chem.269, 24472-24479.

[0463] In some embodiments, the mRNA further comprises a poly-adenylated (poly-A) tail. In some embodiments, the poly-A tail comprises at least 20, 30, 40, 50, 60, 70, 80, 90, or 100 adenines, optionally up to 300 adenines. In some embodiments, the poly-A tail comprises 95, 96, 97, 98, 99, or 100 adenine nucleotides. In some embodiments, the poly-A tail includes non-adenine nucleotides, i.e., is an interrupted poly-A tail. In certain embodiments, the poly- A tail is interrupted by a non-adenine nucleotide about every 40, 50, 60, 70, 80, or 90 nucleotides. In certain embodiments, the poly-A tail is interrupted by a non-adenine nucleotide about every 50 nucleotides. VIII. Ribonucleoprotein complex

[0464] In some embodiments, a composition is encompassed comprising one or more sgRNAs comprising one or more guide sequences from Table 2 or one or more sgRNAs from Table 3 and an RNA-guided DNA binding agent, e.g., a nuclease, such as a Cas nuclease, such as Cas9. In some embodiments, the RNA-guided DNA-binding agent has cleavase activity, which can also be referred to as double-strand endonuclease activity. In someembodiments, the RNA-guided DNA-binding agent comprises a Cas nuclease. Examples of Cas9 nucleases include those of the type II CRISPR systems of S. pyogenes, Neisseria meningitidis, and other prokaryotes as known in the art , and modified (e.g., engineered or mutant) versions thereof.

[0465] In some embodiments, the Cas nuclease is the Cas9 nuclease from Streptococcus pyogenes.

[0466] In some embodiments, the gRNA together with an RNA-guided DNA binding agent is called a ribonucleoprotein complex (RNP). In some embodiments, the RNA-guided DNA binding agent is a Cas nuclease. In some embodiments, the gRNA together with a Cas nuclease is called a Cas RNP. In some embodiments, the RNP comprises Type-I, Type-II, or Type-III components. In some embodiments, the Cas nuclease is the Cas9 protein from the Type-II CRISPR / Cas system. In some embodiment, the gRNA together with Cas9 is called a Cas9 RNP.

[0467] Wild type Cas9 has two nuclease domains: RuvC and HNH. The RuvC domain cleaves the non-target DNA strand, and the HNH domain cleaves the target strand of DNA. In some embodiments, the Cas9 protein comprises more than one RuvC domain or more than one HNH domain. In some embodiments, the Cas9 protein is a wild type Cas9. In each of the composition, use, and method embodiments, the Cas induces a double strand break in target DNA.

[0468] In some embodiments, chimeric Cas nucleases are used, where one domain or region of the protein is replaced by a portion of a different protein. In some embodiments, a Cas nuclease domain may be replaced with a domain from a different nuclease such as Fok1. In some embodiments, a Cas nuclease may be a modified nuclease.

[0469] In other embodiments, the Cas nuclease may be from a Type-I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a component of the Cascade complex of a Type-I CRISPR / Cas system. In some embodiments, the Cas nuclease may be a Cas3 protein. In some embodiments, the Cas nuclease may be from a Type-III CRISPR / Cas system. In some embodiments, the Cas nuclease may have an RNA cleavage activity.

[0470] In some embodiments, the RNA-guided DNA-binding agent has single-strand nickase activity, i.e., can cut one DNA strand to produce a single-strand break, also known as a “nick.” In some embodiments, the RNA-guided DNA-binding agent comprises a Cas nickase. A nickase is an enzyme that creates a nick in dsDNA, i.e., cuts one strand but not the other of the DNA double helix. In some embodiments, a Cas nickase is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which an endonucleolytic active site is inactivated,e.g., by one or more alterations (e.g., point mutations) in a catalytic domain. See, e.g., US Pat. No.8,889,356 for discussion of Cas nickases and exemplary catalytic domain alterations. In some embodiments, a Cas nickase such as a Cas9 nickase has an inactivated RuvC or HNH domain.

[0471] In some embodiments, the RNA-guided DNA-binding agent is modified to contain only one functional nuclease domain. For example, the agent protein may be modified such that one of the nuclease domains is mutated or fully or partially deleted to reduce its nucleic acid cleavage activity. In some embodiments, a nickase is used having a RuvC domain with reduced activity. In some embodiments, a nickase is used having an inactive RuvC domain. In some embodiments, a nickase is used having an HNH domain with reduced activity. In some embodiments, a nickase is used having an inactive HNH domain.

[0472] In some embodiments, a conserved amino acid within a Cas protein nuclease domain is substituted to reduce or alter nuclease activity. In some embodiments, a Cas nuclease may comprise an amino acid substitution in the RuvC or RuvC-like nuclease domain. Exemplary amino acid substitutions in the RuvC or RuvC-like nuclease domain include D10A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015) Cell Oct 22:163(3): 759-771. In some embodiments, the Cas nuclease may comprise an amino acid substitution in the HNH or HNH-like nuclease domain. Exemplary amino acid substitutions in the HNH or HNH-like nuclease domain include E762A, H840A, N863A, H983A, and D986A (based on the S. pyogenes Cas9 protein). See, e.g., Zetsche et al. (2015).

[0473] In some embodiments, an mRNA encoding a nickase is provided in combination with a pair of guide RNAs that are complementary to the sense and antisense strands of the target sequence, respectively. In this embodiment, the guide RNAs direct the nickase to a target sequence and introduce a DSB by generating a nick on opposite strands of the target sequence (i.e., double nicking). In some embodiments, use of double nicking may improve specificity and reduce off-target effects. In some embodiments, a nickase is used together with two separate guide RNAs targeting opposite strands of DNA to produce a double nick in the target DNA. In some embodiments, a nickase is used together with two separate guide RNAs that are selected to be in close proximity to produce a double nick in the target DNA.

[0474] In some embodiments, the RNA-guided DNA-binding agent lacks cleavase and nickase activity. In some embodiments, the RNA-guided DNA-binding agent comprises a dCas DNA-binding polypeptide. A dCas polypeptide has DNA-binding activity while essentially lacking catalytic (cleavase / nickase) activity. In some embodiments, the dCas polypeptide is a dCas9 polypeptide. In some embodiments, the RNA-guided DNA-bindingagent lacking cleavase and nickase activity or the dCas DNA-binding polypeptide is a version of a Cas nuclease (e.g., a Cas nuclease discussed above) in which its endonucleolytic active sites are inactivated, e.g., by one or more alterations (e.g., point mutations) in its catalytic domains. See, e.g., US 20140186958; US 20150166980; and US 20190338308.

[0475] In some embodiments, the RNA-guided DNA-binding agent comprises one or more heterologous functional domains (e.g., is or comprises a fusion polypeptide).

[0476] In some embodiments, the heterologous functional domain may facilitate transport of the RNA-guided DNA-binding agent into the nucleus of a cell. For example, the heterologous functional domain may be a nuclear localization signal (NLS). In some embodiments, the RNA-guided DNA-binding agent may be fused with 1-5 NLS(s). In some embodiments, the RNA-guided DNA-binding agent may be fused with 2, 3, or 4 NLS(s). In some embodiments, the RNA-guided DNA-binding agent may be fused with two NLS(s). In some embodiments, the RNA-guided DNA-binding agent may be fused with one NLS. Where one NLS is used, the NLS may be linked at the N-terminus or the C-terminus of the RNA-guided DNA-binding agent sequence. In some embodiments, the NLS is not linked to the C-terminus. It may also be inserted within the RNA-guided DNA binding agent sequence. In certain circumstances, at least the two NLSs are the same (e.g., two SV40 NLSs). In certain embodiments, at least two different NLSs are present the RNA-guided DNA binding agent. In some embodiments, the RNA-guided DNA-binding agent is fused to two SV40 NLS sequences linked at the carboxy terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with two NLSs, one linked at the N-terminus and one at the C-terminus. In some embodiments, the RNA-guided DNA-binding agent may be fused with 3 NLSs. In some embodiments, the RNA-guided DNA-binding agent may be fused with no NLS.

[0477] In some embodiments, the NLS may be SV40 NLS. Exemplary SV40 NLS sequence may be SV40 NLS, PKKKRKV (SEQ ID NO: 916) or PKKKRRV (SEQ ID NO: 928). In some embodiments, the NLS may be a bipartite sequence, such as the NLS of nucleoplasmin, KRPAATKKAGQAKKKK (SEQ ID NO: 929). In some embodiments, the NLS sequence may comprise LAAKRSRTT (SEQ ID NO: 917), QAAKRSRTT (SEQ ID NO: 918), PAPAKRERTT (SEQ ID NO: 919), QAAKRPRTT (SEQ ID NO: 920), RAAKRPRTT (SEQ ID NO: 921), AAAKRSWSMAA (SEQ ID NO: 922), AAAKRVWSMAF (SEQ ID NO: 923), AAAKRSWSMAF (SEQ ID NO: 924), AAAKRKYFAA (SEQ ID NO: 925), RAAKRKAFAA (SEQ ID NO: 926), or RAAKRKYFAV (SEQ ID NO: 927). The NLS may be a snurportin-1 importin-^ (IBB domain, e.g. an SPN1-imp sequence. See Huber et al.,2002, J. Cell Bio., 156, 467-479. In a specific embodiment, a single PKKKRKV (SEQ ID NO: 916). In some embodiments, the first and second NLS are independently selected from an SV40 NLS, a nucleoplasmin NLS, a bipartite NLS, a c-myc like NLS, and an NLS comprising the sequence KTRAD (SEQ ID NO: 1014). In certain embodiments, the first and second NLSs may be the same (e.g., two SV40 NLSs). In certain embodiments, the first and second NLSs may be different.

[0478] In some embodiments, the first NLS is a SV40NLS and the second NLS is a nucleoplasmin NLS.

[0479] In some embodiments, the SV40 NLS comprises a sequence of PKKKRKVE (SEQ ID NO: 1010) or KKKRKVE (SEQ ID NO: 1011). In some embodiments, the nucleoplasmin NLS comprises a sequence of KRPAATKKAGQAKKKK (SEQ ID NO: 929). In some embodiments, the bipartite NLS comprises a sequence of KRTADGSEFESPKKKRKVE (SEQ ID NO: 1012). In some embodiments, the c-myc like NLS comprises a sequence of PAAKKKKLD (SEQ ID NO: 1013).

[0480] One or more linkers are optionally included at the fusion site of the NLS to the nuclease, or between NLS when more than one is present.

[0481] In some embodiments, one or more NLS(s) according to any of the foregoing embodiments are present in the RNA-guided DNA-binding agent in combination with one or more additional heterologous functional domains. One or more linkers are optionally included at the fusion site.

