Compositions and methods for non-toxic conditioning

Abstract

The invention features compositions and methods for conditioning a patient (e.g., to facilitate transplantation and / or engraftment). The invention provides a base editing strategy targeting cell surface proteins that can be utilized for conditioning.

Description

[0001] Related applications

[0002] This application claims the benefit of U.S. Provisional Application No. 62 / 893,677, filed on January 28, 2019, under 35 U.S.C. 119(e), the entire contents of which are incorporated herein by reference. Technical Field

[0003] The present application relates to compositions and methods for non-toxic conditioning and a base editing strategy targeting cell surface proteins that can be used for such non-toxic conditioning. Background Art

[0004] Gene therapy using autologous hematopoietic stem cells (HSCs) is ideal because it is generally not associated with the requirement to suppress the immune system and reduces the risk of host-versus-graft or graft-versus-host disease. However, ensuring the engraftment of the hematopoietic stem cell transplant in the host remains challenging. Conditioning (HSC removal) before hematopoietic stem cell transplantation (HSCT) is used to promote engraftment. In fact, conditioning efficacy is associated with improved engraftment. One disadvantage of myeloablative (toxic) conditioning is that it can lead to increased chimerism levels.

[0005] Current conditioning regimens for autologous gene therapy for conditions such as thalassemia, sickle cell disease (SCD), or adenosine deaminase deficiency typically involve the use of 2-5 g / kg of intravenous busulfan for 2-4 days. Busulfan is a DNA alkylating drug originally designed to treat blood diseases such as acute myeloid leukemia (AML). However, busulfan carries a significant risk of side effects, including infertility, primary or secondary malignancies, and other acute and chronic toxicities.

[0006] Conditioning prior to HSCT is an unmet medical need. Therefore, there is an urgent need for compositions and methods for conditioning to promote engraftment of hematopoietic stem cell transplants. Summary of the Invention

[0007] As described below, the invention features compositions and methods for non-toxic conditioning.

[0008] In one aspect, the present invention provides a method for producing hematopoietic stem cells or progenitor cells thereof for treating hemoglobinopathies, blood cancers, or myeloproliferative diseases. In some embodiments, the method comprises (a) expressing a nucleobase editing polypeptide in a hematopoietic stem cell or progenitor cell thereof, wherein the nucleobase editing polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase; (b) contacting the hematopoietic stem cell or progenitor cell thereof with a guide RNA, wherein the guide RNA targets a nucleic acid molecule encoding a cell surface protein selected from the group consisting of CD117, CXCR4, CD135, CD90, CD45, and CD34, and introducing a mutation in the cell surface protein.

[0009] In some embodiments, the method comprises (a) expressing a nucleobase editing polypeptide in a hematopoietic stem cell or a progenitor cell thereof comprising a CD117 protein, wherein the nucleobase editing polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase; (b) contacting the hematopoietic stem cell or progenitor cell with a guide RNA capable of targeting a polynucleotide encoding a cell surface protein, thereby producing a hematopoietic stem cell or a progenitor cell thereof for treating a hemoglobinopathy, a blood cancer, or a myeloproliferative disease.

[0010] In some embodiments, the hemoglobinopathy is selected from sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome. In some embodiments, the blood cancer is selected from the group consisting of acute myeloid leukemia, acute lymphocytic leukemia, chronic myeloid leukemia, chronic lymphocytic leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin's lymphoma. In some embodiments, the myeloproliferative disease is myelodysplastic syndrome. In one aspect, the present invention provides a method for manufacturing hematopoietic stem cells or their progenitor cells for treating immunodeficiency. In some embodiments, the immunodeficiency is severe combined immunodeficiency (SCID).

[0011] On the other hand, the present invention provides a method for identifying mutations that change the binding of an antibody to a cell surface protein. In some embodiments, the method includes (a) expressing a nucleobase editing polypeptide in a cell comprising a cell surface protein, wherein the nucleobase editing polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase; (b) contacting the cell with a guide RNA that can target a nucleic acid molecule encoding a cell surface protein and introduce a mutation in the cell surface protein; and (c) contacting the cell with an antibody that specifically binds to a wild-type cell surface protein but exhibits reduced binding to a cell surface protein comprising a mutation, thereby identifying a mutation that changes the binding of the antibody to the cell surface protein.

[0012] In some embodiments, the method further comprises determining the biological activity of the cell. In some embodiments, the cell surface protein is selected from the group consisting of CD117, CXCR4, CD135, CD90, CD45 and CD34. In some embodiments, the cell surface protein is CD117. In some embodiments, the method further comprises contacting the cell with one or more other guide RNAs, which are targeted to cell surface proteins selected from the group consisting of CXCR4, CD135, CD90, CD45, and CD34. In some embodiments, the cell is a hematopoietic stem cell or a progenitor cell thereof. In some embodiments, the mutation is a missense mutation. In some embodiments, the missense mutation cannot change the biological activity of the cell surface protein. In some embodiments, one or more amino acid substitutions are introduced by a nucleobase editing polypeptide and a guide RNA.

[0013] In some embodiments, the method comprises (a) expressing a nucleobase editing polypeptide in a hematopoietic stem cell or a progenitor cell thereof, wherein the nucleobase editing polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase; (b) contacting the cell with a guide RNA that is capable of targeting a nucleic acid molecule encoding a cell surface protein selected from the group consisting of CD117, CXCR4, CD135, CD90, CD45, and CD34 and introducing a mutation in the cell surface protein; and (c) contacting the cell with an antibody that specifically binds to a wild-type cell surface protein but exhibits reduced binding to a cell surface protein comprising a mutation, thereby identifying a mutation that alters binding of the antibody to the cell surface protein.

[0014] Again, on the other hand, the present invention provides a method for base editing a gene encoding a cell surface protein expressed by a hematopoietic stem cell or its progenitor cell. In some embodiments, the method comprises (a) expressing a nucleobase editing polypeptide in a hematopoietic stem cell or its progenitor cell comprising a CD117 protein, wherein the nucleobase editing polypeptide comprises a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase; (b) contacting the cell with a guide RNA capable of targeting a nucleic acid encoding CD117, thereby base editing the gene encoding the cell surface protein.

[0015] In one aspect, the present invention provides a method for conditioning a subject simultaneously with or subsequent to hematopoietic stem cell transplantation (HSCT). In some embodiments, the method comprises (a) expressing a nucleobase editing polypeptide comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase in isolated hematopoietic stem cells of a subject or donor; (b) contacting the hematopoietic stem cells with a guide RNA capable of targeting a nucleic acid molecule encoding a cell surface protein selected from a group consisting of CD117, CXCR4, CD135, CD90, CD45, and CD34, thereby introducing a mutation in the cell surface protein and generating an edited hematopoietic stem cell; (c) administering the edited hematopoietic stem cell to the subject; (d) administering an antibody, an antibody drug conjugate, or a chimeric antigen receptor expressing T cell (CAR-T) that selectively binds to a wild-type cell surface protein to the subject, wherein the administration of step (d) is performed simultaneously with step (c) or subsequent to step (c).

[0016] In some embodiments, the method comprises (a) expressing a nucleobase editing polypeptide comprising a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase in a hematopoietic stem cell of a subject; (b) contacting the hematopoietic stem cell with a guide RNA capable of targeting a nucleic acid molecule encoding a CD117 protein, thereby introducing a mutation in the CD117 protein and generating an edited hematopoietic stem cell; (c) administering the edited hematopoietic stem cell to the subject; (d) administering to the subject an antibody, an antibody drug conjugate or a chimeric antigen receptor-expressing T cell (CAR-T) that selectively binds to a wild-type CD117 protein, wherein the administration of step (d) is performed simultaneously with or subsequent to step (c).

[0017] In some embodiments, the deaminase domain is an adenosine deaminase or a cytidine deaminase. In some embodiments, the adenosine deaminase domain is a TadA deaminase domain. In some embodiments, the adenosine deaminase comprises a TadA*8 variant. In some embodiments, the adenosine deaminase is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24. In some embodiments, the deaminase is a monomer or a heterodimer. In some embodiments, the base editing polypeptide is an ABE8 base editor. In some embodiments, the ABE8 base editor is ABE8.1-m, ABE8.2-m, ABE8.3-m, ABE8.4-m, ABE8.5-m, ABE8.6-m, ABE8.7-m, ABE8.8-m, ABE8.9-m, ABE8.10-m, ABE8.11-m, ABE8.12-m, ABE8.13-m, ABE8.14-m, ABE8.15-m, ABE8.16-m, ABE8.17-m, ABE8.18-m, ABE8.19-m, ABE8.20-m, ABE8.21-m, ABE8.22-m, ABE8.23-m, ABE8.24-m,ABE8.1-d,ABE8.2-d,ABE8.3-d,ABE8.4-d,ABE8.5-d,ABE8.6-d,ABE8.7-d,ABE8.8-d,ABE8.9-d,ABE8.10-d,ABE8.11-d,ABE8.12-d, ABE8.13-d, ABE8.14-d, ABE8.15-d, ABE8.16-d, ABE8.17-d, ABE8.18-d, ABE8.19-d, ABE8.20-d, ABE8.21-d, ABE8.22-d, ABE8.23-d, or ABE8.24-d. In some embodiments, the nucleobase editor polypeptide is an internal base editor (IBE) comprising a deaminase domain inserted at an internal position of napDNAbp. In some embodiments, the nucleobase editor polypeptide further comprises one or more uracil glycosylase inhibitors (UGIs).In some embodiments, the nucleobase editor polypeptide further comprises one or more nuclear localization sequences (NLS).

[0018] In some embodiments, the mutation is a missense mutation. In some embodiments, the missense mutation cannot change the biological activity of the cell surface protein. In some embodiments, the CD117 cell surface protein comprising a mutation can bind to stem cell factor (SCF). In some embodiments, the CD117 cell surface protein comprising a mutation can send a stem cell factor (SCF) signal. In some embodiments, the mutation is at least one amino acid substitution caused by the modification of one or more single target nucleobases. In some embodiments, the single target nucleobase is cytosine (C), and wherein the modification comprises converting the C into thymine (T). In some embodiments, the single target nucleobase is adenosine (A), and wherein the modification comprises converting A into guanine (G). In some embodiments, the at least one amino acid substitution is a naturally occurring mutation. In some embodiments, the at least one amino acid substitution is in domain 1, 2, 3, 4 or 5 of CD117. In some embodiments, the at least one amino acid substitution in CD117 is selected from the group consisting of T13A, S35P, I39V, H40R, K43G, K43R, S44P, D45G, I47V, D52G, E53G, I54V, R55G, L56P, L57P, T59A, F63P, V64A, K65E, K65R, W66R, T67A, D72G, E73G, T7 4A,N75D,N75G,E76G,N77G,N77S,N77Y,K78E,K78R,Q79R,N80G,N80S,E81G,E81D,W82R,I83T,I83 V,T84A,E85G,K86E,E88G,T90A,N99G,K100G,H101R,K116R,V120A,S123P,L124P,Y125H,K127G,K 127R,E128G,D129G,D129E,N130G,N130D,D131G,D131N,T132A,T144A,N145D,N145G,N145Y,N14 5S,Y146C,K149E,K149G,Q152R,K154E,K154G,R161G,F162P,F162L,I163T,I163V,D165G,M171T, I172T,I172V,K173G,S174G,K176G,Q190R,E191G,K193G,V195A,L196P,S197P,E198G,K199G,F20 0P,I201T,I201V,L202P,V213A,V214A,S215P,V216A,K218G,K218R,S220G,Y221C,E225G,E227G,E228G,T230A,S240G,Y243C,K247G,R248G,Q256R,E257G,E257D,K258E,K258G,Y259C,Y259H,N260G,D26 6G,N268D,N268G,Y269C,T276A,I277T,I277V,S279P,R281G,V282A,S285P,N293G,N294G,T295A,F296P, S298P,N300G,N300S,T302A,T303A,T304A,M318V,T322A,V323A,F324L,N326G,D327G,D332G,I334V,K34 2E,K342G,K342R,Q347R,Y350H,M351T,R353G,T354A,T354I,K358G,E360G,D361G,K364G,E366G,N367G,H 378R,T380A,R381G,K383G,T385A,T389A,D398G,V399A,N400G,V407A,Y408H,E414G,I415V,T417A,Y418 C,Y418H,D419G,R420G,L421P,V422A,N423S,M425V,E435G,I438M,D439G,V454A,L455P,V457A,V459A,Q 460R,T461A,N463G,S464P,S465P,F469P,K471E,K471G,L472P,V473A,Q475R,S476G,I478M,I478V,D479G,S481G,F483P,K484G,N486G,N486S,T488A,Y494C,N495D,D496G,K499E,Y503C,Y503H,F504P,and N505G. In some embodiments, the at least one amino acid substitution in CD117 is selected from the group consisting of T13A, I39V, H40R, D45G, D52G, E53G, I54V, R55G, T59A, K65E, K65R, T67A, E76G, N77G, N77S, N77Y, K78E, Q79R, N80G, E81G, E81D, N99G, K100G,H101R,L124P,Y125H,D129E,D129G,N130D,N130G,D131G,D131N,T132A,T144A,N14 5D,N145G,N145Y,N145S,Y146C,F162P,F162L,I163T,I163V,M171T,I172T,V195A,L196P,K199G, I201V, S220G, Y221C, Q256R, E257D, E257G, K258E, Y259C, T303A, T304A, M318V, T322A, V323A, F324L, K342G, K342R, Y350H, M351T, R353G, T354A, T354I, R381G, K383G, T385A, Y418C, D419G, R420G, I438M, D439G, Q460R, T461A, K484G, N486G, and T488A. In some embodiments, at least one amino acid substitution in CD117 is a naturally occurring mutation selected from the group consisting of: T13A, N77S, D129E, N130D, D131N, T144A, Y221C, E257D, T322A, T354I, D419G.,

[0019] In some embodiments, the cell is a hematopoietic stem cell, a common myeloid progenitor cell, a pronormoblast, or an erythrocyte. In some embodiments, the cell is a CD34+ cell. In some embodiments, the cell is from a subject having a hemoglobinopathy, a blood cancer, or a myeloproliferative disorder. In some embodiments, the cell is from a subject having sickle cell disease (SCD). In some embodiments, the cell is from a subject having hereditary persistent fetal hemoglobin elevation (HPFH). In some embodiments, the cell is from a subject having severe combined immunodeficiency (SCID). In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0020] In some embodiments, the adenosine deaminase domain is fused to napDNAbp. In some embodiments, the deaminase domain is inserted into the internal position of napDNAbp. In some embodiments, the napDNAbp is an inactivated nuclease or nickase variant. In some embodiments, the napDNAbp comprises Cas9, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i or Cas12j / CasΦ polynucleotides or a portion thereof. In some embodiments, the napDNAbp comprises Cas9 polynucleotides or a portion thereof. In some embodiments, the napDNAbp comprises inactivated Cas9 (dCas9) or Cas9 nickase (nCas9). In some embodiments, the napDNAbp is Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof. In some embodiments, the napDNAbp comprises an altered protospacer adjacent motif (PAM) specificity. In some embodiments, the altered PAM is specific for the nucleic acid sequence 5'-NGC-3'.

[0021] In some embodiments, the deaminase domain is capable of deaminating cytidine or adenine in DNA. In some embodiments, the deaminase domain is a cytidine deaminase domain. In some embodiments, the cytidine deaminase is an APOBEC deaminase domain or a derivative thereof. In some embodiments, the deaminase domain is an adenosine deaminase domain. In some embodiments, the adenosine deaminase domain is a TadA deaminase domain. In some embodiments, the adenosine deaminase is a TadA*8 variant. In some embodiments, the adenosine deaminase is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24. In some embodiments, the deaminase is a monomer or a heterodimer. In some embodiments, the guide polynucleotide comprises a nucleic acid sequence comprising at least 10 continuous nucleotides complementary to a CD117 nucleic acid sequence. In some embodiments, the one or more guide polynucleotides comprise a nucleic acid sequence selected from Table 23. In some embodiments, the one or more guide polynucleotides comprise a nucleic acid sequence hybridized with the complementary sequence of a CD117 target sequence, and the complementary sequence of the CD117 target sequence is selected from the following groups: CAAGCTATCTTCTTAGGGA; GCAAGCTATCTTCTTAGGGAA; ACTTACGACAGGCTCGTGAA; TGACCAATTATTCCCTCAAG; AGTGACCAATTATTCCCTCA; ACTACAGTATTTGTAAACGA; AAGACAACGACACGCTGGTC; and GGCTGTTATGCACTGATCCG. In some embodiments, guide RNA targets the following scaffold nucleic acid sequences:

[0022] GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGC.

[0023] In some embodiments, the antibody is an anti-CD117 antibody. In some embodiments, the antibody binds to domain 1, 2, 3, 4, or 5 of CD117. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the hemoglobinopathy is sickle cell disease (SCD). In some embodiments, the hemoglobinopathy is hereditary persistent fetal hemoglobin hyperplasia (HPFH). In some embodiments, the sickle cell disease is associated with a mutation in a beta globin (HBB) polynucleotide. In some embodiments, the HPFH is associated with a mutation in a hemoglobin subunit gamma 1 (HBG1) and / or hemoglobin subunit gamma 2 (HBG2) polynucleotide.

[0024] On the other hand, the present invention provides a base editing system comprising a fusion protein or a polynucleotide encoding the fusion protein, wherein the fusion protein comprises a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase domain, and a guide polynucleotide, wherein the guide polynucleotide comprises a nucleic acid sequence selected from Table 23.

[0025] Again, on the other hand, the present invention provides a base editing system comprising a fusion protein or a polynucleotide encoding the fusion protein, wherein the fusion protein comprises a nucleic acid programmable DNA binding protein (napDNAbp) and a deaminase domain, and a guide polynucleotide, wherein the guide polynucleotide hybridizes with a complementary sequence of a target nucleic acid sequence, and the complementary sequence of the target nucleic acid sequence is selected from the following group: CAAGCTATCTTCTTAGGGA; GCAAGCTATCTTCTTAGGGAA; ACTTACGACAGGCTCGTGAA; TGACCAATTATTCCCTCAAG; AGTGACCAATTATTCCCTCA; ACTACAGTATTTGTAAACGA; AAGACAACGACACGCTGGTC; and GGCTGTTATGCACTGATCCG. In some embodiments, the fusion protein further comprises one or more uracil glycosylase inhibitors (UGIs). In some embodiments, the fusion protein further comprises one or more nuclear localization sequences (NLS).

[0026] In some embodiments, the base editing system can modify one or more single target nuclear bases to achieve at least one amino acid substitution in the CD117 polypeptide. In some embodiments, the single target nuclear base is cytosine (C), and wherein the modification comprises converting the C into thymine (T). In some embodiments, the single target nuclear base is adenosine (A), and wherein the modification comprises converting A into guanine (G). In some embodiments, the at least one amino acid substitution is a naturally occurring mutation. In some embodiments, the at least one amino acid substitution is located in domain 1, 2, 3, 4 or 5 of CD117. In some embodiments, the at least one amino acid substitution is selected from the group consisting of: T13A, S35P, I39V, H40R, K43G, K43R, S44P, D45G, I47V, D52G, E53G, I54V, R55G, L56P, L57P, T59A, F63P, V64A, K65E, K65R, W66R, T67A, D72G, E73G, T74A, N75D, N75G, E76G, N77G, N77S, N77Y, K78E, K79G, K80E, K81A, K82A, K83A, K84A, K86A, K87B, K88A, K89A, K90A, K91B, K92A, K93B, K94A, K95C, K96C, K97D, K98E, K99A, K91B 8R,Q79R,N80G,N80S,E81G,E81D,W82R,I83T,I83V,T84A,E85G,K86E,E88G,T90A,N99G,K100G,H101R,K116R,V120A, S123P,L124P,Y125H,K127G,K127R,E128G,D129G,D129E,N130G,N130D,D131G,D131N,T132A,T144A,N145D,N145G,N 145Y,N145S,Y146C,K149E,K149G,Q152R,K154E,K154G,R161G,F162P,F162L,I163T,I163V,D165G,M171T,I172T,I1 72V,K173G,S174G,K176G,Q190R,E191G,K193G,V195A,L196P,S197P,E198G,K199G,F200P,I201T,I201V,L202P,V21 3A,V214A,S215P,V216A,K218G,K218R,S220G,Y221C,E225G,E227G,E228G,T230A,S240G,Y243C,K247G,R248G,Q256 R,E257G,E257D,K258E,K258G,Y259C,Y259H,N260G,D266G,N268D,N268G,Y269C,T276A,I277T,I277V,S279P,R281G,V282A,S285P,N293G,N294G,T295A,F296P,S298P,N300G,N300S,T302A,T303A,T304 A,M318V,T322A,V323A,F324L,N326G,D327G,D332G,I334V,K342E,K342G,K342R,Q3 47R,Y350H,M351T,R353G,T354A,T354I,K358G,E360G,D361G,K364G,E366G,N367G, H378R,T380A,R381G,K383G,T385A,T389A,D398G,V399A,N400G,V407A,Y408H,E414 G,I415V,T417A,Y418C,Y418H,D419G,R420G,L421P,V422A,N423S,M425V,E435G,I4 38M,D439G,V454A,L455P,V457A,V459A,Q460R,T461A,N463G,S464P,S465P,F469P, K471E, K471G, L472P, V473A, Q475R, S476G, I478M, I478V, D479G, S481G, F483P, K484G, N486G, N486S, T488A, Y494C, N495D, D496G, K499E, Y503C, Y503H, F504P, and N505G. In some embodiments, the at least one amino acid substitution in CD117 is selected from the group consisting of T13A, I39V, H40R, D45G, D52G, E53G, I54V, R55G, T59A, K65E, K65R, T67A, E76G, N77G, N77S, N77Y, K78E, Q79R, N80G, E81G, E81D, N99G, K100G, H101R, L124P, Y125H, D129E, D129G, N130D, N130G, D131G, D131N, T132A, T14 4A,N145D,N145G,N145Y,N145S,Y146C,F162P,F162L,I163T,I163V,M171T,I172T,V195A,L196P,K199G,I201V,S220G,Y221C,Q256 R,E257D,E257G,K258E,Y259C,T303A,T304A,M318V,T322A,V323A,F324L,K342G,K342R,Y350H,M351T,R353G,T354A,T354I,R381G,K383G, T385A, Y418C, D419G, R420G, I438M, D439G, Q460R, T461A, K484G, N486G, and T488A. In some embodiments, the at least one amino acid substitution is a naturally occurring mutation selected from the group consisting of: T13A, N77S, D129E, N130D, D131N, T144A, Y221C, E257D, T322A, T354I, D419G.

[0027] In some embodiments, the napDNAbp is an inactivated nuclease or nickase variant. In some embodiments, the napDNAbp includes Cas9, Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i or Cas12j / CasΦ polynucleotides or a portion thereof. In some embodiments, the napDNAbp includes Cas9 polynucleotides or a portion thereof. In some embodiments, the napDNAbp includes inactivated Cas9 (dCas9) or Cas9 nickase (nCas9). In some embodiments, the napDNAbp is Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1Cas9 (St1Cas9), Streptococcus pyogenes Cas9 (SpCas9) or a variant thereof. In some embodiments, the napDNAbp includes a protospacer adjacent motif (PAM) specificity with a change. In some embodiments, the altered PAM is specific for the nucleic acid sequence 5'-NGC-3'. In some embodiments, the deaminase domain is capable of deaminating cytidine or adenine in DNA. In some embodiments, the deaminase domain is a cytidine deaminase domain. In some embodiments, the cytidine deaminase is an APOBEC deaminase or a derivative thereof. In some embodiments, the deaminase domain is an adenosine deaminase domain. In some embodiments, the adenosine deaminase domain is a TadA deaminase domain. In some embodiments, the adenosine deaminase is a TadA*8 variant. In some embodiments, the adenosine deaminase is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.21, TadA*8.22, TadA*8.23, or TadA*8.24. In some embodiments, the deaminase is a monomer or a heterodimer.

[0028] On the one hand, the present invention provides polynucleotides encoding any base editor system as provided herein. On the other hand, the present invention provides cells manufactured by any method provided herein. Again, on the one hand, the present invention provides cells manufactured by introducing any base editor system or any polynucleotide provided herein into a cell or its progenitor cell. In some embodiments, the cell is manufactured in vitro or in vitro. In some embodiments, the cell is a hematopoietic stem cell, a common myeloid progenitor cell, a proerythrocyte, or an erythrocyte. In some embodiments, the cell is a CD34+ cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell.

[0029] In one aspect, the invention provides isolated cells or populations of cells that have been proliferated or expanded as described herein.

[0030] In yet another aspect, the present invention provides a pharmaceutical composition comprising an effective amount of any of the cells provided herein.

[0031] On the other hand, the present invention provides a method for treating a subject suffering from a hemoglobinopathy, a blood cancer, or a myeloproliferative disease. In some embodiments, the method includes administering to the subject any pharmaceutical composition provided herein. In some embodiments, the method includes administering to the subject a conditioning regimen comprising an antibody, an antibody drug conjugate, or a chimeric antigen receptor expressing T cell, wherein the T cell selectively binds to a cell surface protein and any cell provided herein, thereby treating a hemoglobinopathy, a blood cancer, or a myeloproliferative disease. In some embodiments, the cell surface protein is a CD117 antibody. In some embodiments, the antibody, antibody drug conjugate, or chimeric antigen receptor expressing T cell selectively binds to domain 1, 2, 3, 4, or 5 of CD117. In some embodiments, the antibody is a monoclonal antibody. In some embodiments, the administration of the antibody is sequential or simultaneous. In some embodiments, the hemoglobinopathy is sickle cell disease or hereditary persistent fetal hemoglobin hyperlipidemia (HPFH). On the other hand, the present invention provides a method for treating a subject suffering from an immunodeficiency. In some embodiments, the immunodeficiency is severe combined immunodeficiency (SCID).

[0032] In some embodiments, the cells are autologous to the subject. In some embodiments, the cells are allogeneic to the subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is human.

[0033] In one aspect, the present invention provides a kit comprising any cell provided herein, any base editor system provided herein, any polynucleotide provided herein, or any pharmaceutical composition provided herein. In some embodiments, the kit includes written instructions for using the kit to treat a hemoglobinopathy, a blood cancer, a myeloproliferative disease, or an immunodeficiency.

[0034] The description and examples herein describe embodiments of the present disclosure in detail. Any composition or method provided herein can be combined with one or more of any other composition and method provided herein. It should be understood that the present disclosure is not limited to the specific embodiments described herein and therefore can change. Those skilled in the art will recognize that there are multiple variations and modifications to the present disclosure, and these variations and modifications are included within the scope thereof.

[0035] Unless otherwise indicated, the practice of some embodiments disclosed herein employs conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics, and recombinant DNA, which are within the skill of the art. See, for example, Sambrook and Green, Molecular Cloning: A Laboratory Manual, 4th Edition (2012); the series Current Protocols in Molecular Biology (FM Ausubel, et al. eds.); the series Methods In Enzymology (Academic Press, Inc.), PCR 2: A Practical Approach (MJ MacPherson, BD Hames and GR Taylor eds. (1995)), Harlow and Lane, eds. (1988) Antibodies, A Laboratory Manual, and Culture of Animal Cells: A Manual of Basic Technique and Specialized Applications, 6th Edition (RI Freshney, ed. (2010)).

[0036] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.

[0037] Although various features of the present disclosure may be described in the context of a single embodiment, these features may also be provided separately or in any suitable combination. Conversely, although the present disclosure may be described herein in the context of separate embodiments for clarity, the present disclosure may also be implemented in a single embodiment. The section headings used herein are for organizational purposes only and should not be construed as limiting the subject matter described.

[0038] The features of the present disclosure are set forth with particularity in the appended claims.A better understanding of the features and advantages of the present invention will be obtained by referring to the following detailed description which sets forth illustrative embodiments in which the principles of the present disclosure are utilized, and in view of the accompanying drawings as described below.

[0039] definition

[0040] The following definitions supplement those in the art and are specific to the current application and are not attributed to any related or unrelated cases, such as any commonly owned patents or applications. Although any methods and materials similar or equivalent to those described herein can be used to test the practice of the present disclosure, preferred materials and methods are described herein. Therefore, the terminology used herein is for the purpose of describing specific embodiments only and is not intended to be limiting.

[0041] Unless otherwise defined, all technical and scientific terms used herein have the meanings commonly understood by those skilled in the art to which the invention belongs. The following references provide general definitions of many of the terms used in the present invention for those skilled in the art: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). As used herein, the following terms have the meanings assigned to them below, unless otherwise indicated.

[0042] In this application, unless otherwise specifically stated, the use of the singular includes the plural. It must be noted that the singular forms "a," "an," and "the" used in this specification include plural referents unless the context clearly dictates otherwise. In this application, the use of "or" means "and / or" unless otherwise stated. In addition, the use of the term "including," as well as other forms such as "include," "includes," and "included," is not limiting.

[0043] As used in this specification and claims, the terms "comprising" (and any form of comprising, such as "comprise" and "comprises"), "having" (and any form of having, such as "have" and "has"), "including" (and any form of including, such as "includes" and "include"), or "containing" (and any form of containing, such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed in this specification can be implemented with respect to any method or combination of the present disclosure, and vice versa. In addition, the compositions of the present disclosure can be used to implement the methods of the present disclosure.

[0044] The term "about" or "approximately" means within an acceptable error range for a particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the measurement system. For example, according to the practice in the art, "about" can mean within 1 standard deviation or more than 1 standard deviation. Alternatively, "about" can mean a range of up to 20%, up to 10%, up to 5%, or up to 1% of a given value. Alternatively, particularly with respect to biological systems or processes, the term can mean within an order of magnitude, e.g., within 5-fold, within 2-fold, of a value. Where specific values ​​are described in the application and claims, the term "about" should be assumed to be within an acceptable error range for the specific value unless otherwise indicated.

[0045] References in the specification to "some embodiments," "an embodiment," "one embodiment," or "other embodiments" mean that a particular feature, structure, or characteristic described in connection with the examples is included in at least some, but not necessarily all, embodiments of the present disclosure.

[0046] "Adenosine deaminase" refers to a polypeptide or fragment thereof that can catalyze the hydrolytic deamination of adenine or adenosine. In some embodiments, the deaminase or deaminase domain is an adenosine deaminase that catalyzes the hydrolytic deamination of adenosine to inosine or the hydrolytic deamination of deoxyadenosine to deoxyinosine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenine or adenosine in deoxyribonucleic acid (DNA). The adenosine deaminase provided herein (e.g., engineered adenosine deaminase, evolved adenosine deaminase) can be from any organism, such as bacteria.

[0047] In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not exist in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring deaminase. In some embodiments, the adenosine deaminase is from a bacterium such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Clostridium crescentus. In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the TadA deaminase is an Escherichia coli TadA (ecTadA) deaminase or a fragment thereof.

[0048] For example, deaminase domains are described in International PCT Application Nos. PCT / 2017 / 045381 (WO 2018 / 027078) and PCT / US2016 / 058344 (WO 2017 / 070632), each of which is herein incorporated by reference in its entirety. Also see Komor, AC, et al., “Programmable base editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, AC, et al., “Improved base excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:Abase editors with higher efficiency and product purity” Science Advances 3:eaao4774(2017)), and Rees, HA, et al., “Base editing: precision chemistry on the genome and transcriptome of living cells." Nat Rev Genet. 2018 Dec; 19(12): 770-788. doi: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0049] Wild-type TadA (wt) adenosine deaminase has the following sequence (also referred to as the TadA reference sequence):

[0050] MSEVEFSHEYWMRHALTLAKRAWDEREVPVGAVLVHNNRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTLEPCVMCAGAMIHSRIGRVVFGARDAKTGAAGSLMDVLHHPGMNHRVEITEGILADECAALLSDFFRMRRQEIKAQKKAQSSTD.

[0051] In some embodiments, the adenosine deaminase comprises an alteration in the following sequence:

[0052] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0053] (also known as TadA*7.10).

[0054] In some embodiments, TadA*7.10 comprises at least one alteration. In some embodiments, TadA*7.10 comprises alterations at amino acids 82 and / or 166. In specific embodiments, variants of the above sequences comprise one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. The alteration Y123H refers to the reversion of the alteration H123Y in TadA*7.10 back to Y123H TadA (wt). In other embodiments, variants of the TadA*7.10 sequence comprise a combination of alterations selected from the following groups: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y12 3H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T16 6R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R and I76Y+V82S+Y123H+Y147R+Q154R.

[0055] In other embodiments, the present invention provides adenosine deaminase variants comprising deletions, such as TadA*8, comprising a C-terminal deletion starting at residues 149, 150, 151, 152, 153, 154, 155, 156, or 157, relative to the corresponding mutations in TadA*7.10, a TadA reference sequence, or another TadA. In other embodiments, the adenosine deaminase variant is a TadA (e.g., TadA*8) monomer comprising one or more of the following alterations: Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R, relative to the corresponding mutations in TadA*7.10, a TadA reference sequence, or another TadA. In other embodiments, the adenosine deaminase variant is a TadA (e.g., TadA*8) monomer comprising the following changes: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y123H+Y147R; V8 2S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R and I76Y+V82S+Y123H+Y147R+Q154R, relative to the corresponding mutations in TadA*7.10, the TadA reference sequence or another TadA 7.

[0056] In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains, each domain having one or more of the following changes Y147T, Y147R, Q154S, Y123H, V82S, T166R and / or Q154R relative to the corresponding mutations in TadA*7.10, a TadA reference sequence, or another TadA. In other embodiments, the adenosine deaminase variant is a homodimer comprising two adenosine deaminase domains (e.g., TadA*8), each domain having a combination of alterations selected from the following groups: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Y12 3H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R and I76Y+V82S+Y123H+Y147R+Q154R, relative to the corresponding mutations in TadA*7.10, the TadA reference sequence or another TadA 7.

[0057] In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type TadA adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising one or more of the following changes Y147T, Y147R, Q154S, Y123H, V82S, T166R and / or Q154R relative to the corresponding mutations in TadA*7.10, a TadA reference sequence, or another TadA. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a wild-type TadA adenosine deaminase domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising a combination of changes selected from the following groups: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+Q154S; 2S+Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R and I76Y+V82S+Y123H+Y147R+Q154R, relative to the corresponding mutations in TadA*7.10, the TadA reference sequence or another TadA 7.

[0058] In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising one or more of the following changes Y147T, Y147R, Q154S, Y123H, V82S, T166R and / or Q154R relative to the corresponding mutations in TadA*7.10, a TadA reference sequence, or another TadA. In other embodiments, the adenosine deaminase variant is a heterodimer comprising a TadA*7.10 domain and an adenosine deaminase variant domain (e.g., TadA*8) comprising a combination of alterations selected from the group consisting of: Y147T+Q154R; Y147T+Q154S; Y147R+Q154S; V82S+Q154S; V82S+Y147R; V82S+Q154R; V82S+Y123H; I76Y+V82S; V82S+Y123H+Y147T; V82S+ Y123H+Y147R; V82S+Y123H+Q154R; Y147R+Q154R+Y123H; Y147R+Q154R+I76Y; Y147R+Q154R+T166R; Y123H+Y147R+Q154R+I76Y; V82S+Y123H+Y147R+Q154R or I76Y+V82S+Y123H+Y147R+Q154R, relative to the corresponding mutations in TadA*7.10, a TadA reference sequence or another TadA 7. In one embodiment, the adenosine deaminase is TadA*8 comprising or consisting essentially of the following sequence or a fragment thereof having adenosine deaminase activity:

[0059] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD.

