Compositions and methods for multiplexed base editing in hematopoietic cells

Abstract

When a cancer patient is administered an anti-cancer therapy that targets a lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)), for example in the form of an immunotherapeutic agent, the therapy can deplete not only cancer cells expressing the lineage-specific cell surface antigen, but also non-cancer cells expressing the lineage-specific cell surface antigen in an "on-target, off-tumor" effect. The present disclosure provides, for example, novel cells having modifications (e.g., insertions or deletions) in endogenous lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) genes. The present disclosure also provides compositions, e.g., gRNAs, that can be used to make such modifications.

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 343,408, filed May 18, 2022, U.S. Provisional Patent Application No. 63 / 278,375, filed November 11, 2021, and U.S. Provisional Patent Application No. 63 / 244,219, filed September 14, 2021, the entire contents of each of which are incorporated herein by reference. [Background technology]

[0002] When cancer patients are administered anti-cancer therapy that targets lineage-specific cell surface antigens (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)), for example, in the form of immunotherapeutic agents, the therapy can deplete not only cancer cells that express the lineage-specific cell surface antigen, but also non-cancer cells that express the lineage-specific cell surface antigen in an "on-target, off-tumor" effect. Certain non-cancer hematopoietic cells can be targeted by such anti-cancer therapeutics because they typically express CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), and loss of non-cancer CD33 (Siglec-3)+, CLL-1+, CD123+, CD327 (Siglec-6)+, and / or CD312 (EMR2)+ cells can deplete and impair a patient's hematopoietic system. To address this depletion, subjects can be administered rescue cells (e.g., hematopoietic stem cells (HSCs) and / or hematopoietic progenitor cells (HPCs)) containing modifications in the CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes, e.g., gene editing resulting in rescue cells with reduced or eliminated expression of the respective genes, or modifications of epitopes in the proteins encoded by the respective genes that reduce binding of therapeutic agents to the proteins. These CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)-modified cells can thus be resistant to anti-cancer therapy and thus be able to repopulate the hematopoietic system during or after anti-cancer therapy. HSCs and HPCs can be modified using various gene editing techniques, including, for example, CRISPR / Cas technology. However, conventional CRISPR / Cas technology is associated with certain limitations, such as off-target editing effects (OTEs), chromosomal rearrangements, and genotoxicity due to simultaneous double-strand break (DSB) induction at multiple loci. The present disclosure addresses the need for safe and effective methods for achieving gene editing, including multiplex editing of cells for therapeutic applications. Summary of the Invention

[0003] Provided herein are therapeutic modalities involving multiplexed modification of endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes, as well as strategies, compositions, and methods for making and using them. Aspects of the present disclosure are directed to the modification of DNA, such as multiplexed modification of DNA in cells using one or more guide RNAs (gRNAs) directed to a nuclease-damaging enzyme or partial nuclease-damaging enzyme (e.g., an RNA-guided CRISPR / Cas protein) fused to a base editor, e.g., a deaminase that targets and deaminates specific nucleobases, e.g., cytosine or adenosine nucleobases of C or A nucleotides, resulting in a C to T nucleotide change or an A to G nucleotide change at a targeted position on the DNA via cellular mismatch repair mechanisms, where the base editor provides the editing event.

[0004] Certain aspects of the present disclosure provide methods of multiplexed base editing, e.g., methods using certain gRNAs and / or gene editing enzymes (e.g., RNA-guided CRISPR / Cas proteins, base editors, etc.) provided herein, to generate genetically engineered cells, e.g., cells with one, two, or multiple modifications in genes encoding cell surface antigens. In some embodiments, the multiplexed base editing methods provided herein can be used to generate genetically engineered cells with modifications in multiple genes encoding cell surface antigens. Thus, use of the methods provided herein can enable the efficient removal of one, two, or multiple cell surface antigens from cells for therapeutic applications, such as immunotherapy.

[0005] Without wishing to be bound by theory, removing cell surface antigens by multiplex base editing of the genomes of hematopoietic stem and progenitor cells (HSPCs) in allogeneic transplants is a novel and advantageous approach for enabling targeted post-transplant therapy in diseases such as acute myeloid leukemia (AML). In some embodiments, the present disclosure provides methods that enable compatible therapeutic modalities that specifically target leukemic cells while protecting target antigen-null allogeneic cells. However, given that one of the known obstacles in the treatment of AML is tumor antigen heterogeneity, modifying, e.g., removing, a single surface target, may not be sufficient to achieve efficacy in AML and avoid potential antigen escape. In such instances, combination therapy targeting multiple antigens, e.g., multiple cell surface antigens, may provide greater efficacy in AML treatment and help avoid potential antigen escape. Accordingly, aspects of the present disclosure provide a multiplex base editing approach for simultaneously inducing gene knockout (KO) of clinically relevant AML surface antigens in CD34+ HSPCs from healthy donors using cytosine base editors (CBEs). Such methods can enable the administration of combination targeted therapeutics with on-target, reduced off-tumor toxicity to AML patients. Furthermore, co-delivery of base editing guides by the methods provided herein can preserve the health, proliferation, and stemness of HSPCs, which may facilitate the processing and manufacturing of combination-targeted cells for therapeutic applications. In some embodiments, the methods provided herein can achieve high base editing efficiency, robust surface protein knockout (KO), and detection of balanced translocations in multiplex-edited cells. In some embodiments, the present disclosure provides methods for multiplex base editing of one, two, or multiple cell surface targets (e.g., cell surface antigens) in CD34+ HSPCs, thereby providing a beneficial, safe, and effective alternative for engineering next-generation cell transplants to treat AML patients.Accordingly, aspects of the present disclosure provide methods for multiplex base editing in human hematopoietic stem and progenitor cells (HSPCs) that enable efficient removal of one, two, or multiple cell surface antigens in acute myeloid leukemia (AML) immunotherapy.

[0006] In some embodiments, the present disclosure provides methods of multiplex base editing to modify, e.g., remove, one, two, or more cell surface antigens. Exemplary cell surface antigens include, but are not limited to, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CD13, CD14, CD15, CD16a, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO , CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD 60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65s, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD 74, CD75, CD75s, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85a, CD85b, CD85c, CD85d, CD85e, CD85f, CD85g, CD85h, CD85i, CD85j, CD85k, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD10 6, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b,CD122、CD123、CD124、CD125、CD126、CD127、CD128a、CD128b、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CD146、CD147、CD148、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CD156c、CD157、CD158a、CD158b1、CD158b2、CD158c、CD158d、CD158e1、CD158e2、CD158f、CD158g、CD158h、CD158i、CD158j、CD158k、CD159a、CD159c、CD160、CD161、CD162、CD163、CD164、CD165、CD166、CD167a、CD167b、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CD198、CD199、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210a、CD210b、CD212、CD213a1、CD213a2、CD215、CD217、CD218a、CD218b、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD236、CD238、CD239、CD240CE、CD240D、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CD248、CD249、CD252、CD253、CD254、CD256、CD257、CD258、CD261、CD262、CD263、CD264、CD265、CD266、CD267、CD268、CD269, CD270, CD271, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD 281, CD282, CD283, CD284, CD286, CD288, CD289, CD290, CD292, CDw293, CD294, CD295, CD29 6, CD297, CD298, CD299, CD300a, CD300c, CD300d, CD300e, CD300f, CD300g, CD301, CD302, C D303, CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD309, CD312, CD314 , CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, and CD371, or any combination thereof.

[0007] In some embodiments, the present disclosure provides multiplex base editing methods for modifying one, two, three, four, or more cell surface antigens selected from the group consisting of, for example, CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), CD312 (EMR2), and any combination thereof.

[0008] In some embodiments, the cell surface antigen is CD33 (Siglec-3).

[0009] In some embodiments, the cell surface antigen is CLL-1.

[0010] In some embodiments, the cell surface antigen is CD123.

[0011] In some embodiments, the cell surface antigen is CD327 (Siglec-6).

[0012] In some embodiments, the cell surface antigen is CD312 (EMR2).

[0013] Certain aspects of the present disclosure provide methods of multiplex base editing, e.g., methods using certain gRNAs and / or gene editing enzymes provided herein to generate genetically engineered cells, e.g., cells with multiple modifications in endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes.

[0014] Without wishing to be bound by theory, CRISPR / Cas technology may be associated with various limitations, including off-target editing effects (OTEs), chromosomal rearrangements, and genotoxicity due to the simultaneous induction of double-strand breaks (DSBs) at multiple loci. OTEs can include unintended point mutations, deletions, insertions, inversions, and translocations at or near the target sequence. Chromosomal translocations can occur when DNA ends from double-strand breaks (DSBs) on two different chromosomes are improperly joined.

[0015] The multiplexed base editing methods provided herein have advantages over conventional CRISPR / Cas technologies at least because the base editing substantially reduces the frequency of DSB formation compared to conventional CRISPR / Cas technologies, such that the methods described herein can be used without significant risk of translocations.

[0016] The multiplexed base editing methods provided herein can be used to produce engineered cells with a lower overall translocation rate compared to the use of conventional CRISPR / Cas technology. Such cells can have 0% translocations, as assessed by a translocation analysis assay such as a RhampSeq assay. In certain embodiments, cells produced by the multiplexed base editing methods provided herein can have 0% translocations or undetectable levels of translocations and an on-target editing efficiency of at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% or more, for modifications in the endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes, for example.

[0017] In certain embodiments, on-target editing efficiency is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% or greater, e.g., for modification of endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes. In certain embodiments, on-target editing efficiency is for knockout of endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes.

[0018] In certain embodiments, multiplexed base editing may involve comboplexing a base editor and a CRISPR nuclease, such as a CRISPR nuclease including a Cas9 or Cas12a nuclease, without any risk of translocation. In certain embodiments, multiplexed base editing may involve simultaneous delivery of a cytosine base editor (CBE) and a Cpfl nuclease. In certain embodiments, multiplexed base editing may involve simultaneous delivery of a cytosine base editor (CBE) and / or an adenine base editor (ABE). In certain embodiments, multiplexed base editing does not significantly affect cell viability and / or cell proliferation.

[0019] In some embodiments, multiplex modifications of endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes occur simultaneously.

[0020] In particular, provided herein are methods for multiplex base editing, the methods including: (i) providing a cell; and (ii) introducing into the cell: (a) one or more guide RNAs (gRNAs) targeting CD33 (Siglec-3), one or more gRNAs targeting CLL-1, one or more gRNAs targeting CD123, one or more gRNAs targeting CD327 (Siglec-6), and / or one or more gRNAs targeting CD312 (EMR2); and (b) a base editor that binds to the one or more gRNAs, wherein the one or more gRNAs are configured to provide editing events within the same or different target domains, thereby producing a genetically engineered cell.