[0482] In some embodiments, the heterologous functional domain may be capable of modifying the intracellular half-life of the RNA-guided DNA binding agent. In some embodiments, the half-life of the RNA-guided DNA binding agent may be increased. In some embodiments, the half-life of the RNA-guided DNA-binding agent may be reduced. In some embodiments, the heterologous functional domain may be capable of increasing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may be capable of reducing the stability of the RNA-guided DNA-binding agent. In some embodiments, the heterologous functional domain may act as a signal peptide for protein degradation. In some embodiments, the protein degradation may be mediated by proteolytic enzymes, such as, for example, proteasomes, lysosomal proteases, or calpain proteases. In some embodiments, the heterologous functional domain may comprise a PEST sequence. In some embodiments, the RNA-guided DNA-binding agent may be modified by addition of ubiquitin or a polyubiquitin chain. In some embodiments, the ubiquitin may be a ubiquitin-like protein (UBL). Non-limiting examples of ubiquitin-like proteins include smallubiquitin-like modifier (SUMO), ubiquitin cross-reactive protein (UCRP, also known as interferon-stimulated gene-15 (ISG15)), ubiquitin-related modifier-1 (URM1), neuronal- precursor-cell-expressed developmentally downregulated protein-8 (NEDD8, also called Rub1 in S. cerevisiae), human leukocyte antigen F-associated (FAT10), autophagy-8 (ATG8) and -12 (ATG12), Fau ubiquitin-like protein (FUB1), membrane-anchored UBL (MUB), ubiquitin fold-modifier-1 (UFM1), and ubiquitin-like protein-5 (UBL5).

[0483] In some embodiments, the heterologous functional domain may be a marker domain. Non-limiting examples of marker domains include fluorescent proteins, purification tags, epitope tags, and reporter gene sequences. In some embodiments, the marker domain may be a fluorescent protein. Non-limiting examples of suitable fluorescent proteins include green fluorescent proteins (e.g., GFP, GFP-2, tagGFP, turboGFP, sfGFP, EGFP, Emerald, Azami Green, Monomeric Azami Green, CopGFP, AceGFP, ZsGreen1 ), yellow fluorescent proteins (e.g., YFP, EYFP, Citrine, Venus, YPet, PhiYFP, ZsYellow1), blue fluorescent proteins (e.g., EBFP, EBFP2, Azurite, mKalamal, GFPuv, Sapphire, T-sapphire,), cyan fluorescent proteins (e.g., ECFP, Cerulean, CyPet, AmCyan1, Midoriishi-Cyan), red fluorescent proteins (e.g., mKate, mKate2, mPlum, DsRed monomer, mCherry, mRFP1, DsRed-Express, DsRed2, DsRed-Monomer, HcRed-Tandem, HcRed1, AsRed2, eqFP611, mRasberry, mStrawberry, Jred), and orange fluorescent proteins (mOrange, mKO, Kusabira-Orange, Monomeric Kusabira-Orange, mTangerine, tdTomato) or any other suitable fluorescent protein. In other embodiments, the marker domain may be a purification tag or an epitope tag. Non-limiting exemplary tags include glutathione-S-transferase (GST), chitin binding protein (CBP), maltose binding protein (MBP), thioredoxin (TRX), poly(NANP), tandem affinity purification (TAP) tag, myc, AcV5, AU1, AU5, E, ECS, E2, FLAG, HA, nus, Softag 1, Softag 3, Strep, SBP, Glu-Glu, HSV, KT3, S, S1, T7, V5, VSV-G, 6xHis, 8xHis, biotin carboxyl carrier protein (BCCP), poly-His, and calmodulin. Non-limiting exemplary reporter genes include glutathione-S-transferase (GST), horseradish peroxidase (HRP), chloramphenicol acetyltransferase (CAT), beta-galactosidase, beta-glucuronidase, luciferase, or fluorescent proteins.

[0484] In further embodiments, the heterologous functional domain may be an effector domain. When the RNA-guided DNA-binding agent is directed to its target sequence, e.g., when a Cas nuclease is directed to a target sequence by a gRNA, the effector domain may modify or affect the target sequence. In some embodiments, the effector domain may be chosen from a nucleic acid binding domain, a nuclease domain (e.g., a non-Cas nuclease domain), an epigenetic modification domain, a transcriptional activation domain, and atranscriptional repressor domain. In some embodiments, the heterologous functional domain is a nuclease, such as a FokI nuclease. See, e.g., US Pat. No.9,023,649. In some embodiments, the heterologous functional domain is a transcriptional activator or repressor. See, e.g., Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression,” Cell 152:1173-83 (2013); Perez-Pinera et al., “RNA-guided gene activation by CRISPR-Cas9-based transcription factors,” Nat. Methods 10:973-6 (2013); Mali et al., “CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering,” Nat. Biotechnol.31:833-8 (2013); Gilbert et al., “CRISPR-mediated modular RNA-guided regulation of transcription in eukaryotes,” Cell 154:442-51 (2013). As such, the RNA-guided DNA-binding agent essentially becomes a transcription factor that can be directed to bind a desired target sequence using a guide RNA.

[0485] In some embodiments, the heterologous functional domain is a deaminase, such as a cytidine deaminase or an adenine deaminase. In certain embodiments, the heterologous functional domain is a C to T base converter (cytidine deaminase), such as an apolipoprotein B mRNA editing enzyme (APOBEC) deaminase.

[0486] In some embodiments, the heterologous functional domain comprises a APOBEC3 deaminase. In some embodiments, the APOBEC3 deaminase is APOBEC3A (A3A). In some embodiments, the A3A is a human A3A. In some embodiments, the A3A is a wild-type A3A. IX. Determination of Efficacy of Guide RNAs

[0487] In some embodiments, the efficacy of a guide RNA is determined when delivered or expressed together with other components (e.g., an RNA-guided DNA binding agent) forming an RNP. In some embodiments, the guide RNA is expressed together with an RNA- guided DNA binding agent, such as a Cas protein, e.g., Cas9. In some embodiments, the guide RNA is delivered to or expressed in a cell line that already stably expresses an RNA- guided DNA nuclease, such as a Cas nuclease or nickase, e.g., Cas9 nuclease or nickase. In some embodiments the guide RNA is delivered to a cell as part of a RNP. In some embodiments, the guide RNA is delivered to a cell along with a mRNA encoding an RNA- guided DNA nuclease, such as a Cas nuclease or nickase, e.g., Cas9 nuclease or nickase.

[0488] As described herein, use of an RNA-guided DNA nuclease and a guide RNA disclosed herein can lead to DSBs, SSBs, or site-specific binding that results in nucleic acid modification in the DNA or pre-mRNA which can produce errors in the form of insertion / deletion (indel) mutations upon repair by cellular machinery. Many mutations dueto indels alter the reading frame, introduce premature stop codons, or induce exon skipping and, therefore, produce a non-functional protein.

[0489] In some embodiments, the efficacy of particular guide RNAs is determined based on in vitro models. In some embodiments, the in vitro model is T cell line. In some embodiments, the in vitro model is HEK293 T cells. In some embodiments, the in vitro model is HEK293 cells stably expressing Cas9 (HEK293_Cas9). In some embodiments, the in vitro model is a lymphoblastoid cell line. In some embodiments, the in vitro model is primary human T cells. In some embodiments, the in vitro model is primary human B cells. In some embodiments, the in vitro model is primary human peripheral blood lymphocytes. In some embodiments, the in vitro model is primary human peripheral blood mononuclear cells.

[0490] In some embodiments, the number of off-target sites at which a deletion or insertion occurs in an in vitro model is determined, e.g., by analyzing genomic DNA from the cells transfected in vitro with Cas9 mRNA and the guide RNA. In some embodiments, such a determination comprises analyzing genomic DNA from cells transfected in vitro with Cas9 mRNA, the guide RNA, and a donor oligonucleotide. Exemplary procedures for such determinations are provided in the working examples below.

[0491] In some embodiments, the efficacy of particular gRNAs is determined across multiple in vitro cell models for a guide RNA selection process. In some embodiments, a cell line comparison of data with selected guide RNAs is performed. In some embodiments, cross screening in multiple cell models is performed.

[0492] In some embodiments, the efficacy of a guide RNA is evaluated by on target cleavage efficiency. In some embodiments, the efficacy of a guide RNA is measured by percent editing at the target location. In some embodiments, deep sequencing may be utilized to identify the presence of modifications (e.g., insertions, deletions) introduced by gene editing. Indel percentage can be calculated from next generation sequencing “NGS.”

[0493] In some embodiments, the efficacy of a guide RNA is measured by the number or frequency of indels at off-target sequences within the genome of the target cell type. In some embodiments, efficacious guide RNAs are provided which produce indels at off target sites at very low frequencies (e.g., <5%) in a cell population or relative to the frequency of indel creation at the target site. Thus, the disclosure provides for guide RNAs which do not exhibit off-target indel formation in the target cell type (e.g., T cells or B cells), or which produce a frequency of off-target indel formation of <5% in a cell population or relative to the frequency of indel creation at the target site. In some embodiments, the disclosure provides guide RNAs which do not exhibit any off target indel formation in the target cell type (e.g., Tcells or B cells). In some embodiments, guide RNAs are provided which produce indels at less than 5 off-target sites, e.g., as evaluated by one or more methods described herein. In some embodiments, guide RNAs are provided which produce indels at less than or equal to 4, 3, 2, or 1 off-target site(s) e.g., as evaluated by one or more methods described herein. In some embodiments, the off-target site(s) does not occur in a protein coding region in the target cell (e.g., T cells or B cells) genome.

[0494] In some embodiments, linear amplification is used to detect gene editing events, such as the formation of insertion / deletion (“indel”) mutations, translocations, and homology directed repair (HDR) events in target DNA. For example, linear amplification with a unique sequence-tagged primer and isolating the tagged amplification products (herein after referred to as “UnIT,” or “Unique Identifier Tagmentation” method) may be used.

[0495] In some embodiments, the efficacy of a guide RNA is measured by the number of chromosomal rearrangements within the target cell type. Kromatid dGH assay may used to detect chromosomal rearrangements, including e.g., translocations, reciprocal translocations, translocations to off-target chromosomes, deletions (i.e., chromosomal rearrangements where fragments were lost during the cell replication cycle due to the editing event). In some embodiments, the target cell type has less than 10, less than 8, less than 5, less than 4, less than 3, less than 2, or less than 1 chromosomal rearrangement. In some embodiments, the target cell type has no chromosomal rearrangements. X. Delivery of Compositions

[0496] Lipid nanoparticles (LNPs) are a well-known means for delivery of nucleotide and protein cargo and may be used for delivery of the guide RNAs, compositions, or pharmaceutical formulations disclosed herein. In some embodiments, the LNP compositions deliver nucleic acid, protein, or nucleic acid together with protein.