[0060] In some embodiments, the TadA*8 is truncated. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 N-terminal amino acid residues relative to full-length TadA*8. In some embodiments, the truncated TadA*8 lacks 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 C-terminal amino acid residues relative to full-length TadA*8. In some embodiments, the adenosine deaminase variant is full-length TadA*8.

[0061] In certain embodiments, the adenosine deaminase heterodimer comprises a TadA*8 domain and an adenosine deaminase domain selected from:

[0062] Staphylococcus aureus (S. aureus) TadA:

[0063] MGSHMTNDIYFMTLAIEEAKKAAQLGEVPIGAIITKDDEVIARAHNLRETLQQPTAHAEHIAIERAAKVLGSWRLEGCTLYVTLEPCVMCAGTIVMSRIPRVVYGADDPKGGCSGSLMNLLQQSNFNHRAIVDKGVLKEACSTLLTTFFKNLRANKKSTN

[0064] Bacillus subtilis TadA:

[0065] MTQDELYMKEAIKEAKKAEEKGEVPIGAVLVINGEIIARAHNLRETEEQRSIAHAEMLVIDEACKALGTWRLEGATLYVTLEPCPMCAGAVVLSRVEKVVFGAFDPKGGCSGTLMNLLQEERFNHQAEVVSGVLEEECGGMLSAFFRELRKKKKAARKNLSE

[0066] Salmonella typhimurium TadA:

[0067] MPPAFITGVTSLSDVELDHEYWMRHALTLAKRAWDEREVPVGAVLVHNHRVIGEGWNRPIGRHDPTAHAEIMALRQGGLVLQNYRLLDTTLYVTLEPCVMCAGAMVHSRIGRVVFGARDAKTGAAGSLIDVLHHPGMNHRVEIIEGVLRDECATLLSDFFRMRRQEIKALKKADRAEGAGPAV Shewanella putrefaciens TadA:

[0068] MDEYWMQVAMQMAEKAEAAGEVPVGAVLVKDGQQIATGYNLSISQHDPTAHAEILCLRSAGKKLENYRLLDATLYITLEPCAMCAGAMVHSRIARVVYGARDEKTGAAGTVVNLLQHPAFNHQVEVTSGVLAEACSAQLSRFFKRRRDEKKALKLAQRAQQGIE

[0069] Haemophilus influenzae F3031 TadA:

[0070] MDAAKVRSEFDEKMMRYALELADKAEALGEIPVGAVLVDDARNIIGEGWNLSIVQSDPTΑΗAEIIALRNGAKNIQNYRLLNSTLYVTLEPCTMCAGAILHSRIKRLVFGASDYKTGAIGSRFHFFDDYKMNHTLEITSGVLAEECSQKLSTFFQKRREEKKIEKALLKSLSDK

[0071] Caulobacter crescentus TadA:

[0072] MRTDESEDQDHRMMRLALDAARAAAEAGETPVGAVILDPSTGEVIATAGNGPIAAHDPTAHAEIAAMRAAAAKLGNYRLTDLTLVVTLEPCAMCAGAISHARIGRVVFGADDPKGGAVVHGPKFFAQPTCHWRPEVTGGVLADESADLLRGFFRARRKAKI

[0073] Geobacter sulfurreducens TadA:

[0074] MSSLKKTPIRDDAYWMGKAIREAAKAAARDEVPIGAVIVRDGAVIGRGHNLREGSNDPSAHAEMIAIRQAARRSANWRLTGATLYVTLEPCLMCMGAIILARLERVVFGCYDPKGGAAGSLYDLSADPRLNHQVRLSPGVCQEECGTMLSDFFRDLRRRKKAKATPALFIDERKVPPEP

[0075] TadA*7.10

[0076] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD.

[0077] An “adenosine deaminase base editor 8 (ABE8) polypeptide” or “ABE8” refers to a base editor as defined herein comprising an adenosine deaminase variant comprising an alteration at amino acid position 82 and / or 166 of the following reference sequence:

[0078] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD

[0079] In some embodiments, ABE8 comprises further alterations relative to the reference sequence, as described herein.

[0080] "Adenosine deaminase base editor 8 (ABE8) polypeptide" refers to a polynucleotide encoding ABE8.

[0081] "Administering" herein refers to providing one or more compositions described herein to a patient or subject. For example, but not limited to, administering a composition, such as by injection, can be by intravenous (iv), subcutaneous (sc), intradermal (id), intraperitoneal (ip), or intramuscular (im) injection. One or more of these routes can be used. Parenteral administration can be, for example, by bolus injection or gradual infusion over time. Alternatively, or concurrently, administration can be by oral route.

[0082] "Agent" refers to any small molecule compound, antibody, nucleic acid molecule or polypeptide, or fragments thereof.

[0083] "Alteration" refers to a change (increase or decrease) in the structure, expression level, or activity of a gene or polypeptide as detected by standard art-known methods, such as those described herein. As used herein, alteration includes a 10% change in expression level, a 25% change, a 40% change, and a 50% or greater change in expression level.

[0084] "Ameliorate" means to reduce, inhibit, attenuate, reduce, arrest, or stabilize the development or progression of a disease.

[0085] An "analog" is a molecule that is not identical but has similar functional or structural features. For example, a polypeptide analog retains the biological activity of the corresponding naturally occurring polypeptide while having certain biochemical modifications that enhance the analog's function relative to the naturally occurring polypeptide. Such biochemical modifications may increase the analog's protease resistance, membrane permeability, or half-life without altering, for example, ligand binding. Analogs may include unnatural amino acids.

[0086] As used herein, the term "antibody" refers to an immunoglobulin molecule that specifically binds to a specific antigen or immunoreacts with a specific antigen, including polyclonal, monoclonal, genetically engineered and other modified forms of antibodies, including but not limited to chimeric antibodies, humanized antibodies, heterologous conjugate antibodies (e.g., bispecific, trispecific and tetraspecific antibodies, bispecific, trispecific and tetraspecific antibodies) and antigen-binding fragments of antibodies, including, for example, Fab', F(ab')2, Fab, Fv, rIgG and scFv fragments. Unless otherwise indicated, the term "monoclonal antibody" (mAb) includes both complete molecules and antibody fragments (including, for example, Fab and F(ab')2 fragments) that can specifically bind to a target protein. As used herein, Fab and F(ab')2 fragments refer to antibody fragments that lack the Fc fragment of a complete antibody. Examples of these antibody fragments are described herein.

[0087] "Base editor (BE)" or "nucleobase editor polypeptide (NBE)" refers to an agent that binds to a polynucleotide and has nucleobase modification activity. In various embodiments, the base editor comprises a nucleobase modification polypeptide (e.g., a deaminase) and a polynucleotide programmable nucleotide binding domain bound to a guide polynucleotide (e.g., a guide RNA). In various embodiments, the agent is a biomolecule complex comprising a protein domain with base editing activity, i.e., capable of modifying bases (e.g., DNA) within a nucleic acid molecule (e.g., A, T, C, G, or U). In some embodiments, the polynucleotide programmable DNA binding domain is fused or connected to a deaminase domain. In one embodiment, the agent is a fusion protein comprising one or more domains with base editing activity. In another embodiment, the protein domain with base editing activity is connected to a guide RNA (e.g., through an RNA binding motif on a guide RNA and an RNA binding domain fused to a deaminase). In some embodiments, the domain with base editing activity is capable of deaminating bases within a nucleic acid molecule. In some embodiments, the base editor is capable of deaminating one or more bases within a DNA molecule. In some embodiments, the base editor is capable of deaminating cytosine (C) or adenosine (A) in DNA. In some embodiments, the base editor is capable of deaminating cytosine (C) and adenosine (A) in DNA. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenosine base editor (ABE). In some embodiments, the base editor is an adenosine base editor (ABE) and a cytidine base editor (CBE). In some embodiments, the base editor is a nuclease-free Cas9 (dCas9) fused to an adenosine deaminase. In some embodiments, Cas9 is a circular replacement Cas9 (e.g., spCas9 or saCas9). Circular replacement Cas9s are known in the art and are described in, for example, Oakes et al., Cell 176, 254–267, 2019. In some embodiments, the base editor is fused to a base excision repair inhibitor, such as a UGI domain or a dISN domain. In some embodiments, the fusion protein comprises a Cas9 nickase and a base excision repair inhibitor, such as a UGI or dISN domain, fused to a deaminase. In other embodiments, the base editor is a base-free base editor.

[0088] In some embodiments, adenosine deaminase is evolved from TadA. In some embodiments, the polynucleotide programmable DNA binding domain is a CRISPR-related (e.g., Cas or Cpf1) enzyme. In some embodiments, the base editor is a catalytically inactive Cas9 (dCas9) fused to a deaminase domain. In some embodiments, the base editor is a Cas9 nickase (nCas9) fused to a deaminase domain. In some embodiments, the base editor is fused to a base excision repair inhibitor (BER). In some embodiments, the base excision repair inhibitor is a uracil DNA glycosylase inhibitor (UGI). In some embodiments, the base excision repair inhibitor is an inosine base excision repair inhibitor. The details of the base editor are described in International PCT Application Nos. PCT / 2017 / 045381 (WO2018 / 027078) and PCT / US2016 / 058344 (WO2017 / 070632), each of which is incorporated herein by reference in its entirety. Also see Komor, AC, et al., “Programmable base editing of a target base in genomic DNA without double-stranded DNA cleavage” Nature 533, 420-424 (2016); Gaudelli, NM, et al., “Programmable base editing of A·T to G·C in genomic DNA without DNA cleavage” Nature 551, 464-471 (2017); Komor, AC, et al., "Improved base excisionrepair inhibition and bacteriophage Mu Gam protein yields C:G-to-T:A baseeditors with higher efficiency and product purity" Science Advances3:eaao4774 (2017), and Rees, HA, et al., "Base editing: precision chemistry on the genome and transcriptome of living cells." Nat Rev Genet. 2018 Dec; 19(12): 770-788. doi: 10.1038 / s41576-018-0059-1, the entire contents of which are incorporated herein by reference.

[0089] In some embodiments, the base editor is generated by cloning an adenosine deaminase variant (e.g., TadA*8) into a scaffold comprising a circularly permuted Cas9 (e.g., saCAS9) and a bipartite nuclear localization sequence (e.g., ABE8). Circularly permuted Cas9s are known in the art and described, for example, in Oakes et al., Cell 176, 254–267, 2019. Exemplary circularly permuted sequences are as follows, where the bold sequences represent sequences derived from Cas9, the italic sequences represent linker sequences, and the underlined sequences represent the bipartite nuclear localization sequence.

[0090] CP5 (with MSP “NGC = Pam variant conventional Cas9-like NGG with mutations” PID = Protein Interaction Domain and “D10A” nickase):

[0091]

[0092]

[0093] In some embodiments, the ABE8 is selected from the base editors of Tables 10, 11 or 13 below. In some embodiments, ABE8 contains an adenosine deaminase variant evolved from TadA. In some embodiments, the adenosine deaminase variant of the ABE8 is the TadA*8 variant as described in Tables 8, 10, 11 or 13 below. In some embodiments, the adenosine deaminase variant is a TadA*7.10 variant (e.g., TadA*8) comprising one or more alterations selected from the group consisting of Y147T, Y147R, Q154S, Y123H, V82S, T166R, and / or Q154R. In various embodiments, ABE8 comprises a TadA*7.10 variant (e.g., TadA*8) having a combination of alterations selected from the group: Y147T + Q154R; Y147T + Q154S; Y147R + Q154S; V82S + Q154S; V82S + Y147R; V82S + Q154R; V82S + Y123H; I76Y + V82S; V82S + Y123H + Y147T; V82S + Y123H + Y147R; V82S + Y123H + Q154R; Y147R + Q154R + Y123H; Y147R + Q154R + I76Y; Y147R + Q154R + T166R; Y123H + Y147R + Q154R + I76Y; V82S + Y123H + Y147R + Q154R; and I76Y + V82S + Y123H + Y147R + Q154R.

[0094] In some embodiments, ABE8 is a monomeric construct. In some embodiments, ABE8 is a heterodimeric construct. In some embodiments, the ABE8 base editor comprises the sequence:

[0095] MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCTFFRMPRQVFNAQKKAQSSTD

[0096] For example, the adenine base editor ABE used in the base editing compositions, systems, and methods described herein has a nucleic acid sequence (8877 base pairs) (Addgene, Watertown, MA.; Komor NM et al., 2017, Sci Adv., 30; 3(8): 2017 Nov 23; 551(7681): 464-471. doi: 10.1038 / nature24644; Koblan LW, et al., Nat Biotechnol. 2018 Oct; 36(9): 843-846. doi: 10.1038 / nbt.4172.) is provided below. Also included are polynucleotide sequences having at least 95% or greater identity to the ABE nucleic acid sequence.

[0097]

[0098] For example, the cytidine base editor (CBE) used in the base editing compositions, systems, and methods described herein has the following nucleic acid sequence (8877 base pairs) (Addgene, Watertown, MA; Komor AC et al., 2017, Sci Adv., 30; 3(8):eaao4774. doi: 10.1126 / sciadv.aao4774) provided below. Also included are polynucleotide sequences having at least 95% or greater identity to the BE4 nucleic acid sequence.

[0099]

[0100]

[0101]

[0102] In some embodiments, the cytidine base editor is BE4 having a nucleic acid sequence selected from one of the following:

[0103] Original BE4 nucleic acid sequence:

[0104]

[0105] BE4 codon optimized 1 nucleic acid sequence:

[0106]

[0107] BE4 codon optimized 2 nucleic acid sequence:

[0108]

[0109] "Base editing activity" refers to an action to chemically change a base within a polynucleotide. In one embodiment, a first base is converted to a second base. In one embodiment, the base editing activity is cytidine deaminase activity, for example, converting the target C·G to T·A. In another embodiment, the base editing activity is adenosine or adenine deaminase activity, for example, converting the target A·T to C·G. In another embodiment, the base editing activity is cytidine deaminase activity, for example, converting the target C·G to T·A, and adenosine or adenine deaminase activity, for example, converting A·T to G·C.

[0110] The term "base editor system" refers to a system for editing the nucleobases of a target nucleotide sequence. In various embodiments, the base editor (BE) system comprises (1) a polynucleotide programmable nucleotide binding domain, a deaminase domain (e.g., cytidine deaminase or adenosine deaminase) for deaminating the nucleobases in the target nucleotide sequence; (2) one or more guide polynucleotides (e.g., guide RNA) bound to the polynucleotide programmable nucleotide binding domain. In various embodiments, the base editor (BE) system comprises a nucleobase editor domain selected from adenosine deaminase or cytidine deaminase, and a domain with nucleic acid sequence-specific binding activity. In some embodiments, the base editor system comprises (1) a base editor (BE) comprising a polynucleotide programmable DNA binding domain and a deaminase domain for deaminating one or more nucleobases in a target nucleotide sequence; and (2) one or more guide RNAs bound to the polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable acid binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the base editor is a cytidine base editor (CBE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE). In some embodiments, the base editor is an adenine or adenosine base editor (ABE) or a cytidine base editor (CBE).

[0111] The term "Cas9" or "Cas9 domain" refers to an RNA-guided nuclease comprising a Cas9 protein or a fragment thereof (e.g., a protein comprising an active, inactive, or partially active DNA cleavage domain of Cas9, and / or a gRNA binding domain of Cas9). Cas9 nucleases are also sometimes referred to as casn1 nucleases or CRISPR (clustered regularly interspaced short palindromic repeats)-associated nucleases. An exemplary Cas9 is Streptococcus pyogenes Cas9 (spCas9), the amino acid sequence of which is provided below:

[0112] (Single underline: HNH domain; double underline: RuvC domain)

[0113] The term "conservative amino acid substitution" or "conservative mutation" refers to the replacement of an amino acid with another amino acid having common properties. A functional approach to defining common properties between individual amino acids is to analyze the normalized frequency of amino acid changes between corresponding proteins from homologous organisms (Schulz, GE and Schirmer, RH, Principles of Protein Structure, Springer-Verlag, New York (1979)). Based on such an analysis, groups of amino acids can be defined in which the amino acids within a group are preferentially interchangeable with each other and are therefore most similar to each other in terms of their effect on the overall protein structure (Schulz, GE and Schirmer, RH, supra). Non-limiting examples of conservative mutations include amino acid substitutions of amino acids, such as lysine for arginine and vice versa, to maintain a positive charge; glutamic acid for aspartic acid and vice versa, to maintain a negative charge; serine for threonine, to maintain a free -OH; and glutamine for asparagine, to maintain a free -NH2.

[0114] As used interchangeably herein, the terms "coding sequence" or "protein coding sequence" refer to a polynucleotide segment that encodes a protein. A coding sequence may also be referred to as an open reading frame. The region or sequence has a start codon near the 5' end and a stop codon near the 3' end. Useful stop codons for the base editors described herein include the following:

[0115]

[0116] As used herein, the terms "conditioning" and "conditioning" refer to the process in which a patient is prepared to receive a transplant containing hematopoietic stem cells. Such a procedure promotes the implantation of hematopoietic stem cell transplants (e.g., inferred from the continued increase in the number of live hematopoietic stem cells in blood samples isolated from patients who have undergone a conditioning procedure and subsequent hematopoietic stem cell transplant). According to the methods described herein, the patient can be conditioned for hematopoietic stem cell transplantation by administering antibodies or antigen-binding fragments thereof that can bind to antigens expressed by hematopoietic stem cells, such as CD117, CXCR4, CD135, CD90, CD45, and / or CD34. It is expected that such antibodies act by complement-mediated cytotoxicity and antibody-dependent cell-mediated cytotoxicity. As described herein, the transplanted cells have been edited so that the antibody no longer binds to antigens (e.g., CD117, CXCR4, CD135, CD90, CD45, and / or CD34). Administration of antibodies, antigen-binding fragments thereof, drug-antibody conjugates, or chimeric antigen receptor-expressing T cells (CAR-T) that bind to one or more antigens (e.g., CD117, CXCR4, CD135, CD90, CD45, CD34) to patients in need of hematopoietic stem cell transplantation can promote the engraftment of hematopoietic stem cell transplants, for example, by selectively depleting endogenous hematopoietic stem cells, thereby creating a gap that is filled by an exogenous hematopoietic stem cell transplant.

[0117] "Cytidine deaminase" refers to a polypeptide or fragment thereof that can catalyze a deamination reaction that converts an amino group to a carbonyl group. In one embodiment, the cytidine deaminase converts cytosine to uracil or converts 5-methylcytosine to thymine. PmCDA1 from sea lamprey (Petromyzon marinus cytosine deaminase 1), AID (activation-induced cytidine deaminase; AICDA) from mammals (e.g., humans, pigs, cattle, horses, monkeys, etc.), and APOBEC are exemplary cytidine deaminases.

[0118] As used herein, the term "deaminase" or "deaminase domain" refers to a protein or enzyme that catalyzes a deamination reaction. In some embodiments, the deaminase or deaminase domain is a cytidine deaminase, which catalyzes the hydrolytic deamination of cytidine or deoxycytidine to uridine or deoxyuridine, respectively. In some embodiments, the deaminase or deaminase domain is a cytosine deaminase, which catalyzes the hydrolytic deamination of cytosine to uracil. In some embodiments, the deaminase is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenine to hypoxanthine. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine or adenine (A) to inosine (I). In some embodiments, the deaminase or deaminase domain is an adenosine deaminase, which catalyzes the hydrolytic deamination of adenosine or deoxyadenosine to inosine or deoxyinosine, respectively. In some embodiments, the adenosine deaminase catalyzes the hydrolytic deamination of adenosine in deoxyribonucleic acid (DNA). Adenosine deaminases (e.g., engineered adenosine deaminases, evolved adenosine deaminases) provided herein can be from any organism, such as bacteria. In some embodiments, the adenosine deaminase is from bacteria, such as Escherichia coli, Staphylococcus aureus, Salmonella typhimurium, Shewanella putrefaciens, Haemophilus influenzae, or Clostridium crescentum. In some embodiments, the adenosine deaminase is a TadA deaminase. In some embodiments, the deaminase or deaminase domain is a variant of a naturally occurring deaminase from an organism such as a human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse. In some embodiments, the deaminase or deaminase domain does not exist in nature. For example, in some embodiments, the deaminase or deaminase domain is at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.1%, at least 99.2%, at least 99.3%, at least 99.4%, at least 99.5%, at least 99.6%, at least 99.7%, at least 99.8%, or at least 99.9% identical to a naturally occurring deaminase.

[0119] "Detection" refers to identifying the presence, absence, or amount of an analyte to be detected. In one embodiment, sequence changes in a polynucleotide or polypeptide are detected. In another embodiment, the presence of an indel is detected.

[0120] "Detectable label" refers to a composition that, when attached to a molecule of interest, renders the latter detectable by spectroscopic, photochemical, biochemical, immunochemical, or chemical means. For example, useful labels include radioisotopes, magnetic beads, metal beads, colloidal particles, fluorescent dyes, electron-dense reagents, enzymes (e.g., as commonly used in enzyme-linked immunosorbent assays (ELISAs)), biotin, digoxigenin, or haptens.

[0121] "Disease" refers to any condition or disorder that damages or interferes with the normal function of a cell, tissue, or organ. Exemplary diseases include those amenable to treatment with hematopoietic stem cell transplantation, such as thalassemia, sickle cell disease (SCD), or adenosine deaminase deficiency.

[0122] An “effective amount” refers to the amount of an agent or active compound (such as a base editor described herein) required to improve the symptoms of a disease relative to an untreated patient or an individual without the disease (ie, a healthy person), or the amount of an agent or active compound sufficient to induce a desired biological response. The effective amount of the active compound used to practice the present invention to treat a disease varies according to the mode of administration, the age, weight, and general health of the subject. Ultimately, the attending physician or veterinarian will determine the appropriate amount and dosage regimen. This amount is referred to as an “effective” amount. In one embodiment, the effective amount is the amount of the base editor of the present invention sufficient to introduce a change in a gene of interest into a cell (eg, a cell in vitro or in vivo). In one embodiment, the effective amount is the amount of the base editor required to achieve a therapeutic effect. This therapeutic effect does not need to be sufficient to change the pathogenic genes in all cells of the subject, tissue, or organ, but only needs to change the pathogenic genes present in about 1%, 5%, 10%, 25%, 50%, 75% or more cells in the subject, tissue, or organ. In one embodiment, the effective amount is sufficient to improve one or more symptoms of the disease.

[0123] In some embodiments, an effective amount of a fusion protein provided herein, such as an effective amount of a base editor comprising an nCas9 domain and a deaminase domain (e.g., adenosine deaminase, cytidine deaminase), refers to an amount sufficient to induce editing of a target site that is specifically bound and edited by a base editor as described herein. As will be appreciated by those skilled in the art, the effective amount of an agent (e.g., a fusion protein) can vary according to various factors, for example, according to the desired biological response, for example, according to the specific allele, genome, or target site being edited, according to the cell or tissue being targeted, and / or according to the agent being used.

[0124] In some embodiments, an effective amount of a fusion protein provided herein, e.g., an effective amount of a fusion protein comprising an nCas9 domain and a deaminase domain, can refer to an amount of the fusion protein sufficient to induce editing of a target site that is specifically bound and edited by the fusion protein described herein. As will be appreciated by those skilled in the art, the effective amount of an agent (e.g., a fusion protein, a nuclease, a hybrid protein, a protein dimer, a complex of a protein complex (or protein dimer) and a polynucleotide, or a polynucleotide) can vary depending on various factors, e.g., depending on the desired biological response, e.g., depending on the specific allele, genome, or target site being edited, depending on the cell or tissue being targeted, and / or depending on the agent being used.

[0125] "Fragment" refers to a portion of a polypeptide or nucleic acid molecule. The portion comprises at least about 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or 90% of the full length of a reference nucleic acid molecule or polypeptide. A fragment can comprise 10, 20, 30, 40, 50, 60, 70, 80, 90, or 100, 200, 300, 400, 500, 600, 700, 800, 900, or 1000 nucleotides or amino acids.

[0126] "Guide RNA" or "gRNA" refers to a polynucleotide that is specific for a target sequence and can form a complex with a polynucleotide programmable nucleotide binding domain protein (e.g., Cas9 or Cpf1). In one embodiment, the guide polynucleotide is a guide RNA (gRNA). The gRNA can exist as a complex of two or more RNAs or as a single RNA molecule. A gRNA that exists as a single RNA molecule may be referred to as a single guide RNA (sgRNA), but "gRNA" is used interchangeably to refer to a guide RNA that exists as a single molecule or a complex of two or more molecules. Typically, a gRNA that exists as a single RNA species contains two domains: (1) a domain that has homology to the target nucleic acid (e.g., guides the binding of the Cas9 complex to the target); and (2) a domain that binds to the Cas9 protein. In some embodiments, domain (2) corresponds to a sequence called tracrRNA and contains a stem-loop structure. For example, in some embodiments, domain (2) is identical or homologous to the tracrRNA provided in Jinek et al., Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Other examples of gRNAs (e.g., those comprising domain 2) can be found in “Switchable Cas9 Nucleases and Uses Thereof” (US20160208288) and “Delivery System For Functional Nucleases” (US 9,737,604), the entire contents of which are incorporated herein by reference in their entirety. In some embodiments, a gRNA comprises two or more of domains (1) and (2) and can be referred to as an “extended gRNA”. As described herein, an extended gRNA will bind to two or more Cas9 proteins and bind to a target nucleic acid at two or more different regions. The gRNA comprises a nucleotide sequence complementary to the target site that mediates the binding of the nuclease / RNA complex to the target site, providing sequence specificity of the nuclease:RNA complex.

[0127] As used herein, the term "hematopoietic stem cell" ("HSC") refers to immature blood cells with the ability to self-renew and differentiate into mature blood cells, including but not limited to granulocytes (e.g., promyelocytes, neutrophils), eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), platelets (e.g., megakaryocytes, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (e.g., NK cells, B cells, and T cells). Such cells can include CD34+ cells. CD34+ cells are immature cells that express the CD34 cell surface marker. In humans, CD34+ cells are considered to include a subpopulation of cells having the above-mentioned stem cell properties, while in mice, HSC is CD34-. In addition, HSC also refers to long-term repopulation HSC (LT-HSC) and short-term repopulation HSC (ST-HSC). LT-HSCs and ST-HSCs are distinguished based on their functional potential and expression of cell surface markers. For example, human HSCs are CD34+, CD38-, CD45RA-, CD90+, CD49F+, and lin- (negative for mature lineage markers, including CD2, CD3, CD4, CD7, CD8, CD10, CD11B, CD19, CD20, CD56, and CD235A). In mice, bone marrow LT-HSCs are CD34-, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, CD48-, and lin- (negative for mature lineage markers, including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, and IL7ra), while ST-HSCs are CD34+, SCA-1+, C-kit+, CD135-, Slamfl / CD150+, and lin- (negative for mature lineage markers, including Ter119, CD11b, Gr1, CD3, CD4, CD8, B220, and IL7ra). Furthermore, under homeostatic conditions, ST-HSCs are less quiescent and more proliferative than LT-HSCs. However, LT-HSCs have greater self-renewal potential (i.e., they can survive throughout adulthood and can be serially transplanted into successive recipients), whereas ST-HSCs have limited self-renewal (i.e., they can only survive for a limited time and do not have serial transplantation potential). Any of these HSCs can be used in the methods described herein. ST-HSCs are particularly useful because they are highly proliferative and can therefore generate differentiated progeny more quickly.

[0128] As used herein, the term "hematopoietic stem cell functional potential" refers to the functional properties of hematopoietic stem cells, including 1) multipotency (referring to the ability to differentiate into multiple different blood lineages, including but not limited to granulocytes (e.g., promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (e.g., reticulocytes, erythrocytes), platelets (e.g., megakaryocytes, platelet-producing megakaryocytes, platelets), monocytes (e.g., monocytes, macrophages), dendritic cells, microglia, osteoclasts, and lymphocytes (such as NK cells, B cells and T cells), 2) self-renewal (referring to the ability of hematopoietic stem cells to produce daughter cells with equal potential as the parent cell, and this ability can be repeatedly presented throughout the life cycle of an individual without exhaustion), and 3) the ability of hematopoietic stem cells or their progeny to be reintroduced into a transplant recipient, where they then return to the hematopoietic stem cell niche and re-establish productive and sustained hematopoiesis.

[0129] "Heterodimer" refers to a fusion protein comprising two domains, such as a wild-type TadA domain and a variant TadA domain (e.g., TadA*8) or two variant TadA domains (e.g., TadA*7.10 and TadA*8 or two TadA*8 domains).

[0130] "Hybridization" refers to hydrogen bonding between complementary nucleobases, which can be Watson-Crick, Hoogsteen or reversed Hoogsteen hydrogen bonding. For example, adenine and thymine are complementary nucleobases that pair by forming hydrogen bonds.

[0131] By "increase" is meant a positive change of at least 10%, 25%, 50%, 75% or 100%.

[0132] The terms "inhibitor of base repair," "base repair inhibitor," "IBR," or other grammatical equivalents refer to proteins that can inhibit the activity of nucleic acid repair enzymes, such as base excision repair enzymes. In some embodiments, the IBR is an inhibitor of inosine base excision repair. Exemplary inhibitors of base repair include inhibitors of APE1, Endo III, Endo IV, Endo V, Endo VIII, Fpg, hOGG1, hNEIL1, T7 Endol, T4PDG, UDG, hSMUG1, and hAAG. In some embodiments, the base repair inhibitor is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is an inhibitor of Endo V or hAAG. In some embodiments, the IBR is catalytically inactive EndoV or catalytically inactive hAAG. In some embodiments, the base repair inhibitor is catalytically inactive EndoV or catalytically inactive hAAG. In some embodiments, the base repair inhibitor is a uracil glycosylase inhibitor (UGI). UGI refers to a protein that can inhibit uracil-DNA glycosylase base excision repair enzymes. In some embodiments, the UGI domain comprises a wild-type UGI or a fragment of wild-type UGI. In some embodiments, the UGI protein provided herein includes a fragment of UGI and a protein homologous to UGI or a UGI fragment. In some embodiments, the base repair inhibitor is an inhibitor of inosine base excision repair. In some embodiments, the base repair inhibitor is a "catalytically inactive inosine-specific nuclease" or "dead inosine-specific nuclease." Without wishing to be bound by any particular theory, a catalytically inactive inosine glycosylase (e.g., alkyladenine glycosylase (AAG)) can bind to inosine but cannot create a baseless site or remove inosine, thereby sterically blocking the newly formed inosine moiety from DNA damage / repair mechanisms. In some embodiments, a catalytically inactive inosine-specific nuclease is able to bind to inosine in a nucleic acid but does not cleave the nucleic acid. Non-limiting exemplary catalytically inactive inosine-specific nucleases include catalytically inactive alkyladenine glycosylase (AAG nuclease), e.g., from human, and catalytically inactive endonuclease V (EndoV nuclease), e.g., from Escherichia coli. In some embodiments, the catalytically inactive AAG nuclease comprises an E125Q mutation or a corresponding mutation in another AAG nuclease.

[0133] An "intein" is a protein fragment that can self-excise and connect the remaining fragment (extein) with a peptide bond in a process called protein splicing. Inteins are also called "protein introns." The process in which an intein self-excises and connects the remaining part of a protein is referred to herein as "protein splicing" or "intein-mediated protein splicing." In some embodiments, the intein of a precursor protein (a protein containing an intein before intein-mediated protein splicing) comes from two genes. Such inteins are referred to herein as split inteins (e.g., split intein-N and split intein-C). For example, in cyanobacteria, DnaE of the catalytic subunit a of DNA polymerase III is encoded by two independent genes, dnaE-n and dnaE-c. The intein encoded by the dnaE-n gene may be referred to herein as "intein-N." The intein encoded by the dnaE-c gene may be referred to herein as "intein-C."

[0134] Other intein systems can also be used. For example, synthetic inteins based on the dnaE intein, Cfa-N (e.g., split intein-N), and Cfa-C (e.g., split intein-C) intein pairs have been described (e.g., in Stevens et al., J Am Chem Soc. 2016 Feb. 24; 138(7):2162-5, incorporated herein by reference). Non-limiting examples of intein pairs that can be used in accordance with the present disclosure include: Cfa DnaE intein, Ssp GyrB intein, Ssp DnaX intein, Ter DnaE3 intein, Ter ThyX intein, Rma DnaB intein, and Cne Prp8 intein (e.g., as described in U.S. Pat. No. 8,394,604, incorporated herein by reference).

[0135] Exemplary nucleotide and amino acid sequences of inteins are provided.

[0136] DnaE intein-N DNA: TGCCTGTCATACGAAACCGAGATACTGACAGTAGAATATGGCCTTCTGCCAATCGGGAAGATTGTGGAGAAACGGATAGAATGCACAGTTTACTCTGTCGATAACAATGGTAACATTTATACTCAGCCAGTTGCCCAGTGGCACGACCGGGGAGAGCAGGAAGTATTCGAATACTGTCTGGAGGATGGAAGTCTCATTAGGGCCACTAAGGACCACAAATTTATGACAGTCGATGGCCAGATGCTGCCTATAGACGAAATCTTTGAGCGAGAGTTGGACCTCATGCGAGTTGACAACCTTCCTAAT

[0137] DnaE intein-N protein: CLSYETEILTVEYGLLPIGKIVEKRIECTVYSVDNNGNIYTQPVAQWHDRGEQEVFEYCLEDGSLIRATKDHKFMTVDGQMLPIDEIFERELDLMRVDNL PN

[0138] DnaE intein-C DNA: ATGATCAAGATAGCTACAAGGAAGTATCTTGGCAAACAAAACGTTTATGATATTGGAGTCGAAAGAGATCACAACTTTGCTCTGAAGAACGGATTCATAG CTTCTAAT

[0139] Intein-C: MIKIATRKYLGKQNVYDIGVERDHNFALKNGFIASN

[0140] Cfa-N DNA: TGCCTGTCTTATGATACCGAGATACTTACCGTTGAATATGGCTTCTTGCCTATTGGAAAGATTGTCGAAGAGAGAATTGAATGCACAGTATATACTGTAGACAAGAATGGTTTCGTTTACACACAGCCCATTGCTCAATGGCACAATCGCGGCGAACAAGAAGTATTTGAGTACTGTCTCGAGGATGGAAGCATCATACGAGCAACTAAAGATCATAAATTCATGACCACTGACGGGCAGATGTTGCCAATAGATGAGATATTCGAGCGGGGCTTGGATCTCAAACAAGTGGATGGATTGCCA

[0141] Cfa-N protein:

[0142] CLSYDTEILTVEYGFLPIGKIVEERIECTVYTVDKNGFVYTQPIAQWHNRGEQEVFEYCLEDGSIIRATKDHKFMTTDGQMLPIDEIFERGLDLKQVDGLP

[0143] Cfa-C DNA: ATGAAGAGGACTGCCGATGGATCAGAGTTTGAATCTCCCAAGAAGAAGAGGAAAGTAAAGATAATATCTCGAAAAAGTCTTGGTACCCAAAATGTCTATGATATTGGAGTGGAGAAAGATCACAACTTCCTTCTCAAGAACGGTCTCGTAGCCAGCAAC

[0144] Cfa-C protein:

[0145] MKRTADGSEFESPKKKRKVKIISRKSLGTQNVYDIGVEKDHNFLLKNGLVASN

[0146] Intein-N and intein-C can be fused to the N-terminal portion of split-Cas9 and the C-terminal portion of split-Cas9, respectively, for connecting the N-terminal portion of split-Cas9 and the C-terminal portion of split-Cas9. For example, in some embodiments, intein-N is fused to the C-terminus of the N-terminal portion of split-Cas9, i.e., forming a structure of N-[N-terminal portion of split-Cas9]-[intein-N]--C. In some embodiments, intein-C is fused to the N-terminus of the C-terminal portion of split-Cas9, i.e., forming a structure of N-[intein-C]--[C-terminal portion of split-Cas9]-C. Intein-mediated protein splicing mechanisms for connecting proteins to which inteins are fused (e.g., split-Cas9) are known in the art, for example, as described in Shah et al., Chem Sci. 2014; 5(1):446-461, incorporated herein by reference. Methods for designing and using inteins are known in the art and are described in, for example, WO2014004336, WO2017132580, US20150344549, and US20180127780, each of which is herein incorporated by reference in its entirety.