[0021] In particular, provided herein are methods for producing genetically engineered cells, the methods including: (i) providing a cell; and (ii) introducing into the cell: (a) one or more guide RNAs (gRNAs) targeting CD33 (Siglec-3), one or more gRNAs targeting CLL-1, one or more gRNAs targeting CD123, one or more gRNAs targeting CD327 (Siglec-6), and / or one or more gRNAs targeting CD312 (EMR2); and (b) a base editor that binds to the one or more gRNAs, wherein the one or more gRNAs are configured to provide editing events within the same or different target domains, thereby producing a genetically engineered cell.

[0022] In particular, provided herein are methods for multiplex base editing, the methods including: (i) providing a cell; and (ii) introducing into the cell: (a) one or more guide RNAs (gRNAs) targeting CD33 (Siglec-3), one or more gRNAs targeting CLL-1, one or more gRNAs targeting CD123, one or more gRNAs targeting CD327 (Siglec-6), and / or one or more gRNAs targeting CD312 (EMR2); and (b) a base editor that binds to the one or more gRNAs, wherein the one or more gRNAs are configured to provide editing events within different target domains, thereby producing a genetically engineered cell.

[0023] In particular, provided herein are methods for multiplex base editing, the methods including: (i) providing a cell; and (ii) introducing into the cell: (a) one or more guide RNAs (gRNAs) targeting CD33, (b) one or more gRNAs targeting CLL-1 and / or one or more gRNAs targeting CD123, and (c) a base editor that binds to the one or more gRNAs, wherein the one or more gRNAs are configured to provide editing events within different target domains, thereby producing a genetically engineered cell.

[0024] In particular, provided herein are methods for producing genetically engineered cells, the methods including: (i) providing a cell; and (ii) introducing into the cell: (a) one or more guide RNAs (gRNAs) targeting CD33, (b) one or more gRNAs targeting CLL-1 and / or one or more gRNAs targeting CD123, and (c) a base editor that binds to the one or more gRNAs, wherein the one or more gRNAs are configured to provide editing events within different target domains, thereby producing a genetically engineered cell.

[0025] In particular, provided herein are methods for multiplex base editing, the methods including: (i) providing a cell; and (ii) introducing into the cell: (a) one or more guide RNAs (gRNAs) targeting CD33 (Siglec-3), (b) one or more gRNAs targeting CLL-1, one or more gRNAs targeting CD123, one or more gRNAs targeting CD327 (Siglec-6), and / or one or more gRNAs targeting CD312 (EMR2); and (c) a base editor that binds to the one or more gRNAs, wherein the one or more gRNAs are configured to provide editing events within different target domains, thereby producing a genetically engineered cell.

[0026] In particular, provided herein are methods for producing genetically engineered cells, the methods including: (i) providing a cell; and (ii) introducing into the cell: (a) one or more guide RNAs (gRNAs) targeting CD33 (Siglec-3), (b) one or more gRNAs targeting CLL-1, one or more gRNAs targeting CD123, one or more gRNAs targeting CD327 (Siglec-6), and / or one or more gRNAs targeting CD312 (EMR2); and (c) a base editor that binds to the one or more gRNAs, wherein the one or more gRNAs are configured to provide editing events within the same or different target domains, thereby producing a genetically engineered cell.

[0027] In particular, provided herein are methods for triplex base editing, the methods including: (i) providing a cell; and (ii) introducing into the cell (a) a plurality of gRNAs configured to provide simultaneous editing events in at least three different genomic targets, and (d) a base editor that binds to the plurality of gRNAs, thereby producing a genetically engineered cell.

[0028] In particular, provided herein are methods for triplex base editing, the methods including: (i) providing a cell; and (ii) introducing into the cell: (a) one or more gRNAs targeting CD33 (Siglec-3), (b) one or more gRNAs targeting CLL1, (c) one or more gRNAs targeting CD123, and (d) a base editor that binds to the one or more gRNAs, wherein the one or more gRNAs are configured to provide simultaneous editing events in at least three different target domains, thereby producing a genetically engineered cell.

[0029] In particular, provided herein are methods for producing a genetically engineered cell, the methods including: (i) providing a cell; and (ii) introducing into the cell: (a) a plurality of gRNAs configured to provide simultaneous editing events in at least three different genomic targets; and (d) a base editor that binds to the plurality of gRNAs, thereby producing the genetically engineered cell.

[0030] In particular, provided herein are methods for producing genetically engineered cells, the methods including: (i) providing a cell; and (ii) introducing into the cell: (a) one or more gRNAs targeting CD33 (Siglec-3), (b) one or more gRNAs targeting CLL1, (c) one or more gRNAs targeting CD123, and (d) a base editor that binds to the one or more gRNAs, wherein the one or more gRNAs are configured to provide simultaneous editing events in at least three different target domains, thereby producing a genetically engineered cell.

[0031] In particular, provided herein are methods for quadroplex base editing, the methods including: (i) providing a cell; and (ii) introducing into the cell (a) a plurality of gRNAs configured to provide simultaneous editing events in at least four different genomic targets, and (d) a base editor that binds to the plurality of gRNAs, thereby producing a genetically engineered cell.

[0032] In particular, provided herein are methods for quadroplex base editing, the methods including: (i) providing a cell; and (ii) introducing into the cell: (a) one or more gRNAs targeting CD33 (Siglec-3), (b) one or more gRNAs targeting CLL1, (c) one or more gRNAs targeting CD123, (d) one or more gRNAs targeting CD312 (EMR2), and (e) a base editor that binds to the one or more gRNAs, wherein the one or more gRNAs are configured to provide simultaneous editing events in at least four different target domains, thereby producing a genetically engineered cell.

[0033] In particular, provided herein are methods for producing a genetically engineered cell, the methods including: (i) providing a cell; and (ii) introducing into the cell: (a) a plurality of gRNAs configured to provide simultaneous editing events in at least four different genomic targets; and (d) a base editor that binds to the plurality of gRNAs, thereby producing the genetically engineered cell.

[0034] In particular, provided herein are methods for producing genetically engineered cells, the methods including: (i) providing a cell; and (ii) introducing into the cell: (a) one or more gRNAs targeting CD33 (Siglec-3), (b) one or more gRNAs targeting CLL1, (c) one or more gRNAs targeting CD123, (d) one or more gRNAs targeting CD312 (EMR2); and (e) a base editor that binds to the one or more gRNAs, wherein the one or more gRNAs are configured to provide simultaneous editing events in at least four different target domains, thereby producing a genetically engineered cell.

[0035] In some embodiments, multiplex modification of DNA comprises cycling or repeating steps of DNA modification on a cell to create a cell with multiple modifications of DNA within the cell, hi other embodiments, multiplex modification of DNA does not comprise cycling or repeating steps of DNA modification on a cell to create a cell with multiple modifications of DNA within the cell.

[0036] Some embodiments of the present disclosure provide, e.g., novel cells, e.g., HSCs or HPCs, having a modification (e.g., multiple modifications) in the endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes. Some embodiments of the present disclosure provide, e.g., novel cells, e.g., HSCs or HPCs, having a modification (e.g., substitution, insertion, or deletion) in the endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes. Some aspects of the present disclosure provide, e.g., novel cells, e.g., HSCs or HPCs, that have modifications (e.g., stop codons or mutant splice sites) in endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes.

[0037] Some aspects of the present disclosure provide cell populations comprising a plurality of genetically engineered hematopoietic stem or progenitor cells, for example, at least a portion of the cells comprise (i) an edited CD33 gene and an edited CLL-1 gene, (ii) an edited CD33 gene and an edited CD123 gene, or (iii) an edited CD33 gene, an edited CLL-1 gene, and an edited CD123 gene.

[0038] Some aspects of the present disclosure provide cell populations comprising a plurality of genetically engineered hematopoietic stem or progenitor cells, for example, at least a portion of the cells comprising: (i) an edited CD33 (Siglec-3) gene, (ii) an edited CLL-1 gene, (iii) an edited CD123 gene, (iv) an edited CD327 (Siglec-6) gene, (v) an edited CD312 (EMR2) gene, (vi) an edited CD33 (Siglec-3) gene and an edited CLL-1 gene, (vii) an edited CD33 (Siglec-3) gene and an edited CD123 gene, (viii) an edited CD33 (Siglec-3) gene and an edited CD327 (Siglec-6) gene, (ix) an edited CD33 (Siglec-3) gene and an edited CD312 (EMR2) gene. R2) gene, (x) an edited CD33 (Siglec-3) gene, an edited CLL-1 gene, and an edited CD123 gene, (xi) an edited CD33 (Siglec-3) gene, an edited CLL-1 gene, an edited CD123 gene, and an edited CD327 (Siglec-6) gene, (xii) an edited CD33 (Siglec-3) gene, an edited CLL-1 gene, an edited CD123 gene, an edited CD327 (Siglec-6) gene, and an edited CD312 (EMR2) gene, or (xiii) an edited CD33 (Siglec-3) gene, an edited CLL-1 gene, an edited CD123 gene, an edited CD327 (Siglec-6) gene, and / or an edited CD312 (EMR2) gene.

[0039] Such novel cells may have 0% translocations, as assessed, for example, by a translocation analysis assay such as a RhampSeq assay. In certain embodiments, novel cells (e.g., HSCs or HPCs) produced by the multiplex base editing methods provided herein may have 0% translocations, or undetectable levels of translocations, and an on-target editing efficiency of at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% or more, for modifications (e.g., multiple modifications) in endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes. In certain embodiments, on-target editing efficiency is at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% or more, e.g., for a modification (e.g., multiple modifications) of an endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) gene.

[0040] Some embodiments of the present disclosure also provide compositions that can be used to make such modifications, e.g., gene editing enzymes, gRNAs, and combinations thereof. Some embodiments of the present disclosure provide methods of using the compositions provided herein, e.g., using certain gRNAs provided to generate genetically engineered cells, e.g., cells with modifications in the endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes.