[0497] In some embodiments, the present disclosure provides a method for delivering any one of the gRNAs disclosed herein to a subject, wherein the gRNA is formulated as an LNP. In some embodiments, the LNP comprises the gRNA and a Cas9 or an mRNA encoding Cas9.

[0498] In some embodiments, the present disclosure provides a composition comprising any one of the gRNAs disclosed and an LNP. In some embodiments, the composition further comprises a Cas9 or an mRNA encoding Cas9.

[0499] In some embodiments, the LNP compositions comprise cationic lipids. In some embodiments, the LNP compositions comprise (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate) or another ionizable lipid. See, e.g., lipids of WO / 2017 / 173054 and references described therein. In some embodiments, the LNP compositions comprise molar ratios of a cationic lipid amine to RNA phosphate (N:P) of about 4.5, 5.0, 5.5, 6.0, or 6.5. In some embodiments, the term cationic and ionizable in the context of LNP lipids is interchangeable, e.g., wherein ionizable lipids are cationic depending on the pH.

[0500] In some embodiments, the LNP comprises a lipid component, and the lipid component comprises: about 35 mol % Lipid A; about 15 mol % neutral lipid (e.g., distearoylphosphatidylcholine (DSPC)); about 47.5 mol % helper lipid (e.g., cholesterol); and about 2.5 mol % stealth lipid (e.g., 1,2-dimyristoyl-rac-glycero-3-methylpolyoxyethylene glycol 2000 (PEG2k-DMG)), and wherein the N / P ratio of the LNP composition is about 3-7.

[0501] In some embodiments, the LNP comprises a lipid component, and the lipid component comprises ionizable lipid ((9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3- ((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate), cholesterol, DSPC, and PEG2k-DMG in a molar ratio of 50% ionizable lipid, 38% cholesterol, 9% DSPC, and 3% PEG2k-DMG.

[0502] In some embodiments, the gRNAs disclosed herein are formulated as LNP compositions for use in preparing a medicament for treating a disease or disorder.

[0503] Electroporation is a well-known means for delivery of cargo, and any electroporation methodology may be used for delivery of any one of the gRNAs disclosed herein. In some embodiments, electroporation may be used to deliver any one of the gRNAs disclosed herein and Cas9 or an mRNA encoding Cas9.

[0504] In some embodiments, the present disclosure comprises a method for delivering any one of the gRNAs disclosed herein to an ex vivo cell, wherein the gRNA is formulated as an LNP or not formulated as an LNP. In some embodiments, the LNP comprises the gRNA and a Cas9 or an mRNA encoding Cas9.

[0505] In some embodiments, the guide RNA compositions described herein, alone or encoded on one or more vectors, are formulated in or administered via a lipid nanoparticle; see e.g., WO / 2017 / 173054 and WO 2019 / 067992, the contents of which are hereby incorporated by reference in their entirety.

[0506] In certain embodiments, the present disclosure comprises DNA or RNA vectors encoding any of the guide RNAs comprising any one or more of the guide sequences described herein. In some embodiments, in addition to guide RNA sequences, the vectors further comprise nucleic acids that do not encode guide RNAs. Nucleic acids that do not encode guide RNA include, but are not limited to, promoters, enhancers, regulatory sequences, and nucleic acids encoding an RNA-guided DNA nuclease, which can be a nuclease such as Cas9. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, or a crRNA and trRNA. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a sgRNA and an mRNA encoding an RNA-guided DNA nuclease, which can be a Cas nuclease, such as Cas9. In some embodiments, the vector comprises one or more nucleotide sequence(s) encoding a crRNA, a trRNA, and an mRNA encoding an RNA-guided DNA nuclease, which can be a Cas protein, such as, Cas9. In one embodiment, the Cas9 is from S. pyogenes (i.e., Spy Cas9). In some embodiments, the nucleotide sequence encoding the crRNA, trRNA, or crRNA and trRNA (which may be a sgRNA) comprises or consists of a guide sequence flanked by all or a portion of a repeat sequence from a naturally-occurring CRISPR / Cas system. The nucleic acid comprising or consisting of the crRNA, trRNA, or crRNA and trRNA may further comprise a vector sequence wherein the vector sequence comprises or consists of nucleic acids that are not naturally found together with the crRNA, trRNA, or crRNA and trRNA.

[0507] In some embodiments, the components can be introduced into a cell as naked nucleic acid, as nucleic acid complexed with an agent such as a liposome or poloxamer, or they can be delivered by viral vectors (e.g., adenovirus, AAV, herpesvirus, retrovirus, lentivirus). Methods and compositions for non-viral delivery of nucleic acids include electroporation, lipofection, microinjection, biolistics, virosomes, liposomes, immunoliposomes, LNPs, polycation or lipid:nucleic acid conjugates, naked nucleic acid (e.g., naked DNA / RNA), artificial virions, and agent-enhanced uptake of DNA. Sonoporation using, e.g., the Sonitron 2000 system (Rich-Mar) can also be used for delivery of nucleic acids. A. Exemplary Cell Types

[0508] In some embodiments, methods and compositions disclosed herein genetically modify a cell. In some embodiments, the cell is an allogeneic cell. In some embodiments the cell is a human cell. In some embodiments the genetically modified cell is referred to as anengineered cell. An engineered cell refers to a cell (or progeny of a cell) comprising an engineered genetic modification, e.g. that has been contacted with a genomic editing system and genetically modified by the genomic editing system. The terms “engineered cell” and “genetically modified cell” are used interchangeably throughout. The engineered cell may be any of the exemplary cell types disclosed herein.

[0509] In some embodiments, the cell is an immune cell. As used herein, “immune cell” refers to a cell of the immune system, including e.g., a lymphocyte (e.g., T cell, B cell, natural killer cell (“NK cell”, and NKT cell, or iNKT cell)), monocyte, macrophage, mast cell, dendritic cell, or granulocyte (e.g., neutrophil, eosinophil, and basophil). In some embodiments, the cell is a primary immune cell. In some embodiments, the immune system cell may be selected from CD3+, CD4+and CD8+T cells, regulatory T cells (Tregs), B cells, NK cells, and dendritic cells (DC). In some embodiments, the immune cell is allogeneic.

[0510] In some embodiments, the cell is a lymphocyte. In some embodiments, the cell is an adaptive immune cell. In some embodiments, the cell is a T cell. In some embodiments, the cell is a B cell. In some embodiments, the cell is a NK cell. In some embodiments, the lymphocyte is allogeneic.

[0511] As used herein, a T cell can be defined as a cell that expresses a T cell receptor (“TCR” or “^^ TCR” or “^δ TCR”), however in some embodiments, the TCR of a T cell may be genetically modified to reduce its expression (e.g., by genetic modification to the TRAC or TRBC genes), therefore expression of the protein CD3 may be used as a marker to identify a T cell by standard flow cytometry methods. CD3 is a multi-subunit signaling complex that associates with the TCR. Thus, a T cell may be referred to as CD3+. In some embodiments, a T cell is a cell that expresses a CD3+ marker and either a CD4+ or CD8+ marker. In some embodiments, the T cell is allogeneic.

[0512] In some embodiments, the T cell expresses the glycoprotein CD8 and therefore is CD8+ by standard flow cytometry methods and may be referred to as a “cytotoxic” T cell. In some embodiments, the T cell expresses the glycoprotein CD4 and therefore is CD4+ by standard flow cytometry methods and may be referred to as a “helper” T cell. CD4+ T cells can differentiate into subsets and may be referred to as a Th1 cell, Th2 cell, Th9 cell, Th17 cell, Th22 cell, T regulatory (“Treg”) cell, or T follicular helper cells (“Tfh”). Each CD4+ subset releases specific cytokines that can have either proinflammatory or anti-inflammatory functions, survival or protective functions. A T cell may be isolated from a subject by CD4+ or CD8+ selection methods.

[0513] In some embodiments, the T cell is a memory T cell. In the body, a memory T cell has encountered antigen. A memory T cell can be located in the secondary lymphoid organs (central memory T cells) or in recently infected tissue (effector memory T cells). A memory T cell may be a CD8+ T cell. A memory T cell may be a CD4+ T cell.

[0514] As used herein, a “central memory T cell” can be defined as an antigen-experienced T cell, and for example, may express CD62L and CD45RO. A central memory T cell may be detected as CD62L+ and CD45RO+ by Central memory T cells also express CCR7, therefore may be detected as CCR7+ by standard flow cytometry methods.

[0515] As used herein, an “early stem-cell memory T cell” (or “Tscm”) can be defined as a T cell that expresses CD27 and CD45RA, and therefore is CD27+ and CD45RA+ by standard flow cytometry methods. A Tscm does not express the CD45 isoform CD45RO, therefore a Tscm will further be CD45RO- if stained for this isoform by standard flow cytometry methods. A CD45RO- CD27+ cell is therefore also an early stem-cell memory T cell. Tscm cells further express CD62L and CCR7, therefore may be detected as CD62L+ and CCR7+ by standard flow cytometry methods. Early stem-cell memory T cells have been shown to correlate with increased persistence and therapeutic efficacy of cell therapy products.

[0516] In some embodiments, the cell is a B cell. As used herein, a “B cell” can be defined as a cell that expresses CD19 or CD20, or B cell mature antigen (“BCMA”), and therefore a B cell is CD19+, or CD20+, or BCMA+ by standard flow cytometry methods. A B cell is further negative for CD3 and CD56 by standard flow cytometry methods. The B cell may be a plasma cell. The B cell may be a memory B cell. The B cell may be a naïve B cell. The B cell may be IgM+, or has a class-switched B cell receptor (e.g., IgG+, or IgA+). In some embodiments, the B cell is allogeneic.

[0517] In some embodiments, the cell is a mononuclear cell, such as from bone marrow or peripheral blood. In some embodiments, the cell is a peripheral blood mononuclear cell (“PBMC”). In some embodiments, the cell is a PBMC, e.g. a lymphocyte or monocyte. In some embodiments, the cell is a peripheral blood lymphocyte (“PBL”). In some embodiments, the mononuclear cell is allogeneic.

[0518] Cells used in ACT or tissue regenerative therapy are included, such as stem cells, progenitor cells, and primary cells. Stem cells, for example, include pluripotent stem cells (PSCs); induced pluripotent stem cells (iPSCs); embryonic stem cells (ESCs); mesenchymal stem cells (MSCs, e.g., isolated from bone marrow (BM), peripheral blood (PB), placenta, umbilical cord (UC) or adipose); hematopoietic stem cells (HSCs; e.g. isolated from BM or UC); neural stem cells (NSCs); tissue specific progenitor stem cells (TSPSCs); and limbalstem cells (LSCs). Progenitor and primary cells include mononuclear cells (MNCs, e.g., isolated from BM or PB); endothelial progenitor cells (EPCs, e.g. isolated from BM, PB, and UC); neural progenitor cells (NPCs); and tissue-specific primary cells or cells derived therefrom (TSCs) including chondrocytes, myocytes, and keratinocytes. Cells for organ or tissue transplantations such as islet cells, cardiomyocytes, thyroid cells, thymocytes, neuronal cells, skin cells, and retinal cells are also included.