[0147] The terms "isolated," "purified," or "biologically pure" refer to materials that are free, to varying degrees, from components that normally accompany them in their native state. "Isolated" refers to a degree of separation from the original source or surrounding environment. "Purified" refers to a degree of separation greater than isolated. A "purified" or "biologically pure" protein is sufficiently free of other materials such that any impurities do not substantially affect the biological properties of the protein or cause other adverse consequences. That is, a nucleic acid or peptide of the invention is purified if it is substantially free of cellular material, viral material, or culture medium when produced by recombinant DNA technology, or substantially free of chemical precursors or other chemicals when chemically synthesized. Purity and homogeneity are typically determined using analytical chemistry techniques, such as polyacrylamide gel electrophoresis or high performance liquid chromatography. The term "purified" can mean that a nucleic acid or protein produces essentially a single band in an electrophoretic gel. For proteins that can be modified (e.g., phosphorylated or glycosylated), different modifications may result in different isolated proteins that can be purified separately.

[0148] An "isolated polynucleotide" refers to a nucleic acid (e.g., DNA) that is free of the genes that flank the genes in the naturally occurring genome of the organism from which the nucleic acid molecule of the invention is derived. Thus, the term includes, for example, recombinant DNA that is integrated into a vector; into an autonomously replicating plasmid or virus; or into the genomic DNA of a prokaryotic or eukaryotic organism; or that exists as an independent molecule (e.g., cDNA or genomic or cDNA fragments generated by PCR or restriction endonuclease digestion) independent of other sequences. In addition, the term includes RNA molecules transcribed from a DNA molecule, as well as recombinant DNA that is part of a hybrid gene encoding an additional polypeptide sequence.

[0149] "Isolated polypeptide" refers to a polypeptide of the present invention that has been separated from naturally associated components. Generally, a polypeptide is isolated when it is at least 60% (by weight) free of proteins and naturally occurring organic molecules. Preferably, the polypeptide of the present invention is at least 75% by weight, more preferably at least 90% by weight, and most preferably at least 99% by weight. The isolated polypeptide of the present invention can be obtained, for example, by extraction from a natural source, by expression of a recombinant nucleic acid encoding such a polypeptide; or by chemically synthesizing the protein. Purity can be measured by any suitable method, such as column chromatography, polyacrylamide gel electrophoresis, or by HPLC analysis.

[0150] "CD117 (C-kit; SCFR) polypeptide" refers to a polypeptide or fragment thereof that has at least about 95% amino acid sequence identity to the amino acid sequence of an anti-CD117 antibody provided by GenBank Accession No. NP_000213. In some embodiments, the CD117 polypeptide or fragment thereof has SCF signaling activity. The sequence of an exemplary CD117 polypeptide is provided below:

[0151] >NP_000213.1 Mast / stem cell growth factor receptor Kit homolog 1 precursor

[0152] [Homo sapiens]

[0153] MRGARGAWDFLCVLLLLLRVQTGSSQPSVSPGESPPSIHPGKSDLIVRVGDEIRLLCTDPGFVKWTFEILDETNENKQNEWITEKAEATNTGKYTCTNKHGLSNSIYVFVRDPAKLFLVDRSLYGKEDNDTLVRCPLTDPEVTNYSLKGCQGKPLPKDLRFIPDPKAGIMIKSVKRAYHRCLHCSVDQEGKSVLSEKFILKVRPAFKAVPVVSVSKASYLLREGEEFTVTTCTIKDVSSSVYSTWKRENSQTKLQEKYNSWHHGDFNYERQATLTISSARVNDSGVFMCYANNTFGSANVTTTLEVVDKGFINIFPMINTTVFVNDGENVDLIVEYEAFPKPEHQQWIYMNRTFTDKWEDYPKSENESNIRYVSELHLTRLKGTEGGTYTFLVSNSDVNAAIAFNVYVNTKPEILTYDRLVNGMLQCVAAGFPEPTIDWYFCPGTEQRCSASVLPVDVQTLNSSGPPFGKLVVQSSIDSSAFKHNGT VECKAYNDVGKTSAYFNFAFKGNNKEQIHPHTLFTPLLIGFVIVAGMMCIIVMILTYKYLQKPMYEVQWKVVEEINGNNYVYIDPTQLPYDHKWEFPNRLSFGKTLGAGAFGKVVEATAYGLIKSDAAMTVAVKMLKPSAHLTEREALMSELKVLSYLGNHMIVNLLGACTIGGPTLVITEYCCYGDLNLNFLRKRDSFICSKQEDHAEAALYKNLLHSKSCSDSTNEYMDMKPGVSYVV PTKADKRRSVRIGSYIERDVTPAIMEDDELALDLEDLLSFSYQVAKGMAFLASKNCIHRDLAARNILLTHGRITKICDFGLARDIKNDSNYVVKGNARLPVKWMAPESIFNCVYTFESDVWSYGIFLWELFSLGSSPYPGMPVDSKFYKMIKEGFRMLSPEHAPAEMYDIMCWDADPLKRPTFKQIVQLIEKQISESTNHIYSNLANCSPNRQKPVVDHSVRINSVGSTASSSQPLLVHDDV

[0154] "CD117 polynucleotide" refers to a nucleic acid molecule encoding a CD117 polypeptide. The sequence of an exemplary CD117 polynucleotide is provided below:

[0155] >NM_000222.2 Homo sapiens KIT proto-oncogene, receptor tyrosine kinase (KIT), transcript variant 1, mRNA

[0156]

[0157] A "CXC chemokine receptor type 4 (CXCR4) polypeptide" refers to a polypeptide or fragment thereof that has at least about 95% amino acid sequence identity to the amino acid sequence that binds to an anti-CXCR4 antibody provided by GenBank Accession No. NP_001008540. The sequence of an exemplary CXCR4 polypeptide is provided below:

[0158] >NP_001008540.1C-XC chemokine receptor type 4 isoform a [Homo sapiens]

[0159] MSIPLPLLQIYTSDNYTEEMGSGDYDSMKEPCFREENANFNKIFLPTIYSIIFLTGIVGNGLVILVMGYQKKLRSMTDKYRLHLSVADLLFVITLPFWAVDAVANWYFGNFLCKAVHVIYTVNLYSSVLILAFISLDRYLAIVHATNSQRPRKLLAEKVVYVGVWIPALLLTIPDFIF ANVSEADDRYICDRFYPNDLWVVVFQFQHIMVGLILPGIVILSCYCIIISKLSHSKGHQKRKALKTTVILAFFACWLPYYIGISIDSFILLEIIKQGCEFENTVHKWISITEALAFFHCCLNPILYAFLGAKFKTSAQHALTSVSRGSSLKILSKGKRGGHSSVSTESESSSFHSS

[0160] "CXCR4 polynucleotide" refers to a nucleic acid molecule that encodes a CXCR4 polypeptide. The sequence of an exemplary CXCR4 polynucleotide is provided below:

[0161] >NM_003467.2 Homo sapiens CXC motif chemokine receptor 4 (CXCR4), transcript variant 2, mRNA

[0162]

[0163] "CD135 polypeptide" refers to a polypeptide or fragment thereof having about 95% amino acid sequence identity to the amino acid sequence of an anti-CD135 antibody provided by GenBank Accession No. NP_004110. The sequence of an exemplary CD135 polypeptide is provided below:

[0164] >NP_004110.2 Receptor tyrosine kinase FLT3 precursor [Homo sapiens]

[0165] MPALARDGGQLPLLVVFSAMIFGTITNQDLPVIKCVLINHKNNDSSVGKSSSYPMVSESPEDLGCALRPQSSGTVYEAAAVEVDVSASITLQVLVDAPGNISCLWVFKHSSLNCQPHFDLQNRGVVSMVILKMTETQAGEYLLFIQSEATNYTILFTVSIRNTLLYTLRRPYFRKMENQDALVCISESVPEPIVEWVLCDSQGESCKEESPAVVKKEEKVLHELFGTDIRCCARNELGRECTRLFTIDLNQTPQTTLPQLFLKVGEPLWIRCKAVHVNHGFGLTWELENKALEEGNYFEMSTYSTNRTMIRILFAFVSSVARNDTGYYTCSSSKHPSQSALVTIVEKGFINATNSSEDYEIDQYEEFCFSVRFKAYPQIRCTWTFSRKSFPCEQKGLDNGYSISKFCNHKHQPGEYIFHAENDDAQFTKMFTLNIRRKPQVLAEASASQASCFSDGYPLPSWTWKKCSDKSPNCTEEITEGVWNRKANRKVFGQWVSSSTLNMSEAIKGFLVKCCAYNSLGTSCETILLNSPGPFPFIQDNISFYATIGVCLLFIVVLTLLICHKYKKQFRYESQLQMVQVTGSSDNEYFYVDFREYEYDLKWEFPRENLEFGKVLGSGAFGKVMNATAYGISKTGVSIQVAVKMLKEKADSSEREALMSELKMMTQLGSHENIVNLLGACTLSGPIYLIFEYCCYGDLLNYLRSKREKFHRTWTEIFKEHNFSFYPTFQSHPNSSMPGSREVQIHPDSDQISGLHGNSFHSEDEIEYENQKRLEEEEDLNVLTFEDLLCFAYQVAKGMEFLEFKSCVHRDLAARNVLVTHGKVVKICDFGLARDIMSDSNYVVRGNARLPVKWMAPESLFEGIYTIKSDVWSYGILLWEIFSLGVNPYPGIPVDANFYKLIQNGFKMDQPFYATEEIYIIMQSCWAFDSRKRPSFPNLTSFLGCQLADAEEAMYQNVDGRVSECPHTYQNRRPFSREMDLGLLSPQAQVEDS

[0166] "CD135 polynucleotide" refers to a nucleic acid molecule encoding a CD135 polypeptide. The sequence of an exemplary CD135 polynucleotide is provided below:

[0167]

[0168] A "CD90 polypeptide" refers to a polypeptide or fragment thereof that has about 95% amino acid sequence identity to the amino acid sequence that binds to an anti-CD90 antibody provided in GenBank Accession No. NP_001298089. The sequence of an exemplary CD90 polypeptide is provided below:

[0169] >NP_001298089.1thy-1 membrane glycoprotein homologue 1 preproprotein [Homo sapiens]

[0170] MNLAISIALLLTVLQVSRGQKVTSLTACLVDQSLRLDCRHENTSSSPIQYEFSLTRETKKHVLFGTVGVPEHTYRSRTNFTSKYNMKVLYLSAFTSKDEGTYTCALHHSGHSPPISSQNVTVLRDKLVKCEGISLLAQNTSWLLLLLLLSLSLLQATDFMSL

[0171] "CD90 polynucleotide" refers to a nucleic acid molecule that encodes a CD90 polypeptide. The sequence of an exemplary CD90 polynucleotide is provided below:

[0172] >NM_006288.5 Homo sapiens Thy-1 cell surface antigen (THY1), transcript variant 1, mRNA

[0173]

[0174] A "CD45 polypeptide" refers to a polypeptide or fragment thereof that has about 95% amino acid sequence identity to the amino acid sequence provided in GenBank Accession No. NP_001254727 that binds to an anti-CD45 antibody. The sequence of an exemplary CD45 polypeptide is provided below:

[0175] >NP_001254727.1 Receptor-type tyrosine protein phosphatase C homologue 5 precursor [Homo sapiens]

[0176] MTMYLWLKLLAFGFAFLDTEVFVTGQSPTPSPTGHLQAEEQGSQSKSPNLKSREADSSAFSWWPKAREEPLTNHWSKSKSPKAEELGV

[0177] "CD45 polynucleotide" refers to a nucleic acid molecule that encodes a CD45 polypeptide. The sequence of an exemplary CD45 polynucleotide is provided below:

[0178] >NM_001267798.2 Homo sapiens protein tyrosine phosphatase receptor type C (PTPRC), transcript variant 5, mRNA

[0179]

[0180] "CD34 polypeptide" refers to a polypeptide or fragment thereof that has about 95% amino acid sequence identity to the amino acid sequence of an anti-CD34 antibody binding polypeptide provided by GenBank Accession No. NP_001020280. The sequence of an exemplary CD34 polypeptide is provided below:

[0181] >NP_001020280.1 Hematopoietic progenitor cell antigen CD34 isoform precursor [Homo sapiens]

[0182] MLVRRGARAGPRMPRGWTALCLLSLLPSGFMSLDNNGTATPELPTQGTFSNVSTNVSYQETTTPSTLGSTSLHPVSQHGNEATTNITETTVKFTSTSVITSVYGNTNSSVQSQTSVISTVFTTPANVSTPETTLKPSLSPGNVSDLSTTTSLATSPTKPYTSSSPILSDIKAEIKCSGIREVKLTQGICLE QNKTSSCAEFKKDRGEGLARVLCGEEQADADAGAQVCSLLLAQSEVRPQCLLLVLANRTEISSKLQLMKKHQSDLKKLGILDFTEQDVASHQSYSQKTLIALVTSGALLAVLGITGYFLMNRRSWSPTGERLGEDPYYTENGGGQGYSSGPGTSPEAQGKASVNRGAQENGTGQATSRNGHSARQHVVADTEL

[0183] "CD34 polynucleotide" refers to a nucleic acid molecule that encodes a CD34 polypeptide. The sequence of an exemplary CD34 polynucleotide is provided below:

[0184] >NM_001025109.2 Homo sapiens CD34 molecule (CD34), transcript variant 1, mRNA

[0185]

[0186] A "stem cell factor (SCF) polypeptide" refers to a polypeptide or fragment thereof that has at least about 95% amino acid sequence identity to the amino acid sequence provided in GenBank Accession No. NP_000890 that plays a role in hematopoiesis. In some embodiments, the SCF polypeptide or fragment thereof binds to CD117. The sequence of an exemplary SCF polypeptide is provided below:

[0187] >NP_000890.1Kit ligand isomer b precursor [Homo sapiens]

[0188] MKKTQTWILTCIYLQLLLFNPLVKTEGICRNRVTNNVKDVTKLVANLPKDYMITLKYVPGMDVLPSHCWISEMVVQLSDSLTDLLDKFSNISEGLSNYSIIDKLVNIVDDLVECVKENSSKDLKKSFKSPEPRLFT PEEFFRIFNRSIDAFKDFVVASETSDCVVSSTLSPEKDSRVSVTKPFMLPPVAASSLRNDSSSSNRKAKNPPGDSSLHWAAMALPALFSLIIGFAFGALYWKKRQPSLTRAVENIQINEEDNEISMLQEKEREFQEV

[0189] "SCF polynucleotide" refers to a nucleic acid molecule that encodes an SCF polypeptide. The sequence of an exemplary SCF polynucleotide is provided below:

[0190] >NM_003994.5 Homo sapiens KIT ligand (KITLG), transcript variant a, mRNA

[0191]

[0192] As used herein, the term "linker" may refer to a covalent linker (e.g., a covalent bond), a non-covalent linker, a chemical group, or a molecule that connects two molecules or parts (e.g., two components of a protein complex or a ribonucleic acid complex), or two domains of a fusion protein, such as a polynucleotide programmable DNA binding domain (e.g., dCas9) and a deaminase domain (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase). The linker can connect different components of the base editor system or different parts of the components. For example, in some embodiments, the linker can connect the guide polynucleotide binding domain of the polynucleotide programmable nucleotide binding domain and the catalytic domain of the deaminase. In some embodiments, the linker can connect the CRISPR polypeptide and the deaminase. In some embodiments, the linker can connect Cas9 and the deaminase. In some embodiments, the linker can connect dCas9 and the deaminase. In some embodiments, the linker can connect nCas9 and the deaminase. In some embodiments, the linker can connect the guide polynucleotide and the deaminase. In some embodiments, the linker can connect the deamination component and the polynucleotide programmable nucleotide binding component of the base editor system. In some embodiments, the linker can connect the RNA binding portion of the deamination component and the polynucleotide programmable nucleotide binding component of the base editor system. In some embodiments, the linker can connect the RNA binding portion of the deamination component and the RNA binding portion of the polynucleotide programmable nucleotide binding component of the base editor system. The linker can be located between or on both sides of two groups, molecules or other parts and connected to each by a covalent bond or non-covalent interaction, thereby connecting the two. In some embodiments, the linker can be an organic molecule, group, polymer or chemical part. In some embodiments, the linker can be a polynucleotide. In some embodiments, the linker can be a DNA linker. In some embodiments, the linker can be an RNA linker. In some embodiments, the linker can comprise an aptamer capable of binding to a ligand. In some embodiments, the ligand can be a carbohydrate, peptide, protein or nucleic acid. In some embodiments, the linker can comprise an aptamer that can be derived from a riboswitch. The riboswitch from which the aptamer is derived can be selected from the group consisting of a theophylline riboswitch, a thiamine pyrophosphate (TPP) riboswitch, an adenosylcobalamin (AdoCbl) riboswitch, an S-adenosylmethionine (SAM) riboswitch, a SAH riboswitch, a flavin mononucleotide (FMN) riboswitch, a tetrahydrofolate riboswitch, a lysine riboswitch, a glycine riboswitch, a purine riboswitch, a GlmS riboswitch, and a pre-queosine 1 (PreQ1) riboswitch. In some embodiments, the linker can comprise an aptamer that binds to a polypeptide or protein domain, such as a polypeptide ligand.In some embodiments, the polypeptide ligand can be a K homology (KH) domain, an MS2 coat protein domain, a PP7 coat protein domain, an SfMu Com coat protein domain, a sterile alpha motif, a telomerase Ku binding motif and Ku protein, a telomerase Sm7 binding motif and Sm7 protein, or an RNA recognition motif. In some embodiments, the polypeptide ligand can be part of a base editor system component. For example, a nucleobase editing component can include a deaminase domain and an RNA recognition motif.

[0193] In some embodiments, the joint can be an amino acid or multiple amino acids (e.g., a peptide or protein). In certain embodiments, the length of the joint can be about 5-100 amino acids, such as about 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 20-30, 30-40, 40-50, 50-60, 60-70, 70-80, 80-90, or 90-100 amino acid lengths. In certain embodiments, the length of the joint can be about 100-150, 150-200, 200-250, 250-300, 300-350, 350-400, 400-450, or 450-500 amino acids. Longer or shorter joints are also contemplated.

[0194] In some embodiments, the linker connects the gRNA binding domain of the RNA programmable nuclease, including the Cas9 nuclease domain and the catalytic domain of the nucleic acid editing protein (e.g., cytidine or adenosine deaminase). In some embodiments, the linker connects dCas9 and the nucleic acid editing protein. For example, the linker is located between or on both sides of two groups, molecules or other parts and is covalently bonded to each, thereby connecting the two. In some embodiments, the linker can be an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker is an amino acid or multiple amino acids (e.g., a peptide or protein). In some embodiments, the linker can be about 5-200 amino acids in length, e.g., 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 35, 45, 50, 55, 60, 60, 65, 70, 70, 75, 80, 85, 90, 90, 95, 100, 101, 102, 103, 104, 105, 110, 120, 130, 140, 150, 160, 175, 180, 190 or more amino acids in length.

[0195] In some embodiments, the domain of the base editor is fused via a linker comprising the following amino acid sequence: SGGSSGSETPGTSESATPESSGGS, SGGSSGGSSGSETPGTSESATPESSGGSSGGS, or GGSGGSPGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATSGGSGGS. In some embodiments, the domain of the base editor is fused via a linker comprising the amino acid sequence SGSETPGTSESATPES, which may also be referred to as an XTEN linker. In some embodiments, the linker is 24 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPES. In some embodiments, the linker is 40 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGS. In some embodiments, the linker is 64 amino acids in length. In some embodiments, the linker comprises the amino acid sequence SGGSSGGSSGSETPGTSESATPESSGGSSGGSSGGSSGGSSGSETPGTSESATPESSGGSSGGS. In some embodiments, the linker is 92 amino acids in length. In some embodiments, the linker comprises the amino acid sequence PGSPAGSPTSTEEGTSESATPESGPGTSTEPSEGSAPGSPAGSPTSTEEGTSTEPSEGSAPGTSTEPSEGSAPGTSESATPESGPGSEPATS.

[0196] "Makassar" or "Hb G-Makassar" refers to human beta-hemoglobin variant, G-Makassar variant, or mutation (HB Makassar variant) of human hemoglobin (Hb), an asymptomatic, naturally occurring variant (E6A) of hemoglobin. Hb G-Makassar was first identified in Indonesia. (Mohamad, A Set al., 2018, Hematol. Rep., 10(3):7210 (doi:10.4081 / hr.2018.7210). Hb G-Makassar migrates slower when subjected to electrophoresis. The Makassar β-hemoglobin variant has an anatomical abnormality at the β-6 or A3 position, where the glutamyl residue is usually replaced by an alanyl residue. A single amino acid substitution to valine in the gene encoding the β-6 glutamyl residue of the β-globin subunit results in sickle cell disease. Conventional procedures, such as isoelectric focusing, electrophoretic separation of hemoglobin by cation exchange high performance liquid chromatography (HPLC), and cellulose acetate electrophoresis, are unable to separate the HbG-Makassar and HbS globin forms because they are found to have identical properties when analyzed by these methods. Therefore, Hb G-Makassar and HbS have been mistakenly identified and misidentified by those skilled in the art, leading to misdiagnosis of sickle cell disease (SCD). In one embodiment, the valine at amino acid position 6 that causes sickle cell disease is replaced by alanine, thereby producing an Hb variant (HbMakassar) that does not produce a sickle cell phenotype. In some embodiments, the Val Ala (GTGGCG) substitution (i.e., Hb Makassar variant) can occur using an A·T to G·C base editor (ABE).

[0197] Thus, the present invention includes compositions and methods for base-editing the thymidine (T) base in the codon for the sixth amino acid of the sickle cell variant of β-globin (Sickle HbS; E6V) to a cytidine (C), thereby replacing valine with alanine at that amino acid position (V6A). Substituting alanine for valine at position 6 of HbS results in a β-globin variant that does not have a sickle cell phenotype (e.g., does not have the potential to aggregate like the pathogenic variant HbS). Thus, the compositions and methods of the present invention are useful for treating sickle cell disease (SCD).

[0198] "Marker" refers to any protein or polynucleotide having an alteration in expression level or activity that is associated with a disease or condition.

[0199] As used herein, the term "mutation" refers to the replacement of a residue within a sequence (e.g., a nucleic acid or amino acid sequence) by another residue, or the deletion or insertion of one or more residues within the sequence. Mutations are generally described herein by identifying the original residue followed by the position of the residue in the sequence and by the identity of the newly replaced residue. Various methods for performing amino acid substitutions (mutations) provided herein are well known in the art and are provided by, for example, Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)).

[0200] In some embodiments, the currently disclosed base editors can effectively generate "expected mutations," such as point mutations, in nucleic acids (e.g., nucleic acids within the genome of a subject) without generating a large number of unintended mutations, such as unexpected point mutations. In some embodiments, the expected mutation is a mutation generated by a specific base editor (e.g., a cytidine base editor or an adenosine base editor) in conjunction with a guide polynucleotide (e.g., a gRNA), which is specifically designed to generate the expected mutation.

[0201] Typically, mutations generated or identified in a sequence (e.g., an amino acid sequence as described herein) are numbered relative to a reference (or wild-type) sequence, i.e., a sequence that does not contain the mutation. Those skilled in the art will readily understand how to determine the position of a mutation in an amino acid or nucleic acid sequence relative to a reference sequence.

[0202] The term "non-conservative mutation" refers to amino acid substitutions between different groups, for example, tryptophan to lysine, or serine to phenylalanine, etc. In this case, the non-conservative amino acid substitution preferably does not interfere with or inhibit the biological activity of the functional variant. Non-conservative amino acid substitutions can enhance the biological activity of the functional variant, thereby increasing the biological activity of the functional variant compared to the wild-type protein.

[0203] The terms "nuclear localization sequence," "nuclear localization signal," or "NLS" refer to an amino acid sequence that facilitates the import of proteins into the nucleus. Nuclear localization sequences are known in the art and are described, for example, in Plank et al., International PCT Application No. PCT / EP2000 / 011690, filed November 23, 2000, and published as WO / 2001 / 038547 on May 31, 2001, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In other embodiments, the NLS is an optimized NLS, such as described by Koblan et al., Nature Biotech. 2018 doi:10.1038 / nbt.4172. In some embodiments, the NLS comprises the amino acid sequence KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRK, PKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETY.

[0204] The terms "nucleobase," "nitrogenous base," or "base" are used interchangeably herein and refer to nitrogenous biological compounds that form nucleosides, which in turn are components of nucleotides. The ability of nucleobases to form base pairs and stack on each other directly leads to long helical structures, such as ribonucleic acid (RNA) and deoxyribonucleic acid (DNA). The five nucleobases—adenine (A), cytosine (C), guanine (G), thymine (T), and uracil (U)—are referred to as primary or canonical. Adenine and guanine are derived from purines, while cytosine, uracil, and thymine are derived from pyrimidines. DNA and RNA may also contain other (non-primary) modified bases. Non-limiting exemplary modified nucleobases may include hypoxanthine, xanthine, 7-methylguanine, 5,6-dihydrouracil, 5-methylcytosine (m5C), and 5-hydromethylcytosine. Hypoxanthine and xanthine can be produced by the presence of mutagens, and they are both produced by deamination (replacing an amine group with a carbonyl group). Hypoxanthine can be modified from adenine. Xanthine can be modified from guanine. Uracil can be produced by deamination of cytosine. "Nucleoside" consists of a nucleobase and a five-carbon sugar (ribose or deoxyribose). Examples of nucleosides include adenosine, guanosine, uridine, cytidine, 5-methyluridine (m5U), deoxyadenosine, deoxyguanosine, thymidine, deoxyuridine and deoxycytidine. Examples of nucleosides with modified nucleobases include inosine (I), xanthine (X), 7-methylguanosine (m7G), dihydrouridine (D), 5-methylcytidine (m5C) and pseudouridine (Ψ). "Nucleotide" consists of a nucleobase, a five-carbon sugar (ribose or deoxyribose) and at least one phosphate group.

[0205] As used herein, the terms "nucleic acid" and "nucleic acid molecule" refer to compounds comprising a core base and an acidic moiety, such as a polymer of nucleosides, nucleotides or nucleotides. Typically, polymeric nucleic acids, such as nucleic acid molecules comprising three or more nucleotides are linear molecules in which adjacent nucleotides are interconnected by phosphodiester bonds. In some embodiments, "nucleic acid" refers to a single nucleic acid residue (such as a nucleotide and / or nucleoside). In some embodiments, "nucleic acid" refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms "oligonucleotide" and "polynucleotide" are used interchangeably to refer to a polymer (such as a string of at least three nucleotides) of nucleotides. In some embodiments, "nucleic acid" includes RNA and single-stranded and / or double-stranded DNA. Nucleic acid can be naturally occurring, such as in the context of a genome, transcript, mRNA, tRNA, rRNA, siRNA, snRNA, plasmid, cosmid, chromosome, chromatid or other naturally occurring nucleic acid molecules. On the other hand, nucleic acid molecules can be non-naturally occurring molecules, for example recombinant DNA or RNA, artificial chromosome, engineered genome or its fragment, or synthetic DNA, RNA, DNA / RNA hybrid or comprise non-naturally occurring nucleotides or nucleosides.In addition, term " nucleic acid ", " DNA ", " RNA " and / or similar terms comprise nucleic acid analogs, for example have the analogs of other skeletons except the phosphodiester backbone. Nucleic acid can be from natural source purification, use recombinant expression system to produce and optionally purifying, chemosynthesis etc. In suitable cases, for example in the situation of chemosynthetic molecule, nucleic acid can comprise nucleoside analogs, for example have the analog or sugar and backbone modification of chemically modified base.Unless otherwise indicated, otherwise nucleotide sequence is presented with 5 ' to 3 ' direction. In some embodiments, the nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxyguanosine, and deoxycytidine); nucleoside analogs (e.g., 2-aminoadenosine, 2-thiothymidine, inosine, pyrrolopyrimidine, 3-methyladenosine, 5-methylcytidine, 2-aminoadenosine, C5-bromouridine, C5-fluorouridine, C5-iodouridine, C5-propynyl-uridine, C5-propynyl-cytidine, C5-methyl cytidine, 2-aminoadenosine, 7-deazaadenosine, 7-deazaguanosine, 8-oxoadenosine, 8-oxoguanosine, O(6)-methylguanosine, and 2-thiocytidine); chemically modified bases; biologically modified bases (e.g., methylated bases); inserted bases; modified sugars (e.g., 2'-fluororibose, ribose, 2'-deoxyribose, arabinose, and hexose); and / or modified phosphate groups (e.g., phosphorothioate and 5'-N-phosphoramidite linkages).

[0206] The term "nucleic acid programmable DNA binding protein" or "napDNAbp" can be used interchangeably with "polynucleotide programmable nucleotide binding domain" to refer to a protein associated with a nucleic acid (e.g., DNA or RNA), such as a guide nucleic acid or a guide polynucleotide (e.g., gRNA), that guides the napDNAbp to a specific nucleic acid sequence. In some embodiments, the polynucleotide programmable nucleic acid binding domain is a polynucleotide programmable DNA binding domain. In some embodiments, the polynucleotide programmable nucleic acid binding domain is a polynucleotide programmable RNA binding domain. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 protein. The Cas9 protein can be associated with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, napDNAbp is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactive Cas9 (dCas9). Non-limiting examples of nucleic acid-programmable DNA-binding proteins include Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, and Cas12j / CasΦ.Non-limiting examples of Cas enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas8a, Cas8b, Cas8c, Cas9 (also known as Csn1 or Csx12), Cas10, Cas10d, Cas12a / Cpf1, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, Cas12j / CasΦ, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csn1, Csn2, Csn3, Csn4, Csn5e, Csn1, Csn2, Csn3, Csn4, Csn5e, Csn1, Csn2, Csn3, Csn4, Csn5e, Csn1 c1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx1S, Csx11, Csf1, Csf2, CsO, Csf4, Csd1, Csd2, Cst1, Cst2, Csh1, Csh2, Csa1, Csa2, Csa3, Csa4, Csa5, type II Cas effector protein, type V Cas effector protein, type VI Cas effector protein, CARF, DinG, homologs thereof, or modified or engineered versions thereof. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, although they may not be specifically listed in the present disclosure. See, e.g., Makarova et al., "Classification and Nomenclature of CRISPR-Cas Systems: Where from Here?" CRISPR J. 2018 Oct; 1: 325-336. doi: 10.1089 / crispr.2018.0033; Yan et al., "Functionally diverse type V CRISPR-Cas systems" Science. 2019 Jan 4; 363(6422): 88-91. doi: 10.1126 / science.aav7271, the entire contents of which are incorporated herein by reference.

[0207] As used herein, the term "nucleobase editing domain" or "nucleobase editing protein" refers to a protein or enzyme that catalyzes the modification of nucleobases in RNA or DNA, such as the deamination of cytosine (or cytidine) to uracil (or uridine) or thymine (or thymidine) and adenine (or adenosine) to hypoxanthine (or inosine), as well as non-templated nucleotide additions and insertions. In some embodiments, the nucleobase editing domain is a deaminase domain (e.g., an adenine deaminase or adenosine deaminase; or a cytidine deaminase or a cytosine deaminase). In some embodiments, the nucleobase editing domain is a plurality of deaminase domains (e.g., an adenine deaminase or adenosine deaminase and a cytidine or cytosine deaminase). In some embodiments, the nucleobase editing domain may be a naturally occurring nucleobase editing domain. In some embodiments, the nucleobase editing domain may be a nucleobase editing domain that is modified or evolved from a naturally occurring nucleobase editing domain. The nucleobase editing domain can be from any organism, such as bacteria, human, chimpanzee, gorilla, monkey, cow, dog, rat, or mouse.

[0208] As used herein, "obtaining" as in "obtaining an agent" includes synthesizing, purchasing, or otherwise obtaining the agent.

[0209] As used herein, "patient" or "subject" refers to a mammalian subject or individual diagnosed with, at risk of having or developing, or suspected of having or developing a disease or condition. In some embodiments, the term "patient" refers to a mammalian subject with a higher than average likelihood of developing a disease or condition. Exemplary patients can be humans, non-human primates, cats, dogs, pigs, cows, cats, horses, camels, llamas, goats, sheep, rodents (e.g., mice, rabbits, rats, or guinea pigs), and other mammals that can benefit from the therapies disclosed herein. Exemplary human patients can be male and / or female.

[0210] A "patient in need thereof" or "subject in need thereof" as used herein refers to a patient diagnosed with, at risk of or having, scheduled to have, or suspected of having a disease or condition.

[0211] The terms "disease-causing mutation," "pathogenic variant," "disease envelope mutation," "pathogenic variant," "deleterious mutation," or "susceptibility mutation" refer to a genetic alteration or mutation that increases an individual's susceptibility or predisposition to a disease or disorder. In some embodiments, the pathogenic mutation comprises a substitution of at least one wild-type amino acid with at least one pathogenic amino acid in the protein encoded by the gene.

[0212] The terms "protein," "peptide," "polypeptide," and their grammatical equivalents are used interchangeably herein to refer to polymers of amino acid residues linked together by peptide (amide) bonds. These terms refer to proteins, peptides, or polypeptides of any size, structure, or function. Typically, a protein, peptide, or polypeptide is at least three amino acids in length. A protein, peptide, or polypeptide may refer to a single protein or a collection of proteins. One or more amino acids in a protein, peptide, or polypeptide may be modified, for example, by adding chemical entities such as carbohydrate groups, hydroxyl groups, phosphate groups, farnesyl groups, isofarnesyl groups, fatty acid groups, linkers for conjugation, functionalization, or other modifications, and the like. A protein, peptide, or polypeptide may also be a single molecule or may be a multimolecular complex. A protein, peptide, or polypeptide may be simply a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. As used herein, the term "fusion protein" refers to a hybrid polypeptide comprising protein domains from at least two different proteins. A protein can be located at the amino terminal (N-terminal) portion or the carboxyl terminal (C-terminal) protein of the fusion protein, thereby forming an amino terminal fusion protein or a carboxyl terminal fusion protein, respectively. The protein can include different domains, for example, a nucleic acid binding domain (for example, a gRNA binding domain of Cas9 that guides the protein to bind to the target site) and a nucleic acid cleavage domain, or a catalytic domain of a nucleic acid editing protein. In some embodiments, the protein includes a protein portion, such as an amino acid sequence constituting a nucleic acid binding domain, and an organic compound, such as a compound that can act as a nucleic acid cleavage agent. In some embodiments, the protein forms a complex or associates with nucleic acids such as RNA or DNA. Any protein provided herein can be produced by any method known in the art. For example, the protein provided herein can be produced by recombinant protein expression and purification, which is particularly suitable for fusion proteins comprising peptide linkers. Methods for recombinant protein expression and purification are well known, including those described in Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY (2012)), the entire contents of which are incorporated by reference.