[0041] Some aspects of the present disclosure provide genetically engineered cells having modifications in endogenous cell surface antigen genes. In some embodiments, the genetically engineered cells may express, but are not limited to, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CD13, CD14, CD15, CD16a, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD40, CD50, CD60, CD70, CD80, CD90, CD100, CD111, CD112, CD113, CD114, CD15, CD16a, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD40, CD50, CD60, CD70, CD80, CD90, CD101, CD112, CD113, CD114, CD15, CD16a, CD16b, CD17, CD18, CD19, CD202, CD213, CD224, CD25, CD26, CD27, CD28, CD29, CD30, CD403, CD404, CD405, CD406, CD407, CD408, CD409, CD410, CD411, CD412, CD413, CD414, CD415, CD416, CD417, CD418, CD D31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45 RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD6 0c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65s, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85a, CD85b, CD85c, CD85d, CD85e, CD85f, CD85g, CD85h, CD85i, CD85j, CD85k, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD122, CD123,CD124、CD125、CD126、CD127、CD128a、CD128b、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CD146、CD147、CD148、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CD156c、CD157、CD158a、CD158b1、CD158b2、CD158c、CD158d、CD158e1、CD158e2、CD158f、CD158g、CD158h、CD158i、CD158j、CD158k、CD159a、CD159c、CD160、CD161、CD162、CD163、CD164、CD165、CD166、CD167a、CD167b、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CD198、CD199、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210a、CD210b、CD212、CD213a1、CD213a2、CD215、CD217、CD218a、CD218b、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD236、CD238、CD239、CD240CE、CD240D、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CD248、CD249、CD252、CD253、CD254、CD256、CD257、CD258、CD261、CD262、CD263、CD264、CD265、CD266、CD267、CD268、CD269、CD270、CD271, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD28 3, CD284, CD286, CD288, CD289, CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, C D299, CD300a, CD300c, CD300d, CD300e, CD300f, CD300g, CD301, CD302, CD303, CD304, CD305 , CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD309, CD312, CD314, CD315, CD316, CD317 , CD318, CD319, CD320, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, or any combination thereof. In some embodiments, the genetically engineered cells have reduced expression levels of one or more gene products of cell surface antigens (e.g., mRNA, protein, or a combination thereof). In some embodiments, the genetically engineered cells lack one or more gene products of cell surface antigens (e.g., mRNA, protein, or a combination thereof). In some embodiments, the detection of one or more genetic modifications and / or reduced expression levels described herein may be based on one or more measurements or assays, such as, for example, a quantitative or semi-quantitative value of the expression of a single gene, reflecting, for example, a signal obtained from a quantitative or semi-quantitative assay that detects the abundance of a gene product (e.g., a protein or nucleic acid transcript encoded by the gene). Suitable assays for detecting gene expression products are well known to those of skill in the art and include, for example, Western blots, ELISAs,These include RT-PCR (e.g., end-point RT-PCR, real-time PCR, or qPCR), protein or nucleic acid microarrays, and massively parallel sequencing assays. However, any suitable assay may be used, based on hybridization, specific binding (e.g., antibody binding), or any other technique. Certain aspects of the present disclosure provide methods of using the compositions provided herein, e.g., using certain gRNAs and / or gene editing enzymes provided to generate genetically engineered cells, e.g., cells with multiple modifications in the endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes.

[0042] Some aspects of the present disclosure provide methods of administering genetically engineered cells provided herein, e.g., cells having modifications in endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes, to a subject in need thereof. In some embodiments, the subject has or has been diagnosed with cancer or a precancerous condition. In some embodiments, the subject has a hematological malignancy. In some embodiments, the precancerous condition is myelodysplastic syndrome. In some embodiments, the cancer or precancerous condition is characterized by expression of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) on the surface of malignant cells in the subject.

[0043] Some aspects of the present disclosure provide strategies, compositions, methods, and therapeutic modalities for treating patients who have cancer and are receiving or in need of anti-CD33 (Siglec-3), anti-CLL-1, anti-CD123, anti-CD327 (Siglec-6), and / or anti-CD312 (EMR2) therapy; in some embodiments, the subject has or has been diagnosed with cancer or a precancerous condition. In some embodiments, the subject has a hematological malignancy. In some embodiments, the precancerous condition is myelodysplastic syndrome. In some embodiments, the cancer or precancerous condition is characterized by expression of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) on the surface of malignant cells in the subject.