[0519] In some embodiments, the cell is a human cell, such as a cell isolated from a human subject. In some embodiments, the cell is isolated from human donor PBMCs or leukopaks. In some embodiments, the cell is from a subject with a condition, disorder, or disease. In some embodiments, the cell is from a human donor with Epstein Barr Virus (“EBV”).

[0520] In some embodiments, the methods are carried out ex vivo. As used herein, “ex vivo” refers to an in vitro method wherein the cell is capable of being transferred into a subject, e.g. as an ACT therapy. In some embodiments, an ex vivo method is an in vitro method involving an ACT therapy cell or cell population.

[0521] In some embodiments, the cell is from a cell line. In some embodiments, the cell line is derived from a human subject. In some embodiments, the cell line is a lymphoblastoid cell line (“LCL”). The cell may be cryopreserved and thawed. The cell may not have been previously cryopreserved.

[0522] In some embodiments, the cell is from a cell bank. In some embodiments, the cell is genetically modified and then transferred into a cell bank. In some embodiments the cell is removed from a subject, genetically modified ex vivo, and transferred into a cell bank. In some embodiments, a genetically modified population of cells is transferred into a cell bank. In some embodiments, a genetically modified population of immune cells is transferred into a cell bank. In some embodiments, a genetically modified population of immune cells comprising a first and second subpopulations, wherein the first and second sub-populations have at least one common genetic modification and at least one different genetic modification are transferred into a cell bank. B. Therapeutic Methods and Uses

[0523] Any of the engineered cells and compositions described herein can be used in a method of treating a variety of diseases and disorders, as described herein. In some embodiments, the genetically modified cell (engineered cell) or population of genetically modified cells (engineered cells) and compositions may be used in methods of treating a variety of diseases and disorders. In some embodiments, a method of treating any one of thediseases or disorders described herein is encompassed, comprising administering any one or more composition described herein.

[0524] In some embodiments, the present disclosure provides a method of treating a subject in need thereof that includes administering engineered cells prepared by a method of preparing cells described herein, for example, a method of any of the aforementioned aspects and embodiments of methods of preparing cells.

[0525] In some embodiments, the methods and compositions described herein may be used to treat diseases or disorders in need of delivery of a therapeutic agent.

[0526] In some embodiments, the methods comprise administering to a subject a composition comprising an engineered cell described herein, wherein the cell produces, secretes, or expresses a polypeptide (e.g., a targeting receptor) useful for treatment of a disease or disorder in a subject. In some embodiments, the cell acts as a cell factory to produce a soluble polypeptide. In some embodiments, the cell acts as a cell factory to produce an antibody. In some embodiments, the cell continuously secretes the polypeptide in vivo. In some embodiments, the cell continuously secretes the polypeptide following transplantation in vivo for at least 1, 2, 3, 4, 5, or 6 weeks. In some embodiments, the cell continuously secretes the polypeptide following transplantation in vivo for more than 6 weeks. In some embodiments, the soluble polypeptide (e.g., an antibody) is produced by the cell at a concentration of at least 102, 103, 104, 105, 106, 107, or 108copies per day. In some embodiments, the polypeptide is an antibody and is produced by the cell at a concentration of at least 108copies per day.

[0527] In some embodiments, the present disclosure provides a method of preparing engineered cells (e.g., a population of engineered cells). The population of engineered cells may be used for immunotherapy.

[0528] In some embodiments, the present disclosure provides a method of providing an immunotherapy in a subject, the method including administering to the subject an effective amount of an engineered cell (or population of engineered cells) as described herein, for example, a cell of any of the aforementioned cell aspects and embodiments.

[0529] Immunotherapy is the treatment of disease by activating or suppressing the immune system. Immunotherapies designed to elicit or amplify an immune response are classified as activation immunotherapies. Cell-based immunotherapies have been demonstrated to be effective in the treatment of some cancers. Immune effector cells such as lymphocytes, macrophages, dendritic cells, natural killer (NK) cells, cytotoxic T lymphocytes (CTLs), T helper cells, B cells, or their progenitors such as hematopoietic stem cells (HSC) or induced pluripotent stem cells (iPSC) can be programmed to act in response to abnormal antigensexpressed on the surface of tumor cells. Thus, cancer immunotherapy allows components of the immune system to destroy tumors or other cancerous cells.

[0530] Cell-based immunotherapies have also been demonstrated to be effective in the treatment of autoimmune diseases or transplant rejection. Immune effector cells such as regulatory T cells (Tregs) or mesenchymal stem cells can be programmed to act in response to autoantigens or transplant antigens expressed on the surface of normal tissues. Immunotherapy can also be useful for the treatment of chronic infectious disease, e.g., hepatitis B and C virus infection, human immunodeficiency virus (HIV) infection, tuberculosis infection, and malarial infection. Immune effector cells comprising a targeting receptor such as a transgenic TCR or CAR are useful in immunotherapies, such as those described herein.

[0531] While transient TGFBR2 expression plays a key role in promoting a normal immune response, chronic TGFBR2 expression has been implicated in T-cell exhaustion, the response of T cells to chronic antigen stimulation (van Gisbergen et al.2009; Yang et al.2014). Accordingly, in certain embodiments, the present disclosure provides improved methods and compositions for enhancing the immune response by reducing chronic TGFBR2-mediated aberrant immune responses such as T-cell exhaustion.

[0532] In some embodiments, the population of engineered cells exhibits increased expansion relative to a population of unmodified cells expressing TGFBR2. In some embodiments, the population of cells exhibits reduced exhaustion relative to a population of unmodified cells expressing TGFBR2. In some embodiments, the population of cells exhibits an increased percentage of stem cell-like memory T-cells (Tscms) relative to a population of unmodified cells expressing TGFBR2.

[0533] In some embodiments, the population of cells exhibits increased durability relative to a population of unmodified cells expressing TGFBR2. In some embodiments, the population of cells exhibits increased persistence relative to a population of unmodified cells expressing TGFBR2. In some embodiments, the population of cells exhibits reduced fratricide relative to a population of unmodified cells expressing TGFBR2. In some embodiments, the population of cells exhibits increased cytotoxicity relative to a population of unmodified cells expressing TGFBR2. In some embodiments, the population of cells exhibits a reduced tumor volume relative to a population of unmodified cells expressing TGFBR2. In some embodiments, the population of cells leads to a reduced cancer cell area relative to a population of unmodified cells expressing TGFBR2. In some embodiments, the population of cells leads to increased tumor clearance relative to a population of unmodified cells expressing TGFBR2.

[0534] In another aspect, the present disclosure provides a method of preparing cells (e.g., a population of cells) for immunotherapy, the method including: (a) modifying cells by reducing or eliminating expression of TGFBR2 protein, for example, by introducing into said cells a gRNA molecule (as described herein), as disclosed herein; and (b) expanding said cells.

[0535] In another aspect, the present disclosure provides a method of preparing cells (e.g., a population of cells) for immunotherapy, the method including: (a) modifying cells by reducing or eliminating expression of TGFBR2, for example, by introducing into said cells a gRNA molecule (as described herein), or by reducing or eliminating expression of one or more components of T cell receptor, by introducing into said cells more than one gRNA molecule, as disclosed herein; and (b) expanding said cells.

[0536] Cells of the present disclosure are suitable for further engineering, e.g., by introduction of a heterologous sequence coding for a targeting receptor, e.g. a polypeptide that mediates TCR / CD3 zeta chain signaling. In some embodiments, the polypeptide is a targeting receptor selected from a non-endogenous TCR sequence and a non-endogenous CAR sequence. In some embodiments, the polypeptide is a wild-type or variant TCR. Cells according to the present disclosure may also be suitable for further engineering by introduction of a heterologous sequence coding for an alternative antigen binding moiety, e.g., by introduction of a heterologous sequence coding for an alternative (non-endogenous) T cell receptor, e.g., a chimeric antigen receptors (CAR) engineered to target a specific protein. CAR are also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors).

[0537] In some embodiments, the methods comprise administering to a subject a composition comprising an engineered cell described herein as an adoptive cell transfer therapy. In some embodiments, the engineered cell is an allogeneic cell.

[0538] In some embodiments of the methods, the method includes administering a lymphodepleting agent or immunosuppressant prior to administering to the subject an effective amount of the engineered cell (or engineered cells) as described herein, for example, a cell of any of the aforementioned cell aspects and embodiments. In another aspect, the invention provides a method of preparing engineered cells (e.g., a population of engineered cells).

[0539] In some embodiments, the engineered cells can be used to treat cancer, infectious diseases, inflammatory diseases, autoimmune diseases, cardiovascular diseases, neurological diseases, ophthalmologic diseases, renal diseases, liver diseases, musculoskeletal diseases,red blood cell diseases, or transplant rejections. In some embodiments, the engineered cells can be used in cell transplant, e.g., to the heart, liver, lung, kidney, pancreas, skin, or brain. (See e.g., Deuse et al., Nature Biotechnology 37:252-258 (2019).)

[0540] In some embodiments, the engineered cells can be used as a cell therapy comprising an allogeneic stem cell therapy. In some embodiments, the cell therapy comprises induced pluripotent stem cells (iPSCs). iPSCs may be induced to differentiate into other cell types including e.g., cardiomyocytes, beta islet cells, neurons, and blood cells. In some embodiments, the cell therapy comprises hematopoietic stem cells. In some embodiments, the allogeneic stem cell transplant comprises allogeneic bone marrow transplant. In some embodiments, the stem cells comprise pluripotent stem cells (PSCs). In some embodiments, the stem cells comprise induced embryonic stem cells (ESCs).

[0541] The engineered cells disclosed herein are suitable for further engineering, e.g., by introduction of further edited, or modified genes or alleles. Cells of the invention may also be suitable for further engineering by introduction of an exogenous nucleic acid encoding e.g., a targeting receptor, e.g., a TCR, CAR, UniCAR. CARs are also known as chimeric immunoreceptors, chimeric T cell receptors or artificial T cell receptors. In some embodiments, the TCR is a wild-type or variant TCR.

[0542] In some embodiments, the cell therapy is a transgenic T cell therapy. In some embodiments, the cell therapy comprises a Wilms’ Tumor 1 (WT1) targeting transgenic T cell. In some embodiments, the cell therapy comprises a targeting receptor or a donor nucleic acid encoding a targeting receptor of a commercially available T cell therapy, such as a CAR T cell therapy. There are number of targeting receptors currently approved for cell therapy. The cells and methods provided herein can be used with these known constructs. Commercially approved cell products that include targeting receptor constructs for use as cell therapies include e.g., Kymriah® (tisagenlecleucel); Yescarta® (axicabtagene ciloleucel); Tecartus™ (brexucabtagene autoleucel); Tabelecleucel (Tab-cel®); Viralym-M (ALVR105); and Viralym-C.