[0213] The polypeptides and proteins disclosed herein (including functional parts and functional variants thereof) may comprise synthetic amino acids in place of one or more naturally occurring amino acids. Such synthetic amino acids are known in the art and include, for example, aminocyclohexanecarboxylic acid, norleucine, α-amino n-decanoic acid, homoserine, S-acetylaminomethyl-cysteine, trans-3- and trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine, 4-chlorophenylalanine, 4-carboxyphenylalanine, β-phenylserine β-hydroxyphenylalanine, phenylglycine, α-naphthylalanine, cyclohexylalanine, cyclohexylglycine, dihydroindole-2-tetrahydrotetrahydro, 3- Quinoline, 3-hydroxyphenylalanine, 3-2-hydroxyphenylalanine-carboxylic acid, aminomalonic acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine, N', N'-dibenzyl-lysine, 6-hydroxylysine, ornithine, α-aminocyclopentanecarboxylic acid, α-aminocyclohexanecarboxylic acid, α-aminocycloheptanecarboxylic acid, α-(2-amino-2-norbornane)-carboxylic acid, α, γ-diaminobutyric acid, α, β-diaminopropionic acid, homophenylalanine and α-tert-butylglycine. Polypeptides and proteins can be associated with post-translational modifications of one or more amino acids of the polypeptide construct. Non-limiting examples of post-translational modifications include phosphorylation, acylation (including acetylation and formylation), glycosylation (including N-linked and O-linked), amidation, hydroxylation, alkylation (including methylation and ethylation), ubiquitination, addition of pyrrolidone carboxylic acid, disulfide bond formation, sulfation, myristoylation, palmitoylation, prenylation, farnesylation, geranylation, glycosylation, fatty acylation, and iodination.

[0214] As used herein, the term "recombinant" in the context of a protein or nucleic acid refers to a protein or nucleic acid that does not occur in nature but is a product of human engineering. For example, in some embodiments, a recombinant protein or nucleic acid molecule comprises an amino acid or nucleotide sequence that comprises at least one, at least two, at least three, at least four, at least five, at least six, or at least seven mutations compared to any naturally occurring sequence.

[0215] "Decrease" refers to a negative change of at least 10%, 25%, 50%, 75% or 100%.

[0216] "Reference" refers to a standard or control condition. In one embodiment, the reference is a wild-type or healthy cell. In other embodiments and without limitation, the reference is an untreated cell that has not been subjected to the test condition, or has been subjected to a placebo or saline, culture medium, buffer, and / or a control vector that does not contain the polynucleotide of interest.

[0217] A "reference sequence" is a defined sequence used as a basis for sequence comparison. A reference sequence can be a subset or the entirety of a particular sequence; for example, a fragment of a full-length cDNA or gene sequence, or a complete cDNA or gene sequence. For polypeptides, the length of a reference polypeptide sequence is typically at least about 16 amino acids, at least about 20 amino acids, at least about 25 amino acids, about 35 amino acids, about 50 amino acids, or about 100 amino acids. For nucleic acids, the length of a reference nucleic acid sequence is typically at least about 50 nucleotides, at least about 60 nucleotides, at least about 75 nucleotides, about 100 nucleotides, or about 300 nucleotides, or any integer therebetween. In some embodiments, the reference sequence is the wild-type sequence of a protein of interest. In other embodiments, the reference sequence is a polynucleotide sequence encoding a wild-type protein.

[0218] The terms "RNA programmable nuclease" and "RNA-guided nuclease" are used with (e.g., bound to or associated with) one or more RNAs that are not targets for cleavage. In some embodiments, when a complex is formed with an RNA, the RNA programmable nuclease may be referred to as a nuclease:RNA complex. Typically, the bound RNA is referred to as a guide RNA (gRNA). In some embodiments, the RNA programmable nuclease is a (CRISPR-associated system) Cas9 endonuclease, such as Cas9 from Streptococcus pyogenes (Csn1) (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C, Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia H.G., Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III."DeltchevaE.,Chylinski K.,Sharma CM.,Gonzales K.,Chao Y.,Pirzada ZA,Eckert MR,VogelJ.,Charpentier E.,Nature 471:602-607(2011).

[0219] As used herein, the term "scFv" refers to a single-chain Fv antibody in which the variable domains of the heavy and light chains of an antibody have been linked to form a single chain. The scFv fragment comprises a polypeptide chain comprising the variable region of the antibody light chain (VL) (e.g., CDR-L1, CDR-L2, and / or CDR-L3) and the variable region of the antibody heavy chain (VH) (e.g., CDR-H1, CDR-H2, and / or CDR-H3) separated by a linker. The linker connecting the VL and VH regions of the scFv fragment can be a peptide linker composed of protein amino acids. Alternative linkers can be used to increase the resistance of the scFv fragment to proteolytic degradation (e.g., linkers containing D-amino acids), to enhance the solubility of the scFv fragment (e.g., hydrophilic linkers such as linkers containing polyethylene glycol or polypeptides containing repeated glycine and serine residues), to improve the biophysical stability of the molecule (e.g., linkers containing cysteine ​​residues that form intramolecular or intermolecular disulfide bonds), or to reduce the immunogenicity of the scFv fragment (e.g., linkers containing glycosylation sites). It will also be understood by those of ordinary skill in the art that the variable regions of the scFv molecules described herein can be modified so that their amino acid sequences differ from the antibody molecules from which they are derived. For example, nucleotide or amino acid substitutions (e.g., in CDR and / or framework residues) that result in conservative substitutions or changes in amino acid residues can be made to maintain or enhance the ability of the scFv to bind to the antigen recognized by the corresponding antibody.

[0220] "Selectively binds" refers to specifically binding to a wild-type form of a cell surface protein, but exhibiting reduced binding or an inability to bind to a cell surface protein comprising a mutation.

[0221] The term "single nucleotide polymorphism (SNP)" is a variation of a single nucleotide occurring at a specific position in the genome, wherein each variation is present to a certain extent (e.g., >1%) in the population. For example, at a specific base position in the human genome, a C nucleotide may occur in most individuals, but in a minority of individuals, the position is occupied by an A. This means that a SNP exists at the specific position, and the two possible nucleotide variations, C or A, are called alleles at the position. SNPs are the basis for differences in disease susceptibility. The severity of the disease and the way our bodies respond to treatment are also manifestations of genetic variation. SNPs can fall into the coding region of a gene, the non-coding region of a gene, or the intergenic region (the region between genes). In some embodiments, due to the degeneracy of the genetic code, SNPs within the coding sequence do not necessarily change the amino acid sequence of the protein produced. There are two types of SNPs in the coding region: synonymous SNPs and non-synonymous SNPs. Synonymous SNPs do not affect the protein sequence, while non-synonymous SNPs change the amino acid sequence of the protein. There are two types of non-synonymous SNPs: missense and nonsense. SNPs that aren't located in protein-coding regions can still affect gene splicing, transcription factor binding, messenger RNA degradation, or the sequence of non-coding RNAs. Gene expression affected by these SNPs is called an eSNP (expression SNP) and can be located upstream or downstream of the gene. Single nucleotide variants (SNVs) are variations in a single nucleotide that occur in all cells without any frequency restrictions. Somatic SNVs are also referred to as single nucleotide alterations.

[0222] "Specifically binds" refers to a nucleic acid molecule, polypeptide, or complex thereof (e.g., a nucleic acid programmable DNA binding domain and a guide nucleic acid), compound, or molecule that recognizes and binds to a polypeptide and / or nucleic acid molecule of the invention, but does not substantially recognize and bind to other molecules in a sample, such as a biological sample.

[0223] Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecules that encode a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. A polynucleotide that is "substantially identical" to an endogenous sequence is generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule. Nucleic acid molecules useful in the methods of the present invention include any nucleic acid molecules that encode a polypeptide of the present invention or a fragment thereof. Such nucleic acid molecules need not be 100% identical to an endogenous nucleic acid sequence, but will generally exhibit substantial identity. A polynucleotide that is "substantially identical" to an endogenous sequence is generally capable of hybridizing to at least one strand of a double-stranded nucleic acid molecule.

[0224] "Hybridization" refers to the pairing of complementary polynucleotide sequences (e.g., genes described herein) or portions thereof to form a double-stranded molecule under various stringent conditions (see, e.g., Wahl, G. M. and S. L. Berger (1987) Methods Enzymol. 152:399; Kimmel, A. R. (1987) Methods Enzymol. 152:507).

[0225] For example, stringent salt concentrations are typically less than about 750 mM NaCl and 75 mM trisodium citrate, preferably less than about 500 mM NaCl and 50 mM trisodium citrate, more preferably less than about 250 mM NaCl and 25 mM trisodium citrate. Low stringency hybridization can be obtained in the absence of an organic solvent such as formamide, while high stringency hybridization can be obtained in the presence of at least about 35% formamide, more preferably at least about 50% formamide. Stringent temperature conditions typically include a temperature of at least about 30° C., more preferably at least about 37° C., and most preferably at least about 42° C. For example, sodium dodecyl sulfate (SDS), and the inclusion or exclusion of vector DNA, are well known to those skilled in the art. Different degrees of stringency can be achieved by combining these different conditions as needed. In a preferred embodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mM trisodium citrate, and 1% SDS. In a more preferred embodiment, hybridization will occur in 500 mM NaCl, 50 mM trisodium citrate, 1% SDS, 35% formamide, and 100 μg / ml denatured salmon sperm DNA (ssDNA) at 37° C. In a most preferred embodiment, hybridization will occur in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and 200 μg / ml ssDNA at 42° C. Useful variations on these conditions will be apparent to those skilled in the art.

[0226] For most applications, the post-hybridization wash step will also vary in stringency. Wash stringency conditions can be defined by salt concentration and temperature. As described above, wash stringency can be increased by lowering the salt concentration or increasing the temperature. For example, the stringent salt concentration for the wash step is preferably less than about 30 mM NaCl and 3 mM trisodium citrate, and most preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate. Stringent temperature conditions for the wash step typically include a temperature of at least about 25°C, more preferably at least about 42°C, and even more preferably at least about 68°C. In one embodiment, the wash step will occur at 25°C in 30 mM NaCl, 3 mM trisodium citrate, and 0.1% SDS. In another embodiment, the wash step will be performed at 42°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, the wash step will be performed at 68°C in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Other variations on these conditions will be apparent to those skilled in the art. Hybridization techniques are well known to those skilled in the art and are described, for example, in Benton and Davis (Science 196:180, 1977); Grunstein and Hogness (Proc. Natl. Acad. Sci., USA 72:3961, 1975); Ausubel et al. (Current Protocols in Molecular Biology, Wiley Interscience, New York, 2001); Berger and Kimmel (Guide to Molecular Cloning Techniques, 1987, Academic Press, New York); and Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, New York.

[0227] "Split" means to separate into two or more fragments.

[0228] "Split Cas9 protein" or "split Cas9" refers to a Cas9 protein provided as an N-terminal fragment and a C-terminal fragment encoded by two separate nucleotide sequences. The polypeptides corresponding to the N-terminal portion and the C-terminal portion of the Cas9 protein can be spliced ​​to form a "reconstructed" Cas9 protein. In certain embodiments, the Cas9 protein is divided into two fragments within the disordered region of the protein, for example, as described in Nishimasu et al., Cell, Volume 156, Issue 5, pp. 935-949, 2014, or as described in Jiang et al. (2016) Science 351: 867-871. PDB file: 5F9R, each of which is incorporated herein by reference. In some embodiments, the protein is split into two fragments at any C, T, A, or S between about amino acids A292-G364, F445-K483, or E565-T637 within the SpCas9 region, or any other Cas9, Cas9 variant (e.g., nCas9, dCas9), or other napDNAbp. In some embodiments, the protein is split into two fragments at SpCas9 T310, T313, A456, S469, or C574. In some embodiments, the process of splitting a protein into two fragments is referred to as "splitting" the protein.

[0229] In other embodiments, the N-terminal portion of the Cas9 protein comprises amino acids 1-573 or 1-637 of Streptococcus pyogenes Cas9 wild type (SpCas9) (NCBI Reference Sequence: NC_002737.2, Uniprot Reference Sequence: Q99ZW2) and the C-terminal portion of the Cas9 protein comprises amino acids 574-1368 or 638-1368 of the SpCas9 wild type.

[0230] The C-terminal portion of the split Cas9 can be connected to the N-terminal portion of the split Cas9 to form a complete Cas9 protein. In some embodiments, the C-terminal portion of the Cas9 protein starts where the N-terminal portion of the Cas9 protein ends. Therefore, in some embodiments, the C-terminal portion of the split Cas9 comprises a portion of amino acids (551-651)-1368 of spCas9. "(551-651)-1368" refers to the amino acids starting from amino acids 551-651 (inclusive) and ending at amino acid 1368.For example, the C-terminal portion of the split Cas9 can include a portion of any of the amino acids of spCas9: 551-1368, 552-1368, 553-1368, 554-1368, 555-1368, 556-1368, 557-1368, 558-1368, 559-1368, 560-1368, 561-1368, 562-1368, 563-1368, 564-1368, 565-1368, 566-1368, 567-1368, 568-1368, 569-1368, 570-1368, 571-1368, 572-1368, 573-1368 3-1368, 574-1368, 575-1368, 576-1368, 577-1368, 578-1368, 579-1368, 580-1368, 581-1368, 582-1368, 583-1368, 584-1368, 585-1368, 586-1368, 587-1368, 588-1368, 589-1368, 590-1368, 591-1368, 592-1368, 593-1368, 594-1368, 595-1368, 596-1368, 597-1368, 598-1368, 599- 1368, 600-1368, 601-1368, 602-1368, 603-1368, 604-1368, 605-1368, 606-1368, 607-1368, 608-1368, 609-1368, 610-1368, 611-1368, 612-1368, 613-1368, 614-1368, 615-1368, 616-1368, 617-1368, 618-1368, 619-1368, 620-1368, 621-1368, 622-1368, 623-1368, 624-1368, 625-1368 68, 626-1368, 627-1368, 628-1368, 629-1368, 630-1368, 631-1368, 632-1368, 633-1368, 634-1368, 635-1368, 636-1368, 637-1368, 638-1368, 639-1368, 640-1368, 641-1368, 642-1368, 643-1368, 644-1368, 645-1368, 646-1368, 647-1368, 648-1368, 649-1368, 650-1368, or 651-1368. In some embodiments, the C-terminal portion of the split Cas9 comprises a portion of 574-1368 or 638-1368 of the SpCas9 protein.

[0231] "Substantially identical" refers to a polypeptide or nucleic acid molecule that is substantially identical to a reference amino acid sequence (e.g., any of the amino acid sequences described herein) or nucleic acid sequence (e.g., any of the nucleic acid sequences described herein). In one embodiment, such a sequence is at least 60%, 80%, 85%, 90%, 95%, or even 99% identical to the sequence used for comparison at the amino acid level or the nucleic acid level.

[0232] Sequence identity is typically determined using sequence analysis software (e.g., Sequence Analysis Software Package of the Genetics Computer Group, University of Wisconsin Biotechnology Center, 1710 University Avenue, Madison, Wis. 53705, BLAST, BESTFIT, GAP, or PILEUP / PRETTYBOX programs). Such software matches identical or similar sequences by assigning degrees of homology to various substitutions, deletions, and / or other modifications. Conservative substitutions typically include substitutions within the following groups: glycine, alanine; valine, isoleucine, leucine; aspartic acid, glutamic acid, asparagine, glutamine; serine, threonine; lysine, arginine; and phenylalanine, tyrosine. In an exemplary method for determining the degree of identity, the BLAST program can be used, where probability scores between e-3 and e-100 indicate closely related sequences.

[0233] For example, COBALT is used with the following parameters:

[0234] a)alignment parameters:Gap penalties-11,-1and End-Gap penalties-5,-1,

[0235] b)CDD Parameters: Use RPS BLAST on; Blast E-value 0.003; Find Conservedcolumns and Recompute on, and

[0236] c) Query Clustering Parameters: Use query clusters on; Word Size 4; Maxcluster distance 0.8; Alphabet Regular.

[0237] For example, EMBOSS is used with the following parameters:

[0238] a)Matrix:BLOSUM62;

[0239] b) GAP OPEN: 10;

[0240] c) GAP EXTEND: 0.5;

[0241] d)OUTPUT FORMAT:pair;

[0242] e)END GAP PENALTY:false;

[0243] f)END GAP OPEN:10; and

[0244] g)END GAP EXTEND:0.5.

[0245] The term "target site" refers to a sequence within a nucleic acid molecule that is deaminated by a deaminase (e.g., a cytidine or adenine deaminase) or a fusion protein comprising a deaminase (e.g., a dCas9-adenosine deaminase fusion protein or base editor disclosed herein).

[0246] Because RNA-programmable nucleases such as Cas9 use RNA:DNA hybrids to target DNA cleavage sites, these proteins are in principle able to target any sequence specified by the guide RNA. Methods for using RNA programmable nucleases (e.g., Cas9) for site-specific cleavage (e.g., modifying genomes) are known in the art (see, e.g., Cong, L. et al., Multiplex genome engineering using CRISPR / Cas systems. Science 339, 819-823 (2013); Mali, P. et al., RNA-guided human genome engineering via Cas9. Science 339, 823-826 (2013); Hwang, WY et al., Efficient genome editing in zebrafish using a CRISPR-Cas system. Nature biotechnology 31, 227-229 (2013); Jinek, M. et al., RNA-programmed genome editing in human cells. eLife 2, e00471 (2013); Dicarlo, JE et al., Genome engineering in Saccharomyces cerevisiae using CRISPR-Cas systems. Nucleic acids research (2013); Jiang, W. et al. RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nature biotechnology 31, 233-239 (2013); the entire contents of which are incorporated herein by reference).

[0247] As used herein, the terms "treat," "treating," "treatment," and the like refer to reducing or ameliorating a condition and / or symptoms associated therewith or obtaining a desired pharmacological and / or physiological effect. It should be understood that, although not excluded, treating a condition or disorder does not require the complete elimination of the condition, disorder, or symptoms associated therewith. In some embodiments, the effect is therapeutic, that is, but not limited to, the effect partially or completely reduces, attenuates, eliminates, alleviates, mitigates, reduces the intensity of the disease and / or adverse symptoms attributable to the disease, or cures the disease and / or adverse symptoms. In some embodiments, the effect is preventative, that is, the effect protects or prevents the occurrence or recurrence of a disease or disorder. To this end, the presently disclosed methods comprise administering a therapeutically effective amount of a composition as described herein.

[0248] "Uracil glycosylase inhibitor" or "UGI" refers to an agent that inhibits the uracil excision repair system. In one embodiment, the agent is a protein or fragment thereof that binds to the host uracil-DNA glycosylase and prevents the removal of uracil residues from DNA. In one embodiment, UGI is a protein, fragment or domain thereof that is capable of inhibiting the uracil-DNA glycosylase base excision repair enzyme. In some embodiments, the UGI domain comprises wild-type UGI or a modified version thereof. In some embodiments, the UGI domain comprises a fragment of the exemplary amino acid sequence set forth below. In some embodiments, the amino acid sequence comprised by the UGI fragment comprises at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or 100% identical to the exemplary UGI sequence provided below. In some embodiments, UGI comprises an amino acid sequence homologous to the exemplary UGI amino acid sequence or a fragment thereof, as described below. In some embodiments, the UGI or a portion thereof is at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.9%, or at least 99% or 100% identical to a wild-type UGI or a UGI sequence or a portion thereof, as described below. An exemplary UGI comprises the following amino acid sequence:

[0249] >splP14739IUNGI_BPPB2 uracil-DNA glycosylase inhibitor

[0250] MTNLSDIIEKETGKQLVIQESILMLPEEVEEVIGNKPESDILVHTAYDESTDENV MLLTSDAPEYKPWALVIQDSNGENKIKML.

[0251] 41, 42, 43, 44, 45, 46, 47, 48, 49, or 50.

[0252] The recitation of a list of chemical groups in any definition of a variable herein includes definitions of the variable as any single group or combination of listed groups. The recitation of an embodiment of a variation or aspect herein includes that embodiment as any single embodiment or in combination with any other embodiment or portion thereof.

[0253] All terms are intended to be understood in the same manner as those understood by those skilled in the art. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.

[0254] DNA editing has emerged as a viable means to alter disease states by correcting pathogenic mutations at the genetic level. Until recently, all DNA editing platforms functioned by inducing DNA double-strand breaks (DSBs) at specific genomic loci and relying on endogenous DNA repair pathways to determine product outcomes in a semi-stochastic manner, thereby generating complex populations of genetic products. While precise, user-defined repair outcomes can be achieved through the homology-directed repair (HDR) pathway, numerous challenges have hindered the efficient use of HDR for repair in therapeutically relevant cell types. In practice, the pathway is inefficient relative to the competing, error-prone non-homologous end-joining pathway. Furthermore, HDR is strictly restricted to the G1 and S phases of the cell cycle, preventing precise repair of DSBs in post-mitotic cells. Consequently, it has proven difficult or impossible to efficiently alter genomic sequence in these populations in a user-defined, programmable manner. BRIEF DESCRIPTION OF THE DRAWINGS

[0255] Figure 1 Schematic diagram depicting that the anti-CD117 antibody AMG 191 (humanized SR1) can be used for conditioning patients prior to hematopoietic stem cell transplantation.

[0256] Figure 2 Schematic diagram depicting that the anti-CD117 antibody SR1 (AMG191) blocks stem cell factor (SCF) binding to CD117 in vivo and depletes human hematopoietic stem and progenitor cells (HSPCs) and myelodysplastic syndrome (MDS) cells.

[0257] Figure 3 This is a schematic diagram describing a non-toxic conditioning strategy that enables hematopoietic stem cell transplantation (HSCT) with autologous gene-edited cells for patients with blood disorders. Stem cell factor (SCF) drives HSC self-renewal and differentiation into progenitor cells. Administration of anti-CD117 antibodies blocks SCF binding to CD117, thereby depleting the patient's HSCs and progenitor cells (conditioning). Autologous gene-edited HSCs are transplanted into the patient. Base editing is used to create amino acid substitutions in CD117 that prevent anti-CD117 antibodies from binding to the edited cells but do not interfere with normal SCF signaling. The gene-edited cells compete with residual host HSCs to repopulate the bone marrow (BM). The anti-CD117 antibody blocks SCF binding to wild-type (WT) CD117 but not to CD117-edited HSCs. Thus, natural wild-type HSCs are targeted by the anti-CD117 antibody, but gene-edited HSCs are not.

[0258] Figure 4 Schematic diagram depicting SCF binding to wild-type and gene-edited HSCs. Both cells express CD117, which is activated by SCF binding.

[0259] Figure 5 Schematic diagram depicting the effects of anti-CD117 antibodies on wild-type and gene-edited HSCs. Binding of anti-CD117 antibodies to wild-type CD117 disrupts SCF binding and leads to inhibition of SCF signaling in wild-type cells. In contrast, gene-edited HSCs are unresponsive to anti-CD117 antibodies because the amino acid substitutions introduced into CD117 prevent binding of anti-CD117 antibodies but do not interfere with normal SCF binding and signaling.

[0260] Figure 6 Table depicting sequence reads from target site base-edited cells from Makassar-WT guide (top) and HPFH guide (bottom).

[0261] Figure 7A and 7B is a diagram depicting the edits on the target ( Figure 7A ) and the insertion rate of five (5) guides (ABE8.8) ( Figure 7B ). 7B) of five (5) guides (ABE8.8). HPFH guide was used as a control.

[0262] Figure 8A and 8B is a diagram depicting the edits on the target ( Figure 8A) and the insertion rate of eleven (11) guides (ABE8.8) ( Figure 8B ). 8B) of eleven (11) guides (ABE8.8). HPFH guide was used as a control.

[0263] Figure 9A and 9B is a diagram depicting the edits on the target ( Figure 9A ) and the insertion rate of nine (9) guides (ABE8.8) ( Figure 9B ). 9B) of nine (9) guides (ABE8.8). HPFH guide was used as a control.

[0264] Figure 10 is a graph depicting on-target editing of four (4) guides for ABE8.8 and thirteen (13) guides for IBE-NGC (Makassar).

[0265] Figures 11A to 11T is a schematic diagram depicting the locations of targeting mutations that guide activity of anti-CD117 antibodies.

[0266] Figures 12A to 12N Depicted is a guide for introducing naturally occurring mutations into the CD117 target sequence. Figure 12A is a schematic diagram depicting the CD117 target nucleotide sequence and corresponding amino acid sequence used to direct hybridization of cc-102 and cc-103. Figure 12B and 12C is a description of the different base pair positions along the CD117 target site for the guide cc-102 ( Figure 12B ) or guide cc-103 (Figure 12) editing efficiency table. Figure 12C is a schematic diagram depicting the CD117 target nucleotide sequence and corresponding amino acid sequence used to direct cc-78 hybridization. Figure 12E is a table describing the editing efficiency for the guide cc-78 at different base pair positions along the CD117 target site. Figure 12F is a schematic diagram depicting the CD117 target sequence and the corresponding amino acid sequence used to direct hybridization of cc-87 and cc-89. Figure 12G and Figure 12H is a description of the different base pair positions along the CD117 target site for the guide cc-87 ( Figure 12G ) or guide cc-89( Figure 12H )'s editing efficiency table. Figure 12I is a schematic diagram depicting the CD117 target sequence and the corresponding amino acid sequence used to direct cc-110 hybridization. Figure 12J is a table describing the editing efficiency for the guide cc-110 at different base pair positions along the CD117 target site. Figure 12Kis a schematic diagram depicting the CD117 target sequence and the corresponding amino acid sequence used to direct cc-146 hybridization. Figure 12L is a table describing the editing efficiency for the guide cc-146 at different base pair positions along the CD117 target site. Figure 12M is a schematic diagram depicting the CD117 target sequence and the corresponding amino acid sequence used to direct cc-182 hybridization. Figure 12N is a table describing the editing efficiency for the guide cc-182 at different base pair positions along the CD117 target site.

[0267] Figure 13A and Figure 13B is a diagram depicting multi-way editing. Figure 13A Graphs depicting c-KIT editing using a c-KIT and HPFH dual guide (day 3), a c-KIT and HPFH dual guide (day 5), or a c-KIT guide alone without an HPFH guide. Figure 13B Graph depicting HPFH editing using dual guides of c-KIT and HPFH at 72 or 120 hours. HPFH alone was used as a control. DETAILED DESCRIPTION

[0268] The present invention provides a base editing strategy targeting cell surface proteins (e.g., one or more of CD117, CXCR4, CD135, CD90, CD45, CD34), which can be used for non-toxic conditioning.

[0269] The present invention is based at least in part on the following discovery: base editing can be used to produce a single base substitution in the CD117 gene to cause an amino acid substitution, thereby changing the epitope binding domain in the encoded protein, thereby preventing anti-CD117 antibodies, antibody drug conjugates (ADC) or chimeric antigen receptor (CAR)-T cells from binding to CD117 on edited cells. Advantageously, this change in CD117 does not change stem cell factor (SCF) binding or CD117 biological activity. Therefore, the present invention provides post-HSCT use of anti-CD117 therapies, as well as therapies targeting other antigens expressed on the surface of hematopoietic stem cells. It does not bind to or deplete CD117 edited cells. Therefore, gene-edited cells can be expanded in vivo. Advantageously, this allows cancer treatment to be performed in the absence of hematopoietic toxicity.

[0270] Prior art conditioning approaches are limited to administering the conditioning regimen prior to HSC transplantation (i.e., the patient cannot be re-medicated to expand the edited cells in vivo or to continue treatment for the malignancy). To avoid donor-affected / edited cells, the half-life of the antibody needs to be short so that it can be cleared from the body prior to transplantation. This ensures that the antibody does not target the transplanted cells. There are currently no methods / therapies that allow for selective targeting of patient HSCs. This applies to all current conditioning steps (e.g., Abs, ADCs, CAR-T cells). Therefore, clinical trial design is extremely challenging for conditioning prior to HSCT and must consider the number / concentration of doses versus HSC depletion versus hematopoietic failure. However, studies have shown that effective antibody-based conditioning can only be achieved with multiple doses in immunocompetent patients (unlike SCID trials).

[0271] Therefore, the conditioning method of the present invention was developed to overcome the limitations of previous methods. The conditioning method of the present invention has several advantages. The methods described herein provide selective targeting of endogenous HSCs while retaining edited HSCs. Therefore, antibody or ADC therapy can continue to be administered after HSCT to amplify gene-edited cells in vivo or to treat malignant diseases by repeated administration. This minimizes the risk of killing edited cells. Edited cells will allow the administration of antibodies or ADCs in the absence of Fc modification to reduce their half-life. This may enable the use of antibodies with longer half-lives, such as AMG191, and simplify the development of ADCs. Clinical trial design is also simplified - HSCs can be injected before or simultaneously with conditioning with little or no risk of depletion (for example, in patients with MDS or AML). These methods can benefit all patients, regardless of their immune status.

[0272] In one embodiment, CD117 is changed (for example, using base editing) in cells for transplantation to prevent the binding of anti-CD117 antibodies, but does not interfere with normal SCF signaling. Using base editing, changes in bases can be produced to cause amino acid substitutions in CD117 to occur. Stem cell factor (SCF) drives HSC self-renewal and differentiation into progenitor cells. Administration of anti-CD117 antibodies can block the binding of SCF to CD117, thereby consuming HSC and progenitor cells (conditioning) in the patient's body. Autologous gene-edited HSCs are transplanted into the patient's body. Genetically edited cells compete with residual host HSCs to repopulate bone marrow (BM). Anti-CD117 antibodies can block the binding of SCF to wild-type (WT) CD117, but cannot bind to HSCs with edited CD117. Therefore, natural wild-type HSCs are targeted by anti-CD117 antibodies, but gene-edited HSCs do not. Therefore, both cells express the CD117 polypeptide activated by SCF binding. However, binding of anti-CD117 antibodies to wild-type CD117 disrupts SCF binding and leads to inhibition of SCF signaling in wild-type cells. In contrast, gene-edited HSCs are unresponsive to anti-CD117 antibodies because the amino acid substitutions introduced into CD117 prevent binding of anti-CD117 antibodies but do not interfere with normal SCF binding and signaling.

[0273] In various embodiments, the antibodies of the present invention are currently used in the clinic (for example, if the edited epitope is necessary for the binding of AMG 191). In one embodiment, CD117 is changed to resemble mouse CD117, which does not cross-react with AMG191 or SR1. Because mouse SCF activates mouse and human cells, interspecies variation of the epitope provides a potential variant with SCF activity while having reduced or no anti-CD117 binding. In order to develop new antibodies, ADC, CAR-T cells, etc. can target new epitopes in one of the extracellular domains of CD117 (D1-D5). Therefore, methods for identifying candidate agents for selectively depleting or ablating endogenous stem cell populations are also within the scope of the present invention. Such a method may comprise the following steps: (a) contacting a sample comprising a stem cell population with a test agent (such as an antibody); and (b) detecting whether one or more cells of the stem cell population are depleted or ablated from the sample; wherein, after the contact step, the depletion or ablation of one or more cells of the stem cell population will identify the test agent as a candidate agent. In a further step, the edited cells are identified as not being similarly depleted or ablated by the agent. In some embodiments, the cells are contacted with the test agent for at least about 2-24 hours.

[0274] Illustrative, but non-limiting, examples of compositions and methods for treating hemoglobinopathies are described in International Publication No. WO2020168133, which is herein incorporated by reference in its entirety.

[0275] Treatment

[0276] Method and composition disclosed herein can be used for conditioning the tissue (for example, bone marrow) of subject to implant or transplant, and after this conditioning, stem cell group is applied to subject.The cell (for example, HSC) of transplantation can be autologous cell or allogeneic cell.In some aspects, stem cell group comprises exogenous stem cell group.In some embodiments, stem cell group comprises endogenous stem cell (for example, having been genetically modified to correct disease or genetic defect endogenous stem cell) of subject.Preferably, such method and composition can be used for treating such disease and can not cause the toxicity observed in response to traditional conditioning therapy (for example radiation).

[0277] Hematopoietic stem cell transplantation therapy can be applied to the subject in need of treatment, to fill or refill one or more blood cell types.Hematopoietic stem cells usually show pluripotency, and therefore can be differentiated into a variety of different blood lineages, including but not limited to granulocytes (for example, promyelocytes, neutrophils, eosinophils, basophils), erythrocytes (for example, reticulocytes, erythrocytes), platelets (for example, megakaryocytes, platelet-producing megakaryocytes, platelets), monocytes (for example, monocytes, macrophages), dendritic cells, microglia, osteoclasts and lymphocytes (for example, NK cells, B cells and T cells).Hematopoietic stem cells also have the ability of self-renewal, so it is possible to produce daughter cells with equal potential as mother cells, and also have the ability to reintroduce transplant recipients, so they home to hematopoietic stem cell locations and rebuild productive and persistent hematopoiesis.

[0278] Therefore, hematopoietic stem cells can be applied to patients with defects or deletions in one or more cell types of the hematopoietic lineage to rebuild defective or missing cell populations in vivo, thereby treating pathologies associated with defects or depletion in endogenous blood cell populations. Therefore, the compositions and methods described herein can be used to treat non-malignant hemoglobinopathies (e.g., hemoglobinopathies selected from the group consisting of sickle cell anemia, thalassemia, Fanconi anemia, aplastic anemia, and Wiskott-Aldrich syndrome). Additionally or alternatively, the compositions and methods described herein can be used to treat malignancies or proliferative diseases, such as blood cancers, myeloproliferative diseases. In the case of cancer treatment, the compositions and methods described herein can be applied to patients to consume endogenous hematopoietic stem cell populations before hematopoietic stem cell transplantation therapy, in which case the transplanted cells can home to the resulting location by the step of endogenous cell depletion and establish productive hematopoietic function. In turn, this can rebuild the cell populations exhausted during cancer cell eradication, such as during systemic chemotherapy. Exemplary hematological cancers that can be treated using the compositions and methods described herein include, but are not limited to, acute myeloid leukemia, acute lymphoid leukemia, chronic myeloid leukemia, chronic lymphoid leukemia, multiple myeloma, diffuse large B-cell lymphoma, and non-Hodgkin lymphoma, as well as other cancerous conditions including neuroblastoma.

[0279] The antibodies, antigen-binding fragments thereof, and ligands described herein can be administered to patients (e.g., human patients suffering from cancer, autoimmune diseases, or those requiring hematopoietic stem cell transplantation) in a variety of dosage forms. For example, the antibodies, antigen-binding fragments thereof, and ligands described herein can be administered to patients suffering from cancer, autoimmune diseases, or those requiring hematopoietic stem cell transplantation in the form of an aqueous solution, such as an aqueous solution containing one or more pharmaceutically acceptable excipients. Pharmaceutically acceptable excipients used with the compositions and methods described herein include viscosity modifiers. The aqueous solution can be sterilized using techniques known in the art.

[0280] The antibodies, Fabs and ligands described herein can be administered by a variety of routes, such as oral, transdermal, subcutaneous, intranasal, intravenous, intramuscular, intraocular or parenteral. The most appropriate route of administration in any given case will depend on the specific antibody, Fab or ligand being administered, the patient, the pharmaceutical formulation method, the method of administration (e.g., time of administration and route of administration), the patient's age, body weight, sex, severity of the disease being treated, the patient's diet and the patient's excretion rate.

[0281] Nucleobase editors

[0282] Disclosed herein are base editors or nuclear base editors for editing, modifying or changing the target nucleotide sequence of a polynucleotide. A nuclear base editor or base editor comprising a polynucleotide programmable nucleotide binding domain and a nuclear base editing domain (e.g., adenosine deaminase, cytidine deaminase) is described herein. The polynucleotide programmable nucleotide binding domain, when bound to a bound guide polynucleotide (e.g., gRNA), can specifically bind to a target polynucleotide sequence (i.e., by a complementary base pairing sequence between the bases of the bound guide nucleic acid and the bases of the target polynucleotide), thereby positioning the base editor to the target nucleic acid sequence that needs to be edited. In some embodiments, the target polynucleotide sequence comprises single-stranded DNA or double-stranded DNA. In some embodiments, the target polynucleotide sequence comprises RNA. In some embodiments, the target polynucleotide sequence comprises a DNA-RNA hybrid.