[0044] Enumerated Embodiments 1. gRNAs containing a targeting domain that binds to a target domain in Tables 1-19. 2. A gRNA comprising a targeting domain that binds to a target domain comprising any one of the nucleic acid sequences of SEQ ID NOs: 1 to 2021. 3. gRNAs containing a targeting domain capable of directing editing of a target domain in Tables 1-19. 4. A gRNA comprising a targeting domain, wherein the targeting domain is: A gRNA comprising any one of the nucleic acid sequences of SEQ ID NOs: 1 to 2021. 5. The gRNA of any one of the preceding embodiments, which binds to a target domain in the CD33 (Siglec-3) gene. 6. The gRNA of any one of the preceding embodiments, which binds to a target domain in the CLL-1 gene. 7. The gRNA of any one of the preceding embodiments, which binds to a target domain in the CD123 gene. 8. The gRNA of any one of the preceding embodiments, which binds to a target domain in the CD327 (Siglec-6) gene. 9. The gRNA of any one of the preceding embodiments, which binds to a target domain in the CD312 (EMR2) gene. 10. The gRNA of any one of the preceding embodiments, which binds to a target domain in the CD327 (Siglec-6) gene. 11. The gRNA of any one of the preceding embodiments, wherein the targeting domain is configured to provide an editing event within the target domain under conditions suitable for the gRNA to complex with a gene-editing enzyme, thereby forming a gRNA:enzyme complex, and for the gRNA:enzyme complex to bind to the target domain in the target nucleic acid molecule. 12. The gRNA of embodiment 11, wherein the gene-editing enzyme comprises an endonuclease. 13. The gRNA of embodiment 12, wherein the endonuclease comprises a Cas endonuclease. 14. The gRNA of embodiment 12 or 13, wherein the endonuclease comprises a catalytically inactive Cas molecule. 15. A gRNA according to any one of embodiments 12 to 14, wherein the endonuclease comprises a dead Cas (dCas). 16. The gRNA of embodiment 15, wherein the endonuclease comprises dead Cas9 (dCas9). 17. The gRNA of any one of embodiments 12 to 14, wherein the endonuclease comprises nickase (nCas). 18. The gRNA of embodiment 17, wherein the endonuclease comprises nCas9. 19. A gRNA described in any one of embodiments 12 to 18, wherein the endonuclease comprises a dCas or nCas fused to one or more uracil glycosylase inhibitor (UGI) domains. 20. The gRNA of any one of embodiments 12 to 19, wherein the endonuclease comprises a dCas or nCas fused to a base editor (BE). 21. The gRNA of any one of embodiments 12 to 20, wherein the endonuclease comprises a dCas or nCas fused to an adenine base editor (ABE). 22. The gRNA of embodiment 21, wherein the ABE comprises an adenine deaminase enzyme. 23. The gRNA of any one of embodiments 12 to 20, wherein the endonuclease comprises a dCas or nCas fused to a cytosine base editor (CBE). 24. The gRNA of embodiment 23, wherein the CBE comprises a cytidine deaminase enzyme. 25. A gRNA described in any one of embodiments 11 to 24, wherein the nucleic acid molecule is contained in the genomic DNA of the cell. 26. The gRNA of embodiment 25, wherein the cell is a mammalian cell. 27. The gRNA of embodiment 25 or 26, wherein the cell is a human cell. 28. The gRNA of embodiment 25 or 26, wherein the cell is a CD34+ cell. 29. The gRNA of embodiment 25 or 26, wherein the cell is a hematopoietic cell. 30. The gRNA of embodiment 25 or 26, wherein the cell is a hematopoietic stem cell. 31. The gRNA of embodiment 25 or 26, wherein the cell is a hematopoietic progenitor cell. 32. The gRNA of embodiment 25 or 26, wherein the cell is an immune effector cell. 33. The gRNA of embodiment 25 or 26, wherein the cell is a lymphocyte. 34. The gRNA of embodiment 25 or 26, wherein the cell is a T lymphocyte. 35. The gRNA of embodiment 25 or 26, wherein the cell is a natural killer (NK) cell. 36. The gRNA of embodiment 25 or 26, wherein the cell is a stem cell. 37. The gRNA of embodiment 36, wherein the stem cell is an embryonic stem cell (ESC), an induced pluripotent stem cell (iPSC), a mesenchymal stem cell, or a tissue-specific stem cell. 38. A gRNA according to any one of embodiments 11 to 37, wherein the editing event comprises a chemical change to a nucleic acid base. 39. The gRNA of embodiment 38, wherein the editing event comprises deamination of cytosine. 40. The gRNA of embodiment 38, wherein the editing event comprises adenine deamination. 41. The gRNA of embodiment 38, wherein the editing event comprises a nucleobase transition. 42. The gRNA of embodiment 38, wherein the editing event comprises a nucleobase transversion. 43. The gRNA of embodiment 38, wherein the editing event comprises converting a cytosine-guanine (CG) base pair to a thymine-adenine (TA) base pair within the target nucleic acid molecule. 44. The gRNA of embodiment 38, wherein the editing event comprises converting a thymine-adenine (TA) base pair to a cytosine-guanine (CG) base pair within the target nucleic acid molecule. 45. The gRNA of embodiment 38, wherein the editing event comprises introducing a premature stop codon into the target nucleic acid molecule. 46. ​​The gRNA of embodiment 38, wherein the editing event comprises introducing a splice site into the target nucleic acid molecule. 47. The gRNA of embodiment 38, wherein the editing event comprises disrupting a splice site within the target nucleic acid molecule. 48. A gRNA described in any one of embodiments 38 to 47, wherein the target nucleic acid molecule comprises a chromosomal or genomic DNA molecule. 49. A gRNA described in any one of embodiments 38 to 47, wherein the target nucleic acid molecule comprises a targeting domain. 50. The gRNA of embodiment 49, wherein the targeting domain of the gRNA base-pairs (with full or partial complementarity) to a sequence of a double-stranded target nucleic acid molecule that is complementary to the sequence of the targeting domain, the strand being complementary to the strand containing the PAM sequence. 51. The gRNA of embodiment 50, wherein the targeting domain of the gRNA does not comprise a PAM sequence. 52. The gRNA of embodiment 50, wherein the PAM position can be 5' or 3' of the target domain sequence. 53. The gRNA of embodiment 51, wherein the position of the targeted nucleobase in the target domain is about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleobases from the PAM. 54. A gRNA described in any one of embodiments 11 to 53, wherein the editing event reduces the activity of CD33 (Siglec-3) in the cell. 55. A gRNA described in any one of embodiments 11 to 54, wherein the editing event reduces the expression level of a nucleic acid encoding CD33 (Siglec-3) in the cell. 56. A gRNA described in any one of embodiments 11 to 55, wherein the editing event reduces the expression level of CD33 (Siglec-3) protein in the cell. 57. A gRNA described in any one of embodiments 11 to 56, wherein the editing event reduces or abolishes the expression of full-length CD33 (Siglec-3) RNA or CD33 (Siglec-3) protein in the cell. 58. A gRNA described in any one of embodiments 11 to 57, wherein the editing event reduces the activity of CLL-1 in the cell. 59. A gRNA described in any one of embodiments 11 to 58, wherein the editing event reduces the expression level of a nucleic acid encoding CLL-1 in the cell. 60. A gRNA described in any one of embodiments 11 to 59, wherein the editing event reduces the expression level of CLL-1 protein in the cell. 61. A gRNA described in any one of embodiments 11 to 60, wherein the editing event reduces or abolishes the expression of full-length CLL-1 RNA or CLL-1 protein in the cell. 62. A gRNA described in any one of embodiments 11 to 61, wherein the editing event reduces the activity of CD123 in the cell. 63. A gRNA described in any one of embodiments 11 to 62, wherein the editing event reduces the expression level of a nucleic acid encoding CD123 in the cell. 64. A gRNA described in any one of embodiments 11 to 63, wherein the editing event reduces the expression level of CD123 protein in the cell. 65. A gRNA according to any one of embodiments 11 to 64, wherein the editing event reduces or abolishes the expression of full-length CD123 RNA or CD123 protein in the cell. 66. A gRNA described in any one of embodiments 11 to 65, wherein the editing event reduces the activity of CD327 (Siglec-6) in the cell. 67. A gRNA described in any one of embodiments 11 to 66, wherein the editing event reduces the expression level of a nucleic acid encoding CD327 (Siglec-6) in the cell. 68. A gRNA described in any one of embodiments 11 to 67, wherein the editing event reduces the expression level of CD327 (Siglec-6) protein in the cell. 69. A gRNA described in any one of embodiments 11 to 68, wherein the editing event reduces or abolishes the expression of full-length CD327 (Siglec-6) RNA or CD327 (Siglec-6) protein in the cell. 70. A gRNA described in any one of embodiments 11 to 69, wherein the editing event reduces the activity of CD312 (EMR2) in the cell. 71. A gRNA described in any one of embodiments 11 to 70, wherein the editing event reduces the expression level of a nucleic acid encoding CD312 (EMR2) in the cell. 72. A gRNA described in any one of embodiments 11 to 71, wherein the editing event reduces the expression level of CD312 (EMR2) protein in the cell. 73. A gRNA described in any one of embodiments 11 to 72, wherein the editing event reduces or abolishes the expression of full-length CD312 (EMR2) RNA or CD312 (EMR2) protein in the cell. 74. A gRNA described in any one of embodiments 25 to 73, wherein the cell expresses a truncated form of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) RNA or protein. 75. The gRNA of embodiment 74, wherein the truncated form of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) RNA or protein is expressed at a level equal to or greater than the level of full-length CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) RNA or protein in non-edited cells. 76. The gRNA of embodiment 75, wherein the function or activity of a truncated form of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) RNA or protein is impaired or abolished. 77. The gRNA of embodiment 76, wherein the function or activity comprises binding to an antibody or a chimeric antigen receptor (CAR). 78. The gRNA of any one of the preceding embodiments, wherein the targeting domain is 16 nucleotides or more in length. 79. The gRNA of any one of the preceding embodiments, wherein the targeting domain is about 16 to about 30 nucleotides in length. 80. The gRNA of any one of the preceding embodiments, wherein the targeting domain is 30 nucleotides in length. 81. The gRNA of any one of the preceding embodiments, wherein the targeting domain is 21 nucleotides in length. 82. The gRNA of any one of the preceding embodiments, wherein the targeting domain is 20 nucleotides in length. 83. The gRNA of any one of the preceding embodiments, wherein the targeting domain comprises the sequence of any one of SEQ ID NOs: 1-2021 or its reverse complement, or a sequence having at least 90% or 95% identity to SEQ ID NOs: 1-2021, or a sequence having no more than 1, 2, or 3 mutations to SEQ ID NOs: 1-2021. 