[0543] In some embodiments, the methods provide for administering the engineered cells to a subject, wherein the administration is an injection. In some embodiments, the methods provide for administering the engineered cells to a subject, wherein the administration is an intravascular injection or infusion. In some embodiments, the methods provide for administering the engineered cells to a subject, wherein the administration is a single dose.

[0544] In some embodiments, the methods provide for reducing a sign or symptom associated of a subject’s disease treated with a composition disclosed herein. In someembodiments, the subject has a response to treatment with a composition disclosed herein that lasts more than one week. In some embodiments, the subject has a response to treatment with a composition disclosed herein that lasts more than two weeks. In some embodiments, the subject has a response to treatment with a composition disclosed herein that lasts more than three weeks. In some embodiments, the subject has a response to treatment with a composition disclosed herein that lasts more than one month.

[0545] In some embodiments, the methods provide for administering the engineered cells to a subject, and wherein the subject has a response to the administered cell that comprises a reduction in a sign or symptom associated with the disease treated by the cell therapy. In some embodiments, the subject has a response that lasts more than one week. In some embodiments, the subject has a response that lasts more than one month. In some embodiments, the subject has a response that lasts for at least 1-6 weeks.

[0546] This description and exemplary embodiments should not be taken as limiting. For the purposes of this specification and appended claims, unless otherwise indicated, all numbers expressing quantities, percentages, or proportions, and other numerical values used in the specification and claims, are to be understood as being modified in all instances by the term “about,” to the extent they are not already so modified. Accordingly, unless indicated to the contrary, the numerical parameters set forth in the following specification and attached claims are approximations that may vary depending upon the desired properties sought to be obtained and tolerances accepted within the art. At the very least, and not as an attempt to limit the application of the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques.

[0547] References: • Molecular and Biological Characterization of a Ligand for CD27 Defines a New Family of Cytokines with Homology to Tumor Necrosis Factor, Cell.1993 May 7;73(3):447-56. doi: 10.1016 / 0092-8674(93)90133-b. PMID: 8387892. • Protective CD8 T Cell Memory Is Impaired during Chronic TGFBR2-Driven Costimulation, J Immunol.2009 May 1;182(9):5352-62. doi: 10.4049 / jimmunol.0802809. PMID: 19380782. • TGF- upregulates TGFBR2 expression and induces exhaustion of effector memory T cells in B-cell non-Hodgkin!s lymphoma. Leukemia.2014 Sep;28(9):1872-84. doi: 10.1038 / leu.2014.84. Epub 2014 Feb 26. PMID: 24569779; PMCID: PMC4145058.TCP00-7500-55110.o Ntekco DyenrottA 2 21TCP00-7500-55110.o Ntekco DyenrottA 3 21TCP00-7500-55110.o Ntekco DyenrottA 4 21TCP00-7500-55110.o Ntekco DyenrottA 5 21TCP00-7500-55110.o Ntekco DyenrottA 6 21TCP00-7500-55110.o Ntekco DyenrottA 7 21TCP00-7500-55110.o Ntekco DyenrottA 8 21TCP00-7500-55110.o Ntekco DyenrottA 9 21TCP00-7500-55110.o Ntekco DyenrottA 0 31TCP00-7500-55110.o Ntekco DyenrottA 1 31TCP00-7500-55110.o Ntekco DyenrottA 2 31TCP00-7500-55110.o Ntekco DyenrottA 3 31TCP00-7500-55110.o Ntekco DyenrottA 4 31TCP00-7500-55110.o Ntekco DyenrottA 5 31TCP00-7500-55110.o Ntekco DyenrottA 6 31TCP00-7500-55110.o Ntekco DyenrottA 7 31TCP00-7500-55110.o Ntekco DyenrottA 8 31TCP00-7500-55110.o Ntekco DyenrottA 9 31TCP00-7500-55110.o Ntekco DyenrottA 0 41TCP00-7500-55110.o Ntekco DyenrottA 1 41TCP00-7500-55110.o Ntekco DyenrottA 2 41TCP00-7500-55110.o Ntekco DyenrottA 3 41TCP00-7500-55110.o Ntekco DyenrottA 4 41TCP00-7500-55110.o Ntekco DyenrottA 5 41TCP00-7500-55110.o Ntekco DyenrottA 6 41TCP00-7500-55110.o Ntekco DyenrottA 7 41TCP00-7500-55110.o Ntekco DyenrottA 8 41TCP00-7500-55110.o Ntekco DyenrottA 9 41TCP00-7500-55110.o Ntekco DyenrottA 0 51TCP00-7500-55110.o Ntekco DyenrottA 1 51TCP00-7500-55110.o Ntekco DyenrottA 2 51TCP00-7500-55110.o Ntekco DyenrottA 3 51Table 10A. ADDITIONAL NME GUIDE RNA SEQUENCESTable 10B. ADDITIONAL SPY GUIDE RNA SEQUENCES* The guide sequence disclosed in this Table may be unmodified, modified with the exemplary modification pattern shown in the Table, or modified with a different modification pattern disclosed herein or available in the art. Throughout this application, the terms “mA,” “mC,” “mU,” or “mG” may be used to denote a nucleotide that has been modified with 2’-O-Me. Throughout this application, the terms A*, C*, U*, or G* may be used to denote a nucleotide that is linked to the next (e.g., 3') nucleotide with a phosphorothioate (PS) bond.XI. EXAMPLES

[0548] The following examples are provided to illustrate certain disclosed embodiments and are not to be construed as limiting the scope of this disclosure in any way. Example 1: General Methods

[0549] Generally, unless otherwise indicated, guide RNAs used throughout the Examples identified as “GXXXXXX” refer to modified sgRNA formats such as those shown in the Tables provided herein. 1.1 In vitro transcription (“IVT”) of mRNA

[0550] Capped and polyadenylated mRNA containing N1-methyl pseudo-U was generated by in vitro transcription using a linearized plasmid DNA template and T7 RNA polymerase. The linearized plasmid DNA containing a T7 promoter, and a sequence for transcription was linearized by restriction endonuclease digestion followed by heat inactivation of the reaction mixture and purified from enzyme and buffer salts. Messenger RNA was synthesized and purified using standard techniques known in the art.

[0551] Messenger RNA were generated from plasmid DNA encoding an open reading frame according to sequences included in the Additional Sequences Table 10. When sequences are referred to below with respect to mRNAs, it is understood that Ts should be replaced with Us (e.g., N1-methyl pseudouridines as described above). Messenger RNAs used in the Examples include a 5’ cap and a 3’ polyadenylation region, e.g., up to 100 nts. Guide RNAs were chemically synthesized by methods known in the art. 1.2 T cell Preparation

[0552] T cells were isolated from commercially obtained donor apheresis and cryopreserved. Upon thaw, T cells were plated at a density of 1.0 x 10^6cells / mL in T cell growth media (TCGM) composed of CTS OpTmizer T Cell Expansion SFM and T Cell Expansion Supplement (ThermoFisher Cat. A1048501) containing 5% human AB serum (GeminiBio, Cat.100-512), 1X Penicillin-Streptomycin (ThermoFisher, 15140122), 1X Glutamax (ThermoFisher, 35050061), and 10 mM HEPES (ThermoFisher, 15630106) further supplemented with 200 U / mL recombinant human interleukin-2 (Peprotech, Cat.200-02), 5 ng / ml recombinant human interleukin-7 (Peprotech, Cat.200-07), and 5 ng / ml recombinant human interleukin-15 (Peprotech, Cat.200-15). T cells were activated with TransAct™ (1:100 dilution, Miltenyi Biotec, Cat.130-111-160) prior to transfection.1.3 LNP formulation

[0553] In general, lipid components were dissolved in 100% ethanol at various molar ratios. The RNA cargos (e.g., Cas9 mRNA and sgRNA) were dissolved in 25 mM citrate buffer, 100 mM NaCl, pH 5.0, resulting in a concentration of RNA cargo of approximately 0.45 mg / mL.

[0554] The lipid nanoparticles (LNPs) contained ionizable lipid ((9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate), also herein called Lipid A. LNPs contain a molar ratio of lipids of 35 Lipid A: 47 cholesterol: 15 DSPC:2.51,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol-2000 (PEG2K-DMG) (e.g., catalog # GM-020 from NOF, Tokyo, Japan). LNPs were formulated with a lipid amine to RNA phosphate (N:P) molar ratio of about 6, and a ratio of gRNA to mRNA of 1:1 by weight.

[0555] LNPs were prepared using a cross-flow technique utilizing impinging jet mixing of the lipid in ethanol with two volumes of RNA solutions and one volume of water. The lipids in ethanol were mixed through a mixing cross with the two volumes of RNA solution. A fourth stream of water was mixed with the outlet stream of the cross through an inline tee (See WO2016010840 Fig.2.). The LNPs were held for 1 hour at room temperature, and further diluted with water (approximately 1:1 v / v). Diluted LNPs were buffer exchanged into 50 mM Tris, 45 mM NaCl, 5% (w / v) sucrose, pH 7.5 (TSS) and concentrated as needed by methods known in the art. The resulting mixture was then filtered using a 0.2 ^m sterile filter. The final LNP was stored at 4°C or -80°C until further use. 1.4 Next-generation sequencing (“NGS”) and analysis for on-target cleavage efficiency.

[0556] Genomic DNA was extracted using QuickExtract™ DNA Extraction Solution (Lucigen, Cat. No. QE09050) according to manufacturer's protocol.

[0557] To quantitatively determine the efficiency of editing at the target location in the genome, deep sequencing was utilized to identify the presence of insertions, deletions, and substitution introduced by gene editing. PCR primers were designed around the target site within the gene of interest (e.g., TGFBR2) and the genomic area of interest was amplified. Primer sequence design was done as is standard in the field. Additional PCR was performed according to the manufacturer's protocols (Illumina) to add chemistry for sequencing. The amplicons were sequenced on an Illumina MiSeq instrument. The reads were aligned to thehuman reference genome (e.g., hg38) after eliminating those having low quality scores. The following analysis was performed for the detection of indels or determination of base editor activity. 1.4.1 Indel analysis

[0558] The resulting files containing the reads were mapped to the reference genome (BAM files), where reads that overlapped the target region of interest were selected and the number of wild type reads versus the number of reads which contain an insertion or deletion (“indel”) was calculated.