[0283] Polynucleotide programmable nucleotide binding domain

[0284] It should be understood that the polynucleotide programmable nucleotide binding domain can also include nucleic acid programmable proteins that bind RNA. For example, the polynucleotide programmable nucleotide binding domain can be associated with a nucleic acid that guides the polynucleotide programmable nucleotide binding domain to the RNA. Other nucleic acid programmable DNA binding proteins are also within the scope of the present disclosure, although they are not specifically listed in the present disclosure.

[0285] The polynucleotide programmable nucleotide binding domain of the base editor itself may include one or more domains. For example, the polynucleotide programmable nucleotide binding domain may include one or more nuclease domains. In some embodiments, the nuclease domain of the polynucleotide programmable nucleotide binding domain may include an endonuclease or an exonuclease. In this article, the term "exonuclease" refers to a protein or polypeptide capable of digesting nucleic acids (e.g., RNA or DNA) from free ends, and the term "endonuclease" refers to a protein or polypeptide capable of catalyzing (e.g., cutting) nucleic acids (e.g., DNA or RNA) inside. In some embodiments, an endonuclease can cut a single strand of a double-stranded nucleic acid. In some embodiments, an endonuclease can cut a double strand of a double-stranded nucleic acid. In some embodiments, the polynucleotide programmable nucleotide binding domain may be a deoxyribonuclease. In some embodiments, the polynucleotide programmable nucleotide binding domain may be a ribonuclease.

[0286] In some embodiments, the nuclease domain of a polynucleotide programmable nucleotide binding domain can cut zero, one or two chains of a target polynucleotide. In some cases, the polynucleotide programmable nucleotide binding domain may include a nickase domain. In this article, the term "nickase" refers to a polynucleotide programmable nucleotide binding domain comprising a nuclease domain that can cut only one chain of the two chains in a double-stranded nucleic acid molecule (e.g., DNA). In some embodiments, a nickase can be derived from a polynucleotide programmable nucleotide binding domain of complete catalytic activity (e.g., natural) form by introducing one or more mutations into an active polynucleotide programmable nucleotide binding domain. For example, in the case where a polynucleotide programmable nucleotide binding domain includes a nickase domain derived from Cas9, the nickase domain derived from Cas9 can include a D10A mutation and histidine at position 840. In such cases, residue H840 retains catalytic activity and can thus cut a single strand of a nucleic acid duplex. In another example, the nickase domain derived from Cas9 can include an H840A mutation, and the amino acid residue at position 10 is still D. In some embodiments, by removing all or part of the nuclease domain that is not needed for nickase activity, nickase can be derived from a polynucleotide programmable nucleotide binding domain with a fully catalytically active (e.g., native) form. For example, in the case where the polynucleotide programmable nucleotide binding domain comprises a nickase domain derived from Cas9, the nickase domain derived from Cas9 may comprise all or part of a deletion of a RuvC domain or a HNH domain.

[0287] The amino acid sequence of an exemplary catalytically active Cas9 is as follows:

[0288]

[0289] The base editor comprising a polynucleotide programmable nucleotide binding domain comprising a nickase domain is therefore able to produce single-stranded DNA breaks (nicks) at a specific polynucleotide target sequence (e.g., determined by the complementary sequence of the bound guide nucleic acid). In some embodiments, the chain of the nucleic acid duplex target polynucleotide sequence cut by a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain) is a chain that is not edited by a base editor (i.e., the chain cut by the base editor is opposite to the chain comprising the base to be edited). In other embodiments, a base editor comprising a nickase domain (e.g., a Cas9-derived nickase domain) can cut the chain of a DNA molecule that is targeted for editing. In such cases, the non-targeting chain is not cut.

[0290] Also provided herein is a base editor comprising a catalytically dead polynucleotide programmable nucleotide binding domain (i.e., unable to cut the target polynucleotide sequence). In this article, the terms "catalytically dead (catalytically dead)" and "nuclease dead (nuclease dead)" are used interchangeably to refer to a polynucleotide programmable nucleotide binding domain having one or more mutations and / or deletions that result in its inability to cut a nucleic acid chain. In some embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain base editor may lack nuclease activity due to specific point mutations in one or more nuclease domains. For example, in the case where the base editor comprises a Cas9 domain, Cas9 may comprise a D10A mutation and an H840A mutation. Such mutations inactivate two nuclease domains, resulting in loss of nuclease activity. In other embodiments, the catalytically dead polynucleotide programmable nucleotide binding domain may comprise one or more deletions of all or part of a catalytic domain (e.g., RuvC1 and / or HNH domain). In a further embodiment, the catalytically dead polynucleotide programmable nucleotide binding domain comprises a point mutation (e.g., D10A or H840A) and a deletion of all or part of a nuclease domain.

[0291] Also contemplated herein are mutations of polynucleotide programmable nucleotide binding domains that are capable of producing catalytically dead polynucleotide programmable nucleotide binding domains from a previous functional version of the polynucleotide programmable nucleotide binding domain. For example, in the case of catalytically dead Cas9 ("dCas9"), variants with mutations other than D10A and H840A are provided, which result in nuclease-inactivated Cas9. For example, such mutations include other amino acid replacements at D10 and H840, or other replacements within the Cas9 nuclease domain (e.g., replacements in the HNH nuclease subdomain and / or RuvC1 subdomain). Based on the present disclosure and knowledge in the art, other suitable dCas9 domains without nuclease activity will be apparent to those skilled in the art and are within the scope of the present disclosure. Such additional exemplary suitable nuclease-inactive Cas9 domains include, but are not limited to, D10A / H840A, D10A / D839A / H840A, and D10A / D839A / H840A / N863A mutant domains (see, e.g., Prashant et al., CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nature Biotechnology. 2013; 31(9):833-8338, incorporated by reference in its entirety.

[0292] Non-limiting examples of polynucleotide programmable nucleotide binding domains that can be incorporated into base editors include CRISPR protein-derived domains, restriction nucleases, meganucleases, TAL nucleases (TALENs), and zinc finger nucleases (ZFNs). In some cases, the base editor comprises a polynucleotide programmable nucleotide binding domain comprising a natural or modified protein or portion thereof that is capable of binding to a nucleic acid sequence (i.e., clustered regularly spaced short palindromic repeats) during CRISPR-mediated nucleic acid modification via a bound guide nucleic acid. This protein is referred to herein as a "CRISPR protein." Thus, disclosed herein are base editors comprising a polynucleotide-programmable nucleotide-binding domain comprising all or a portion of a CRISPR protein (i.e., base editors comprising all or a portion of a CRISPR protein as a domain, also referred to as "CRISPR protein-derived domains"). The CRISPR protein-derived domains incorporating the base editors can be modified compared to a wild-type or naturally occurring version of the CRISPR protein. For example, as described below, the CRISPR protein-derived domains can comprise one or more mutations, insertions, deletions, rearrangements, and / or recombinations relative to a wild-type or naturally occurring form of the CRISPR protein.

[0293] CRISPR is an adaptive immune system that provides protection against mobile genetic elements (viruses, transposable elements, and conjugative plasmids). The CRISPR cluster contains a spacer, a sequence complementary to the preceding mobile element, and a target invading nucleic acid. The CRISPR cluster is transcribed and processed into CRISPR RNA (crRNA). In the type II CRISPR system, the correct processing of pre-crRNA requires transcoded small RNA (tracrRNA), endogenous ribonuclease 3 (rnc), and Cas9 protein. tracrRNA serves as a guide for ribonuclease 3 to assist in the processing of pre-crRNA. Subsequently, Cas9 / crRNA / tracrRNA endonucleases linear or circular dsDNA targets that are complementary to the spacer. The target chain that is not complementary to the crRNA is first cut by endonucleolysis and then trimmed 3′-5′ by exonucleolysis. In nature, DNA binding and cleavage generally require proteins and two types of RNA. However, single guide RNA ("sgRNA", or simply "gRNA") can be engineered to integrate aspects of crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J.A., Charpentier E. Science 337:816-821 (2012), the entire contents of which are incorporated herein by reference. Cas9 recognizes a short motif within the CRISPR repeat sequence (PAM or protospacer adjacent motif) to help distinguish self from non-self.

[0294] In some embodiments, the methods described herein can utilize engineered Cas proteins. A guide RNA (gRNA) is a short synthetic RNA consisting of a scaffold sequence required for Cas binding and a user-defined ~20 nucleotide spacer that defines the genomic (or polynucleotide, such as DNA or RNA) target to be modified. Thus, technicians can change the genomic or polynucleotide target of the Cas protein by changing the target sequence present in the gRNA. The specificity of the Cas protein depends in part on the specificity of the gRNA targeting sequence for the genomic nucleotide targeting sequence compared to the rest of the genome.

[0295] In some embodiments, the gRNA scaffold sequence is as follows: GUUUUAGAGC UAGAAAUAGC AAGUUAAAAUAAGGCUAGUC CGUUAUCAAC UUGAAAAAGU GGCACCGAGU CGGUGCUUUU.

[0296] In one embodiment, the RNA scaffold comprises a stem-loop. In one embodiment, the RNA scaffold comprises a nucleic acid sequence:

[0297] GUUUUUGUACUCUCAAGAUUUAAGUAACUGUACAACGAAACUUACACAGUUACUUAAAUCUUGCAGAAGCUACAAAGAUAAGGCUUCAUGCCGAAAUCAACACCCUGUCAUUUUAUGGCAGGGUG.

[0298] In one embodiment, the RNA scaffold comprises the nucleic acid sequence:

[0299] GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCCGGUGCUUUU.

[0300] In some embodiments, the Streptococcus pyogenes sgRNA scaffold polynucleotide sequence is as follows:

[0301] GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUGAAAAAGUGGCACCGAGUCGGUGC.

[0302] In one embodiment, the S. aureus scaffold polynucleotide sequence is as follows:

[0303] GUUUUAGUACUCUGUAAUGAAAAUUACAGAAUCUACUAAAACAAGGCAAAAUGCCGUGUUUAUCUCGUCAACUUGUUGGCGAGA.

[0304] In one embodiment, the BhCas12b sgRNA scaffold has the following polynucleotide sequence:

[0305] GUUCUGTCUUUUGGUCAGGACAACCGUCUAGCUAUAAGUGCUGCAGGGUGUGAGAAACUCCUAUUGCUGGACGAUGUCUCUUACGAGGCAUUAGCAC.

[0306] In one embodiment, the BvCas12b sgRNA scaffold has the following polynucleotide sequence: GACCUAUAGGGUCAAUGAAUCUGUGCGUGUGCCAUAAGUAAUUAAAAAUUACCCACCACAGGAGCACCUGAAAACAGGUGCUUGGCAC.

[0307] In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is an endonuclease (e.g., a deoxyribonuclease or a ribonuclease) that can bind to a target polynucleotide when bound to a bound guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is a nickase that can bind to a target polynucleotide when bound to a bound guide nucleic acid. In some embodiments, the CRISPR protein-derived domain incorporated into the base editor is a catalytic death domain that can bind to a target polynucleotide when bound to a bound guide nucleic acid. In some embodiments, the target polynucleotide bound by the CRISPR protein-derived domain of the base editor is DNA. In some embodiments, the target polynucleotide bound by the CRISPR protein-derived domain of the base editor is RNA.

[0308] Cas proteins useful herein include Class 1 and Class 2. Non-limiting examples of Cas proteins include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas5d, Cas5t, Cas5h, Cas5a, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 or Csx12), Cas10, Csy1, Csy2, Csy3, Csy4, Cse1, Cse2, Cse3, Cse4, Cse5e, Csc1, Csc2, Csa5, Csn1, Csn2, Csm1, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, C Cas12a / Cpf1, Cas12b / C2c1, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i and Cas12j / CasΦ, CARF, DinG, homologs thereof, or modified versions thereof. Unmodified CRISPR enzymes can have DNA cleavage activity, such as Cas9, which has two functional endonuclease domains: RuvC and HNH. The CRISPR enzyme can direct cleavage of one or both strands at a target sequence, e.g., within the target sequence and / or within the complement of the target sequence. For example, the CRISPR enzyme can direct cleavage of one or both strands within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence.

[0309] Vectors encoding a CRISPR enzyme that is mutated relative to the corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence can be used. Cas9 can refer to a polypeptide having at least or at least about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., Cas9 from Streptococcus pyogenes). Cas9 can refer to a polypeptide having at most or at most about 50%, 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity and / or sequence homology to a wild-type exemplary Cas9 polypeptide (e.g., from Streptococcus pyogenes). Cas9 can refer to a wild-type or modified form of a Cas9 protein, which can include amino acid changes, such as deletions, insertions, substitutions, variants, mutations, fusions, chimeras, or any combination thereof.

[0310] In some embodiments, the CRISPR protein-derived domain of the base editor can include a CRISPR protein-derived domain from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma syrphidicola (NCBI Refs: NC_021284.1); Prevotella intermedia (NCBI Refs: NC_017861.1); Spiroplasma taiwanense, China (NCBI Refs: NC_021846.1); Streptococcus iniae (NCBI Refs: NC_021314.1); Belliella baltica (NCBI Refs: NC_021285.1); Refs: NC_018010.1); Psychroflexus torquisI (NCBI Ref: NC_018721.1); Streptococcus thermophilus (NCBI Refs: YP_820832.1); Listeria innocua (NCBI Refs: NP_472073.1); Campylobacter jejuni (NCBI Refs: YP_002344900.1); Neisseria meningitidis (NCBI Refs: YP_002342100.1), Streptococcus pyogenes, or Staphylococcus aureus.

[0311] Cas9 domains of nucleobase editors

[0312] The Cas9 nuclease sequence and structure are well known to those skilled in the art (see, e.g., “Complete genome sequence of an M1 strain of Streptococcus pyogenes.” Ferretti et al., JJ, McShan WM, Ajdic DJ, Savic DJ, Savic G., Lyon K., Primeaux C., Sezate S., Suvorov AN, Kenton S., Lai HS, Lin SP, Qian Y., Jia HG, Najar FZ, Ren Q., Zhu H., Song L., White J., Yuan X., Clifton SW, Roe BA, McLaughlin RE, Proc. Natl. Acad. Sci. USA 98:4658-4663 (2001); “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” Deltcheva E., Chylinski J., et al., “CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III.” K., Sharma C. M., Gonzales K., Chao Y., Pirzada Z. A, Eckert MR, Vogel J., Charpentier E., Nature 471:602-607 (2011); and "A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity." Jinek M., Chylinski K., Fonfara I., Hauer M., Doudna J. A., Charpentier E. Science 337:816-821 (2012), the entire contents of each of which are incorporated herein by reference. Cas9 orthologs have been described in various species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus.Other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, “The tracrRNA and Cas9 families of type II CRISPR-Cas immunity systems” (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0313] In some aspects, nucleic acid programmable DNA binding protein (napDNAbp) is a Cas9 domain. Non-limiting exemplary Cas9 domains are provided herein. The Cas9 domain can be a nuclease activity Cas9 domain, a nuclease inactive Cas9 domain or a Cas9 nickase. In some embodiments, the Cas9 domain is a domain with nuclease activity. For example, the Cas9 domain can be a Cas9 domain that cuts two chains of a double-stranded nucleic acid (e.g., two chains of a double-stranded DNA molecule). In some embodiments, the Cas9 domain comprises any one of the amino acid sequences described herein. In some embodiments, the amino acid sequence included in the Cas9 domain is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% or 99.5% identical to any one of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 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 mutations compared to any of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises a polypeptide having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any of the amino acid sequences described herein.

[0314] In some embodiments, proteins comprising Cas9 fragments are provided. For example, in some embodiments, the protein comprises one of two Cas9 domains: (1) the gRNA binding domain of Cas9; or (2) the DNA cleavage domain of Cas9. In some embodiments, proteins comprising Cas9 or a fragment thereof are referred to as "Cas9 variants". Cas9 variants have homology to Cas9 or a fragment thereof. For example, a Cas9 variant is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to wild-type Cas9. In some embodiments, the Cas9 variant can 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 wild-type Cas9. In some embodiments, the Cas9 variant comprises a fragment of Cas9 (e.g., a gRNA binding domain or a DNA cleavage domain) such that the fragment is at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 96% identical, at least about 97% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical to the corresponding fragment of wild-type Cas9. In some embodiments, the fragment is at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% identical, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% the amino acid length of the corresponding wild-type Cas9. In some embodiments, the fragment is at least 100 amino acids long. In some embodiments, the fragment is at least 100, 150, 200, 250, 300, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000, 1050, 1100, 1150, 1200, 1250, or 1300 amino acids in length.

[0315] In some embodiments, the Cas9 fusion proteins provided herein comprise the full-length amino acid sequence of a Cas9 protein, such as one of the Cas9 sequences provided herein. However, in other embodiments, the fusion proteins provided herein do not comprise the full-length Cas9 sequence, but rather comprise only one or more fragments thereof. Exemplary amino acid sequences of suitable Cas9 domains and Cas9 fragments are provided herein, and other suitable sequences of Cas9 domains and fragments will be apparent to those skilled in the art.

[0316] The Cas9 protein can be associated with a guide RNA that guides the Cas9 protein to a specific DNA sequence that is complementary to the guide RNA. In some embodiments, the polynucleotide programmable nucleotide binding domain is a Cas9 domain, such as a nuclease-active Cas9, a Cas9 nickase (nCas9), or a nuclease-inactivated Cas9 (dCas9). Examples of nucleic acid programmable DNA-binding proteins include, but are not limited to, Cas9 (e.g., dCas9 and nCas9), CasX, CasY, Cpf1, Cas12b / C2C1, Cas12c / C2C3, and Cas12j / CasΦ.

[0317] In some embodiments, the wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes (NCBI Reference Sequence: NC_017053.1, nucleotide and amino acid sequences below).

[0318]

[0319]

[0320] (Single underline: HNH domain; double underline: RuvC domain)

[0321] In some embodiments, the wild-type Cas9 corresponds to or comprises the following nucleotide and / or amino acid sequence:

[0322]

[0323]

[0324] (Single underline: HNH domain; double underline: RuvC domain).

[0325] In some embodiments, wild-type Cas9 corresponds to Cas9 from Streptococcus pyogenes

[0326] (NCBI reference sequence: NC_002737.2 (nucleotide sequence below); and Uniprot reference sequence: Q99ZW2 (amino acid sequence below):

[0327]

[0328]

[0329] (Single underline: HNH domain; double underline: RuvC domain)

[0330] In some embodiments, Cas9 refers to Cas9 from Corynebacterium ulcerans (NCBI Refs: NC_015683.1, NC_017317.1); Corynebacterium diphtheria (NCBI Refs: NC_016782.1, NC_016786.1); Spiroplasma yrphidicola (NCBI Refs: NC_021284.1); Prevotella intermedia (NCBI Refs: NC_017861.1); Spiroplasma taiwanense, China (NCBI Refs: NC_021846.1); Streptococcus iniae (NCBI Refs: NC_021314.1); Belliella balticola (NCBI Refs: NC_021284.1); baltica)(NCBI Refs:NC_018010.1); Psychroflexus torqueI(NCBI Refs:NC_018721.1); Streptococcus thermophilus(NCBI Refs:YP_820832.1); Listeria innocua(NCBI Refs:NP_472073.1); Campylobacter jejuni(NCBI Refs:YP_002344900.1); Neisseria meningitidis(NCBI Refs:YP_002342100.1), or Cas9 from any other organism.

[0331] It will be understood that additional Cas9 proteins (e.g., nuclease-dead Cas9 (dCas9), Cas9 nickase (nCas9), or nuclease-active Cas9), including variants and homologs thereof, are within the scope of the present disclosure. Exemplary Cas9 proteins include, but are not limited to, those provided below. In some embodiments, the Cas9 protein is a nuclease-inactive Cas9 (dCas9). In some embodiments, the Cas9 protein is a Cas9 nickase (nCas9). In some embodiments, the Cas9 protein is a Cas9 with nuclease activity.

[0332] In some embodiments, the Cas9 domain is a nuclease-inactivated domain (dCas9). For example, the dCas9 domain can bind to a double-stranded nucleic acid molecule (e.g., through a gRNA molecule) without cutting any chain of the double-stranded nucleic acid molecule. In some embodiments, the nuclease-inactivated dCas9 domain comprises a D10X mutation and an H840X mutation of an amino acid sequence described herein, or a corresponding mutation in any amino acid sequence provided herein, wherein X is any amino acid change. In some embodiments, the nuclease-inactivated dCas9 domain comprises a D10A mutation and an H840A mutation of an amino acid sequence described herein, or a corresponding mutation in any amino acid sequence provided herein. As an example, a nuclease-inactive Cas9 domain is contained in the amino acid sequence listed in the cloning vector pPlatTET-gRNA2 (accession number BAV54124).

[0333] The amino acid sequence of an exemplary catalytically inactive Cas9 (dCas9) is as follows:

[0334]

[0335] (See, e.g., Qi et al., “Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression.” Cell. 2013; 152(5): 1173-83, which is incorporated herein by reference in its entirety).

[0336] In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase, referred to as a "nCas9" protein (for "nickase" Cas9). Nuclease-inactivated Cas9 proteins are interchangeably referred to as "dCas9" proteins (for nuclease-"dead" Cas9) or catalytically inactivated Cas9. Methods for producing Cas9 proteins (or fragments thereof) with inactive DNA cleavage domains are known (see, e.g., Jinek et al., Science. 337: 816-821 (2012); Qi et al. "Repurposing CRISPR as an RNA-guided Platform for Sequence-Specific Control of Gene Expression" (2013) Cell. 28; 152 (5): 1173-83, the entire contents of which are incorporated herein by reference). For example, the DNA cleavage domain of Cas9 is known to include two subdomains, the HNH nuclease subdomain and the RuvC1 subdomain. The HNH subdomain cleaves the strand complementary to the gRNA, while the RuvC1 subdomain cleaves the non-complementary strand. Mutations within these subdomains can silence the nuclease activity of Cas9. For example, mutations D10A and H840A completely inactivate the nuclease activity of Streptococcus pyogenes Cas9 (Jinek et al., Science. 337:816-821 (2012); Qi et al., Cell. 28:152(5):1173-83 (2013)).

[0337] In some embodiments, the dCas9 domain comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 99.5% identical to any of the Cas9 domains described herein. In some embodiments, the Cas9 domain comprises an amino acid sequence having 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 mutations compared to any of the amino acid sequences described herein. In some embodiments, the Cas9 domain comprises a polypeptide having at least 10, at least 15, at least 20, at least 30, at least 40, at least 50, at least 60, at least 70, at least 80, at least 90, at least 100, at least 150, at least 200, at least 250, at least 300, at least 350, at least 400, at least 500, at least 600, at least 700, at least 800, at least 900, at least 1000, at least 1100, or at least 1200 identical contiguous amino acid residues compared to any of the amino acid sequences described herein.

[0338] In some embodiments, the dCas9 corresponds to or comprises, in part or in whole, a Cas9 amino acid sequence having one or more mutations that inactivate Cas9 nuclease activity. For example, in some embodiments, the dCas9 domain comprises D10A and H840A mutations or corresponding mutations in another Cas9.

[0339] In some embodiments, the dCas9 comprises the amino acid sequence of dCas9(D10A and H840A):

[0340]

[0341] (Single underline: HNH domain; double underline: RuvC domain).

[0342] In some embodiments, the Cas9 domain comprises a D10A mutation, while the residue at position 840 remains a histidine in the amino acid sequence provided above or at the corresponding position in any amino acid sequence provided herein.

[0343] In other embodiments, dCas9 variants having mutations other than D10A and H840A are provided, which, for example, result in nuclease-inactivated Cas9 (dCas9). For example, such mutations include other amino acid replacements at D10 and H840, or other replacements within the Cas9 nuclease domain (e.g., replacements in the HNH nuclease subdomain and / or the RuvC1 subdomain). In some embodiments, variants or homologs of dCas9 are provided, which are at least about 70% identical, at least about 80% identical, at least about 90% identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, at least about 99.5% identical, or at least about 99.9% identical. In some embodiments, variants or homologs having shorter or longer lengths of about 5 amino acids, about 10 amino acids, about 15 amino acids, about 20 amino acids, about 25 amino acids, about 30 amino acids, about 40 amino acids, about 50 amino acids, about 75 amino acids, about 100 amino acids or more are provided.

[0344] In some embodiments, the Cas9 domain is a Cas9 nickase. Cas9 nickase can be a Cas9 protein that can only cut a chain of a double-stranded nucleic acid molecule (e.g., double-stranded DNA molecule). In some embodiments, Cas9 nickase cuts the target chain of a double-stranded nucleic acid molecule, meaning that Cas9 nickase cuts the chain that is base-paired (complementary) with the gRNA (e.g., sgRNA) bound to Cas9. In some embodiments, Cas9 nickase includes a D10A mutation and has histidine at position 840. In some embodiments, Cas9 nickase cuts the non-target, non-base editing chain of a double-stranded nucleic acid molecule, meaning that Cas9 nickase cuts the chain that is not base-paired with the gRNA (e.g., sgRNA) bound to Cas9. In some embodiments, Cas9 nickase includes an H840A mutation and has an aspartic acid residue or a corresponding mutation at position 10. In some embodiments, the Cas9 nickase comprises an amino acid sequence that is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% or 99.5% identical to any Cas9 nickase described herein. Based on this disclosure and knowledge in the art, other suitable Cas9 nickases will be apparent to those skilled in the art and are within the scope of this disclosure.

[0345] The amino acid sequence of an exemplary catalytically active Cas9 nickase (nCas9) is as follows:

[0346]

[0347] In some embodiments, Cas9 refers to Cas9 from archaea (e.g., nanoarchaea), which constitute the domain and kingdom of unicellular prokaryotic microorganisms. In some embodiments, the programmable nucleotide binding protein can be a CasX or CasY protein, which has been described in, for example, Burstein et al., "New CRISPR-Cas systems from uncultivated microbes." Cell Res. 2017 Feb 21. doi: 10.1038 / cr.2017.21, the entire contents of which are incorporated herein by reference. Using genome-resolved metagenomics, many CRISPR-Cas systems have been identified, including Cas9, which was first reported in the field of archaea. This divergent Cas9 protein was found in the rarely studied nanoarchaea as part of an active CRISPR-Cas system. In bacteria, two previously unknown systems, CRISPR-CasX and CRISPR-CasY, were found, which are among the most compact systems discovered to date. In some embodiments, in the base editor systems described herein, Cas9 is replaced by CasX or a variant of CasX. In some embodiments, in the base editor systems described herein, Cas9 is replaced by CasY or a variant of CasY. It should be understood that other RNA-guided DNA binding proteins can be used as nucleic acid programmable DNA binding proteins (napDNAbp) and are within the scope of the present disclosure.

[0348] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) or any fusion protein provided herein can be CasX or CasY protein. In some embodiments, napDNAbp is CasY protein. In some embodiments, napDNAbp is CasY protein. In some embodiments, the amino acid sequence included in napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to naturally occurring CasX or CasY protein. In some embodiments, the programmable nucleotide binding protein is a naturally occurring CasX or CasY protein. In some embodiments, the amino acid sequence included in the programmable nucleotide binding protein is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to any CasX or CasY protein described herein. It will be appreciated that CasX and CasY from other bacterial species may also be used in accordance with the present disclosure.

[0349] An exemplary CasX ((uniprot.org / uniprot / F0NN87; uniprot.org / uniprot / F0NH53)tr|F0NN87|F0NN87_SULIHCRISPR-associatedCasx protein OS=Sulfolobus icelandica (strainHVE10 / 4) GN=SiH_0402PE=4SV=1) amino acid sequence is as follows:

[0350] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYEFGRSPGMVERTRRVKLE VEPHYLIIAAAGWVLTRLGKAKVSEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVRIYTISDAVGQNPTTINGGFSIDLTKLLEKRYLLSERLEAIARNALSISSNMRERYIVLANYIYEYLTG SKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0351] An exemplary CasX (>tr|F0NH53|F0NH53_SULIR CRISPR associated protein, CasxOS=Icelandic Sulfolobus (strain REY15A) GN=SiRe_0771 PE=4SV=1) amino acid sequence is as follows:

[0352] MEVPLYNIFGDNYIIQVATEAENSTIYNNKVEIDDEELRNVLNLAYKIAKNNEDAAAERRGKAKKKKGEEGETTTSNIILPLSGNDKNPWTETLKCYNFPTTVALSEVFKNFSQVKECEEVSAPSFVKPEFYKFGRSPGMVERTRRVKLEVEPHYLIMAAAGWVLTRLGKAKV SEGDYVGVNVFTPTRGILYSLIQNVNGIVPGIKPETAFGLWIARKVVSSVTNPNVSVVSIYTISDAVGQNPTTINGGFSIDLTKLLEKRDLLSERLEAIARNALSISSMRERYIVLANYIYEYLTGSKRLEDLLYFANRDLIMNLNSDDGKVRDLKLISAYVNGELIRGEG.

[0353] Delta bacteriumCasX

[0354] MEKRINKIRKKLSADNATKPVSRSGPMKTLLVRVMTDDLKKRLEKRRKKPEVMPQVISNNAANNLRMLLDDYTKMKEAILQVYWQEFKDDHVGLMCKFAQPASKKIDQNKLKPEMDEKGNLTTAGFACSQCGQPLFVYKLEQVSEKGKAYTNYFGRCNVAEHEKLILLAQLKPVKDSDEAVTYSLGKFGQRALDFYSIHVTKESTHPVKPLAQIAGNRYASGPVGKALSDACMGTIASFLSKYQDIIIEHQKVVKGNQKRLESLRELAGKENLEYPSVTLPPQPHTKEGVDfAYNEVIARVRMWVNLLWQKLKLSRDDAKPLLRLKGFPSFPVVERRENEVDWWNTINEVKKLIDAKRDMGRVFWSGVTAEKRNTILEGYNYLPNENDHKKREGSLENPKKPAKRQFGDLLLYLEKKYAGDWGKVFDEAWERIDKKIAGLTSHIEREEARNAEDAQSKAVLTDWLRAKASFVLERLKEMDEKEFYACEIQLQ KWYGDLRGNPFAVEAENRVVDISGFSIGSDGHSIQYRNLLAWKYLENGKREFYLLMNYGKKGRIRFTDGTDIKKSGKWQGLLYGGGGKAKVIDLTFDPDDEQLIILPLAFGTRQGREFIWNDLLSLETGLIKLANGRVIEKTIYNKKIGRDEPALFVALTFERREVVDPSNIKPVNLIGVARGENIPAVIALTDPEGCPLPEFKDSSGGPTDILRIGEGYKEKQRAIQAAKEVEQRRAGGYSRKFASKSRNLADDMVRNSARDLFYHAVTHDAVLVFANLSRGFGRQGKRTFMTERQYTKMEDWLTAKLAYEGLTSKTYLSKTLAQYTSKTCSNCGFTITYADMDVMLVRLKKTSDGWATTLNNKELKAEYQITYYNRYKRQTVEKELSAELDRLSEESGNNDISKWTKGRRDEALFLLKKRFSHRPVQEQFVCLDCGHEVHAAEQAALNIARSWLFLNSNSTEFKSYKSGKQPFVGAWQAFYKRRLKEVWKPNA

[0355] Exemplary CasY

[0356] The amino acid sequence of the CRISPR-associated protein CasY (uncultured Parcubacteria) is as follows:

[0357]

[0358] In some embodiments, nucleic acid programmable DNA binding protein (napDNAbp) is a single effector of a microbial CRISPR-Cas system. Single effectors of microbial CRISPR-Cas systems include, but are not limited to, Cas9, Cpf1, Cas12b / C2c1, and Cas12c / C2c3. Typically, microbial CRISPR-Cas systems are divided into Class 1 and Class 2 systems. Class 1 systems have multi-subunit effector complexes, while Class 2 systems have single protein effectors. For example, Cas9 and Cpf1 are Class 2 effectors. In addition to Cas9 and Cpf1, three different Class 2 CRISPR-Cas systems (Cas12b / C2c1 and Cas12c / C2c3) are described by Shmakov et al. in “Discovery and Functional Characterization of Diverse Class 2 CRISPR Cas Systems”, Mol. Cell, 2015 Nov. 5; 60(3): 385-397, the entire contents of which are incorporated herein by reference. The effectors of two of these systems, Cas12b / C2c1 and Cas12c / C2c3, contain a RuvC-like endonuclease domain associated with Cpf1. The third system contains an effector with two predicted HEPN RNase domains. Unlike the CRISPR RNA produced by Cas12b / C2c1, which relies on both CRISPR RNA and tracrRNA for DNA cleavage, the production of mature CRISPR RNA is independent of tracrRNA.

[0359] It has been reported that the crystal structure of Alicyclobaccillus acidoterrastris Cas12b / C2c1 (AacC2c1) is complexed with a chimeric single-molecule guide RNA (sgRNA). See, e.g., Liu et al., “C2c1-sgRNA Complex Structure Reveals RNA-guided DNA Cleavage Mechanism”, Mol. Cell, 2017 Jan. 19; 65(2): 310-322, the entire contents of which are incorporated herein by reference. A crystal structure has also been reported in Alicyclobaccillus acidoterrastris C2c1 bound to target DNA in a ternary complex. See, e.g., Yang et al., “PAM-dependent Target DNA Recognition and Cleavage by C2C1 CRISPR-Cas endonuclease”, Cell, 2016 Dec. 15; 167(7): 1814-1828, the entire contents of which are incorporated herein by reference. The catalytically competent conformation of AacC2c1, encompassing both the target and non-target DNA strands, has been independently trapped within a single RuvC catalytic pocket, and Cas12b / C2c1-mediated cleavage results in a seven-nucleotide staggered break of the target DNA. Structural comparisons between the Cas12b / C2c1 ternary complex and previously identified Cas9 and Cpf1 counterparts demonstrate the diversity of mechanisms employed by the CRISPR-Cas9 system.

[0360] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) or any fusion protein provided herein can be Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, napDNAbp is Cas12b / C2c1 protein. In some embodiments, napDNAbp is Cas12c / C2c3 protein. In some embodiments, the amino acid sequence included in napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, napDNAbp is naturally occurring Cas12b / C2c1 or Cas12c / C2c3 protein. In some embodiments, the amino acid sequence comprised by napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to any of the napDNAbp sequences provided herein. It should be understood that Cas12b / C2c1 or Cas12c / C2c3 from other bacterial species can also be used according to the present disclosure.

[0361] A Cas12b / C2c1 ((uniprot.org / uniprot / T0D7A2#2) sp|T0D7A2|C2C1_ALIAGCRISPR-associated endonuclease C2c1 OS=Alicyclobacillus acidophilus (strain ATCC 49025 / DSM3922 / CIP 106132 / NCIMB 13137 / GD3B) GN=c2c1 PE=1SV=1) amino acid sequence is as follows:

[0362]

[0363] BhCas12b (Bacillus subtilis) NCBI Reference Sequence: WP_095142515

[0364]

[0365] In some embodiments, Cas12b is BvCas12B, which is a variant of BhCas12b and comprises the following changes relative to BhCas12B: S893R, K846R, and E837G.