84. The gRNA of any one of the preceding embodiments, wherein the targeting domain comprises at least 16 contiguous nucleotides of any one of SEQ ID NOs: 1-2021 and / or is base-paired to or complementary to at least 10 nucleotides of the targeting domain of any one of SEQ ID NOs: 1-2021. 85. The gRNA of any one of the preceding embodiments, which is a single guide RNA (sgRNA). 86. The gRNA of any one of the preceding embodiments, comprising one or more chemical modifications. 87. The gRNA of any one of the preceding embodiments, which binds to a base editor. 88. The gRNA of embodiment 87, wherein the base editor is a cytosine base editor (CBE). 89. The gRNA of embodiment 88, wherein the CBE is CBE1, CBE2, CBE3, or CBE4. 90.CBE was used to identify nCas9-2xUGI, BE4-rAPOBEC1, BE4-rAPOBEC1 K34A H122A, and BE4-PpAPOBEC1. BE4-PpAPOBEC1 R33A, BE4-PpAPOBEC1 H122A, BE4-RrA3F, BE4-AmAPOBEC1, and 90. The gRNA of embodiment 88 or 89, selected from the group consisting of BE4-SsAPOBEC3B. 91. A gRNA according to any one of embodiments 88 to 90, wherein the CBE is CBE-PpAPOBEC1 WT. 92. The gRNA of embodiment 87, wherein the base editor is an adenine base editor (ABE). 93. The gRNA of embodiment 92, wherein ABE is ABE1, ABE2, ABE3, ABE4, ABE5, ABE6, ABE7, or ABE8. 94. The gRNA of embodiment 92 or 93, wherein ABE is selected from the group consisting of ABE7.10-m, ABE7.10-d, ABE8.8-m, ABE8.8-d, ABE8.13-m, ABE8.13-d, ABE8.17-m, ABE8.17-d, ABE8.20-m, and ABE8.20-d. 95. The gRNA of any one of embodiments 92 to 94, wherein the ABE is ABE8. 96. The gRNA of embodiment 87, wherein the base editor is a wild-type base editor. 97. A ribonucleoprotein (RNP) complex comprising a gRNA according to any one of embodiments 1 to 96 and a base editor. 98. The gRNA of embodiment 97, wherein the base editor is a cytosine base editor (CBE). 99. The gRNA of embodiment 98, wherein the CBE is CBE1, CBE2, CBE3, or CBE4. 100. The gRNA of embodiment 98 or 99, wherein the CBE is selected from the group consisting of nCas9-2xUGI, BE4-rAPOBEC1, BE4-rAPOBEC1 K34A H122A, BE4-PpAPOBEC1, BE4-PpAPOBEC1 R33A, BE4-PpAPOBEC1 H122A, BE4-RrA3F, BE4-AmAPOBEC1, and BE4-SsAPOBEC3B. 101. The gRNA of embodiment 98, wherein the CBE is CBE-PpAPOBEC1 WT. 102. The gRNA of embodiment 97, wherein the base editor is an adenine base editor (ABE). 103. The gRNA of embodiment 102, wherein ABE is ABE1, ABE2, ABE3, ABE4, ABE5, ABE6, ABE7, or ABE8. 104. The gRNA of embodiment 102 or 103, wherein ABE is selected from the group consisting of ABE7.10-m, ABE7.10-d, ABE8.8-m, ABE8.8-d, ABE8.13-m, ABE8.13-d, ABE8.17-m, ABE8.17-d, ABE8.20-m, and ABE8.20-d. 105. The gRNA of embodiment 102, wherein ABE is ABE8. 106. The gRNA of embodiment 97, wherein the base editor is a wild-type base editor. 107. A composition, comprising a preformed complex comprising a base editor and a gRNA according to any one of embodiments 1 to 96. 108. A mixture comprising an mRNA encoding a base editor and a gRNA according to any one of embodiments 1 to 96. 109. A method for base editing, comprising: contacting a target domain in a double-stranded DNA molecule with a complex comprising a base editor and a guide RNA (gRNA) of any one of embodiments 1 to 96; The method, wherein the base editor is a CBE or ABE that has higher on-target editing efficiency compared to the variant base editor. 110. The method of embodiment 109, wherein the base editor is a wild-type base editor. 111. The method of embodiment 110, wherein the wild-type base editor comprises BE4-PpAPOBEC. 112. The method of embodiment 109, wherein the variant base editor comprises BE4-PpAPOBEC1 R33A. 113. The method of any one of embodiments 109-112, wherein the double-stranded DNA molecule is intracellular. 114. The method of embodiment 113, comprising contacting the cell with a gRNA and an mRNA encoding a base editor. 115. The method of embodiment 114, wherein the mRNA encoding the base editor is chemically modified to improve expression of the encoded base editor. 116. The method of 115, wherein the chemically modified mRNA comprises a 5-methoxyuridine modification. 117. The method of embodiment 115, wherein the chemically modified mRNA comprises an N1-methylpseudouridine modification. 118. The method of any one of embodiments 114 to 117, comprising contacting the cell with a ribonucleoprotein (RNP) complex comprising a gRNA and a base editor. 119. A method for multiplex base editing, comprising: (i) providing cells; (ii) to a cell, (a) one or more guide RNAs (gRNAs) targeting CD33 (Siglec-3), one or more gRNAs targeting CLL-1, one or more gRNAs targeting CD123, one or more gRNAs targeting CD327 (Siglec-6), and / or one or more gRNAs targeting CD312 (EMR2); and (b) introducing a base editor that binds to the one or more gRNAs; The method, wherein one or more gRNAs are configured to provide editing events within the same or different target domains, thereby producing a genetically engineered cell. 120. A method for producing a genetically engineered cell, comprising: (i) providing cells; (ii) to a cell, (a) one or more guide RNAs (gRNAs) targeting CD33 (Siglec-3), one or more gRNAs targeting CLL-1, one or more gRNAs targeting CD123, one or more gRNAs targeting CD327 (Siglec-6), and / or one or more gRNAs targeting CD312 (EMR2); and (b) introducing a base editor that binds to the one or more gRNAs; The method, wherein one or more gRNAs are configured to provide editing events within the same or different target domains, thereby producing a genetically engineered cell. 121. A method for multiplex base editing, comprising: (i) providing cells; (ii) to a cell, (a) one or more guide RNAs (gRNAs) targeting CD33; (b) one or more gRNAs targeting CLL-1 and / or one or more gRNAs targeting CD123, and (c) introducing a base editor that binds to the one or more gRNAs; The method, wherein one or more gRNAs are configured to provide editing events within different target domains, thereby producing a genetically engineered cell. 122. A method for producing a genetically engineered cell, comprising: (i) providing cells; (ii) to a cell, (a) one or more guide RNAs (gRNAs) targeting CD33; (b) one or more gRNAs targeting CLL-1 and / or one or more gRNAs targeting CD123, and (c) introducing a base editor that binds to the one or more gRNAs; The method, wherein one or more gRNAs are configured to provide editing events within different target domains, thereby producing a genetically engineered cell. 123. A method for multiplex base editing, comprising: (i) providing cells; (ii) to a cell, (a) one or more guide RNAs (gRNAs) targeting CD33 (Siglec-3); (b) one or more gRNAs targeting CLL-1, one or more gRNAs targeting CD123, one or more gRNAs targeting CD327 (Siglec-6), and / or one or more gRNAs targeting CD312 (EMR2); and (c) introducing a base editor that binds to the one or more gRNAs; The method, wherein one or more gRNAs are configured to provide editing events within the same or different target domains, thereby producing a genetically engineered cell. 124. A method for producing a genetically engineered cell, comprising: (i) providing cells; (ii) to a cell, (a) one or more guide RNAs (gRNAs) targeting CD33 (Siglec-3); (b) one or more gRNAs targeting CLL-1, one or more gRNAs targeting CD123, one or more gRNAs targeting CD327 (Siglec-6), and / or one or more gRNAs targeting CD312 (EMR2); and (c) introducing a base editor that binds to the one or more gRNAs; The method, wherein one or more gRNAs are configured to provide editing events within the same or different target domains, thereby producing a genetically engineered cell. 125. A method for triplex base editing, comprising: (i) providing cells; (ii) to a cell, (a) a plurality of gRNAs configured to provide simultaneous editing events in at least three different genomic targets; and (d) introducing a base editor that binds to the plurality of gRNAs, thereby producing a genetically engineered cell. 126. A method for triplex base editing, comprising: (i) providing cells; (ii) to a cell, (a) one or more gRNAs targeting CD33 (Siglec-3); (b) one or more gRNAs targeting CLL1; (c) one or more gRNAs targeting CD123, and (d) introducing a base editor that binds to the one or more gRNAs; The method, wherein the one or more gRNAs are configured to provide simultaneous editing events in at least three different target domains, thereby producing a genetically engineered cell. 127. A method for producing a genetically engineered cell, comprising: (i) providing cells; (ii) to a cell, (a) a plurality of gRNAs configured to provide simultaneous editing events in at least three different genomic targets; and (d) introducing a base editor that binds to the plurality of gRNAs, thereby producing a genetically engineered cell. 128. A method for producing a genetically engineered cell, comprising: (i) providing cells; (ii) to a cell, (a) one or more gRNAs targeting CD33 (Siglec-3); (b) one or more gRNAs targeting CLL1; (c) one or more gRNAs targeting CD123, and (d) introducing a base editor that binds to the one or more gRNAs; The method, wherein the one or more gRNAs are configured to provide simultaneous editing events in at least three different target domains, thereby producing a genetically engineered cell. 129. A method for quadroplex base editing, comprising: (i) providing cells; (ii) to a cell, (a) a plurality of gRNAs configured to provide simultaneous editing events in at least four different genomic targets; and (d) introducing a base editor that binds to the plurality of gRNAs, thereby producing a genetically engineered cell. 130. A method for quadroplex base editing, comprising: (i) providing cells; (ii) to a cell, (a) one or more gRNAs targeting CD33 (Siglec-3); (b) one or more gRNAs targeting CLL1; (c) one or more gRNAs targeting CD123; (d) one or more gRNAs targeting CD312 (EMR2); (e) introducing a base editor that binds to the one or more gRNAs; The method, wherein the one or more gRNAs are configured to provide simultaneous editing events in at least four different target domains, thereby producing a genetically engineered cell. 131. A method for producing a genetically engineered cell, comprising: (i) providing cells; (ii) to a cell, (a) a plurality of gRNAs configured to provide simultaneous editing events in at least four different genomic targets; and (d) introducing a base editor that binds to the plurality of gRNAs, thereby producing a genetically engineered cell. 132. A method for producing a genetically engineered cell, comprising: (i) providing cells; (ii) to a cell, (a) one or more gRNAs targeting CD33 (Siglec-3); (b) one or more gRNAs targeting CLL1; (c) one or more gRNAs targeting CD123; (d) one or more gRNAs targeting CD312 (EMR2); (e) introducing a base editor that binds to the one or more gRNAs; The method, wherein the one or more gRNAs are configured to provide simultaneous editing events in at least four different target domains, thereby producing a genetically engineered cell. 133. The method of any one of the preceding embodiments, wherein the one or more guide RNAs (gRNAs) comprise a gRNA described in any one of embodiments 1-96. 