[0559] The editing percentage (e.g., the “editing efficiency” or “indel percent”) is defined as the total number of sequence reads with insertions or deletions (“indels”) over the total number of sequence reads, including wild type. Insertions and deletions were scored in a ~20 bp region centered on the predicted Cas9 cleavage site. Indel percentage is defined as the total number of sequencing reads with one or more base inserted or deleted within the ~20 bp scoring region divided by the total number of sequencing reads, including wild type. 1.4.2 Detection of base editor activity

[0560] Reads that overlapped the target region of interest were re-aligned to the local genome sequence to improve the alignment. Then the number of wild-type reads versus the number of reads which contain C-to-T mutations, C-to-A / G mutations or indels was calculated. Insertions and deletions were scored in a ~20 bp region centered on the predicted Cas9 cleavage site. Indel percentage is defined as the total number of sequencing reads with one or more base inserted or deleted within the ~20 bp scoring region divided by the total number of sequencing reads, including wild type. C-to-T mutations or C-to-A / G mutations were scored in a 40 bp region including 10 bp upstream and 10 bp downstream of the 20 bp sgRNA target sequence. The C-to-T editing percentage is defined as the total number of sequencing reads with either one or more C-to-T mutations within the ~40 bp region divided by the total number of sequencing reads, including wild type. The percentage of C-to-A / G mutations are calculated similarly. a. 1.5 Flow cytometry

[0561] Edited T cells were phenotyped by flow cytometry to determine phosphorylated SMAD2 and SMAD3 protein (pSMAD2 / 3) expression. T cells were incubated with 10 ng / ml TGFB1 (Millipore Sigma, Cat # 11412272001) for 30 minutes at 37 C. Cells were thenwashed and stained for live dead (Thermo Fisher, Cat # L34955) and CD3 positivity (BD Biosciences Cat # 560176). The cells were washed and incubated in 1X Lyse / Fix buffer (BD Biosciences, Cat # 558049) for 10 minutes at 37C. The cells were washed with buffer and again with HBSS (Millipore Sigma, Cat # H6648). The cells were resuspended in 1X cold PERM buffer III (BD Biosciences, Cat # 558050) for 30 minutes at 4C. The cells were washed twice with FACS bufffer (PBS with 2% FBS and 2 mM EDTA) and stained with pSMAD2 / 3 antibody (BD Biosciences, Cat # 562586) at 1:200 dilution for 30 minutes at room temperature. After the incubation, the cells were washed and resuspended with FACS buffer. Cells were then processed on a Cytoflex flow cytometer (Beckman Coulter) and analyzed using the FlowJo™ software package. T cells were gated based on size, shape, viability, CD3 and pSMAD2 / 3 positivity. Example 2. TGFBR2 Disruption Guide Screening with SpyCas9

[0562] Initial guide selection was performed in silico using a human reference genome (e.g., hg38) and user defined genomic regions of interest (e.g., TGFBR2 protein coding exons), for identifying PAMs in the regions of interest. Guide RNA molecules were further selected and rank-ordered based on a number of criteria known in the art (e.g., GC content, predicted on- target activity, and potential off-target activity).

[0563] Guide RNAs were designed toward TGFBR2 (ENSG00000XXX) corresponding genomic coordinates are provided (Table 2). TGFBR2 guide RNAs were screened in T cells for editing efficacy by NGS and loss of phosphorylated SMAD2 / 3 (pSMAD2 / 3) intracellular expression by flow cytometry. In T cells with TGFBR2 gene knock out, SMAD2 / 3 cannot be phosphorylated in the presence of TGFB1.

[0564] Seventy-two hours post activation, T cells from a single donor apheresis were harvested and resuspended at a concentration of 8.35 x 10^6T cells / mL in P3 electroporation buffer (Lonza Catalog # V4SP-3960). T cells were co-electroporated with sgRNA targeting the TGFBR2 locus and mRNA encoding SpyCas9. Cas9 electroporation mix was prepared with 1 x 10^5T cells, 10 ng / µL of Spy Cas9 mRNA and 1 uM of sgRNA in a final volume of 20 µL of P3 electroporation buffer. The mixture was transferred to the corresponding wells of a 96-well Nucleofector™ plate (Lonza Catalog # V4SP-3960). Cells were electroporated in duplicate using Lonza shuttle 96w using manufacturer’s pulse code. Immediately post electroporation, cells were recovered in TCGM and incubated at 37oC for 15 minutes. Electroporated T cells were subsequently cultured in TCGM containing 5% human AB serum and cytokines as listed in T cell preparation. The plates were incubated at 37ºC 5% CO2.

[0565] On day 3 post-electroporation, T cells samples were harvested and analyzed by NGS as described in Example 1. On day 7 post-electroporation, T cells were phenotyped by flow cytometry as described in Example 1. Table 11 and Fig.1 show the mean percent editing of TGFBR2 as a percent of total NGS reads and mean percentage of pSMAD2 / 3 negative cells.

[0566] Data values that could not be detected due to experimental failure are denoted as “ND” and those uncalculatable due to limited sample size as “-“. Table 11. Mean percent indels and mean percentage of cells negative for phosphorylated SMAD2 / 3 follow editing at TGFB2.Example 3. Dose Response Analysis for Select TGFBR2 Guides

[0567] A subset of TGFBR2 guide sequences evaluated in Example 2 were assessed for dose responsive editing in a different guide RNA format. Guides were screened in T cells by assessing editing frequency by NGS and loss of phosphorylated SMAD2 / 3 (pSMAD2 / 3) intracellular expression by flow cytometry. In T cells with TGFBR2 gene knock out, SMAD2 / 3 cannot be phosphorylated in the presence of TGFB1. A dose response assayassessed the impact of increasing amounts of pre-mixed LNPs encapsulating mRNA encoding SpyCas9 and an sgRNA targeting TGFBR2 or control locus as described in Table 13. T cells from three donor aphereses were prepared and activated as described in Example 1 using 1:50 TransACT for activation for 30 minutes.

[0568] Directly following activation, T cells were collected by centrifugation, resuspended, and plated at 100,000 cells / well in T cell growth media (TCGM). LNPs were prepared as described in Example 1 with a molar ratio of lipids of 35 Lipid A: 47 cholesterol: 15 DSPC:2.5 PEG2k-DMG and a ratio of gRNA to mRNA of 1:1 by weight. LNPs were incubated with 2.5 ug / ml ApoE (Peprotech, Cat.350-02) in TCGM with 2.5% human AB serum at 37C for about 5 minutes. These LNPs were added to cells for transfection using total RNA cargo / ml doses as described in Table 13. On day 3 post transfection, T cells were harvested for NGS analysis to determine percent editing at TGFBR2. On day 7 post transfection, T cells were assayed for functional TGFB signaling by flow cytometry as described in Example 1. Table 13 and Figs.2A-2B show the mean percent indel formation at TGFBR2 and mean percentage of T cells negative for phosphorylated SMAD2 / 3 for duplicate samples from a single, representative donor. Table 13. Mean percent indels at TGFBR2 and mean percentage of pSMAD2 / 3 negative T cells using cells from a representative donorExample 4. Dose Response Analysis for Select TGFBR2 Guides in multiple donors

[0569] TGFBR2 guide RNAs were screened for editing efficacy in T cells by assessing editing frequency by NGS following lipid nanoparticle (LNP) delivery. A dose response assay assessed the impact of increasing amounts of LNPs co-formulated with a fixed concentration of mRNA encoding SpyCas9 and a sgRNA. 4.1 sgRNA dose-response with LNP formulation

[0570] T cells isolated from human donor aphereses from three donors (6632, W3137, and W0535) were prepared and activated as described in Example 1. LNPs were formulated as described in Example 1.

[0571] Thirty minutes post activation, T cells were centrifuged, resuspended and plated at 50,000 cells / well in 100 ul / well T cell growth media (TCGM). LNPs were prepared as described in Example 1 with a molar ratio of lipids of 35 Lipid A: 47 cholesterol: 15 DSPC:2.5 PEG2k-DMG and a ratio of gRNA to SpyCas9 mRNA of 1:1 by weight. LNPs were incubated with 2.5 ug / ml ApoE (Peprotech, Cat.350-02) in TCGM with 2.5% human AB serum at 37C for about 5 minutes. LNP formulation containing Spy Cas9 mRNA (SEQ ID NO: 813) and sgRNA targeting TGFBR2 was added to T cells at total RNA cargo / ml noted in Table 14. On day 3 post-editing, T cells were harvested for NGS analysis as described in Example 1. Table 14 and Figs.3A-3C show the mean percent indels in TGFBR2 in each donor.Table 14. Mean percent indels at TGFBR2 in three donors.Example 5. Off-target analysis of TGFBR2 guides

[0572] A biochemical method (See, e.g., Cameron et al., Nature Methods.6, 600-606; 2017) was used to determine potential off-target genomic sites cleaved by Cas9 using guides targeting TGFBR2. Guide RNAs targeting human TGFBR2 were screened using NA24385 genomic DNA (Coriell Institute) alongside three control guides with known off-targetprofiles. Genomic DNA was treated with calf intestinal alkaline phosphatase (CIP) prior to use. The number of cleaved sites detected using 16nM SpyCas9 ribonucleoprotein with a guide RNA:Cas9 ratio of 3:1 in the biochemical assay are shown in Table 15. Table 15 - Count of cleaved sites for TGFBR2 guides predicted by biochemical assay

[0573] To further investigate potential off-target cleavage sites, primary T cells are treated with LNPs comprising Cas9 mRNA and a sgRNA of interest (e.g., a sgRNA having potential off-target sites for evaluation). The primary T cells are then lysed and primers flanking the potential off-target site(s) are used to generate an amplicon for NGS analysis. Identification of indels at a certain level may validate potential off-target site, whereas the lack of indels found at the potential off-target site may indicate a false positive in the off-target assay that was utilized. Example 6: Screening of TGFBR2 Guide RNAs with a SpyCas9 base editor

[0574] Guide RNAs designed toward TGFBR2 (ENSG00000XXX) were tested for editing efficacy with a SpyCas9 base editing system. Guide sequences and corresponding genomic coordinates are provided (Table 2). TGFBR2 guide RNAs were screened for base editing efficacy in T cells by assessing editing frequency by NGS and loss of phosphorylated SMAD2 / 3 (pSMAD2 / 3) intracellular expression by flow cytometry. If TGFBR2 is knocked out, SMAD2 / 3 will not be phosphorylated in presence of TGFB1.