[0366] BvCas12b (Bacillus V3-13) NCBI Reference Sequence: WP_101661451.1

[0367]

[0368] In some embodiments, the nucleic acid programmable DNA binding protein (napDNAbp) or any fusion protein provided herein can be a Cas12j / CasΦ protein. Pausch et al. describe Cas12j / CasΦ, "CRISPR-CasΦfromhuge phages is ahypercompact genome editor", Science, July 17, 2020, Vol. 369, No. 6501, pp. 333-337, which is incorporated herein by reference in its entirety. In some embodiments, the amino acid sequence included in napDNAbp is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to a naturally occurring Cas12j / CasΦ protein. In some embodiments, napDNAbp is a naturally occurring Cas12j / CasΦ protein. In some embodiments, the napDNAbp is a nuclease-inactivated (dead) Cas12j / CasΦ protein. It should be understood that Cas12j / CasΦ from other species can also be used according to the present disclosure.

[0369] An exemplary Cas12j / CasΦ amino acid sequence is as follows:

[0370] >CasΦ-1

[0371] MADTPTLFTQFLRHHLPGQRFRKDILKQAGRILANKGEDATIAFLRGKSEESPPDFQPPVKCPIIACSRPLTEWPIYQASVAIQGYVYGQSLAEFEASDPGCSKDGLLGWFDKTGVCTDYFSVQGLNLIFQNARKRYIGVQTKVTNRNEKRHKKLKRINAKRIAEGLPELTSDEPESALDETGHLIDPPGLNTNIYCYQQVSPKPLALSEVNQLPTAYAGYSTSGDDPIQPMVTKDRLSISKGQPGYIPEHQRALLSQKKHRRMRGYGLKARALLVIVRIQDDWAVIDLRSLLRNAYWRRIVQTKEPSTITKLLKLVTGDPVLDATRMVATFTYKPGIVQVRSAKCLKNKQGSKLFSERYLNETVSVTSIDLGSNNLVAVATYRLVNGNTPELLQRFTLPSHLVKDFERYKQAHDTLEDSIQKTAVASLPQGQQTEIRMWSMYGFREAQERVCQELGLADGSIPWNVMTATSTILTDLFLARGGDPKKCMFTSEPKKKKNSKQVLYKIRDRAWAKMYRTLLSKETREAWNKALWGLKRGSPDYARLSKRKEELARRCVNYTISTAEKRAQCGRTIVALEDLNIGFFHGRGKQEPGWVGLFTRKKENRWLMQALHKAFLELAHHRGYHVIEVNPAYTSQTCPVCRHCDPDNRDQHNREAFHCIGCGFRGNADLDVATHNIAMVAITGESLKRARGSVASKTPQPLAAE*

[0372] >CasΦ-2

[0373] MPKPAVESEFSKVLKKHFPGERFRSSYMKRGGKILAAQGEEAVVAYLQGKSEEEPPNFQPPAKCHVVTKSRDFAEWPIMKASEAIQRYIYALSTTERAACKPGKSSESHAAWFAATGVSNHGYSHVQGLNLIFDHTLGRYDGVLKKVQLRNEKARARLESINASRADEGLPEIKAEEEEVATNETGHLLQPPGINPSFYVYQTISPQAYRPRDEIVLPPEYAGYVRDPNAPIPLGVVRNRCDIQKGCPGYIPEWQREAGTAISPKTGKAVTVPGLSPKKNKRMRRYWRSEKEKAQDALLVTVRIGTDWVVIDVRGLLRNARWRTIAPKDISLNALLDLFTGDPVIDVRRNIVTFTYTLDACGTYARKWTLKGKQTKATLDKLTATQTVALVAIDLGQTNPISAGISRVTQENGALQCEPLDRFTLPDDLLKDISAYRIAWDRNEEELRARSVEALPEAQQAEVRALDGVSKETARTQLCADFGLDPKRLPWDKMSSNTTFISEALLSNSVSRDQVFFTPAPKKGAKKKAPVEVMRKDRTWARAYKPRLSVEAQKLKNEALWALKRTSPEYLKLSRRKEELCRRSINYVIEKTRRRTQCQIVIPVIEDLNVRFFHGSGKRLPGWDNFFTAKKENRWFIQGLHKAFSDLRTHRSFYVFEVRPERTSITCPKCGHCEVGNRDGEAFQCLSCGKTCNADLDVATHNLTQVALTGKTMPKREEPRDAQGTAPARKTKKASKSKAPPAEREDQTPAQEPSQTS

[0374] >CasΦ-3

[0375] *

[0376] >CasΦ-4

[0377] *

[0378] >CasΦ-5MSSLPTPLELLKQKHADLFKGLQFSSKDNKMAGKVLKKDGEEAALAFLSERGVSRGELPNFRPPAKTLVVAQSRPFEEFPIYRVSEAIQLYVYSLSVKELETVPSGSSTKKEHQRFFQDSSVPDFGYTSVQGLNKIFGLARGIYLGVITRGENQLQKAKSKHEALNKKRRASGEAETEFDPTPYEYMTPERKLAKPPGVNHSIMCYVDISVDEFDFRNPDGIVLPSEYAGYCREINTAIEKGTVDRLGHLKGGPGYIPGHQRKESTTEGPKINFRKGRIRRSYTALYAKRDSRRVRQGKLALPSYRHHMMRLNSNAESAILAVIFFGKDWVVFDLRGLLRNVRWRNLFVDGSTPSTLLGMFGDPVIDPKRGVVAFCYKEQIVPVVSKSITKMVKAPELLNKLYLKSEDPLVLVAIDLGQTNPVGVGVYRVMNASLDYEVVTRFALESELLREIESYRQRTNAFEAQIRAETFDAMTSEEQEEITRVRAFSASKAKENVCHRFGMPVDAVDWATMGSNTIHIAKWVMRHGDPSLVEVLEYRKDNEIKLDKNGVPKKVKLTDKRIANLTSIRLRFSQETSKHYNDTMWELRRKHPVYQKLSKSKADFSRRVVNSIIRRVNHLVPRARIVFIIEDLKNLGKVFHGSGKRELGWDSYFEPKSENRWFIQVLHKAFSETGKHKGYYIIECWPNWTSCTCPKCSCCDSENRHGEVFRCLACGYTCNTDFGTAPDNLVKIATTGKGLPGPKKRCKGSSKGKNPKIARSSETGVSVTESGAPKVKKSSPTQTSQSSSQSAP*

[0379] >CasΦ-6

[0380] MNKIEKEKPLAKLMNENFAGLRFPFAIIKQAGKKLLKEGELKTIEYMTGKGSIEPLPNFKPPVKCLIVAKRRDLKYFPICKASCEIQSYVYSLNYKDFMDYFSTPMTSQKQHEEFFKKSGLNIEYQNVAGLNLIFNNVKNTYNGVILKVKNRNEKLKKKAIKNYEFEEIKTFNDDGCLINKPGINNVIYCFQSISPKILKNITHLPKEYNDYDCSVDRNIIQKYVSRLDIPESQPGHVPEWQRKLPEFNNTNNPRRRRKWYSNGRNISKGYSVDQVNQAKIEDSLLAQIKIGEDWIILDIRGLLRDLNRRRELISYKNKLTIKDVLGFFSDYPIIDIKKNLVTFCYKEGVIQVVSQKSIGNKKSKQLLEKLIENKPIALVSIDLGQTNPVSVKISKLNKINKISIESFTYRFLNEEILKEIEKYRKDYDKLELKLINEA

[0381] >CasΦ-7

[0382] MSNTAVSTREHMSNKTTPPSPLSLLLRAHFPGLKFESQDYKIAGKKLRDGGPEAVISYLTGKGQAKLKDVKPPAKAFVIAQSRPFIEWDLVRVSRQIQEKIFGIPATKGRPKQDGLSETAFNEAVASLEVDGKSKLNEETRAAFYEVLGLDAPSLHAQAQNALIKSAISIREGVLKKVENRNEKNLSKTKRRKEAGEEATFVEEKAHDERGYLIHPPGVNQTIPGYQAVVIKSCPSDFIGLPSGCLAKESAEALTDYLPHDRMTIPKGQPGYVPEWQHPLLNRRKNRRRRDWYSASLNKPKATCSKRSGTPNRKNSRTDQIQSGRFKGAIPVLMRFQDEWVIIDIRGLLRNARYRKLLKEKSTIPDLLSLFTGDPSIDMRQGVCTFIYKAGQACSAKMVKTKNAPEILSELTKSGPVVLVSIDLGQTNPIAAKVSRVTQLSDGQLSHETLLRELLSNDSSDGKEIARYRVASDRLRDKLANLAVERLSPEHKSEILRAKNDTPALCKARVCAALGLNPEMIAWDKMTPYTEFLATAYLEKGGDRKVATLKPKNRPEMLRRDIKFKGTEGVRIEVSPEAAEAYREAQWDLQRTSPEYLRLSTWKQELTKRILNQLRHKAAKSSQCEVVVMAFEDLNIKMMHGNGKWADGGWDAFFIKKRENRWFMQAFHKSLTELGAHKGVPTIEVTPHRTSITCTKCGHCDKANRDGERFACQKCGFVAHADLEIATDNIERVALTGKPMPKPESERSGDAKKSVGARKAAFKPEED

[0383] AEAAE*

[0384] >CasΦ-8

[0385] *

[0386] >CasΦ-9

[0387] *

[0388] >CasΦ-10MDMLDTETNYATETPAQQQDYSPKPPKKAQRAPKGFSKKARPEKKPPKPITLFTQKHFSGVRFLKRVIRDASKILKLSESRTITFLEQAIERDGSAPPDVTPPVHNTIMAVTRPFEEWPEVILSKALQKHCYALTKKIKIKTWPKKGPGKKCLAAWSARTKIPLIPGQVQATNGLFDRIGSIYDGVEKKVTNRNANKKLEYDEAIKEGRNPAVPEYETAYNIDGTLINKPGYNPNLYITQSRTPRLITEADRPLVEKILWQMVEKKTQSRNQARRARLEKAAHLQGLPVPKFVPEKVDRSQKIEIRIIDPLDKIEPYMPQDRMAIKASQDGHVPYWQRPFLSKRRNRRVRAGWGKQVSSIQAWLTGALLVIVRLGNEAFLADIRGALRNAQWRKLLKPDATYQSLFNLFTGDPVVNTRTNHLTMAYREGVVNIVKSRSFKGRQTREHLLTLLGQGKTVAGVSFDLGQKHAAGLLAAHFGLGEDGNPVFTPIQACFLPQRYLDSLTNYRNRYDALTLDMRRQSLLALTPAQQQEFADAQRDPGGQAKRACCLKLNLNPDEIRWDLVSGISTMISDLYIERGGDPRDVHQQVETKPKGKRKSEIRILKIRDGKWAYDFRPKIADETRKAQREQLWKLQKASSEFERLSRYKINIARAIANWALQWGRELSGCDIVIPVLEDLNVGSKFFDGKGKWLLGWDNRFTPKKENRWFIKVLHKAVAELAPHRGVPVYEVMPHRTSMTCPACHYCHPTNREGDRFECQSCHVVKNTDRDVAPYNILRVAVEGKTLDRWQAEKKPQAEPDRPMILIDNQES*

[0389] The asterisk (*) in the above sequence represents the STOP codon. Alternatively, CasΦ-1 is also known as Cas12j ortholog 1. Thus, CasΦ-1 - CasΦ-10 can also be called Cas12j orthologs 1 - 10, respectively.

[0390] The Cas9 nuclease has two functional endonuclease domains: RuvC and HNH. Upon target binding, Cas9 undergoes a conformational change that positions the nuclease domains to cleave the opposite strand of the target DNA. The end result of Cas9-mediated DNA cleavage is a double-strand break (DSB) within the target DNA (approximately 3-4 nucleotides upstream of the PAM sequence). The resulting DSB is then repaired via one of two general repair pathways: (1) the efficient but error-prone nonhomologous end joining (NHEJ) pathway; or (2) the less efficient but high-fidelity homology-directed repair (HDR) pathway.

[0391] The "efficiency" of non-homologous end joining (NHEJ) and / or homology-directed repair (HDR) can be calculated by any convenient method. For example, in some cases, efficiency can be expressed as a percentage of successful HDR. For example, Surveyor nuclease assays can be used to generate cleavage products, and the ratio of product to substrate can be used to calculate the percentage. For example, surveyor nuclease can be used to directly cut DNA containing newly integrated restriction sequences as a result of successful HDR. More cleaved substrates indicate a higher HDR percentage (higher HDR efficiency). As an illustrative example, the following equation [(cleavage product) / (substrate plus cleavage product)] (e.g., (b+c) / (a+b+c) can be used to calculate the score (percentage) of HDR, where "a" is the band intensity of the DNA substrate, and "b" and "c" are cleavage products).

[0392] In some cases, efficiency can be expressed as a percentage of successful NHEJ. For example, the T7 endonuclease I assay can be used to generate cleavage products, and the ratio of product to substrate can be used to calculate the NHEJ percentage. T7 endonuclease I cleaves mismatched heteroduplex DNA generated by hybridization of wild-type and mutant DNA chains (NHEJ generates small random insertions or deletions (indels) at the original break site). More cleavage indicates a higher NHEJ percentage (higher NHEJ efficiency). As an illustrative example, the fraction (percentage) of NHEJ can be calculated using the following equation: (1-(1-(b+c) / (a+b+c)) 1 / 2 )×100, where “a” is the band intensity of the DNA substrate, and “b” and “c” are the cleavage products (Ran et al., Cell. 2013 Sep. 12; 154(6): 1380-9; and Ran et al., Nat Protoc. 2013 Nov.; 8(11): 2281–2308).

[0393] The NHEJ repair pathway is the most active repair mechanism, which frequently results in small nucleotide insertions or deletions (indels) at DSB sites. The stochastic nature of NHEJ-mediated DSB repair has important practical implications, as a population of cells expressing Cas9 and gRNA or guide polynucleotides can result in a variety of mutations. In most cases, NHEJ generates small indels in the target DNA, resulting in amino acid deletions, insertions, or frameshift mutations, leading to premature stop codons within the open reading frame (ORF) of the target gene. The ideal end result is a loss-of-function mutation within the target gene.

[0394] While NHEJ-mediated DSB repair often disrupts the open reading frame of a gene, homology-directed repair (HDR) can be used to generate specific nucleotide changes, ranging from single nucleotide changes to large insertions, such as the addition of fluorophores or tags.

[0395] To utilize HDR for gene editing, a DNA repair template containing the desired sequence can be delivered to the cell type of interest using gRNA and Cas9 or Cas9 nickase. The repair template can contain the desired edit as well as additional homologous sequences immediately upstream and downstream of the target (called left and right homology arms). The length of each homology arm depends on the size of the introduced change, with larger insertions requiring longer homology arms. The repair template can be a single-stranded oligonucleotide, a double-stranded oligonucleotide, or a double-stranded DNA plasmid. Even in cells expressing Cas9, gRNA, and an exogenous repair template, the efficiency of HDR is typically low (<10% modified alleles). The efficiency of HDR can be improved by synchronizing the cells, as HDR occurs during the S and G2 phases of the cell cycle. Chemical or genetic inhibition of genes involved in NHEJ can also increase the frequency of HDR.

[0396] In some embodiments, Cas9 is a modified Cas9. A given gRNA targeting sequence can have additional sites throughout the genome where there is partial homology. These sites are called off-target sites and need to be considered when designing the gRNA. In addition to optimizing the gRNA design, the specificity of CRISPR can also be improved by modifying Cas9. Cas9 produces double-strand breaks (DSBs) through the combined activity of two nuclease domains, RuvC and HNH. Cas9 nickase is a D10A mutant of SpCas9 that retains one nuclease domain and produces DNA nicks instead of DSBs. The nickase system can also be combined with HDR-mediated gene editing for specific gene editing.

[0397] In some cases, Cas9 is a variant Cas9 protein. The variant Cas9 polypeptide has an amino acid sequence that differs by one amino acid compared to the amino acid sequence of the wild-type Cas9 protein (e.g., with deletion, insertion, substitution, fusion). In some cases, the variant Cas9 polypeptide has an amino acid change (e.g., deletion, insertion or substitution) that reduces the nuclease activity of the Cas9 polypeptide. For example, in some cases, the variant Cas9 polypeptide has less than 50%, less than 40%, less than 30%, less than 20%, less than 10%, less than 5% or less than 1% of the nuclease activity of the corresponding wild-type Cas9 protein. In some cases, the variant Cas9 protein has no substantial nuclease activity. When the subject Cas9 protein is a variant Cas9 protein without substantial nuclease activity, it can be referred to as "dCas9".

[0398] In some cases, the variant Cas9 protein has reduced nuclease activity. For example, the variant Cas9 protein exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1% of the endonuclease activity of a wild-type Cas9 protein, such as a wild-type Cas9 protein.

[0399] In some cases, the variant Cas9 protein can cut the complementary strand of the guide target sequence, but the ability to cut the non-complementary strand of the double-stranded guide target sequence is reduced. For example, the variant Cas9 protein can have a mutation (amino acid substitution) that reduces the function of the RuvC domain. As a non-limiting example, in some embodiments, the variant Cas9 protein has D10A (aspartic acid to alanine at amino acid position 10) and can therefore cut the complementary strand of the double-stranded guide target sequence but cut the complementary strand of the non-double-stranded guide target sequence (thus when the variant Cas9 protein cuts the double-stranded target nucleic acid, it causes a single-strand break (SSB) instead of a double-strand break (DSB)) (see, e.g., Jinek et al., Science. 2012 Aug. 17; 337(6096): 816-21).

[0400] In some cases, variant Cas9 protein can cut the non-complementary chain of double-stranded guide target sequence, but the ability of cutting the complementary chain of guide target sequence is reduced.For example, variant Cas9 protein can have the mutation (amino acid substitution) that reduces HNH domain (RuvC / HNH / RuvC domain motif) function.As a non-limiting example, in some embodiments, variant Cas9 protein has H840A (histidine to alanine at amino acid position 840) mutation, therefore can cut the non-complementary chain of guide target sequence, but the ability of cutting the complementary chain of guide target sequence is reduced (so as to cause when variant Cas9 protein cuts double-stranded guide target sequence, use SSB instead of DSB).The ability of such Cas9 protein to cut guide target sequence (for example, single-stranded guide target sequence) is reduced, but retains the ability to bind to guide target sequence (for example, single-stranded guide target sequence).

[0401] In some cases, the variant Cas9 protein has a reduced ability to cut the complementary and non-complementary strands of a double-stranded target DNA. As a non-limiting example, in some cases, the variant Cas9 protein comprises both D10A and H840A mutations, such that the ability of the polypeptide to cut the complementary and non-complementary strands of a double-stranded target DNA is reduced. Such Cas9 proteins have a reduced ability to cut target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0402] As another non-limiting example, in some cases, the variant Cas9 protein contains W476A and W1126A mutations, which reduce the ability of the polypeptide to cut the target DNA. Such Cas9 proteins have a reduced ability to cut target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0403] As another non-limiting example, in some cases, the variant Cas9 protein contains P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, such that the ability of the polypeptide to cut the target DNA is reduced. Such Cas9 proteins have a reduced ability to cut target DNA (e.g., single-stranded target DNA), but retain the ability to bind to target DNA (e.g., single-stranded target DNA).

[0404] As another non-limiting example, in some cases, variant Cas9 protein contains H840A, W476A and W1126A mutations, so that the ability of polypeptide cutting target DNA is reduced. The ability of such Cas9 protein cutting target DNA (e.g., single-stranded target DNA) is reduced, but the ability of binding target DNA (e.g., single-stranded target DNA) is retained. As another non-limiting example, in some cases, variant Cas9 protein contains H840A, D10A, W476A and W1126A mutations, so that the ability of polypeptide cutting target DNA is reduced. The ability of such Cas9 protein cutting target DNA (e.g., single-stranded target DNA) is reduced, but the ability of binding target DNA (e.g., single-stranded target DNA) is retained. In some embodiments, variant Cas9 restores catalytic His residues at position 840 in Cas9 HNH domain (A840H).

[0405] As another non-limiting example, in some cases, variant Cas9 protein contains H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, so that the ability of polypeptide to cut target DNA is reduced. The ability of such Cas9 protein to cut target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind target DNA (e.g., single-stranded target DNA) is retained. As another non-limiting example, in some cases, variant Cas9 protein contains D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, so that the ability of polypeptide to cut target DNA is reduced. The ability of such Cas9 protein to cut target DNA (e.g., single-stranded target DNA) is reduced, but the ability to bind target DNA (e.g., single-stranded target DNA) is retained. In some cases, when the variant Cas9 protein comprises W476A and W1126A mutations or when the variant Cas9 protein comprises P475A, W476A, N477A, ​​D1125A, W1126A and D1127A mutations, the variant Cas9 protein does not effectively bind to the PAM sequence. Therefore, in some such cases, when such variant Cas9 protein is used in the binding method, the method does not require a PAM sequence. In other words, in some cases, when such variant Cas9 protein is used in the binding method, the method may include a guide RNA, but the method may be performed in the absence of a PAM sequence (and the specificity of the binding is therefore provided by the targeting fragment of the guide RNA). Other residues can be mutated to achieve the above-mentioned effects (i.e., inactivating one or other nuclease moieties). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986 and / or A987 may be altered (i.e., substituted). In addition, mutations other than alanine substitutions are also suitable.

[0406] In some embodiments, a variant Cas9 protein with reduced catalytic activity (e.g., when the Cas9 protein has D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986 and / or A987 mutations, e.g., D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A and / or D986A), the variant Cas9 protein can still bind to the target DNA in a site-specific manner (because it is still guided to the target DNA sequence by the guide RNA) as long as it retains the ability to interact with the guide RNA.

[0407] In some embodiments, the variant Cas protein can be spCas9, spCas9-VRQR, spCas9-VRER, xCas9(sp), saCas9, saCas9-KKH, spCas9-MQKSER, spCas9-LRKIQK, or spCas9-LRVSQL.

[0408] In some embodiments, a modified SpCas9 comprising amino acid substitutions D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (SpCas9-MQKFRAER) and having specificity for the altered PAM 5'-NGC is used.

[0409] Alternatives to Streptococcus pyogenes Cas9 include RNA-guided endonucleases from the Cpf1 family, which have demonstrated cleavage activity in mammalian cells. CRISPR from Prevotella and Francisella novokiller 1 (CRISPR / Cpf1) is a DNA editing technology similar to the CRISPR / Cas9 system. Cpf1 is an RNA-guided endonuclease of the class II CRISPR / Cas system. This acquired immunity mechanism is present in Prevotella and Francisella novokiller. The Cpf1 gene, associated with the CRISPR locus, encodes an endonuclease that uses a guide RNA to seek out and cleave viral DNA. Cpf1 is a smaller and simpler endonuclease than Cas9, overcoming some of the limitations of the CRISPR / Cas9 system. Unlike the Cas9 nuclease, Cpf1-mediated DNA cleavage results in double-strand breaks with short 3' overhangs. The staggered cleavage pattern of Cpf1 opens up the possibility of targeted gene transfer, similar to traditional restriction enzyme cloning, and can improve gene editing efficiency. Like the aforementioned Cas9 variants and orthologs, Cpf1 can also expand the number of CRISPR-targetable sites to AT-rich regions or AT-rich regions of the genome that lack the NGG PAM sites favored by SpCas9. The Cpf1 locus contains a mixed alpha / beta domain, a RuvC-I followed by a helical region, a RuvC-II, and a zinc finger-like domain. The Cpf1 protein possesses a RuvC-like endonuclease domain similar to the RuvC domain of Cas9. Furthermore, Cpf1 lacks the HNH endonuclease domain, and its N-terminus lacks the α-helical recognition lobe of Cas9. The Cpf1 CRISPR-Cas domain architecture indicates that Cpf1 is functionally unique and is classified as a Class 2, Type V CRISPR system. The Cas1, Cas2, and Cas4 proteins encoded by the Cpf1 locus are more similar to Type I and Type III systems than to those derived from Type II systems. Functional Cpf1 does not require transactivating CRISPR RNA (tracrRNA); therefore, only CRISPR (crRNA) is required. This facilitates genome editing because not only is Cpf1 smaller than Cas9, but its sgRNA molecule is also smaller (approximately half the nucleotides of Cas9). In contrast to the G-rich PAM targeted by Cas9, the Cpf1-crRNA complex cleaves target DNA or RNA by recognizing the protospacer-adjacent motif 5'-YTN-3'. After identifying the PAM, Cpf1 introduces a sticky-end-like DNA double-strand break with a 4- or 5-nucleotide overhang.

[0410] Some aspects of the present disclosure provide fusion proteins, the domains included therein act as nucleic acid programmable DNA binding proteins, which can be used to guide proteins, such as alkali editing, to specific nucleic acid (e.g., DNA or RNA) sequences. In a particular embodiment, the fusion protein comprises a nucleic acid programmable DNA binding protein domain and a deaminase domain. DNA binding proteins include but are not limited to Cas9 (e.g., dCas9 and nCas9), Cas12a / Cpfl, Cas12b / C2cl, Cas12c / C2c3, Cas12d / CasY, Cas12e / CasX, Cas12g, Cas12h, Cas12i, and Cas12j / CasΦ. An example of a programmable polynucleotide binding protein with a PAM specificity different from Cas9 is a clustered regularly interspaced short palindromic repeat sequence from Prevotella and Francisella 1 (Cpf1). Similar to Cas9, Cpf1 is also a Class 2 CRISPR effector. It has been shown that Cpf1 mediates powerful DNA interference, and its characteristics are different from Cas9. Cpf1 is a single RNA-guided endonuclease that lacks tracrRNA and utilizes a T-rich protospacer adjacent motif (TTN, TTTN, or YTN). In addition, Cpf1 cuts DNA by staggered DNA double-strand breaks. Among the 16 Cpf1 family proteins, two enzymes from Aminococcus and Lachnospira were shown to have effective genome editing activity in human cells. Cpf1 proteins are known in the art and have been described previously, for example, Yamano et al., "Crystal structure of Cpf1 in complex with guide RNA and target DNA." Cell (165) 2016, p. 949-962; the entire contents of which are incorporated herein by reference.

[0411] Useful in the present compositions and methods are nuclease-inactivated Cpf1 (dCpf1) variants, which can be used as guide nucleotide sequence-programmable polynucleotide binding protein domains. The Cpf1 protein has a RuvC-like nuclease domain similar to the RuvC domain of Cas9 but without an HNH nuclease domain, and the N-terminus of Cpf1 does not have the α-helical recognition lobe of Cas9. Zetsche et al., Cell, 163, 759-771, 2015 (incorporated herein by reference) show that the RuvC-like domain of Cpf1 is responsible for cutting two DNA chains and inactivating the RuvC-like domain to inactivate the Cpf1 nuclease activity. For example, mutations corresponding to D917A, E1006A or D1255A in Francisella novae Cpf1 inactivate Cpf1 nuclease activity. In some embodiments, dCpf1 of the present disclosure comprises a mutation corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D122. It will be appreciated that any mutation that inactivates the RuvC domain of Cpf1, such as a substitution mutation, deletion, or insertion, can be used in accordance with the present disclosure.

[0412] In some embodiments, the nucleic acid programmable nucleotide binding protein or any fusion protein provided herein can be a Cpf1 protein. In some embodiments, the Cpf1 protein is a Cpf1 nickase (nCpf1). In some embodiments, the Cpf1 protein is a nuclease-inactivated Cpf1 (dCpf1). In some embodiments, the Cpf1, the nCpf1, or the dCpf1 comprises an amino acid sequence that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to any Cpf1 protein described herein. In some embodiments, dCpf1 comprises at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the Cpf1 sequence disclosed herein and comprises mutations corresponding to D917A, E1006A, D1255A, D917A / E1006A, D917A / D1255A, E1006A / D1255A, or D917A / E1006A / D1255A. It should be understood that Cpf1 from other bacterial species can also be used according to the present disclosure.

[0413] The amino acid sequence of wild-type Francisella novicida Cpf1 is shown below. D917, E1006, and D1255 are in bold and underlined.

[0414]

[0415]

[0416] The amino acid sequence of Francisella novicida Cpf1 D917A is shown below (A917, E1006, and D1255 are bold and underlined).

[0417]

[0418] The amino acid sequence of Francisella novicida Cpf1 E1006A is shown below (D917, A1006, and D1255 are bold and underlined).

[0419]

[0420]

[0421] The amino acid sequence of Francisella novicida Cpf1 D1255A is shown below. (The mutation positions of D917, E1006, and A1255 are indicated in bold and underlined.)

[0422]

[0423]

[0424] The amino acid sequence of Francisella novicida Cpf1 D917A / E1006A is shown below (A917, A1006, and D1255 are bold and underlined).

[0425]

[0426]

[0427] The amino acid sequence of Francisella novicida Cpf1 D917A / D1255A is shown below (A917, E1006, and A1255 are bold and underlined).

[0428]

[0429] The amino acid sequence of Francisella novicida Cpf1 E1006A / D1255A is shown below (D917, A1006, and A1255 are bold and underlined).

[0430]

[0431]

[0432] The amino acid sequence of Francisella novicida Cpf1 D917A / E1006A / D1255A is shown below (A917, A1006, and A1255 are bold and underlined).

[0433]

[0434]

[0435] In some embodiments, one of the Cas9 domains present in the fusion protein can be replaced with a guide nucleotide sequence-programmable DNA binding protein domain that has no requirement for a PAM sequence.

[0436] In some embodiments, the Cas domain is a Cas9 domain from Staphylococcus aureus (SaCas9). In some embodiments, the SaCas9 domain is a nuclease-active SaCas9, a nuclease-inactivated SaCas9 (SaCas9d), or a SaCas9 nickase (SaCas9n). In some embodiments, the SaCas9 domain comprises an N579A mutation, or a corresponding mutation in any of the amino acid sequences provided herein.

[0437] In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence with a non-canonical PAM. In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence with a NNGRRT or NNGRRT PAM sequence. In some embodiments, the SaCas9 domain comprises one or more of E781X, N967X, and R1014X mutations, or corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SaCas9 domain comprises one or more of E781K, N967K, and R1014H mutations, or one or more corresponding mutations in any amino acid sequence provided herein. In some embodiments, the SaCas9 domain comprises E781K, N967K, or R1014H mutations, or corresponding mutations in any amino acid sequence provided herein.

[0438] The amino acid sequence of an exemplary SaCas9 is as follows:

[0439]

[0440] In this sequence, residue N579 (underlined and bold) can be mutated (e.g., to A579) to generate a SaCas9 nickase.

[0441] The amino acid sequence of an exemplary SaCas9n is as follows:

[0442]

[0443] In this sequence, residue A579 can be mutated from N579 to generate the SaCas9 nickase, which is underlined and in bold.

[0444] The amino acid sequence of an exemplary SaKKH Cas9 is as follows:

[0445]

[0446]

[0447] Residue A579, which can be mutated from N579 to generate the SaCas9 nickase, is underlined and bolded. Residues K781, K967, and H1014, which can be mutated from E781, N967, and R1014 to generate the SaKKH Cas9, are underlined and italicized.

[0448] High-fidelity Cas9 domain

[0449] Some aspects of the present disclosure provide high-fidelity Cas9 domains. In some embodiments, the high-fidelity Cas9 domain is an engineered Cas9 domain comprising one or more mutations, relative to corresponding wild-type Cas9 domains, which mutations reduce the electrostatic interaction between the sugar-phosphate backbone of the Cas9 domain and DNA. The high-fidelity Cas9 domain reduced by the electrostatic interaction with the sugar-phosphate backbone of DNA can have less off-target effects. In some embodiments, the Cas9 domain (e.g., wild-type Cas9 domain) comprises one or more mutations that reduce the association between the sugar-phosphate backbone of the Cas9 domain and DNA. In some embodiments, the Cas9 domain comprises one or more mutations that reduce the association between the sugar-phosphate backbone of the Cas9 domain and DNA by at least 1%, at least 2%, at least 3%, at least 4%, at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65% or at least 70%.

[0450] In some embodiments, any Cas9 fusion protein provided herein comprises one or more N497X, R661X, Q695X and / or Q926X mutations, or corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, any Cas9 fusion protein provided herein comprises one or more N497A, R661A, Q695A and / or Q926A mutations, or corresponding mutations in any amino acid sequence provided herein. In some embodiments, the Cas9 domain comprises a D10A mutation, or a corresponding mutation in any amino acid sequence provided herein. Cas9 domains with high fidelity are known in the art and are apparent to those skilled in the art. For example, Cas9 domains with high fidelity have been described in Kleinstiver, BP et al., “High-fidelity CRISPR-Cas9 nucleases with nodetectable genome-wide off-target effects.” Nature 529, 490-495 (2016); and Slaymaker, IM, et al., “Rationally engineered Cas9 nucleases with improved specificity.” Science 351, 84-88 (2015); the entire contents of which are incorporated by reference.

[0451] In some embodiments, the modified Cas9 is a high-fidelity Cas9 enzyme. In some embodiments, the high-fidelity Cas9 enzyme is SpCas9 (K855A), eSpCas9 (1.1), SpCas9-HF1, or a super-accurate Cas9 variant (HypaCas9). The modified Cas9 eSpCas9 (1.1) contains an alanine substitution that weakens the interaction between the HNH / RuvC groove and the non-target DNA chain, preventing chain separation and cutting at off-target sites. Similarly, SpCas9-HF1 reduces off-target editing by alanine substitutions that disrupt the interaction of Cas9 with the DNA phosphate backbone. HypaCas9 contains mutations in the REC3 domain (SpCas9N692A / M694A / Q695A / H698A) that increase Cas9 proofreading and target discrimination. All three high-fidelity enzymes produce less off-target editing than wild-type Cas9.

[0452] Exemplary high-fidelity Cas9s are provided below.

[0453] High-fidelity Cas9 domain mutations relative to Cas9 are shown in bold and underlined.

[0454]

[0455] Guide polynucleotide

[0456] In one embodiment, the guide polynucleotide is a guide RNA. The RNA / Cas complex can help guide the Cas protein to the target DNA. Cas9 / crRNA / tracrRNA endonucleolytically cuts a linear or circular dsDNA target that is complementary to the spacer. The target chain that is not complementary to the crRNA is first cut by endonucleolytic cutting and then trimmed 3'-5' by exonucleolytic cutting. In nature, DNA binding and cutting usually require protein and two RNAs. However, single guide RNA ("sgRNA", or simply "gRNA") can be engineered to integrate various aspects of crRNA and tracrRNA into a single RNA species. See, e.g., Jinek M. et al., Science 337:816-821 (2012), the entire contents of which are hereby incorporated by reference. Cas9 recognizes a short motif in the CRISPR repeat sequence (PAM or protospacer adjacent motif) to help distinguish self from non-self. Cas9 nuclease sequence and structure are well known to those skilled in the art (see, e.g., "Complete genome sequence of an M1 strain of Streptococcus pyogenes." Ferretti, JJ et al., Natl. Acad. Sci. USA 98:4658-4663 (2001); "CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III." Deltcheva E. et al., Nature 471:602-607 (2011); and "Programmable dual-RNA-guided d DNA endonuclease in adaptive bacterial immunity." Jinek M. et al., Science 337:816-821 (2012), all of which are incorporated herein by reference). Cas9 orthologs have been described in various species, including but not limited to Streptococcus pyogenes and Streptococcus thermophilus.Based on the present disclosure, other suitable Cas9 nucleases and sequences will be apparent to those skilled in the art, and such Cas9 nucleases and sequences include Cas9 sequences from organisms and loci disclosed in Chylinski, Rhun, and Charpentier, "The tracrRNA and Cas9 families of type II CRISPR-Cas immunitysystems" (2013) RNA Biology 10:5,726-737; the entire contents of which are incorporated herein by reference. In some embodiments, the Cas9 nuclease has an inactive (e.g., inactivated) DNA cleavage domain, i.e., Cas9 is a nickase.