134. The method of any one of the preceding embodiments, resulting in simultaneous editing of one or more target domains within the same gene and / or in different genes. 135. The method of any one of the preceding embodiments, resulting in simultaneous editing of two or more target domains within the same gene and / or different genes. 136. The method of any one of the preceding embodiments, resulting in simultaneous editing of three or more target domains within the same gene and / or different genes. 137. The method of any one of the preceding embodiments, resulting in simultaneous editing of four or more target domains within the same gene and / or different genes. 138. The method of any one of the preceding embodiments, resulting in simultaneous editing of one or more target domains within the CD33 (Siglec-3) gene, the CLL-1 gene, the CD123 gene, the CD327 (Siglec-6) gene, and / or the CD312 (EMR2) gene. 139. The method of any one of the preceding embodiments, wherein one or more gRNAs targeting CD33 (Siglec-3) are designed for use with a cytosine base editor (CBE) and / or an adenine base editor (ABE). 140. The method of any one of the preceding embodiments, wherein one or more gRNAs targeting CD33 (Siglec-3) are designed for use with CBE. 141. The method of any one of the preceding embodiments, wherein one or more gRNAs targeting CD33 (Siglec-3) are designed for use with ABE. 142. The method of any one of the preceding embodiments, wherein one or more gRNAs targeting CLL1 are designed for use with a cytosine base editor (CBE) and / or an adenine base editor (ABE). 143. The method of any one of the preceding embodiments, wherein one or more gRNAs targeting CLL1 are designed for use with a CBE. 144. The method of any one of the preceding embodiments, wherein one or more gRNAs targeting CLL1 are designed for use with ABE. 145. The method of any one of the preceding embodiments, wherein one or more gRNAs targeting CD123 are designed for use with a CBE and / or an ABE. 146. The method of any one of the preceding embodiments, wherein one or more gRNAs targeting CD123 are designed for use with a CBE. 147. The method of any one of the preceding embodiments, wherein one or more gRNAs targeting CD123 are designed for use with ABE. 148. The method of any one of the preceding embodiments, wherein one or more gRNAs targeting EMR2 are designed for use with a CBE and / or an ABE. 149. The method of any one of the preceding embodiments, wherein one or more gRNAs targeting EMR2 are designed for use with CBE. 150. The method of any one of the preceding embodiments, wherein one or more gRNAs targeting EMR2 are designed for use with ABE. 151. The method of any one of the preceding embodiments, comprising contacting the cell with one or more gRNAs and an mRNA encoding a base editor. 152. The method of any one of the preceding embodiments, comprising contacting the cell with a ribonucleoprotein (RNP) complex comprising one or more gRNAs and a base editor. 153. The method of any one of the preceding embodiments, comprising contacting the cell with a gRNA and an mRNA encoding a base editor. 154. The method of any one of the preceding embodiments, wherein the mRNA encoding the base editor is chemically modified to improve expression of the encoded base editor. 155. The method of any one of the preceding embodiments, wherein the chemically modified mRNA comprises a 5-methoxyuridine modification. 156. The method of any one of the preceding embodiments, wherein the chemically modified mRNA comprises an N1-methylpseudouridine modification. 157. The method of any one of the preceding embodiments, wherein the RNP is introduced into the cell via electroporation. 158. The method of any one of the preceding embodiments, wherein the base editor is a wild-type base editor. 159. The method of any one of the preceding embodiments, wherein the base editor is a cytosine base editor (CBE) and / or an adenine base editor (ABE). 160. The method of any one of the preceding embodiments, wherein only the CBE is introduced into the cell. 161. The method of any one of the preceding embodiments, wherein only the ABE is introduced into the cell. 162. The method of any one of the preceding embodiments, wherein both the CBE and the ABE are introduced into the cell. 163. The method of any one of the preceding embodiments, wherein a wild-type base editor is introduced into the cell, and optionally, the wild-type base editor targets cytosine-guanine (CG) base pairs or thymine-adenine (TA) base pairs with greater on-target editing efficiency compared to the variant base editor. 164. The method of any one of the preceding embodiments, wherein the method results in a lower translocation risk compared to variant base editors, and optionally the method results in 0% translocations, or undetectable levels of translocations, and an on-target editing efficiency of at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99% or more for modifications in endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes. 167. The method of any one of the preceding embodiments, wherein the cells are hematopoietic stem or progenitor cells. 168. A genetically engineered hematopoietic stem or progenitor cell produced by the method of any one of the preceding embodiments. 169. A cell population comprising the genetically engineered hematopoietic stem or progenitor cells of a plurality of embodiments 168. 170. A cell population comprising a plurality of genetically engineered hematopoietic stem or progenitor cells, wherein at least a portion of the cells are: (i) the edited CD33 (Siglec-3) gene; (ii) an edited CLL-1 gene; (iii) an edited CD123 gene; (iv) edited CD327 (Siglec-6) gene; (v) edited CD312 (EMR2) gene; (vi) an edited CD33 (Siglec-3) gene and an edited CLL-1 gene; (vii) an edited CD33 (Siglec-3) gene and an edited CD123 gene; (viii) an edited CD33 (Siglec-3) gene and an edited CD327 (Siglec-6) gene; (ix) an edited CD33 (Siglec-3) gene and an edited CD312 (EMR2) gene; (x) an edited CD33 (Siglec-3) gene, an edited CLL-1 gene, and an edited CD123 gene; (xi) an edited CD33 (Siglec-3) gene, an edited CLL-1 gene, an edited CD123 gene, and an edited CD327 (Siglec-6) gene; (xii) an edited CD33 (Siglec-3) gene, an edited CLL-1 gene, an edited CD123 gene, an edited CD327 (Siglec-6) gene, and an edited CD312 (EMR2) gene; or (xiii) A cell population comprising an edited CD33 (Siglec-3) gene, an edited CLL-1 gene, an edited CD123 gene, an edited CD327 (Siglec-6) gene, and / or an edited CD312 (EMR2) gene. 180. The cell population of any one of the preceding embodiments, wherein the CD33 (Siglec-3) gene contains a stop codon or a mutant splice site, but does not contain a frameshift mutation, typically introduced by CRISPR nuclease-mediated non-homologous end joining (NHEJ). 181. The cell population of any one of the preceding embodiments, wherein the CLL-1 gene comprises a stop codon or a mutant splice site, but does not comprise a frameshift mutation, typically introduced by CRISPR nuclease-mediated non-homologous end joining (NHEJ). 182. The cell population of any one of the preceding embodiments, wherein the CD123 gene comprises a stop codon or a mutant splice site, but does not comprise a frameshift mutation, typically introduced by CRISPR nuclease-mediated non-homologous end joining (NHEJ). 183. The cell population of any one of the preceding embodiments, wherein the CD327 (Siglec-6) gene contains a stop codon or a mutant splice site, but does not contain a frameshift mutation, typically introduced by CRISPR nuclease-mediated non-homologous end joining (NHEJ). 184. The cell population of any one of the preceding embodiments, wherein the CD312 (EMR2) gene contains a stop codon or a mutant splice site, but does not contain a frameshift mutation, typically introduced by CRISPR nuclease-mediated non-homologous end joining (NHEJ). 185. The cell population of any one of the preceding embodiments, which expresses less than 30% of the CD33 (Siglec-3) expressed by a wild-type counterpart cell population. 186. The cell population of any one of the preceding embodiments, which expresses less than 30% of the CLL-1 expressed by a wild-type counterpart cell population. 187. The cell population of any one of the preceding embodiments, which expresses less than 30% of the CD123 expressed by a wild-type counterpart cell population. 188. The cell population of any one of the preceding embodiments, which expresses less than 30% of the CD327 (Siglec-6) expressed by a wild-type counterpart cell population. 189. The cell population of any one of the preceding embodiments, which expresses less than 30% of the CD312 (EMR2) expressed by a wild-type counterpart cell population. 190. The cell population of any one of the preceding embodiments, wherein at least a portion of the cells have a gene edit in a gene encoding a lineage-specific cell surface antigen other than CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), or CD312 (EMR2). 191. A gene encoding a lineage-specific cell surface antigen other than CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), or CD312 (EMR2) is 191. The cell population of embodiment 190, wherein the cells are CD19, CD30, CD5, CD6, CD7, CD34, CD38, or BCMA. 192. A method of administering to a subject in need thereof a cell population according to any one of the preceding embodiments, comprising: Optionally, the subject has a hematopoietic malignancy. 193. The method of any one of the preceding embodiments, wherein the hematopoietic malignancy is Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, or multiple myeloma. 194. The method of any one of the preceding embodiments, wherein the leukemia is acute myeloid leukemia (AML), acute lymphocytic leukemia, chronic myeloid leukemia, acute lymphoblastic leukemia or chronic lymphoblastic leukemia, and chronic lymphocytic leukemia. 195. The method of any one of the preceding embodiments, wherein the hematopoietic malignancy comprises acute myeloid leukemia (AML). 196. The method of any one of the preceding embodiments, further comprising administering to the subject an effective amount of an agent that targets CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), wherein the agent comprises an antigen-binding fragment that binds to CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2). 197. The method of any one of the preceding embodiments, wherein the agent targeting CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) is an antibody or a chimeric antigen receptor (CAR). 198. A nucleic acid encoding a gRNA according to any one of embodiments 1 to 96. 199. A kit or composition comprising: a) a gRNA according to any one of embodiments 1 to 96, or a nucleic acid encoding a gRNA; and b) a second gRNA, or a nucleic acid encoding a second gRNA.