[0575] T cells from single donor apheresis were prepared and activated as described in Example 1. Ninety-six hours post T cell activation, T cells were harvested by centrifugation and resuspended at a concentration of 12.5 x 10^6T cells / mL in P3 electroporation buffer (Lonza Catalog # V4SP-3960). T cells were electroporated with sgRNAs targeting TGFBR2,mRNA encoding Spy base editor (SEQ ID NO: 811), and mRNA encoding UGI (SEQ ID NO: 823) as described in Example 2 except the electroporation mix was prepared with 1 x 10^5T cells, 20 ng / µL of mRNA encoding the base editor, 20 ng / µL of mRNA encoding UGI, and 1 uM of sgRNA in a final volume of 20 µL of P3 electroporation buffer. On day 3 post-editing, edited T cell samples were subjected to PCR and NGS analysis as described in Example 1. On day 7 post-editing, T cells were assayed for functional TGFB signaling by flow cytometry as described in Example 1. Table 16 and Fig.4 show the mean percent editing of TGFBR2 as a percent of total NGS reads and the mean percentage of TGFBR2 negative T cells using the pSMAD2 / 3 as a cell marker. Table 16 – Mean percent editing and mean percentage of mean percentage of pSMAD2 / 3 negative T cells.Example 7. In vitro assessment of anti-CD70 CAR constructs with and without IEE edits in 786-O model

[0576] This study compared the efficacy of T cells engineered with construct 5718 or construct 5719 with and without IEE(s) to benchmark construct 4645, in the presence of absence of TGF^ against a 786-O cell line. T cells were engineered with construct 5718 alone, construct 5718 + TGF^R2 KO, construct 5719 alone, construct 5719 + TGF^R2 KO, benchmark construct 4645 alone, or were untreated. 7.1. Engineering T Cells with construct 5718 or 5719, and Immune Enhancing Edits

[0577] T cells were isolated from peripheral blood of healthy human donor 535 with the following MHC I phenotype: HLA-A*02:01:01G, 03:01:01G, HLA-B*07:02:01G, 15:01:01, HLA-C*03:04:01, 07:02:01G, HLA-DRB1*07:01:01, 15:01:01, HLA-DRB4*01:03:01:02N, HLA-DRB5*01:01:01, HLA-DQA1*01:02:01, 02:01:01, HLA-DQB1*03:03:02, 06:02:01, HLA-DPA1-01:03:01, 01:03:01 HLA-DPB1*04:01:01, 04:04:01. Briefly, a leukapheresis pack (HemaCare Technologies) was treated in ammonium chloride RBC lysis buffer (Stemcell Technologies; Cat.07800) for 15 minutes to lyse red blood cells. Peripheral blood mononuclear cell...

Claims

We claim:

1. An engineered cell, comprising a genetic modification within genomic coordinates chr3:30606864-30691614.

2. An engineered cell, which has reduced or eliminated surface expression of TGF^R2 protein relative to an unmodified cell, comprising a genetic modification within genomic coordinates chr3:30606864-30691614.

3. The engineered cell of claim 1 or 2, wherein the genetic modification is within the genomic coordinates targeted by a guide RNA comprising a guide sequence of any one of SEQ ID NOs: 1-117.

4. The engineered cell of any one of claims 1-3, which has reduced or eliminated surface expression of TGF^R2 relative to an unmodified cell, comprising a genetic modification within any one of the genomic coordinates listed in Table 2.

5. The engineered cell of any one of claims 1-4, wherein the genetic modification is within genomic coordinates chosen from: (a) chr3:30672267-30672287; chr3:30644743-30644763; chr3:30671764-30671784; chr3:30672177-30672197; chr3:30606958-30606978; chr3:30606870-30606890; chr3:30606961-30606981; chr3:30606911-30606931; chr3:30606909-30606929; chr3:30606954-30606974; chr3:30606865-30606885; chr3:30606947-30606967; chr3:30606864-30606884; chr3:30606916-30606936; chr3:30606871-30606891; chr3:30606930-30606950; chr3:30606957-30606977; chr3:30623239-30623259; chr3:30623238-30623258; chr3:30623240-30623260; chr3:30623263-30623283; chr3:30644843-30644863; chr3:30644757-30644777; chr3:30650418-30650438; chr3:30650323-30650343; chr3:30650327-30650347; chr3:30650317-30650337; chr3:30650326-30650346; chr3:30650318-30650338; chr3:30650319-30650339; chr3:30650393-30650413; chr3:30672152-30672172; chr3:30672410-30672430; chr3:30672296-30672316; chr3:30671871-30671891; chr3:30672193-30672213; chr3:30671791-30671811; chr3:30672024-30672044; chr3:30671784-30671804; chr3:30672322-30672342; chr3:30672192-30672212; chr3:30672270-30672290; chr3:30671941-30671961; chr3:30671752-30671772; chr3:30672387-30672407; chr3:30671929-30671949; chr3:30672266-30672286; chr3:30672295-30672315;chr3:30672021-30672041; chr3:30671932-30671952; chr3:30671835-30671855; chr3:30672023-30672043; chr3:30671908-30671928; chr3:30672212-30672232; chr3:30671854-30671874; chr3:30671701-30671721; chr3:30672294-30672314; chr3:30672193-30672213; chr3:30671821-30671841; chr3:30672268-30672288; chr3:30672250-30672270; chr3:30672421-30672441; chr3:30672400-30672420; chr3:30672249-30672269; chr3:30672196-30672216; chr3:30672340-30672360; chr3:30671842-30671862; chr3:30671739-30671759; chr3:30674221-30674241; chr3:30674178-30674198; chr3:30674085-30674105; chr3:30674136-30674156; chr3:30674220-30674240; chr3:30674184-30674204; chr3:30688451-30688471; chr3:30688403-30688423; chr3:30688434-30688454; chr3:30688432-30688452; chr3:30688402-30688422; chr3:30688388-30688408; chr3:30688429-30688449; chr3:30688510-30688530; chr3:30688416-30688436; chr3:30691489-30691509; chr3:30691522-30691542; chr3:30691427-30691447; chr3:30691519-30691539; chr3:30691435-30691455; chr3:30691594-30691614; chr3:30691409-30691429; chr3:30691463-30691483; and chr3:30691475-30691495; and (b) chr3:30671764-30671784; chr3:30672177-30672197; chr3:30606958-30606978; chr3:30606961-30606981; chr3:30606916-30606936; chr3:30606957-30606977; chr3:30650418-30650438; chr3:30672193-30672213; chr3:30672024-30672044; chr3:30672023-30672043; chr3:30672421-30672441; chr3:30674178-30674198; chr3:30688510-30688530; chr3:30691409-30691429; chr3:30606962-30606982; chr3:30606974-30606994; chr3:30606975-30606995; chr3:30644885-30644905; chr3:30644893-30644913; chr3:30650250-30650270; chr3:30671618-30671638; chr3:30671763-30671783; chr3:30671983-30672003; chr3:30672088-30672108; chr3:30672094-30672114; chr3:30672099-30672119; chr3:30674083-30674103; chr3:30674198-30674218; chr3:30688476-30688496; chr3:30688481-30688501; chr3:30688490-30688510; chr3:30688491-30688511; chr3:30688507-30688527; chr3:30691396-30691416; chr3:30691397-30691417; chr3:30691412-30691432; chr3:30691413-30691433; chr3:30691582-30691602; and chr3:30691583-30691603.

6. The engineered cell of any one of claims 1-5, wherein the genetic modification is within the genomic coordinates chosen from: chr3:30671941-30671961 and chr3:30671739-30671759.

7. The engineered cell of any one of claims 1-6, wherein the genetic modification is within genomic coordinates targeted by a guide RNA comprising a guide sequence of SEQ ID NO: 43 or 68.

8. The engineered cell of any one of claims 1-5, wherein the genetic modification is within the genomic coordinates chosen from: chr3:30644885-30644905; chr3:30671618-30671638; chr3:30671983-30672003; chr3:30672094-30672114; chr3:30672177-30672197; and chr3:30674198-30674218.

9. The engineered cell of any one of claims 1-5 and 8, wherein the genetic modification is within genomic coordinates targeted by a guide RNA comprising a guide sequence of any one of SEQ ID NOs: 4, 95, 98, 100, 102, and 105.

10. A composition comprising a guide RNA and optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the guide RNA comprises: a. a guide sequence selected from SEQ ID NOs: 1-117; b. a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence selected from SEQ ID NOs: 1-117; c. a guide sequence that is at least 85%, 90%, or 95% identical to a sequence selected from SEQ ID NOs: 1-117; d. a sequence that comprises 10 contiguous nucleotides ±10 nucleotides of a genomic coordinate listed in Table 2; e. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence from (d); or f. a guide sequence that is at least 85%, 90%, or 95% identical to a sequence selected from (d).

11. The composition of claim 10, for use in altering a DNA sequence within the TGF^R2 gene in a cell.

12. A pharmaceutical composition comprising, or use of, the composition of claim 10 for inducing a double stranded break or a single stranded break within a TGF^R2 gene in a cell, modifying the nucleic acid sequence of a TGF^R2 gene in a cell, or reducing expression of a TGF^R2 gene in a cell.

13. A method of making an engineered human cell, which has reduced or eliminated surface expression of TGF^R2 protein relative to an unmodified cell, comprising contacting a cell with the composition of claim 10.

14. A method of reducing surface expression of TGF^R2 protein in a cell relative to an unmodified cell, comprising contacting a cell with a composition comprising a guide RNA and optionally an RNA-guided DNA binding agent or a nucleic acid encoding an RNA-guided DNA binding agent, wherein the guide RNA comprises: a. a guide sequence selected from SEQ ID NOs: 1-117; b. a guide sequence that is at least 17, 18, 19, or 20 contiguous nucleotides of a sequence of selected from SEQ ID NOs: 1-117; c. a guide sequence that is at least 85%, 90%, or 95% identical to a sequence of any one of SEQ ID NOs: 1-117; d. a sequence that comprises 10 contiguous nucleotides ±10 nucleotides of a genomic coordinate listed in Table 2; e. at least 17, 18, 19, or 20 contiguous nucleotides of a sequence from (d); or f. a guide sequence that is at least 85%, 90%, or 95% identical to a sequence selected from (d).

15. The composition, use, or method of any one of claims 10-14, wherein the RNA-guided DNA binding agent is a cleavase and the guide RNA comprises a guide sequence of SEQ ID NO: 43 or 68.

16. The composition, use, or method of any one of claims 10-14, wherein the RNA-guided DNA binding agent is a base editor and wherein the guide RNA comprises a guide sequence of any one of SEQ ID NOs: 4, 95, 98, 100, 102, and 105.

17. A population of cells comprising the engineered cell produced by use of the composition of any one of claims 10-12, 15, and 16, or the method of any one of claims 13-16.

18. A pharmaceutical composition comprising (a) the engineered cell produced by the composition or method of any one of claims 10-16; or (b) the population of cells of claim 17.

19. The engineered cell, population of cells, pharmaceutical composition, or method of any one of claims 1-18, wherein the genetic modification comprises an insertion, a deletion, or a substitution.