[0457] In some embodiments, the guide polynucleotide is at least one single guide RNA ("sgRNA" or "gRNA"). In some embodiments, the guide polynucleotide is at least one tracrRNA. In some embodiments, the guide polynucleotide does not require a PAM sequence to guide the polynucleotide-programmable DNA binding domain (e.g., Cas9 or Cpf1) to the target nucleotide sequence.

[0458] The polynucleotide programmable nucleotide binding domains (e.g., CRISPR-derived domains) of base editors disclosed herein can identify target polynucleotide sequences by associating with guide polynucleotides. Guide polynucleotides (e.g., gRNA) are typically single-stranded and can be programmed to site-specifically bind to (i.e., by complementary base pairing) the target sequence of polynucleotides, thereby directing the base editor bound to the guide nucleic acid to the target sequence. The guide polynucleotide can be DNA. The guide polynucleotide can be RNA. In some cases, the guide polynucleotide includes natural nucleotides (e.g., adenosine). In some cases, the guide polynucleotide includes non-natural (or non-natural) nucleotides (e.g., peptide nucleic acid or nucleotide analogs). In some cases, the length of the targeting region of the guide nucleic acid sequence can be at least 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 amino acids. The targeting region of the guide nucleic acid can be between 10-30 nucleotides in length, or between 15-25 nucleotides in length, or between 15-20 nucleotides in length.

[0459] In some embodiments, the guide polynucleotide comprises two or more separate polynucleotides that can interact with each other through, for example, complementary base pairing (e.g., dual guide polynucleotides). For example, the guide polynucleotide can comprise a CRISPR RNA (crRNA) and a transactivating CRISPR RNA (tracrRNA). For example, the guide polynucleotide can comprise one or more transactivating CRISPR RNAs (tracrRNAs).

[0460] In type II CRISPR systems, CRISPR protein (e.g., Cas9) targeting nucleic acids typically requires a first RNA molecule (crRNA) containing a sequence that recognizes the target sequence and a second RNA molecule (trRNA) containing a sequence that recognizes the target sequence. Complementary base pairing repeats form a scaffold region to stabilize the guide RNA-CRISPR protein complex. Such dual-guide RNA systems can be used as guide polynucleotides to guide the base editors disclosed herein to the target polynucleotide sequence.

[0461] In some embodiments, the base editors provided herein utilize a single guide polynucleotide (e.g., gRNA). In some embodiments, the base editors provided herein utilize dual guide polynucleotides (e.g., dual gRNA). In some embodiments, the base editors provided herein utilize one or more guide polynucleotides (e.g., multiple gRNA). In some embodiments, a single guide polynucleotide is used for different base editors described herein. For example, a single guide polynucleotide can be used for a cytidine base editor and an adenosine base editor.

[0462] In other embodiments, the guide polynucleotide may comprise a polynucleotide targeting portion of a nucleic acid and a scaffold portion of a nucleic acid in a single molecule (i.e., a single molecule guide nucleic acid). For example, a single molecule guide polynucleotide can be a single guide RNA (sgRNA or gRNA). In this article, the term guide polynucleotide sequence encompasses any single, double, or multi-molecule nucleic acid that can interact with a base editor and guide the base editor to a target polynucleotide sequence.

[0463] Typically, a guide polynucleotide (e.g., a crRNA / trRNA complex or gRNA) comprises a "polynucleotide targeting fragment," which includes a sequence capable of recognizing and binding to a target polynucleotide sequence, and a "protein binding fragment," which stabilizes the guide polynucleotide within the polynucleotide programmable nucleotide binding domain assembly of the base editor. In some embodiments, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to a DNA polynucleotide, thereby facilitating the editing of bases in the DNA. In other cases, the polynucleotide targeting segment of the guide polynucleotide recognizes and binds to an RNA polynucleotide, thereby facilitating the editing of bases in the RNA. Herein, a "fragment" refers to a portion or region of a molecule, for example, a continuous stretch of nucleotides in a guide polynucleotide. A fragment may also refer to a region / segment of a complex, such that a segment may include a region of more than one molecule. For example, when the guide polynucleotide comprises a plurality of nucleic acid molecules, the protein binding segment may include, for example, all or part of a plurality of separate molecules that hybridize along a complementary region. In some embodiments, a protein-binding segment of a DNA-targeting RNA comprising two separate molecules can comprise (i) 40-75 base pairs of a first RNA molecule that is 100 base pairs in length; and (ii) 10-25 base pairs of a second RNA molecule that is 50 base pairs in length. Unless expressly defined otherwise in a particular context, the definition of "segment" is not limited to a particular total number of base pairs, is not limited to any particular number of base pairs from a given RNA molecule, is not limited to the number of isolated molecules in a particular complex, and can include regions of RNA molecules of any total length, and can include regions that are complementary to other molecules.

[0464] Guide RNA or guide polynucleotide can comprise two or more RNA, for example CRISPR RNA (crRNA) and trans-activating crRNA (tracrRNA). Guide RNA or guide polynucleotide can sometimes comprise single-stranded RNA or single guide RNA (sgRNA) formed by fusion of a portion (e.g., functional portion) of crRNA and tracrRNA. Guide RNA or guide polynucleotide can also be a dual RNA comprising crRNA and tracrRNA. In addition, crRNA can hybridize with target DNA.

[0465] As described above, the guide RNA or guide polynucleotide can be an expression product. For example, the DNA encoding the guide RNA can be a vector comprising a sequence encoding the guide RNA. The guide RNA or guide polynucleotide can be transferred into the cell by transfecting the cell with an isolated guide RNA or plasmid DNA comprising a sequence encoding the guide RNA and a promoter. The guide RNA or guide polynucleotide can also be transferred into the cell in other ways, such as using viral-mediated gene delivery.

[0466] The guide RNA or guide polynucleotide can be isolated. For example, the guide RNA can be transfected into a cell or organism in the form of isolated RNA. The guide RNA can be prepared by in vitro transcription using any in vitro transcription system known in the art. The guide RNA can be transferred into the cell in the form of isolated RNA rather than in the form of a plasmid containing the coding sequence of the guide RNA.

[0467] A guide RNA or guide polynucleotide can contain three regions: a first region at the 5' end that is complementary to a target site in a chromosomal sequence, a second internal region that forms a stem-loop structure, and a third 3' region that can be single-stranded. The first region of each guide RNA can also be different, so that each guide RNA guides the fusion protein to a specific target site. In addition, the second and third regions of each guide RNA can be identical in all guide RNAs.

[0468] The first region of a guide RNA or a guide polynucleotide can be complementary to the sequence of a target site in a chromosome sequence so that the first region of a guide RNA can be base paired with the target site. In some cases, the first region of a guide RNA can include or be from about 10 nucleotides to 25 nucleotides (i.e., from 10 nucleotides to nucleotides; or from about 10 nucleotides to about 25 nucleotides; or from 10 nucleotides to about 25 nucleotides; or from about 10 nucleotides to 25 nucleotides) or more. For example, the base pairing region between the first region of a guide RNA and the target site in a chromosome sequence can be or can be the length of about 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25 or more nucleotides. Sometimes, the length of the first region of a guide RNA can be or can be about 19, 20 or 21 nucleotides.

[0469] Guide RNA or guide polynucleotide can also comprise the second region that forms secondary structure.For example, the secondary structure formed by guide RNA can comprise stem (or hairpin) and ring.The length of ring and stem can change.For example, the length range of ring can be about 3 to 10 nucleotides, and the length range of stem can be about 6 to 20 base pairs.The stem can comprise one or more 1 to 10 or about 10 nucleotide protrusions.The total length of the second region can be in the length range of about 16 to 60 nucleotides.For example, the length of ring can be or can be about 4 nucleotides, and stem can be or can be about 12 base pairs.

[0470] The guide RNA or guide polynucleotide may also include a third region at the 3' end that may be substantially single-stranded. For example, the third region may sometimes be non-complementary to any chromosomal sequence in the target cell and sometimes be non-complementary to the rest of the guide RNA. Furthermore, the length of the third region may vary. The length of the third region may be greater than or equal to about 4 nucleotides. For example, the total length of the third region may range from about 5 to 60 nucleotides.

[0471] A guide RNA or guide polynucleotide can target any exon or intron of a gene target. In some cases, a guide can target exon 1 or 2 of a gene, and in other cases, a guide can target exon 3 or 4 of a gene. A composition can include multiple guide RNAs that all target the same exon, or in some cases, multiple guide RNAs that target different exons. Exons and introns of a gene can be targeted.

[0472] Guide RNA or guide polynucleotide can target about 20 nucleotides or about 20 nucleotides of nucleic acid sequence.Target nucleic acid can be less than or less than about 20 nucleotides.The length of target nucleic acid can be at least or at least about 5,10,15,16,17,18,19,20,21,22,23,24,25,30 or any position between 1-100 nucleotides.The length of target nucleic acid can be at most or at most about 5,10,5,10,15,16,17,18,19,20,21,22,23,24,25,30,40,50 or any position between 1-100 nucleotides.The target nucleic acid sequence can be or can be about 20 bases at the 5' place of the first nucleotide next to PAM.Guide RNA can target nucleic acid sequence. The target nucleic acid can be at least or at least about 1-10, 1-20, 1-30, 1-40, 1-50, 1-60, 1-70, 1-80, 1-90, or 1-100 nucleotides in length.

[0473] A guide polynucleotide, such as a guide RNA, can refer to a nucleic acid that can hybridize to another nucleic acid, such as a target nucleic acid or a protospacer in the genome of a cell. The guide polynucleotide can be RNA. The guide polynucleotide can be DNA. The guide polynucleotide can be programmed or designed to site-specifically bind to a nucleic acid sequence. The guide polynucleotide can comprise a polynucleotide chain and can be referred to as a single guide polynucleotide. The guide polynucleotide can comprise two polynucleotide chains and can be referred to as a dual guide polynucleotide. The guide RNA can be introduced into the cell as an RNA molecule. For example, the RNA molecule can be transcribed in vitro and / or can be chemically synthesized. The RNA can be transcribed from a synthetic DNA molecule, such as Gene fragment. The guide RNA can then be introduced into the cell as an RNA molecule. The guide RNA can also be introduced into the cell in the form of a non-RNA nucleic acid molecule, such as a DNA molecule. For example, the DNA encoding the guide RNA can be operably linked to a promoter control sequence to express the guide RNA in the cell of interest. The RNA coding sequence can be operably linked to a promoter sequence recognized by RNA polymerase III (Pol III). Plasmid vectors that can be used to express guide RNA include but are not limited to px330 vectors and px333 vectors. In some cases, a plasmid vector (e.g., px333 vector) can contain at least two DNA sequences encoding guide RNA.

[0474] Methods for selecting, designing and verifying guide polynucleotides such as guide RNAs and targeting sequences are described herein and are known to those skilled in the art. For example, in order to minimize the impact of potential substrate promiscuity of the deaminase domain in the nuclear base editor system (e.g., AID domain), the number of residues that may inadvertently become deamination targets (e.g., off-target C residues that may be present on ssDNA within the target nucleic acid locus) can be minimized. In addition, software tools can be used to optimize the gRNA corresponding to the target nucleic acid sequence, for example, to minimize the total off-target activity of the entire genome. For example, for each possible targeting domain selection using Streptococcus pyogenes Cas9, all off-target sequences (before the selected PAM, such as NAG or NGG) can be identified in the genome, containing up to a specific number (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10) of mismatched base pairs. The first region of the gRNA that is complementary to the target site can be identified, and all first regions (e.g., crRNAs) can be ranked according to their total predicted off-target scores; the top-ranked target domains represent those that are likely to have the greatest on-target and least off-target activity. Candidate targeting gRNAs can be functionally evaluated using methods known in the art and / or as described herein.

[0475] As a non-limiting example, a DNA sequence search algorithm can be used to identify the target DNA hybridization sequence in the crRNA of the guide RNA used with Cas9. GRNA design can be performed using a custom gRNA design software based on a public tool cas-offinder, such as Bae S., Park J. and Kim J.-S.Cas-OFFinder: A fast and versatile algorithm that searches for potential off-target sites of Cas9RNA-guided dendonucleases.Bioinformatics 30, 1473-1475 (2014). The software is used to guide scoring after calculating the whole genome off-target tendency. Typically, for guides with a length of 17 to 24, matches from perfect matches to 7 mismatches are considered. Once the off-target site is determined by calculation, a total score is calculated for each guide and summarized in a table output using a web interface. In addition to identifying potential target sites adjacent to the PAM sequence, the software also identifies all PAM adjacent sequences that differ from the selected target site by 1, 2, 3 or more nucleotides. A target nucleic acid sequence, such as the genomic DNA sequence of a target gene, can be obtained and publicly available tools, such as the RepeatMasker program, can be used to screen for repetitive elements. RepeatMasker searches for repetitive elements and low-complexity regions in the input DNA sequence. The output is a detailed annotation of the repeats present in the given query sequence.

[0476] After identification, the first region of the guide RNA, such as crRNA, can be graded based on their distance from the target site, their orthogonality, and the presence of 5' nucleotides to identify sequences that closely match the relevant PAM sequence (e.g., 5'G based on the identification of a close match containing the relevant PAM in the human genome, e.g., NGG PAM of Streptococcus pyogenes, NNGRRT or NNGRRV PAM of Staphylococcus aureus). As used herein, orthogonality refers to the number of sequences in the human genome that contain the least number of mismatches with the target sequence. For example, "high level orthogonality" or "good orthogonality" can refer to a 20-mer targeting domain that does not have the same sequence in the human genome except for the intended target, nor any sequence order that contains one or two mismatches in the target. Targeting domains with good orthogonality can be selected to minimize off-target DNA cutting.

[0477] In some embodiments, the reporter system can be used to detect base editing activity and test candidate guide polynucleotides. In some embodiments, the reporter system can include a reporter gene-based assay, wherein the base editing activity results in the expression of the reporter gene. For example, the reporter system can include a reporter gene comprising an inactivation start codon, such as a mutation from 3'-TAC-5' to 3'-CAC-5' on the template chain. After the target C is successfully deaminated, the corresponding mRNA will be transcribed as 5'-AUG-3' instead of 5'-GUG-3', thereby achieving translation of the reporter gene. Suitable reporter genes are obvious to those skilled in the art. Non-limiting examples of reporter genes include genes encoding green fluorescent protein (GFP), red fluorescent protein (RFP), luciferase, secretory alkaline phosphatase (SEAP), or any other gene whose expression is detectable and obvious to those skilled in the art. The reporter system can be used to test many different gRNAs, for example, to determine which residues of the target DNA sequence the corresponding deaminase will target. sgRNA targeting non-template chains can also be tested to assess the off-target effects of specific base editing proteins (e.g., Cas9 deaminase fusion proteins). In some embodiments, such gRNAs can be designed so that the mutated start codon does not base pair with the gRNA. The guide polynucleotides may include standard ribonucleotides, modified ribonucleotides (e.g., pseudouridine), ribonucleotide isomers, and / or ribonucleotide analogs. In some embodiments, the guide polynucleotides may include at least one detectable label. Detectable labels may be fluorophores (e.g., FAM, TMR, Cy3, Cy5, Texas Red, Oregon Green, Alexa Fluors, Halo tags, or suitable fluorescent dyes), detection labels (e.g., biotin, digoxin, etc.), quantum dots, or gold particles.

[0478] The guide polynucleotide can be chemically synthesized, enzymatically synthesized, or a combination thereof. For example, guide RNA can be synthesized using a standard solid phase synthesis method based on phosphoramidite. Alternatively, guide RNA can be synthesized in vitro by operably linking the DNA encoding the guide RNA to a promoter control sequence recognized by a phage RNA polymerase. Examples of suitable phage promoter sequences include T7, T3, SP6 promoter sequences, or variants thereof. In embodiments where the guide RNA comprises two separate molecules (e.g., crRNA and tracrRNA), crRNA can be chemically synthesized and tracrRNA can be enzymatically synthesized.

[0479] In some embodiments, the base editor system can include multiple guide polynucleotides, such as gRNAs. For example, gRNAs can target one or more target loci (e.g., at least 1 gRNA, at least 2 gRNAs, at least 5 gRNAs, at least 10 gRNAs, at least 20 gRNAs, at least 30 gRNAs, at least 50 gRNAs) included in the base editor system. Multiple gRNA sequences can be arranged in series and preferably separated by directional repeats.

[0480] The DNA sequence encoding the guide RNA or guide polynucleotide can also be part of the vector. In addition, the vector can contain additional expression control sequences (e.g., enhancer sequences, Kozak sequences, polyadenylation sequences, transcription termination sequences, etc.), selection marker sequences (e.g., GFP or antibiotic resistance genes, such as puromycin), replication origins, and the like. The DNA molecule encoding the guide RNA can also be linear. The DNA molecule encoding the guide RNA or guide polynucleotide can also be circular.

[0481] In some embodiments, one or more components of the base editor system may be encoded by a DNA sequence. Such DNA sequences can be introduced into an expression system, such as a cell, together or separately. For example, a DNA sequence encoding a polynucleotide programmable nucleotide binding domain and a guide RNA can be introduced into a cell, and each DNA sequence can be part of a separate molecule (e.g., a vector containing a polynucleotide programmable nucleotide binding domain encoding sequence and a second vector containing a guide RNA encoding sequence) or both can be part of the same molecule (e.g., a vector containing a polynucleotide programmable nucleotide binding domain and a coding (and regulatory) sequence for a guide RNA).

[0482] The guide polynucleotide may comprise one or more modifications to provide a nucleic acid with new or enhanced characteristics. The guide polynucleotide may comprise a nucleic acid affinity tag. The guide polynucleotide may comprise synthetic nucleotides, synthetic nucleotide analogs, nucleotide derivatives, and / or modified nucleotides.

[0483] In some cases, the gRNA or guide polynucleotide may comprise modifications. Modifications may be made at any position of the gRNA or guide polynucleotide. More than one modification may be made to a single gRNA or guide polynucleotide. The gRNA or guide polynucleotide may be quality controlled after modification. In some cases, quality control may include PAGE, HPLC, MS, or any combination thereof.

[0484] Modifications of the gRNA or guide polynucleotide can be substitutions, insertions, deletions, chemical modifications, physical modifications, stabilization, purification, or any combination thereof.

[0485] The gRNA or guide polynucleotide can also be modified with 5' adenylate, 5' guanosine-triphosphate cap, 5' N7-methylguanosine-triphosphate cap, 5' triphosphate cap, 3' phosphate, 3' phosphorothioate, 5' phosphate, 5' modified phosphorothioate, cis-Syn thymidine dimer, trimer, C12 spacer, C3 spacer, C6 spacer, dSpacer, PC spacer, rSpacer, Spacer 18, Spacer 9, 3'-3' modification, 5'-5' modification, no base, acridine, azobenzene, biotin, biotin BB, biotin TEG, cholesterol TEG, desthiobiotin TEG, DNP TEG, DNP-X, DOTA, dT-biotin, bis-biotin, PC biotin, psoralen C2, psoralen C6, TINA, 3' DABCYL, black hole quencher 1, black hole quencher 2, DABCYL SE, dT-DABCYL, IRDye QC-1, QSY-21, QSY-35, QSY-7, QSY-9, carboxyl linker, thiol linker, 2'-deoxyribonucleoside analog purine, 2'-deoxyribonucleoside analog pyrimidine, ribonucleoside analog, 2'-O-methyl ribonucleoside analog, sugar-modified analog, wobble / universal base, fluorescent dye label, 2'-fluoro RNA, 2'-O-methyl RNA, methylphosphonate, phosphodiester DNA, phosphodiester RNA, phosphorothioate DNA, phosphorothioate RNA, UNA, pseudouridine-5'-triphosphate, 5'-methylcytidine-5'-triphosphate, or any combination thereof.

[0486] In some cases, the modification is permanent. In other cases, the modification is temporary. In some cases, the gRNA or guide polynucleotide is modified multiple times. The gRNA or guide polynucleotide modifications can change the physicochemical properties of the nucleotides, such as their conformation, polarity, hydrophobicity, chemical reactivity, base pairing interactions, or any combination thereof.

[0487] The PAM sequence can be any PAM sequence known in the art. Suitable PAM sequences include, but are not limited to, NGG, NGA, NGC, NGN, NGT, NGCG, NGAG, NGAN, NGNG, NGCN, NGCG, NGTN, NNGRRT, NNNRRT, NNGRR(N), TTTV, TYCV, TYCV, TATV, NNNNGATT, NNAGAAW, or NAAAC. Y is a pyrimidine; N is any nucleotide base; and W is A or T.

[0488] The modification can also be a phosphorothioate substitute. In some cases, natural phosphodiester bonds can be easily and rapidly degraded by cellular nucleases; modifications of internucleotide bonds using phosphorothioate (PS) bond substitutes can be more stable to hydrolysis by cellular degradation. The modification can increase the stability of the gRNA or guide polynucleotide. The modification can also enhance biological activity. In some cases, phosphorothioate-enhanced RNA gRNA can inhibit RNase A, RNase T1, calf serum nuclease, or any combination thereof. These properties can make PS-RNA gRNA useful in applications where there is a high likelihood of exposure to nucleases in vivo or in vitro. For example, a phosphorothioate (PS) bond can be introduced between the last 3-5 nucleotides of the 5'- or "-end of the gRNA, which can inhibit exonuclease degradation. In some cases, phosphorothioate bonds can be added throughout the gRNA to reduce attack by endonucleases.

[0489] protospacer-adjacent motif

[0490] The term "protospacer adjacent motif (PAM)" or PAM-like motif refers to a 2-6 base pair DNA sequence that immediately follows the DNA sequence targeted by the Cas9 nuclease in the CRISPR bacterial adaptive immune system. In some embodiments, the PAM can be a 5' PAM (i.e., located upstream of the 5' end of the protospacer). In some embodiments, the PAM can be a 3' PAM (i.e., located downstream of the 5' end of the protospacer).

[0491] The PAM sequence is crucial for target binding, but the exact sequence depends on the type of Cas protein.

[0492] The base editors provided herein may include a CRISPR protein-derived domain that is capable of binding to a nucleotide sequence comprising a canonical or non-canonical protospacer adjacent motif (PAM) sequence. A PAM site is a nucleotide sequence close to a target polynucleotide sequence. Some aspects of the present disclosure provide base editors comprising all or part of a CRISPR protein with different PAM specificities. For example, a typical Cas9 protein, such as Cas9 from Streptococcus pyogenes (spCas9), requires a typical NGG PAM sequence to bind to a specific nucleic acid region, where the "N" in "NGG" is adenine (A), thymine (T), guanine (G) or cytosine (C), and G is guanine. The PAM can be CRISPR protein-specific and can be different between different base editors comprising different CRISPR protein-derived domains. The PAM can be 5' or 3' of the target sequence. The PAM can be upstream or downstream of the target sequence. The length of the PAM can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more nucleotides. Typically, the length of a PAM is between 2 and 6 nucleotides. Several PAM variants are described in Table 1 below.

[0493] Table 1. Cas9 proteins and corresponding PAM sequences

[0494] Variants PAM spCas9 NGG spCas9-VRQR NGA spCas9-VRER NGCG spCas9-MQKFRAER NGC xCas9(sp) NGN saCas9 NNGRRT saCas9-KKH NNNRRT spCas9-MQKSER NGCG spCas9-MQKSER NGCN spCas9-LRKIQK NGTN spCas9-LRVSQK NGTN spCas9-LRVSQL NGTN SpyMacCas9 NAA Cpf1 5'(TTTV)

[0495] In some embodiments, the PAM is NGC. In some embodiments, the NGC PAM is recognized by a Cas9 variant. In some embodiments, the NGC PAM variant comprises one or more amino acid substitutions selected from D1135M, S1136Q, G1218K, E1219F, A1322R, D1332A, R1335E, and T1337R (collectively referred to as "MQKFRAER").

[0496] In some embodiments, the PAM is NGT. In some embodiments, the NGT PAM is recognized by a Cas9 variant. In some embodiments, the NGT PAM variant is generated by targeted mutation at one or more residues 1335, 1337, 1135, 1136, 1218, and / or 1219. In some embodiments, the NGT PAM variant is generated by targeted mutation at one or more residues 1219, 1335, 1337, 1218. In some embodiments, the NGT PAM variant is generated by targeted mutation at one or more residues 1135, 1136, 1218, 1219, and 1335. In some embodiments, the NGT PAM variant is selected from a group of targeted mutations provided in Tables 2 and 3 below.

[0497] Table 2: NGT PAM variant mutations at residues 1219, 1335, 1337, 1218

[0498] Variants E1219V R1335Q T1337 G1218 1 F V T 2 F V R 3 F V Q 4 F V L 5 F V T R 6 F V R R 7 F V Q R 8 F V L R 9 L L T 10 L L R 11 L L Q 12 L L L 13 F I T 14 F I R 15 F I Q 16 F I L 17 F G C 18 H L N 19 F G C A 20 H L N V 21 L A W 22 L A F 23 L A Y 24 I A W 25 I A F 26 I A Y

[0499] Table 3: NGT PAM variant mutations at residues 1135, 1136, 1218, 1219 and 1335

[0500] Variants D1135L S1136R G1218S E1219V R1335Q 27 G 28 V 29 I 30 A 31 W 32 H 33 K 34 K 35 R 36 Q 37 T 38 N 39 I 40 A 41 N 42 Q 43 G 44 L 45 S 46 T 47 L 48 I 49 V 50 N 51 S 52 T 53 F 54 Y 55 N1286Q I1331F

[0501] In some embodiments, the NGT PAM variant is selected from variants 5, 7, 28, 31, or 36 in Tables 2 and 3. In some embodiments, the variant has improved NGT PAM recognition.

[0502] In some embodiments, the NGT PAM variant has a mutation at residues 1219, 1335, 1337, and / or 1218. In some embodiments, the NGT PAM variant having a mutation for improved recognition is selected from the variants provided in Table 4 below.

[0503] Table 4: NGT PAM variant mutations at residues 1219, 1335, 1337, and 1218

[0504] Variants E1219V R1335Q T1337 G1218 1 F V T 2 F V R 3 F V Q 4 F V L 5 F V T R 6 F V R R 7 F V Q R 8 F V L R

[0505] In some embodiments, the NGT PAM is selected from the variants provided in Table 5 below.

[0506] Table 5. NGT PAM variants

[0507]

[0508] In some embodiments, the NGTN variant is variant 1. In some embodiments, the NGTN variant is variant 2. In some embodiments, the NGTN variant is variant 3. In some embodiments, the NGTN variant is variant 4. In some embodiments, the NGTN variant is variant 5. In some embodiments, the NGTN variant is variant 6.

[0509] In some embodiments, the Cas9 domain is a Cas9 domain (SpCas9) from Streptococcus pyogenes. In some embodiments, the SpCas9 domain is a nuclease-active SpCas9, a nuclease-inactivated SpCas9 (SpCas9d) or a SpCas9 nickase (SpCas9n). In some embodiments, SpCas9 comprises a D9X mutation, or a corresponding mutation in any amino acid sequence provided herein, wherein X is any amino acid except D. In some embodiments, SpCas9 comprises a D9A mutation, or a corresponding mutation in any amino acid sequence provided herein. In some embodiments, the SpCas9 domain, SpCas9d domain or SpCas9n domain can bind to a nucleic acid sequence with a non-canonical PAM. In some embodiments, the SpCas9 domain, SpCas9d domain or SpCas9n domain can bind to a nucleic acid sequence with an NGG, NGA or NGCGPAM sequence.

[0510] In some embodiments, the SpCas9 domain comprises one or more of D1135X, R1335X, and T1337X mutations, or corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1135E, R1335Q, and T1337R mutations, or corresponding mutations in any amino acid sequence provided herein. In some embodiments, the SpCas9 domain comprises D1135E, R1335Q, and T1337R mutations, or corresponding mutations in any amino acid sequence provided herein. In some embodiments, the SpCas9 domain comprises one or more of D1135X, R1335X, and T1337T1337X mutations, or corresponding mutations in any amino acid sequence provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1135V, R1335Q, and T1337R mutations, or corresponding mutations in any amino acid sequence provided herein. In some embodiments, the SpCas9 domain comprises D1135V, R1335Q, and T1337R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises one or more of D1135X, G1218X, R1335X, and T1337X mutations, or corresponding mutations in any of the amino acid sequences provided herein, wherein X is any amino acid. In some embodiments, the SpCas9 domain comprises one or more of D1135V, G1218R, R1335Q, and T1337R mutations, or corresponding mutations in any of the amino acid sequences provided herein. In some embodiments, the SpCas9 domain comprises D1135V, G1218R, R1335Q, and T1337R mutations, or corresponding mutations in any of the amino acid sequences provided herein.

[0511] In some embodiments, the amino acid sequence included in the Cas9 domain of any fusion protein provided herein is at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or at least 99.5% identical to the Cas9 polypeptides described herein. In some embodiments, the Cas9 domain of any fusion protein provided herein comprises the amino acid sequence of any Cas9 polypeptide described herein. In some embodiments, the Cas9 domain of any fusion protein provided herein consists of the amino acid sequence of any Cas9 polypeptide described herein.

[0512] In some examples, a PAM recognized by a CRISPR protein-derived domain of a base editor disclosed herein can be provided to the cell on an oligonucleotide that is different from the insert encoding the base editor (e.g., an AAV insert). In such embodiments, providing a PAM on a separate oligonucleotide can allow cleavage of a target sequence that would otherwise not be cleaved because there is no adjacent PAM on the same polynucleotide as the target sequence.

[0513] In one embodiment, Streptococcus pyogenes Cas9 (SpCas9) can be used as a CRISPR nuclease for genome engineering. However, others can also be used. In some embodiments, different nucleases can be used to target certain genomic targets. In some embodiments, synthetic SpCas9 derivatives with non-NGG PAM sequences can be used. In addition, other Cas9 orthologs from different species have been identified, and these "non-SpCas9" can be combined with a variety of PAM sequences that can also be used in the present disclosure. For example, the relatively large SpCas9 (approximately 4kb coding sequence) will result in a plasmid carrying the SpCas9 cDNA being unable to be effectively expressed in cells. In contrast, the coding sequence of Staphylococcus aureus Cas9 (SaCas9) is about 1 kilobase shorter than SpCas9, which may enable it to be effectively expressed in cells. Similar to SpCas9, SaCas9 endonuclease is able to modify target genes in mammalian cells in vitro and in mice. In some embodiments, Cas proteins can target different PAM sequences. In some embodiments, the target gene can be adjacent to, for example, the Cas9 PAM, 5'-NGG. In other embodiments, other Cas9 orthologs may have different PAM requirements. For example, other PAMs, such as those from Streptococcus thermophilus (5'-NNAGAA for CRISPR1 and 5'-NGGNG for CRISPR3) and Neisseria meningitidis (5'-NNNNGATT), may also be adjacent to the target gene.

[0514] In some embodiments, for the Streptococcus pyogenes system, the target gene sequence can be before (i.e., 5' to) 5'-NGG PAM, and the 20-nt guide RNA sequence can be paired with the opposite chain base to mediate the Cas9 cleavage adjacent to the PAM. In some embodiments, the adjacent cutting can be or can be about 3 base pairs upstream of the PAM. In some embodiments, the adjacent cutting can be or can be about 10 base pairs upstream of the PAM. In some embodiments, the adjacent cutting can be or can be about 0-20 base pairs upstream of the PAM. For example, adjacent cutting can be next to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29 or 30 base pairs upstream of the PAM. Adjacent cutting can also be 1 to 30 base pairs downstream of the PAM. The sequence of an exemplary SpCas9 protein capable of binding to a PAM sequence is as follows:

[0515] The amino acid sequence of an exemplary PAM-bound SpCas9 is as follows:

[0516]

[0517] The amino acid sequence of an exemplary PAM-bound SpCas9n is as follows:

[0518]

[0519] The amino acid sequence of an exemplary PAM-bound SpEQR Cas9 is as follows:

[0520] In this sequence, residues E1135, Q1335, and R1337 can be mutated from D1135, R1335, and T1337 to generate SpEQR Cas9, which are underlined and in bold.

[0521] The amino acid sequence of an exemplary PAM binding SpVQR Cas9 is as follows:

[0522] In this sequence, residues V1135, Q1335, and R1337 can be mutated from D1135, R1335, and T1337 to generate SpVQR Cas9, which are underlined and in bold.

[0523] The amino acid sequence of an exemplary PAM binding SpVRER Cas9 is as follows:

[0524]

[0525] In the above sequence, residues V1135, R1218, Q1335, and R1337 can be mutated from D1134, G1217, R1335, and T1337 to generate SpVRER Cas9, which are underlined and in bold.

[0526] In some embodiments, the Cas9 domain is a recombinant Cas9 domain. In some embodiments, the Cas9 domain is a SpyMacCas9 domain. In some embodiments, the SpyMacCas9 domain is a nuclease-active SpyMacCas9, a nuclease-inactivated SpyMacCas9 (SpyMacCas9d), or a SpyMacCas9 nickase (SpyMacCas9n). In some embodiments, the SaCas9 domain, SaCas9d domain, or SaCas9n domain can bind to a nucleic acid sequence with a non-canonical PAM. In some embodiments, the SpyMacCas9 domain, SpCas9d domain, or SpCas9n domain can bind to a nucleic acid sequence with a NAAPAM sequence.

[0527] Exemplary SpyMacCas9

[0528]

[0529] In some cases, variant Cas9 protein includes H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1218A mutations, so that the ability of polypeptide cutting target DNA or RNA is reduced. The ability of such Cas9 protein cutting target DNA (e.g., single-stranded target DNA) is reduced, but the ability of binding target DNA (e.g., single-stranded target DNA) is retained. As another non-limiting example, in some cases, variant Cas9 protein contains D10A, H840A, P475A, W476A, N477A, ​​D1125A, W1126A and D1218A mutations, so that the ability of polypeptide cutting target DNA is reduced. The ability of such Cas9 protein cutting target DNA (e.g., single-stranded target DNA) is reduced, but the ability of binding target DNA (e.g., single-stranded target DNA) is retained. In some cases, when the variant Cas9 protein contains W476A and W1126A mutations or when the variant Cas9 protein contains P475A, W476A, N477A, ​​D1125A, W1126A and D1218A mutations, the variant Cas9 protein does not effectively bind to the PAM sequence. Therefore, in some such cases, when such variant Cas9 proteins are used in the binding method, the method does not require a PAM sequence. In other words, in some cases, when such variant Cas9 proteins are used in the binding method, the method may include a guide RNA, but the method may be performed in the absence of a PAM sequence (and the specificity of the binding is therefore provided by the targeting fragment of the guide RNA). Other residues can be mutated to achieve the above-mentioned effects (i.e., inactivating one or other nuclease moieties). As non-limiting examples, residues D10, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986 and / or A987 may be altered (i.e., substituted). In addition, mutations other than alanine substitutions are also suitable.