[0045] The above summary is meant to illustrate, in a non-limiting manner, some of the embodiments, advantages, features, and uses of the technology disclosed herein. Other embodiments, advantages, features, and uses of the technology disclosed herein will be apparent from the detailed description, drawings, examples, and claims. [Brief explanation of the drawings]

[0046] [Figure 1A] FIG. 1A is a schematic diagram showing an example of precise genome editing using cytosine base editors (CBEs) and adenine base editors (ABEs). [Figure 1B] FIG. 1B is a schematic showing exemplary base editing applications for gene silencing by introduction of stop codons (top right panel), multiplex editing with reduced translocations and low off-target activity (bottom right panel), gene correction (top left panel), and epitope engineering (bottom left panel). [Figure 1C] Figure 1C shows an exemplary multiplex editing strategy for identifying and designating base editing guides for targeting therapeutic genes, including, for example, CD33 and CLL-1. [Figure 2A] FIG. 2A shows exemplary in silico base editor guide design and prioritization, which can be used to achieve gene knockout (KO) using base editors through the introduction of premature stop codons or splice site disruptions. [Figure 2B] FIG. 2B shows exemplary CD33 and CLL-1 base editor candidate guides for inducing protein knockout (KO). [Figure 2C] FIG. 2C is a schematic diagram showing exemplary base editor CD33 and CLL-1 guide binding sites. [Figure 3A] Figure 3A shows a selection of exemplary CBE and ABE constructs with high on-target activity and reduced off-target activity. Adapted from Yu et al., "Cytosine base editors with minimized unguided DNA and RNA off-target events and high on-target activity." Nature Communications, Volume 11, Article number 2052 (2020), and Gaudelli et al., "Directed evolution of adenine base editors with increased activity and therapeutic application." Nature Biotechnology, Volume 38, pages 892-900 (2020), the entire contents of each of which are incorporated herein by reference. [Figure 3B] Figure 3B shows an exemplary base editor delivery in hematopoietic stem and progenitor cells (HSPCs) using mRNA with chemical modifications. [Figure 3C] Figure 3C shows an exemplary engineering protocol for editing HSPCs using a base editor. [Figure 3D] Figure 3D shows an exemplary CD33 CBE guide screen demonstrating high on-target base editing in HSPCs using guides 7, 8, and 17. N1: N1-methylpseudouridine-modified mRNA encoding the base editor; 5-mO: 5-methoxyuridine-modified mRNA encoding the base editor. [Figure 3E]Figure 3E shows an exemplary CLL-1 CBE guide screen demonstrating high on-target base editing in HSPCs using guides 3 and 4. [Figure 4A] FIG. 4A shows an exemplary CD33 / CLL-1 protein knockout (KO) experimental design. [Figure 4B] Figure 4B shows a study outline for evaluating CD33 / CLL-1 protein KO using an exemplary base editor guide and comparing CBE WT and R33A variants. [Figure 4C] Figure 4C shows that cytosine / adenine base editing of the CD33E1 splice site using different guide RNAs efficiently disrupts CD33 expression. Insertion shows the results for guide RNA 17. EP: electroporation. [Figure 4D] FIG. 4D shows an exemplary nonsense-mediated decay mechanism postulated to mediate protein KO when the CD33E1 splice donor site is disrupted by CBE / ABE-CD33sg17. [Figure 4E] Figure 4E shows that cytosine base editing of CLL-1 using various guide RNAs efficiently disrupts protein expression. Insertion shows the results for guide RNA3. [Figure 4F] Figure 4F shows the improvement of base editor technology in terms of gene KO potential. These data show that the exemplary CD33 BE and guide combination can achieve CD33 protein loss in HSPCs or greater than 60% CD33, and the exemplary CLL-1 BE and guide combination can achieve greater than 60% CLL-1 protein loss in HPSCs. [Figure 5A] FIG. 5A is a schematic diagram showing exemplary criteria for selecting and prioritizing base editor guides. [Figure 5B] Figure 5B shows that base editing enables efficient CD33 protein KO using targeted iSTOP guides 7 and 8 and SpliceR guide 17-guide screening 2 CBE_R33A N1mod. [Figure 5C] Figure 5C shows CD33 CBE (and ABEsg17) guide screening demonstrating high on-target base editing in HSPCs using guides 7, 8, and 17. [Figure 5D] Figure 5D shows the results of cell viability and cell proliferation CD33-guided screen 2. [Figure 5E] FIG. 5E shows the results of cell viability and cell proliferation CLL-1 guided screen 2. [Figure 5F] FIG. 5F shows the results of cell viability CD33 / CLL-1 construct comparison. [Figure 5G] FIG. 5G shows the results of cell proliferation CD33 / CLL-1 construct comparison. [Figure 5H] FIG. 5H shows the time course of CLL-1 protein KO. [Figure 6A] Figure 6A shows an exemplary CBE CD33+CLL-1 multiplex base editing and experimental design. [Figure 6B] Figure 6B shows an overview of study groups for dose-titrated multiplex base-edited top CBE CD33 guide with top CLL-1 guide. [Figure 6C] Figure 6C shows that multiplexed base-edited CD34+ cells exhibit efficient cell surface CD33 and CLL-1 protein KO in hPSCs. [Figure 6D] Figure 6D shows that multiplexed base-edited CD34+ cells exhibit efficient CD33 and CLL-1 protein KO in hPSCs. [Figure 6E] Figure 6E shows CD33 protein KO data in hPSCs. Data for CD33g8 and CLL-1g3 are highlighted. An exemplary off-target analysis of CD33sg8 is shown at the bottom left, demonstrating a favorable off-target profile. [Figure 6F] Figure 6F shows flow data demonstrating that approximately 80% of multiplex-edited cells lack CD33 and CLL-1 surface protein expression. [Figure 7]Figure 7 shows data demonstrating that base editing does not affect hPSC cell differentiation. [Figure 8A] Figure 8A shows that combined co-delivery of a cytosine base editor and Cpf1 nuclease allows for single delivery and no risk of translocation because the BE does not make double-strand breaks. [Figure 8B] FIG. 8B shows that viability and cell proliferation are not affected when CBE and AsCpf1 are delivered simultaneously in CD34 cells. [Figure 9A] FIG. 9A is a schematic diagram showing the experimental design for multiplex editing of CD33 and CLL-1 performed using different CD33 guide RNAs (sg7, sg8, or sg17) in combination with CLL-1 guide RNA sg3. [Figure 9B] Figure 9B shows bone marrow in vitro differentiation data assessed by flow cytometry for protein knockout (KO) readout. Combination of base editors (BE) with CD33g8 and CLL-1g3 showed 80% dual surface protein KO. [Figure 9C] Figure 9C shows that no balanced translocations were detected in the multiplexed base-edited samples, as determined by the RhampSeq assay. [Figure 10] FIG. 10 is a schematic showing multiplex base editing using cytosine base editors (CBEs) in CD34+ hematopoietic stem and progenitor cells (HSPCs). [Figure 11]Figure 11A shows a mapping of on-target base editing efficiencies of the top three CBE gene KOs inducing single guides (sg7, sg8, or sg17, tagged g7, g8, or g17) using stop codon and splice disruptor base editing guides on the CD33 locus and Cas9 control binding site (left panel), and three different CBE4 mRNA encoding constructs (right panel). The gray bars represent the Cas9-induced indel frequency on the CD33 locus. Figure 11B shows a mapping of on-target base editing efficiencies of the top two CBE gene KOs inducing single guides (sg3 or sg4, tagged g3 or g4) using stop codon and splice disruptor base editing guides on the CLL-1 locus and Cas9 control binding site (left panel), and three different CBE4 mRNA encoding constructs (right panel). The gray bars represent the Cas9-induced indel frequency on the CLL-1 locus. [Figure 12] Figure 12 shows CD33 (left panel) and CLL-1 (right panel) surface protein expression quantified in edited CD34+ HSPCs by flow cytometry 9 days after electroporation (EP) for three different CD33 gRNAs (sg7, sg8, or sg17, labeled g7, g8, or g17; left panel) or two different CLL-1 gRNAs (sg3 or sg4, labeled g3 or g4). Histograms are color-coded by guide to show the percentage of the positive population for CD33 or CLL-1 edited samples compared to control untreated cells. [Figure 13A] Figure 13A shows the on-target editing efficiency of single and multiplex base-edited cells against CD33 and CLL-1 compared to multiplex Cas9 control-edited cells. [Figure 13B] FIG. 13B shows normalized CD33 and CLL-1 surface protein expression in edited and unedited CD34+ HSPCs using flow cytometry 9 days after EP in myeloid differentiation culture conditions. [Figure 14]Figure 14 shows edited and untreated control HSPCs cultured in differentiation culture conditions to support the growth of multiple progenitor cell lineages. Colony-forming units (CFUs) were measured 14 days after seeding, quantifying erythroid (BFU-E), myeloid (CFU-G / M / GM), and mixed (CFU-GEMM) lineages. [Figure 15A] Figure 15A shows CD34+ HSPCs electroporated with CBE4 encoding mRNA or Cas9 ribonucleoprotein (RNP) complexes using CD33 and CLL-1 synthetic guides and differentiated in vitro to the monocyte lineage. [Figure 15B] Figure 15B shows the on-target editing efficiency of CD33 and CLL-1 in base-edited and Cas9-edited samples collected at different time points after EP (day 2) and throughout monocytic differentiation. Editing efficiency was calculated and annotated using CRISPResso v2.0.30 and the Variant Effector Predictor (VEP). CD33 (upper panel) and CLL-1 (lower panel) protein expression in edited and unedited samples was measured throughout monocytic differentiation using flow cytometry. Bulk populations of the entire multiplexed base-edited cells showed decreased CD33 and CLL-1 expression in monocytic-differentiated CD34+ HSPCs. [Figure 16A] Figure 16A shows the frequency of on-on (CD33-CLL-1 cleavage site) translocation events using a multiplexed rhAmpSeq approach with coverage of 217442, where reads were folded and aligned to the 223 bp junction of the predicted translocation between two different loci. [Figure 16B] Figure 16B shows representative metaphase spreads using directed genomic hybridization (dGH) assays in edited and unedited samples, showing pink (chromosomes 1, 2, 3), yellow (chromosome 12, CLL-1 locus), and green (chromosome 19, CD33 locus) chromosome paints used as normalizers to account for donor variability (dosimetry). [Figure 17A]Figure 17A shows that complexed co-delivery of an adenine base editor (ABE) with gRNAs targeting CD33 and CD123 (e.g., CD33g17 and CD123g18, respectively) enables approximately 90% on-target editing efficiency in CD123. [Figure 17B] Figure 17B shows the off-target profile of adenine base editing with CD123g18. [Figure 17C] Figure 17C is a schematic diagram showing the experimental design for multiplex editing of CD33 and CD123 performed using different CD33 and CD123 guide RNAs in combination with adenine base editors (ABEs). [Figure 18A] Figure 18A shows the on-target editing efficiency of single base-edited cells for CD33 compared to Cas9 control-edited cells. Base editing was performed using different CD33 guide RNAs (e.g., CD33g7, CD33g8, and CD33g17) in combination with an adenine base editor (ABE) or a cytosine base editor (CBE). N1: N1-methylpseudouridine-modified mRNA encoding the base editor; 5-mO: 5-methoxyuridine-modified mRNA encoding the base editor. The combination of an adenine base editor (ABE) and CD33g17 resulted in an on-target editing efficiency of approximately 95% 120 hours after electroporation (EP) of CD34+ HSPCs with 9 μg of 5-methoxyuridine-modified mRNA encoding the adenine base editor (ABE). [Figure 18B]Figure 18B shows the on-target editing efficiency of single base-edited cells against CD33 using different CD33 guide RNAs (e.g., CD33g7, CD33g8, and CD33g17) in combination with an adenine base editor (ABE) or a cytosine base editor (CBE). N1: N1-methylpseudouridine-modified mRNA encoding the base editor; 5-mO: 5-methoxyuridine-modified mRNA encoding the base editor. 