20. The engineered cell, population of cells, pharmaceutical composition, or method of any one of claims 1-19, wherein the genetic modification comprises an indel, a C to T substitution, or an A to G substitution within the genomic coordinates.

21. The engineered cell, population of cells, pharmaceutical composition, or method of any one of claims 1-20, wherein the cells are engineered with a genomic editing system.

22. The engineered cell, population of cells, pharmaceutical composition, or method of any one of claims 3-21, wherein the guide RNA is a dual guide RNA (dgRNA) or a single guide RNA (sgRNA).

23. The engineered cell, population of cells, pharmaceutical composition, or method of claim 22, wherein the sgRNA is a Spy sgRNA.

24. The engineered cell, population of cells, pharmaceutical composition, or method of claim 23, wherein the Spy sgRNA further comprises one or more of: A. a shortened hairpin 1 region, or a substituted and optionally shortened hairpin 1 region, wherein 1. at least one of the following pairs of nucleotides are substituted in hairpin 1 with Watson-Crick pairing nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, or H1-4 and H1-9, and the hairpin 1 region optionally lacks a. any one or two of H1-5 through H1-8, b. one, two, or three of the following pairs of nucleotides: H1-1 and H1-12, H1-2 and H1-11, H1-3 and H1-10, and H1-4 and H1-9, or c. 1-8 nucleotides of hairpin 1 region; or 2. the shortened hairpin 1 region lacks 4-8 nucleotides, preferably 4-6 nucleotides; and a. one or more of positions H1-1, H1-2, or H1-3 is deleted or substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601) or b. one or more of positions H1-6 through H1-10 is substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601); or 3. the shortened hairpin 1 region lacks 5-10 nucleotides, preferably 5-6 nucleotides, and one or more of positions N18, H1-12, or n is substituted relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601); orB. a shortened upper stem region, wherein the shortened upper stem region lacks 1-6 nucleotides and wherein the 6, 7, 8, 9, 10, or 11 nucleotides of the shortened upper stem region include less than or equal to 4 substitutions relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601); or C. a substitution relative to Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601) at any one or more of LS6, LS7, US3, US10, B3, N7, N15, N17, H2-2 and H2-14, wherein the substituent nucleotide is neither a pyrimidine that is followed by an adenine, nor an adenine that is preceded by a pyrimidine; or D. an Exemplary SpyCas9 sgRNA-1 (SEQ ID NO: 601) with an upper stem region, wherein the upper stem modification comprises a modification to any one or more of US1-US12 in the upper stem region.

25. The engineered cell, population of cells, pharmaceutical composition, or method of claim 24, wherein the guide RNA lacks 6 or 8 nucleotides in shortened hairpin 1, and / or wherein H-1 and H-3 are deleted, and / or wherein the guide RNA further comprises a 3’ tail, wherein the 3’ tail is 1-4 nucleotides in length, optionally 1 nucleotide in length.

26. The engineered cell, population of cells, pharmaceutical composition, or method of claim 24, comprising a sequence or modification pattern as set forth in Tables 6-7, wherein the N’s are collectively the guide sequence, N, A, C, G, and U are ribonucleotides (2’-OH), “m” indicates a 2’-O-Me modification, “f” indicates a 2’-fluoro modification, and a “*” indicates a phosphorothioate linkage between nucleotides.

27. The engineered cell, population of cells, pharmaceutical composition, or method of any one of claims 3-26, wherein the guide RNA comprises at least one end modification, optionally wherein the modification comprises a 5’ end modification and / or the modification comprises a 3’ end modification.

28. The engineered cell, population of cells, pharmaceutical composition, or method of claim 27, wherein the guide RNA comprises a modification in a hairpin region, optionally wherein the modification in a hairpin region is also an end modification.

29. The engineered cell, population of cells, pharmaceutical composition, or method of claim 27 or 28, wherein the modification comprises a 2’-O-methyl (2’-O-Me) modified nucleotide, and / or wherein the modification comprises a phosphorothioate (PS) bond between nucleotides, and / or wherein the modification comprises a 2’-O-methyl (2’-O-Me) modified nucleotide with aphosphorothioate (PS) bond to a 3’ adjacent nucleotide, and / or wherein the modification comprises a 2’-fluor (2’F) modified nucleotide, and / or wherein the 5’ end modification comprises a 2’-O-methyl (2’-O-Me) modified nucleotide with a phosphorothioate (PS) bond to a 3’ adjacent nucleotide at nucleotides 1-3 of the 5’ end of the guide sequence.

30. The engineered cell, population of cells, pharmaceutical composition, or method of any one of claims 3-29, wherein the guide RNA is associated with a lipid nanoparticle (LNP), optionally wherein the LNP comprises a cationic lipid, a helper lipid, a neutral lipid, a stealth lipid, or a combination of two or more thereof.

31. The engineered cell, population of cells, pharmaceutical composition, or method of claim 30, wherein the cationic lipid is (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate, and / or wherein the helper lipid is cholesterol, and / or wherein the neutral lipid is 1,2-distearoyl-sn-glycero-3-phosphocholine (DSPC), and / or wherein the stealth lipid is 1,2-dimyristoyl-rac-glycero-3-methoxypolyethylene glycol 2000 (PEG2k-DMG), and / or wherein the LNP comprises (9Z,12Z)-3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy)carbonyl)oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate, DSPC, cholesterol, and PEG2k-DMG.

32. A pharmaceutical composition comprising the engineered cell of any one of claims 1-31.

33. A population of cells comprising the engineered cell of any one of claims 1-31.

34. A pharmaceutical composition comprising a population of cells, wherein the population of cells comprises a plurality of the engineered cell of any one of claims 1-31, optionally wherein the pharmaceutical composition further comprises a pharmaceutical excipient.

35. A method of administering the engineered cell, population of cells, or pharmaceutical composition of any one of claims 1-34 to a subject in need thereof, to a subject as an adoptive cell transfer (ACT) therapy, or to a subject as an immunotherapy.

36. An engineered cell, population of cells, or pharmaceutical composition of any one of claims 1-34, for use as an ACT therapy.

37. A method of treating a disease or disorder comprising administering the engineered cell, population of cells, or pharmaceutical composition of any one of claims 1-34 to a subject in need thereof.

38. The engineered cell, population of cells, pharmaceutical composition, or method of any one of claims 1-37, wherein the engineered cell has reduced surface expression of TGF^R2 protein relative to an unmodified cell.

39. The engineered cell, population of cells, pharmaceutical composition, or method of any one of claims 1-38, wherein the cell comprises an exogenous nucleic acid encoding a targeting receptor that is expressed on the surface of the engineered cell, optionally wherein the targeting receptor is a T cell receptor (TCR) or wherein the targeting receptor is a chimeric antigen receptor (CAR).

40. The engineered cell, population of cells, pharmaceutical composition, or method of claim 39, wherein the targeting receptor is a WT1 TCR, optionally wherein the WT1 TCR comprises the amino acid sequence of SEQ ID NO: 1003, and / or optionally wherein the exogenous nucleic acid encoding the targeting receptor comprises the nucleic acid sequence of SEQ ID NO: 1002.

41. The engineered cell, population of cells, pharmaceutical composition, or method of claim 40, wherein the cell comprises an exogenous nucleic acid encoding a CD8 coreceptor that is expressed on the surface of the engineered cell, optionally wherein the CD8 coreceptor comprises the amino acid sequence of SEQ ID NO: 1005 and / or 1007, and / or optionally wherein the exogenous nucleic acid encoding the CD8 coreceptor comprises the nucleic acid sequence of SEQ ID NO: 1004 and / or 1006.

42. The engineered cell, population of cells, pharmaceutical composition, or method of any one of claims 1-41, wherein the engineered cell further comprises a genetic modification in one or more of CIITA, HLA-A, HLA-B, TRBC, or TRAC gene, and / or wherein the engineered cell further has reduced surface expression of one or more of MHC class II protein, HLA-A, HLA-B, TRBC, or TRAC relative to an unmodified cell.

43. The engineered cell, population of cells, pharmaceutical composition, or method of claim 42, wherein the engineered cell comprises: i. a genetic modification in the HLA-A gene within the genomic coordinates chr6:29942891-29942915 or chr6:29942609-29942633;ii. a genetic modification in the HLA-B gene within the genomic coordinates chr6:31355222-31355246, chr6:31355221-31355245, or chr6:31355205-31355229; iii. a genetic modification in the TRBC gene within the genomic coordinates chr7:142792047-142792067; iv. a genetic modification in the TRAC gene within the genomic coordinates chr14:22547524-22547544, chr14:22550574-22550598, or chr14:22550544-22550568; v. a genetic modification in the CIITA gene within the genomic coordinates chr16:10907504-10907528 or chr16:10906643-10906667; or vi. a combination of two or more of (i)-(v).

44. The engineered cell, population of cells, pharmaceutical composition, or method of claim 42 or 43, wherein the engineered cell comprises at least one genetic modification (i) within the genomic coordinates targeted by a HLA-A guide RNA comprising a guide sequence of SEQ ID NO: 403 or 404; (ii) within the genomic coordinates targeted by a HLA-B guide RNA comprising a guide sequence of SEQ ID NO: 406, 405 or 407; (iii) within the genomic coordinates targeted by a TRBC guide RNA comprising a guide sequence of SEQ ID NO: 414; (iv) within the genomic coordinates targeted by a TRAC guide RNA comprising a guide sequence of SEQ ID NO: 408, 409, or 413; (v) within the genomic coordinates targeted by a CIITA guide RNA comprising a guide sequence of SEQ ID NO: 401 or 402; or (vi) a combination of two or more of (i)-(v).

45. The engineered cell, population of cells, pharmaceutical composition, or method of any one of claims 1-44, wherein the engineered cell is an immune cell, optionally wherein the engineered cell is a lymphocyte.

46. The engineered cell, population of cells, pharmaceutical composition, or method of claim 45, wherein the engineered cell is a T cell, optionally wherein the cell is a CD4+ T cell or a CD8+T cell and / or wherein the cell is a memory T cell.

47. The engineered cell, population of cells, pharmaceutical composition, or method of any one of claims 1-46, wherein the cell is an allogeneic cell.

48. The engineered cell, population of cells, pharmaceutical composition, or method of any one of claims 1-47, for use in administering to a subject as an adoptive cell transfer (ACT) therapy, for use in treating a subject with cancer, for use in treating a subject with an infectious disease, or for use in treating a subject with an autoimmune disease.

49. The population or the pharmaceutical composition of any one of claims 17-48, wherein the population of cells is at least 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% TGF^R2 negative as measured by flow cytometry.

50. The population or pharmaceutical composition of any one of claims 17-49, wherein at least 65%, 70%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% of the population of cells comprises the genetic modification in the TGF^R2 gene, as measured by next-generation sequencing (NGS).

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