[0530] In some embodiments, the CRISPR protein-derived domain of the base editor may comprise all or part of a Cas9 protein having a canonical PAM sequence (NGG). In other embodiments, the Cas9-derived domain of the base editor may employ a non-canonical PAM sequence. Such sequences have been described in the art and are apparent to those skilled in the art. For example, in Kleinstiver, BP et al., “Engineered CRISPR-Cas9 nucleases with altered PAM specificities” Nature 523, 481-485 (2015); Kleinstiver, BP et al., “Broadening the targeting range of Staphylococcus aureus CRISPR-Cas9 by modifying PAMrecognition” Nature Biotechnology 33, 1293-1298 (2015); RT Walton et al., "Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9 variants" Science 10.1126 / science.aba8853 (2020); Hu et al., "Evolved Cas9 variants with broad PAM compatibility and high DNA specificity," Nature, 2018Apr.5,556(7699),57-63; Miller et al., "Continuous evolution of SpCas9 variants compatiblewith Cas9 domains that bind to non-canonical PAM sequences have been described in "non-G PAMs" Nat. Biotechnol., 2020 Apr; 38(4): 471-481, the entire contents of each of which are incorporated herein by reference.Walton et al. "Unconstrained genome targeting with near-PAMless engineered CRISPR-Cas9variants" Science 10.1126 / science.aba8853(2020); Hu et al. "Evolved Cas9variants with broad PAM compatibility and high DNA specificity," Nature, 2018Apr.5,556(7699),57-63; Miller et al., "Continuous evolution of SpCas9 variants compatible with non-G PAMs" Nat. Biotechnol., 2020Apr; 38(4):471-481; the entire contents of each are hereby incorporated by reference.

[0531] Fusion proteins comprising a Cas9 domain and a cytidine deaminase and / or adenosine deaminase

[0532] Some aspects of the present disclosure provide a fusion protein comprising a Cas9 domain or other nucleic acid programmable DNA binding protein and one or more adenosine deaminase domains, cytidine deaminase domains and / or DNA glycosylase domains. It should be understood that the Cas9 domain can be any Cas9 domain or Cas9 protein (e.g., dCas9 or nCas9) provided herein. In some embodiments, any Cas9 domain or Cas9 protein (e.g., dCas9 or nCas9) provided herein can be fused to any cytidine deaminase and adenosine deaminase provided herein. The domains of the base editors disclosed herein can be arranged in any order.

[0533] For example, but not limited to, in some embodiments, the fusion protein comprises the following structure:

[0534] NH2-[cytidine deaminase]-[Cas9 domain]-[adenosine deaminase]-COOH;

[0535] NH2-[adenosine deaminase]-[Cas9 domain]-[cytidine deaminase]-COOH;

[0536] NH2-[adenosine deaminase]-[cytidine deaminase]-[Cas9 domain]-COOH;

[0537] NH2-[cytidine deaminase]-[adenosine deaminase]-[Cas9 domain]-COOH;

[0538] NH2-[Cas9 domain]-[adenosine deaminase]-[cytidine deaminase]-COOH; or

[0539] NH2-[Cas9 domain]-[cytidine deaminase]-[adenosine deaminase]-COOH.

[0540] In some embodiments, the adenosine deaminase of the fusion protein comprises TadA*8 and a cytidine deaminase. In some embodiments, TadA*8 is TadA*8.1, TadA*8.2, TadA*8.3, TadA*8.4, TadA*8.5, TadA*8.6, TadA*8.7, TadA*8.8, TadA*8.9, TadA*8.10, TadA*8.11, TadA*8.12, TadA*8.13, TadA*8.14, TadA*8.15, TadA*8.16, TadA*8.17, TadA*8.18, TadA*8.19, TadA*8.20, TadA*8.2A, or TadA*8.24.

[0541] Exemplary fusion protein constructs include the following:

[0542] NH2-[adenosine deaminase]-[Cas9]-[[cytidine deaminase]-COOH;

[0543] NH2-[cytidine deaminase]-[Cas9]-[adenosine deaminase]-COOH;

[0544] NH2-[TadA*8]-[Cas9]-[cytidine deaminase]-COOH; or

[0545] NH2-[Cytidine deaminase]-[Cas9]-[TadA*8]-COOH.

[0546] In some embodiments, the fusion protein comprising cytidine deaminase, a base editor and adenosine deaminase and napDNAbp (e.g., Cas9 domain) does not include a linker sequence. In some embodiments, there is a linker between the cytidine deaminase and adenosine deaminase domains and napDNAbp. In some embodiments, the "-" used in the above general architecture indicates the presence of an optional linker. In some embodiments, cytidine deaminase and adenosine deaminase and napDNAbp are fused by any linker provided herein. For example, in some embodiments, cytidine deaminase and adenosine deaminase and napDNAbp are fused by any linker provided by the following paragraph entitled "Linker".

[0547] For example, in some embodiments, the cytidine deaminase and adenosine deaminase and the napDNAbp are fused via any of the linkers provided below in the section entitled "Linkers."

[0548] NH2-NLS-[cytidine deaminase]-[Cas9 domain]-[adenosine deaminase]-COOH;

[0549] NH2-NLS-[adenosine deaminase]-[Cas9 domain]-[cytidine deaminase]-COOH;

[0550] NH2-NLS-[adenosine deaminase][cytidine deaminase]-[Cas9 domain]-COOH;

[0551] NH2-NLS-[cytidine deaminase]-[adenosine deaminase]-[Cas9 domain]-COOH;

[0552] NH2-NLS-[Cas9 domain]-[adenosine deaminase]-[cytidine deaminase]-COOH;

[0553] NH2-NLS-[Cas9 domain]-[cytidine deaminase]-[adenosine deaminase]-COOH;

[0554] NH2-[cytidine deaminase]-[Cas9 domain]-[adenosine deaminase]-NLS-COOH;

[0555] NH2-[adenosine deaminase]-[Cas9 domain]-[cytidine deaminase]-NLS-COOH;

[0556] NH2-[adenosine deaminase][cytidine deaminase]-[Cas9 domain]-NLS-COOH;

[0557] NH2-[cytidine deaminase]-[adenosine deaminase]-[Cas9 domain]-NLS-COOH;

[0558] NH2-[Cas9 domain]-[adenosine deaminase]-[cytidine deaminase]-NLS-COOH; or NH2-[Cas9 domain]-[cytidine deaminase]-[adenosine deaminase]-NLS-COOH.

[0559] In some embodiments, the NLS is present in a linker or is flanked by linkers, such as those described herein. In some embodiments, the N-terminal or C-terminal NLS is a bipartite NLS. A bipartite NLS comprises two basic amino acid clusters separated by a relatively short spacer sequence (thus bipartite-2 parts, whereas a monopartite NLS is not). The nucleoplasmin NLS KR[PAATKKAGQA]KKKK is a prototype of a ubiquitous bipartite signal: two basic amino acid clusters separated by a spacer of approximately 10 amino acids. The sequence of an exemplary bipartite NLS is as follows: PKKKRKVEGADKRTADGSEFESPKKKRKV.

[0560] In some embodiments, the fusion protein comprising cytidine deaminase, adenosine deaminase, Cas9 domain and NLS does not comprise a linker sequence. In some embodiments, there is a linker sequence between one or more domains or proteins (e.g., cytidine deaminase, adenosine deaminase, Cas9 domain or NLS).

[0561] It should be understood that fusion protein of the present disclosure can include one or more additional features.For example, in some embodiments, fusion protein can include inhibitor, cytoplasm localization sequence, export sequence, such as nuclear export sequence or other localization sequence, and can be used for dissolving, purifying or detecting the sequence tag of fusion protein.Suitable protein tag provided herein includes but is not limited to biotin carboxylase carrier protein (BCCP) label, myc label, calmodulin label, FLAG label, hemagglutinin (HA) label, polyhistidine label, also referred to as histidine label or His-label, maltose binding protein (MBP)-label, nus-label, glutathione-S-transferase (GST)-label, green fluorescent protein (GFP)-label, thioredoxin-label, S-label, Softags (for example, Softag 1, Softag 3), chain label, biotin ligase label, Flash label, V5 label and SBP label.Other suitable sequences are apparent to those skilled in the art.In some embodiments, fusion protein includes one or more His tags.

[0562] Exemplary, but non-limiting, fusion proteins are described in International PCT Application Nos. PCT / 2017 / 044935 and PCT / US2020 / 016288, each of which is incorporated herein by reference in its entirety.

[0563] Fusion proteins containing a nuclear localization sequence (NLS)

[0564] In some embodiments, the fusion protein provided herein further comprises one or more (e.g., 2, 3, 4, 5) nuclear targeting sequences, such as nuclear localization sequences (NLS). In one embodiment, a dichotomous NLS is used. In some embodiments, the NLS comprises an amino acid sequence that promotes the import of proteins comprising the NLS into the nucleus (e.g., by nuclear transport). In some embodiments, any fusion protein provided herein further comprises a nuclear localization sequence (NLS). In some embodiments, the NLS is fused to the N-terminus of the fusion protein. In some embodiments, the NLS is fused to the C-terminus of the fusion protein. In some embodiments, the NLS is fused to the N-terminus of the Cas9 domain. In some embodiments, the NLS is fused to the C-terminus of the nCas9 domain or the dCas9 domain. In some embodiments, the NLS is fused to the N-terminus of the deaminase. In some embodiments, the NLS is fused to the C-terminus of the deaminase. In some embodiments, the NLS is fused to the fusion protein through one or more linkers. In some embodiments, the NLS is fused to the fusion protein without a linker. In some embodiments, the NLS comprises the amino acid sequence of any NLS sequence provided or mentioned herein. Additional nuclear localization sequences are known in the art and will be apparent to the skilled artisan. For example, NLS sequences are described in Plank et al., PCT / EP2000 / 011690, the contents of which are incorporated herein by reference for their disclosure of exemplary nuclear localization sequences. In some embodiments, the NLS comprises the amino acid sequence PKKKRKVEGADKRTADGSEFESPKKKRKV, KRTADGSEFESPKKKRKV, KRPAATKKAGQAKKKK, KKTELQTTNAENKTKKL, KRGINDRNFWRGENGRKTR, RKSGKIAAIVVKRPRKPKKKRKV, or MDSLLMNRRKFLYQFKNVRWAKGRRETYLC. In some embodiments, the NLS is present in a linker or is flanked by linkers, such as those described herein. In some embodiments, the N-terminal or C-terminal NLS is a bipartite NLS. Bipartite NLSs consist of two basic amino acid clusters separated by a relatively short spacer sequence (thus being bipartite-2, whereas monopartite NLSs are not). The nucleoplasmin NLS, KR[PAATKKAGQA]KKKK, is a prototype of the ubiquitous bipartite signal: two basic amino acid clusters separated by a spacer of approximately 10 amino acids. The sequence of an exemplary bipartite NLS is as follows: PKKKRKVEGADKRTADGSEFESPKKKRKV.

[0565] In some embodiments, the fusion protein of the present invention does not include a linker sequence. In some embodiments, there is a linker sequence between one or more domains or proteins. In some embodiments, the general structure of an exemplary Cas9 fusion protein with an adenosine deaminase or cytidine deaminase and a Cas9 domain comprises any of the following structures, wherein NLS is a nuclear localization sequence (e.g., any NLS provided herein), NH2 is the N-terminus of the fusion protein, and COOH is the C-terminus of the fusion protein:

[0566] NH2-NLS-[adenosine deaminase]-[Cas9 domain]-COOH;

[0567] NH2-NLS[Cas9 domain]-[adenosine deaminase]-COOH;

[0568] NH2-[adenosine deaminase]-[Cas9 domain]-NLS-COOH;

[0569] NH2-[Cas9 domain]-[adenosine deaminase]-NLS-COOH;

[0570] NH2-NLS-[cytidine deaminase]-[Cas9 domain]-COOH;

[0571] NH2-NLS[Cas9 domain]-[cytidine deaminase]-COOH;

[0572] NH2-[Cytidine deaminase]-[Cas9 domain]-NLS-COOH; or

[0573] NH2-[Cas9 domain]-[Cytidine deaminase]-NLS-COOH.

[0574] It should be understood that the fusion protein of the present disclosure can include one or more additional features. For example, in some embodiments, the fusion protein can include an inhibitor, a cytoplasmic localization sequence, an export sequence, such as a nuclear export sequence or other localization sequence, and a sequence tag that can be used for dissolving, purifying or detecting fusion. Protein. Suitable protein tags provided herein include but are not limited to biotin carboxylase carrier protein (BCCP) tags, myc tags, calmodulin tags, FLAG tags, hemagglutinin (HA) tags, polyhistidine tags, also referred to as histidine tags or His- tags, maltose binding protein (MBP)-tags, nus-tags, glutathione-S-transferase (GST)-tags, green fluorescent protein (GFP)-tags, thioredoxin-tags, S-tags, Softags (for example, Softag 1, Softag 3), chain tags, biotin ligase tags, Flash tags, V5 tags and SBP tags. Other suitable sequences are apparent to those skilled in the art. In some embodiments, the fusion protein includes one or more His tags.

[0575] Vectors encoding CRISPR enzymes comprising one or more nuclear localization sequences (NLSs) can be used. For example, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NLSs can be used. The CRISPR enzyme can comprise an NLS at or near the amino terminus, about or more than about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 NLSs at or near the carboxyl terminus, or any combination of these (e.g., one or more NLSs at the amino terminus and one or more NLSs at the carboxyl terminus). When more than one NLS is present, each NLS can be selected independently of the others, such that a single NLS can be present in more than one copy and / or in one or more copies with one or more other NLSs.

[0576] The CRISPR enzyme used in the method can comprise about 6 NLSs, when the amino acid closest to the NLS is within about 50 amino acids of the polypeptide chain from the N- or C-terminus, such as within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 amino acids.

[0577] Fusion proteins with internal insertions

[0578] Fusion proteins are provided herein, which include heterologous polypeptides fused to nucleic acid programmable nucleic acid binding proteins (e.g., napDNAbp). Heterologous polypeptides can be polypeptides not found in natural or wild-type napDNAbp polypeptide sequences. Heterologous polypeptides can be fused to napDNAbp at the C-terminus of napDNAbp, the N-terminus of napDNAbp, or inserted into an internal position of napDNAbp. In some embodiments, heterologous polypeptides are inserted into an internal position of napDNAbp.

[0579] In some embodiments, the heterologous polypeptide is a deaminase or a functional fragment thereof. For example, the fusion protein may comprise a deaminase flanked by an N-terminal fragment and a C-terminal fragment of a Cas9 or Cas12 (e.g., Cas12b / C2c1) polypeptide. The deaminase in the fusion protein may be an adenosine deaminase. In some embodiments, the adenosine deaminase is TadA (e.g., TadA7.10 or TadA*8). In some embodiments, the TadA is TadA*8. The TadA sequence as described herein (e.g., TadA7.10 or TadA*8) is a deaminase suitable for use in the above-mentioned fusion protein.

[0580] The deaminase can be a cyclic displacement deaminase. For example, the deaminase can be a cyclic displacement adenosine deaminase. In some embodiments, the deaminase is a circular TadA that is circularly arranged at amino acid residue 116, as numbered in the TadA reference sequence. In some embodiments, the deaminase is a circular TadA that is circularly arranged at amino acid residue 136, as numbered in the TadA reference sequence. In some embodiments, the deaminase is a circular TadA that is circularly arranged at amino acid residue 65, as numbered in the TadA reference sequence.

[0581] The fusion protein may comprise more than one deaminase. The fusion protein may comprise, for example, 1, 2, 3, 4, 5, or more deaminases. In some embodiments, the fusion protein comprises one deaminase. In some embodiments, the fusion protein comprises two deaminases. The two or more deaminases in the fusion protein may be adenosine deaminase, cytidine deaminase, or a combination thereof. The two or more deaminases may be homodimers. The two or more deaminases may be heterodimers. The two or more deaminases may be inserted in series into napDNAbp. In some embodiments, the two or more deaminases may not be inserted in series into napDNAbp.

[0582] In some embodiments, the napDNAbp in the fusion protein is a Cas9 polypeptide or a fragment thereof. The Cas9 polypeptide may be a variant Cas9 polypeptide. In some embodiments, the Cas9 polypeptide is a Cas9 nickase (nCas9) polypeptide or a fragment thereof. In some embodiments, the Cas9 polypeptide is a nuclease-dead Cas9 (dCas9) polypeptide or a fragment thereof. The Cas9 polypeptide in the fusion protein may be a full-length Cas9 polypeptide. In some cases, the Cas9 polypeptide in the fusion protein may not be a full-length Cas9 polypeptide. The Cas9 polypeptide can be truncated, for example, at the N-terminus or C-terminus relative to the naturally occurring Cas9 protein. The Cas9 polypeptide may be a circularly permuted Cas9 protein. The Cas9 polypeptide may be a fragment, portion, or domain of a Cas9 polypeptide that is still capable of binding to the target polynucleotide and the guide nucleic acid sequence.

[0583] In some embodiments, the Cas9 polypeptide is Streptococcus pyogenes Cas9 (SpCas9), Staphylococcus aureus Cas9 (SaCas9), Streptococcus thermophilus 1 Cas9 (St1Cas9), or a fragment or variant thereof.

[0584] The Cas9 polypeptide of the fusion protein can comprise a polypeptide that is at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to a naturally occurring Cas9 polypeptide.

[0585] The Cas9 polypeptide of the fusion protein may comprise at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or at least 99.5% identical to the Cas9 amino acid sequence set forth below (hereinafter referred to as the "Cas9 reference sequence"):

[0586]

[0587] (Single underline: HNH domain; double underline: RuvC domain).

[0588] Fusion proteins comprising heterologous catalytic domains flanked by N and C-terminal fragments of Cas9 polypeptides can also be used for base editing in the methods described herein. Comprising Cas9 and one or more deaminase domains, such as adenosine deaminase, or fusion proteins comprising adenosine deaminase domains flanked by Cas9 sequences can also be used for highly specific and effective base editing of target sequences. In one embodiment, the chimeric Cas9 fusion protein comprises a heterologous catalytic domain (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) inserted into the Cas9 polypeptide. In some embodiments, the fusion protein comprises an adenosine deaminase domain and a cytidine deaminase domain inserted into Cas9. In some embodiments, adenosine deaminase is fused in Cas9 and cytidine deaminase is fused to the C-terminus. In some embodiments, adenosine deaminase is fused in Cas9 and cytidine deaminase is fused to the N-terminus. In some embodiments, cytidine deaminase is fused in Cas9 and adenosine deaminase is fused to the C-terminus. In some embodiments, a cytidine deaminase is fused within Cas9 and an adenosine deaminase is fused to the N-terminus.

[0589] An exemplary structure of a fusion protein with adenosine deaminase and cytidine deaminase and Cas9 is provided below:

[0590] NH2-[Cas9(adenosine deaminase)]-[cytidine deaminase]-COOH;

[0591] NH2-[cytidine deaminase]-[Cas9(adenosine deaminase)]-COOH;

[0592] NH2-[Cas9(cytidine deaminase)]-[adenosine deaminase]-COOH; or

[0593] NH2-[Adenosine deaminase]-[Cas9(cytidine deaminase)]-COOH.

[0594] In some embodiments, the "-" used in the general scheme above indicates the presence of an optional linker.

[0595] In various embodiments, the catalytic domain has DNA modification activity (e.g., deaminase activity), such as adenosine deaminase activity. In some embodiments, the adenosine deaminase is TadA (e.g., TadA7.10). In some embodiments, the TadA is TadA*8. In some embodiments, TadA*8 is fused within Cas9 and the cytidine deaminase is fused to the C-terminus. In some embodiments, TadA*8 is fused within Cas9 and the cytidine deaminase is fused to the N-terminus. In some embodiments, the cytidine deaminase is fused within Cas9 and TadA*8 is fused to the C-terminus. In some embodiments, the cytidine deaminase is fused within Cas9 and TadA*8 is fused to the N-terminus. Exemplary structures of fusion proteins with TadA*8, cytidine deaminase, and Cas9 are provided below:

[0596] NH2-[Cas9(TadA*8)]-[cytidine deaminase]-COOH;

[0597] NH2-[Cytidine deaminase]-[Cas9]-[Cas9(TadA*8)]-COOH;

[0598] NH2-[Cas9(cytidine deaminase)]-[TadA*8]-COOH; or

[0599] NH2-[TadA*8]-[Cas9(cytidine deaminase)]-COOH.

[0600] In some embodiments, the "-" used in the general scheme above indicates the presence of an optional linker.

[0601] Heterologous polypeptides (e.g., deaminases) can be inserted into suitable positions of napDNAbp (e.g., Cas9 or Cas12 (e.g., Cas12b / C2c1)), for example, so that napDNAbp maintains its binding to target polynucleotides and guide nucleic acids. Deaminases (e.g., adenosine deaminases, cytidine deaminases, or adenosine deaminases and cytidine deaminases) can be inserted into napDNAbp without compromising the function of the deaminases (e.g., base editing activity) or napDNAbp (e.g., the ability to bind to target nucleic acids and guide nucleic acids). Deaminases (e.g., adenosine deaminases, cytidine deaminases, or adenosine deaminases and cytidine deaminases) can be inserted into napDNAbp, such as disordered regions shown by crystallographic studies or regions comprising high temperature factors or B factors. Less ordered, disordered, or unstructured protein regions, such as solvent exposed regions and loops, can be used for insertion without compromising structure or function. Deaminases (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) can be inserted into the flexible loop region or solvent exposed region of napDNAbp. In some embodiments, deaminases (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) are inserted into the flexible loop of Cas9 or Cas12b / C2c1 polypeptide.

[0602] In some embodiments, the insertion position of a deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is determined by B-factor analysis of the crystal structure of a Cas9 polypeptide. In some embodiments, a deaminase (e.g., adenosine deaminase, cytidine deaminase, or adenosine deaminase and cytidine deaminase) is inserted into a Cas9 polypeptide comprising a higher than average B factor (e.g., with total protein or a protein domain comprising a disordered region). The B factor or temperature factor can represent the fluctuation of an atom from its average position (e.g., due to temperature-related atomic vibrations or static disorder in a lattice). The high B factor (e.g., higher than average B factor) of a skeleton atom can indicate a region with relatively high local mobility. Such a region can be used to insert a deaminase without compromising structure or function. The deaminase (e.g., an adenosine deaminase, a cytidine deaminase, or both an adenosine deaminase and a cytidine deaminase) can be inserted at the position of a residue with a Ca atom whose B-factor is 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or greater than 200% greater than the average B-factor of the total protein. The deaminase (e.g., an adenosine deaminase, a cytidine deaminase, or an adenosine deaminase and a cytidine deaminase) can be inserted at a position having a residue with a Cα atom, wherein the residue has a B-factor that is 50%, 60%, 70%, 80%, 90%, 100%, 110%, 120%, 130%, 140%, 150%, 160%, 170%, 180%, 190%, 200%, or greater than 200% greater than the average B-factor of the Cas9 protein domain comprising the residue. Positions of the Cas9 polypeptide comprising a higher than average B-factor can include, for example, residues numbered 768, 792, 1052, 1015, 1022, 1026, 1029, 1067, 1040, 1054, 1068, 1246, 12247 in the Cas9 reference sequence above. Positions of the Cas9 polypeptide comprising above-average B-factors can include, for example, residues numbered 792-872, 792-906, and 2-791 in the above-mentioned Cas9 reference sequence.

[0603] A heterologous polypeptide (e.g., a deaminase) can be inserted into napDNAbp at an amino acid residue selected from the group consisting of amino acid residues 768, 791, 792, 1015, 1016, 1022, 1023, 1026, 1029, 1040, 1052, 1054, 1067, 1068, 1069, 1246, 1247, and 1248 in the Cas9 reference sequence, or the corresponding amino acid residues in another Cas9 polypeptide. In some embodiments, the heterologous polypeptide is inserted into the Cas9 reference sequence at amino acid positions numbered 768 to 769, 791 to 792, 792 to 793, 1015 to 1016, 1022 to 1023, 1026 to 1027, 1029 to 1030, 1040 to 1041, 1052 to 1053, 1054 to 1055, 1067 to 1068, 1068 to 1069, 1247 to 1248, or 1248 to 1249, or their corresponding amino acid positions. In some embodiments, the heterologous polypeptide is inserted into the Cas9 reference sequence at amino acid positions numbered 769 to 770, 792 to 793, 793 to 794, 1016 to 1017, 1023 to 1024, 1027 to 1028, 1030 to 1031, 1041 to 1042, 1053 to 1054, 1055 to 1056, 1068 to 1069, 1069 to 1070, 1248 to 1249, or 1249 to 1250, or their corresponding amino acid positions. In some embodiments, the heterologous polypeptide replaces an amino acid residue selected from the group consisting of: 768, 791, 792, 1015, 1016, 1022, 1023, 1026, 1029, 1040, 1052, 1054, 1067, 1068, 1069, 1246, 1247, and 1248 in the above-mentioned Cas9 reference sequence, or the corresponding amino acid residue in another Cas9 polypeptide. It should be understood that reference to the above-mentioned Cas9 reference sequence with respect to the insertion position is for illustrative purposes. Insertions as discussed herein are not limited to the Cas9 polypeptide sequence of the above-mentioned Cas9 reference sequence, but include insertions at corresponding positions in variant Cas9 polypeptides, such as Cas9 nickase (nCas9), nuclease-dead Cas9 (dCas9), Cas9 variants lacking a nuclease domain, truncated Cas9, or a Cas9 domain lacking a partial or complete HNH domain.

[0604] A heterologous polypeptide (e.g., a deaminase) can be inserted into napDNAbp at an amino acid residue selected from the group consisting of amino acid residues 768, 792, 1022, 1026, 1040, 1068, and 1247 in the Cas9 reference sequence, or corresponding amino acid residues in another Cas9 polypeptide. In some embodiments, the heterologous polypeptide is inserted into the amino acid positions 768 to 769, 792 to 793, 1022 to 1023, 1026 to 1027, 1029 to 1030, 1040 to 1041, 1068 to 1069, or 1247 to 1248 in the Cas9 reference sequence, or their corresponding amino acid positions. In some embodiments, the heterologous polypeptide is inserted into the amino acid positions 769 to 770, 793 to 794, 1023 to 1024, 1027 to 1028, 1030 to 1031, 1041 to 1042, 1069 to 1070, or 1248 to 1249 of the Cas9 reference sequence, or the corresponding amino acid positions thereof. In some embodiments, the heterologous polypeptide replaces an amino acid residue selected from the group consisting of: amino acid residues 768, 792, 1022, 1026, 1040, 1068, and 1247 of the Cas9 reference sequence, or the corresponding amino acid residues in another Cas9 polypeptide.

[0605] A heterologous polypeptide (e.g., a deaminase) can be inserted into napDNAbp at an amino acid residue as described herein or at a corresponding amino acid residue in another Cas9 polypeptide. In one embodiment, a heterol...

Claims

1. A method for producing hematopoietic stem cells or progenitor cells thereof for hematopoietic stem cell transplantation, the method comprising (a) expressing a nucleobase editing polypeptide in a hematopoietic stem cell or a progenitor cell thereof comprising a CD117 protein, wherein the nucleobase editing polypeptide comprises a nucleic acid programmable DNA binding protein and an adenosine deaminase, wherein, The amino acid sequence of the adenosine deaminase is MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 11) or SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, and the amino acid sequence of the adenosine deaminase has the amino acid sequence of SEQ ID NO: 11, wherein the amino acid changes consist of Y123H, Y147R and Q154R; and the nucleic acid programmable DNA binding protein comprises a Cas9 polypeptide; (b) contacting the hematopoietic stem cell or its progenitor cell with a guide RNA that can target a CD117 protein encoded by a polynucleotide, wherein the amino acid sequence of the CD117 protein is as shown in SEQ ID NO: 35, and wherein the guide RNA comprises the nucleotide sequence AAAUAUAAUAGCUGGCAUCA, Thus, a Y259C amino acid substitution is introduced into the CD117 protein, and the CD117 protein comprising the amino acid substitution is able to bind to stem cell factor without interfering with normal stem cell factor signal transduction; And producing the hematopoietic stem cells or progenitor cells thereof for hematopoietic stem cell transplantation.

2. The method of claim 1, wherein the nucleobase editing polypeptide is an internal base editor comprising a deaminase domain inserted at an internal position of a nucleic acid programmable DNA binding protein.

3. The method according to claim 1, wherein: The cells are common myeloid progenitor cells.

4. A method for in vitro base editing of a gene encoding a cell surface protein expressed by a hematopoietic stem cell or a progenitor cell thereof, the method comprising (a) expressing a nucleobase editing polypeptide in a hematopoietic stem cell or a progenitor cell thereof comprising a CD117 protein, wherein the nucleobase editing polypeptide comprises a nucleic acid programmable DNA binding protein and an adenosine deaminase, wherein, The amino acid sequence of the adenosine deaminase is MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 11) or SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, and the amino acid sequence of the adenosine deaminase has the amino acid sequence of SEQ ID NO: 11, wherein the amino acid changes consist of Y123H, Y147R and Q154R; and the nucleic acid programmable DNA binding protein comprises a Cas9 polypeptide; and (b) contacting the cell with a guide RNA capable of targeting a nucleic acid molecule encoding a CD117 protein, wherein the amino acid sequence of the CD117 protein is as shown in SEQ ID NO: 35, and wherein the guide RNA comprises the nucleotide sequence AAAUAUAAUAGCUGGCAUCA, Thus, the Y259C amino acid substitution is introduced into the CD117 protein, and the CD117 protein containing the amino acid substitution can bind to the stem cell factor without interfering with the normal signal transduction of the stem cell factor.

5. The method according to claim 1 or 4, wherein: The nucleobase editing polypeptide further comprises a wild-type adenosine deaminase domain.

6. The method of claim 1 or 4, wherein the nucleobase editing polypeptide further comprises one or more nuclear localization sequences.

7. The method of claim 1 or 4, wherein the cells are from a subject suffering from a hemoglobinopathy, a blood cancer, or a myeloproliferative disorder.

8. The method according to claim 7, wherein: The cells are from a subject suffering from sickle cell disease.

9. The method according to claim 1 or 4, wherein: The cells are mammalian cells.

10. The method according to claim 9, wherein: The cells are human cells.

11. The method according to claim 1 or 4, wherein: The adenosine deaminase is fused to the nucleic acid programmable DNA binding protein.

12. The method according to claim 1 or 4, wherein: The adenosine deaminase is inserted at an internal position of the nucleic acid programmable DNA binding protein.

13. The method according to claim 1 or 4, wherein: The nucleic acid programmable DNA binding protein comprises an inactive Cas9 or a Cas9 nickase.

14. The method according to claim 1 or 4, wherein: The nucleic acid programmable DNA binding protein is Staphylococcus aureus Cas9, Streptococcus thermophilus 1 Cas9 or Streptococcus pyogenes Cas9.

15. The method according to claim 14, wherein: The nucleic acid programmable DNA binding protein comprises a Streptococcus pyogenes Cas9 with an altered protospacer-adjacent motif specificity, wherein the altered protospacer-adjacent motif is specific for the nucleic acid sequence 5'-NGC-3'.

16. Use of hematopoietic stem cells produced by the method according to claim 1 in preparing a medicament for hematopoietic stem cell transplantation, wherein An antibody, an antibody-drug conjugate or a chimeric antigen receptor-expressing T cell that selectively binds to wild-type CD117 is administered to a subject simultaneously with the drug or subsequently to the drug; wherein the amino acid substitution cannot change the biological activity of the CD117 protein, and the antibody is an anti-CD117 antibody.

17. The use according to claim 16, wherein The nucleobase editing polypeptide further comprises a wild-type adenosine deaminase.

18. The use according to claim 16, wherein the nucleobase editing polypeptide is an internal base editor comprising an adenosine deaminase inserted at an internal position of a nucleic acid programmable DNA binding protein.

19. The use according to claim 16, wherein The nucleobase editing polypeptide further comprises one or more nuclear localization sequences.

20. The use according to claim 16, wherein The antibody is a monoclonal antibody.

21. A base editing system for introducing a mutation into a CD117 protein, the base editing system comprising a fusion protein or a polynucleotide encoding the fusion protein, wherein: The fusion protein comprises a nucleic acid programmable DNA binding protein and an adenosine deaminase domain, wherein the amino acid sequence of the adenosine deaminase domain is MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 11) or SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, and the amino acid sequence of the adenosine deaminase has an amino acid change consisting of Y123H, Y147R and Q154R according to SEQ ID NO: 11; and the nucleic acid programmable DNA binding protein comprises a Cas9 polypeptide; and a guide polynucleotide comprising the nucleic acid sequence AAAUAUAAUAGCUGGCAUCA; Wherein, the guide polynucleotide is a guide RNA, which can target the CD117 protein as shown in SEQ ID NO: 35, and Wherein, the mutation is a Y259C amino acid substitution in the CD117 protein.

22. A base editing system for introducing a mutation into a CD117 protein, the base editing system comprising a fusion protein or a polynucleotide encoding the fusion protein, wherein: The fusion protein comprises a nucleic acid programmable DNA binding protein and an adenosine deaminase domain, wherein the amino acid sequence of the adenosine deaminase domain is MSEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD (SEQ ID NO: 11) or SEVEFSHEYWMRHALTLAKRARDEREVPVGAVLVLNNRVIGEGWNRAIGLHDPTAHAEIMALRQGGLVMQNYRLIDATLYVTFEPCVMCAGAMIHSRIGRVVFGVRNAKTGAAGSLMDVLHYPGMNHRVEITEGILADECAALLCYFFRMPRQVFNAQKKAQSSTD, and the amino acid sequence of the adenosine deaminase has an amino acid change consisting of Y123H, Y147R, and Q154R according to SEQ ID NO: 11; and the nucleic acid programmable DNA binding protein comprises a Cas9 polypeptide; and a guide polynucleotide that hybridizes to a complementary sequence of a target nucleic acid sequence, and the complementary sequence of the target nucleic acid sequence is AAATATAATAGCTGGCATCA; Wherein, the guide polynucleotide is a guide RNA, which can target the CD117 protein as shown in SEQ ID NO: 35, and Wherein, the mutation is a Y259C amino acid substitution in the CD117 protein.

23. The base editing system according to claim 21 or 22, wherein: The fusion protein further comprises one or more nuclear localization sequences.

24. The base editing system according to claim 21 or 22, wherein: The nucleic acid programmable DNA binding protein comprises an inactive Cas9 or a Cas9 nickase.

25. The base editing system according to claim 21 or 22, wherein: The nucleic acid programmable DNA binding protein is Staphylococcus aureus Cas9, Streptococcus thermophilus 1 Cas9 or Streptococcus pyogenes Cas9.

26. The base editing system of claim 25, wherein: The nucleic acid programmable DNA binding protein comprises a Streptococcus pyogenes Cas9 with an altered protospacer-adjacent motif specificity, wherein the altered protospacer-adjacent motif is specific for the nucleic acid sequence 5'-NGC-3'.

27. The base editing system according to claim 21 or 22, wherein: The fusion protein further comprises a wild-type adenosine deaminase domain.

28. A polynucleotide encoding a base editing system according to any one of claims 21 to 27.

29. A cell produced by the method of any one of claims 1 to 4.

30. The cell according to claim 29, wherein The cells are in vitro.

31. The cell according to claim 29, wherein The cells are hematopoietic stem cells.

32. The cell according to claim 29, wherein The cells are common myeloid progenitor cells.

33. The cell according to claim 29, wherein The cells are mammalian cells.

34. The cell according to claim 33, wherein The cells are human cells.

35. A pharmaceutical composition comprising a therapeutically effective amount of the cell according to claim 29 or 30.

36. Use of the pharmaceutical composition according to claim 35 in the preparation of a medicament for hematopoietic stem cell transplantation in a subject.

37. The use according to claim 36, wherein The subject has sickle cell disease.

38. The use according to claim 36, wherein The cells are autologous to the subject.

39. The use according to claim 36, wherein The cells are allogeneic to the subject.

40. The use according to claim 36, wherein The subject is a mammal.

41. The use according to claim 40, wherein The mammal is a human.

42. A kit comprising a cell according to any one of claims 29 to 34, a base editing system according to any one of claims 21 to 27, a polynucleotide according to claim 28, or a pharmaceutical composition according to claim 35.

43. The kit of claim 42, further comprising written instructions for using the kit in hematopoietic stem cell transplantation.

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