120 hours after electroporation (EP) of CD34+ HSPCs with 9 μg of 5-methoxyuridine-modified mRNA encoding the adenine base editor (ABE), the combination of adenine base editor (ABE) and CD33g17 resulted in virtually all edits creating splicing-disrupting substitutions. [Figure 18C] Figure 18C shows CD33 surface protein expression in edited and unedited CD34+ HSPCs 120 hours after electroporation (EP). The combination of adenine base editor (ABE) and CD33g17 resulted in a strong loss of CD33 surface protein expression 120 hours after electroporation (EP) of CD34+ HSPCs with 9 μg of 5-methoxyuridine-modified mRNA encoding adenine base editor (ABE) compared to unedited (MockEP). [Figure 19]Figure 19 is a schematic diagram showing the experimental design for adenine base editing (ABE) multiplex editing. CD34+ cells were thawed and rested in culture for 48 hours. Next, for the ABE portion (boxed), ABE CD33g17 was paired with each of the following guide RNAs for multiplex editing: ABE CD123g17, ABE CD123g18, and ABE CD123g21. Two guide RNAs and mRNA encoding adenine base editors (ABEs) were electroporated into the cells. The cells were then cultured in myeloid in vitro differentiation medium. Flow cytometry was performed on days 2 and 9 after electroporation to measure surface protein expression of CD33 and CD123 in the guide-edited cell samples using a cytometer. Cells for gDNA molecular analysis were collected on days 6 and 9 after electroporation. [Figure 20A] Figure 20A shows that complexed co-delivery of an adenine base editor (ABE) with gRNAs targeting CD33 and CD123 (e.g., CD33g17 and CD123g18, respectively) enables approximately 90% on-target editing efficiency in CD123. [Figure 20B] Figure 20B shows the percent chimerism (right panel) and on-target editing input (right panel) after 16 weeks of engraftment in bone marrow (BM) for a no-electroporation control (No EP), a mock-electroporation control (Mock EP Ctl), single ABE gRNA targeting of CD33 or CD123 (e.g., using CD33g17 or CD123g18, respectively), and combined simultaneous ABE targeting of both CD33 and CD123 (e.g., using both CD33g17 and CD123g18). These data demonstrate that multiplex deletion of myeloid antigens by base editing in human hematopoietic stem and progenitor cells (HSPCs) enables the potential for next-generation transplantation for the treatment of acute myeloid leukemia (AML). [Figure 20C] FIG. 20C shows that the frequency of splice site disruption induced by ABE was consistently increased across different groups of the study. [Figure 21A] Figure 21A is a schematic diagram showing the experimental design of in vivo study Viivs 042 to assess the persistence of editing and long-term reconstitution of simultaneously CBE CD33+CLL1 and ABE CD33+CD123 multiplex-edited CD34+ HSPCs in NSG mice. [Figure 21B] FIG. 21B shows the group of Viivs 042 studies and materials production. [Figure 22A] FIG. 22A is a schematic showing the experimental design of the Viivs 042 input material production workflow. [Figure 22B] FIG. 22B is a table detailing exemplary editing conditions for the Viivs 042 study. [Figure 23A] Figure 23A shows cell viability for the Viivs 042 study. These data demonstrate high viability (approximately 90%) between BE single and multiplex conditions. [Figure 23B] Figure 23B shows cell proliferation for the Viivs 042 study. These data show similar cell counts between the BE single and multiplex conditions. [Figure 24A] Figure 24A shows the total editing efficiency of the Viivs 042 study conditions. [Figure 24B] Figure 24B shows the base editing efficiency of the Viivs 042 study condition. These data show that the base editing efficiency in samples taken 48 hours after electroporation (EP) for administration showed the expected alleles, including alleles with stop codon gains and disrupted splice sites. [Figure 25] Figure 25 shows the total editing efficiency of the Viivs 042 study conditions. High total editing was observed in all samples at 48 hours post-EP (treated cells), with a slight increase at 144 hours post-EP. [Figure 26A] FIG. 26A shows the results for colony forming units (CFU) at 200 dilutions. [Figure 26B] FIG. 26B shows the colony forming unit (CFU) results at 400 dilutions. [Figure 27]Figure 27 shows the rate of chimerism after 16 weeks of engraftment in the bone marrow (BM). These data show no impact on chimerism in the edited groups. [Figure 28A] Figures 28A-28C show highly efficient knockout of CD33 (Figure 28A), CLL-1 (Figure 28B), and CD123 (Figure 28C) in the ABE-edited group. [Figure 28B] Figures 28A-28C show highly efficient knockout of CD33 (Figure 28A), CLL-1 (Figure 28B), and CD123 (Figure 28C) in the ABE-edited group. [Figure 28C] Figures 28A-28C show highly efficient knockout of CD33 (Figure 28A), CLL-1 (Figure 28B), and CD123 (Figure 28C) in the ABE-edited group. [Figure 29A] Figures 29A-29H show no effect on lineage reconstitution in the edited population. Figure 29A shows total lineage reconstitution in the edited population. Figures 29B-29H show lineage reconstitution in the edited population across different cell types, including: B lymphocytes (Figure 29B), T lymphocytes (Figure 29C), monocytes (Figure 29D), HSPCs (Figure 29E), granulocytes (Figure 29F), cDCs (Figure 29G), and pDCs (Figure 29H). [Figure 29B] Figures 29A-29H show no effect on lineage reconstitution in the edited population. Figure 29A shows total lineage reconstitution in the edited population. Figures 29B-29H show lineage reconstitution in the edited population across different cell types, including: B lymphocytes (Figure 29B), T lymphocytes (Figure 29C), monocytes (Figure 29D), HSPCs (Figure 29E), granulocytes (Figure 29F), cDCs (Figure 29G), and pDCs (Figure 29H). [Figure 29C]Figures 29A-29H show no effect on lineage reconstitution in the edited population. Figure 29A shows total lineage reconstitution in the edited population. Figures 29B-29H show lineage reconstitution in the edited population across different cell types, including: B lymphocytes (Figure 29B), T lymphocytes (Figure 29C), monocytes (Figure 29D), HSPCs (Figure 29E), granulocytes (Figure 29F), cDCs (Figure 29G), and pDCs (Figure 29H). [Figure 29D] Figures 29A-29H show no effect on lineage reconstitution in the edited population. Figure 29A shows total lineage reconstitution in the edited population. Figures 29B-29H show lineage reconstitution in the edited population across different cell types, including: B lymphocytes (Figure 29B), T lymphocytes (Figure 29C), monocytes (Figure 29D), HSPCs (Figure 29E), granulocytes (Figure 29F), cDCs (Figure 29G), and pDCs (Figure 29H). [Figure 29E] Figures 29A-29H show no effect on lineage reconstitution in the edited population. Figure 29A shows total lineage reconstitution in the edited population. Figures 29B-29H show lineage reconstitution in the edited population across different cell types, including: B lymphocytes (Figure 29B), T lymphocytes (Figure 29C), monocytes (Figure 29D), HSPCs (Figure 29E), granulocytes (Figure 29F), cDCs (Figure 29G), and pDCs (Figure 29H). [Figure 29F] Figures 29A-29H show no effect on lineage reconstitution in the edited population. Figure 29A shows total lineage reconstitution in the edited population. Figures 29B-29H show lineage reconstitution in the edited population across different cell types, including: B lymphocytes (Figure 29B), T lymphocytes (Figure 29C), monocytes (Figure 29D), HSPCs (Figure 29E), granulocytes (Figure 29F), cDCs (Figure 29G), and pDCs (Figure 29H). [Figure 29G]Figures 29A-29H show no effect on lineage reconstitution in the edited population. Figure 29A shows total lineage reconstitution in the edited population. Figures 29B-29H show lineage reconstitution in the edited population across different cell types, including: B lymphocytes (Figure 29B), T lymphocytes (Figure 29C), monocytes (Figure 29D), HSPCs (Figure 29E), granulocytes (Figure 29F), cDCs (Figure 29G), and pDCs (Figure 29H). [Figure 29H] Figures 29A-29H show no effect on lineage reconstitution in the edited population. Figure 29A shows total lineage reconstitution in the edited population. Figures 29B-29H show lineage reconstitution in the edited population across different cell types, including: B lymphocytes (Figure 29B), T lymphocytes (Figure 29C), monocytes (Figure 29D), HSPCs (Figure 29E), granulocytes (Figure 29F), cDCs (Figure 29G), and pDCs (Figure 29H). [Figure 30A] Figures 30A-30E show high levels of CD123 KO in myeloid subpopulations across different cell types, including: monocytes (Figure 30A), granulocytes (Figure 30B), obese / basophils (Figure 30C), cDCs (Figure 30D), and pDCs (Figure 30E). [Figure 30B] Figures 30A-30E show high levels of CD123 KO in myeloid subpopulations across different cell types, including: monocytes (Figure 30A), granulocytes (Figure 30B), obese / basophils (Figure 30C), cDCs (Figure 30D), and pDCs (Figure 30E). [Figure 30C] Figures 30A-30E show high levels of CD123 KO in myeloid subpopulations across different cell types, including: monocytes (Figure 30A), granulocytes (Figure 30B), obese / basophils (Figure 30C), cDCs (Figure 30D), and pDCs (Figure 30E). [Figure 30D] Figures 30A-30E show high levels of CD123 KO in myeloid subpopulations across different cell types, including: monocytes (Figure 30A), granulocytes (Figure 30B), obese / basophils (Figure 30C), cDCs (Figure 30D), and pDCs (Figure 30E). [Figure 30E]Figures 30A-30E show high levels of CD123 KO in myeloid subpopulations across different cell types, including: monocytes (Figure 30A), granulocytes (Figure 30B), obese / basophils (Figure 30C), cDCs (Figure 30D), and pDCs (Figure 30E). [Figure 31A] Figures 31A-31E show low levels of double KO in myeloid subpopulations due to low levels of CLL1 KO across different cell types, including: monocytes (Figure 31A), granulocytes (Figure 31B), obese / basophils (Figure 31C), cDCs (Figure 31D), and pDCs (Figure 31E). [Figure 31B] Figures 31A-31E show low levels of double KO in myeloid subpopulations due to low levels of CLL1 KO across different cell types, including: monocytes (Figure 31A), granulocytes (Figure 31B), obese / basophils (Figure 31C), cDCs (Figure 31D), and pDCs (Figure 31E). [Figure 31C] Figures 31A-31E show low levels of double KO in myeloid subpopulations due to low levels of CLL1 KO across different cell types, including: monocytes (Figure 31A), granulocytes (Figure 31B), obese / basophils (Figure 31C), cDCs (Figure 31D), and pDCs (Figure 31E). [Figure 31D] Figures 31A-31E show low levels of double KO in myeloid subpopulations due to low levels of CLL1 KO across different cell types, including: monocytes (Figure 31A), granulocytes (Figure 31B), obese / basophils (Figure 31C), cDCs (Figure 31D), and pDCs (Figure 31E). [Figure 31E] Figures 31A-31E show low levels of double KO in myeloid subpopulations due to low levels of CLL1 KO across different cell types, including: monocytes (Figure 31A), granulocytes (Figure 31B), obese / basophils (Figure 31C), cDCs (Figure 31D), and pDCs (Figure 31E). [Figure 32] Figure 32 shows on-target editing analysis in bone marrow material across different arms of the study. These data confirm the persistence of editing. [Figure 33]FIG. 33 shows that the stop codon frequency induced by CBE was consistently slightly reduced across the different groups of the study. [Figure 34] FIG. 34 shows that the frequency of splice site disruption induced by ABE was consistently increased across the different groups of the study. [Figure 35] FIG. 35 is a schematic showing in vivo BE multiplex scale-up characterization. [Figure 36] FIG. 36 is a schematic diagram showing the various conditions evaluated for in vivo BE multiplex scale-up. [Figure 37] Figure 37 shows the experimental conditions for in vivo BE multiplex scale-up. [Figure 38A] Figures 38A and 38B show cell counts and viability, respectively, for in vivo BE multiplex scale-up. Cell proliferation was slightly reduced in the 6M cell, 2x dose condition. [Figure 38B] Figures 38A and 38B show cell counts and viability, respectively, for in vivo BE multiplex scale-up. Cell proliferation was slightly reduced in the 6M cell, 2x dose condition. [Figure 39] Figure 39 shows the flow gating strategy for in vivo BE multiplex scale-up. [Figure 40A] Figures 40A and 40B show flow cytometry data for CD33 and CLL-1, respectively. [Figure 40B] Figures 40A and 40B show flow cytometry data for CD33 and CLL-1, respectively. [Figure 41] Figure 41 shows double knockout of CD33 and CLL-1. [Figure 42] Figure 42 shows base editing efficiency with CBE CD33sg8. These data indicate that a 2x dose results in a higher frequency of alleles that lead to premature stop codon formation in CBE CD33g8. [Figure 43]Figure 43 shows base editing efficiency with CBE CLL1g3. These data show that a 2x dose results in a higher frequency of alleles that lead to premature stop codon formation in CBE CLL1g3. [Figure 44] FIG. 44 is a schematic diagram showing the experimental conditions for CBE and ABE editing of EMR2 and CD33. [Figure 45] Figure 45 is a schematic showing the CD33 and EMR2 guide screening landscape. [Figure 46] FIG. 46 is a schematic diagram showing the experimental design of EMR2-guided screening and protein KO evaluation. [Figure 47A] Figures 47A and 47B show cell viability and cell counts for CBE and ABE editing of EMR2 and CD33, respectively. [Figure 47B] Figures 47A and 47B show cell viability and cell counts for CBE and ABE editing of EMR2 and CD33, respectively. [Figure 48A] Figures 48A and 48B show reduced surface expression of EMR2, respectively. These data demonstrate that ABE EMR2 guides exhibit potent protein KO 6 days after EP. [Figure 48B] Figures 48A and 48B show reduced surface expression of EMR2, respectively. These data demonstrate that ABE EMR2 guides exhibit potent protein KO 6 days after EP. [Figure 49A] Figures 49A and 49B show that the EMR2 experimental conditions resulted in different levels of protein KO 6 days after EP, respectively. [Figure 49B] Figures 49A and 49B show that the EMR2 experimental conditions resulted in different levels of protein KO 6 days after EP, respectively. [Figure 50A] Figures 50A and 50B show reduced surface expression of CD33, respectively. These data demonstrate that ABE CD33 guides exhibit potent protein KO 6 days after EP. [Figure 50B]Figures 50A and 50B show reduced surface expression of CD33, respectively. These data demonstrate that ABE CD33 guides exhibit potent protein KO 6 days after EP. [Figure 51A] Figures 51A and 51B show that the CD33 experimental conditions resulted in different levels of protein KO 6 days after EP, respectively. [Figure 51B] Figures 51A and 51B show that the CD33 experimental conditions resulted in different levels of protein KO 6 days after EP, respectively. [Figure 52A] Figures 52A and 52B show the total editing efficiency and base editing efficiency by ABE, respectively. ABE-guided screening in HSPCs showed high editing at various sites in the CD33 and EMR2 loci and low frequencies of bystander editing. All experimental conditions showed good viability (90%) and cell proliferation compared to the MockEP control. [Figure 52B] Figures 52A and 52B show the total editing efficiency and base editing efficiency by ABE, respectively. ABE-guided screening in HSPCs showed high editing at various sites in the CD33 and EMR2 loci and low frequencies of bystander editing. All experimental conditions showed good viability (90%) and cell proliferation compared to the MockEP control. [Figure 53] Figure 53 shows the editing efficiency of ABE CD33 gRNA. [Figure 54] Figure 54 shows the editing efficiency of ABE and CBE EMR2 gRNAs. [Figure 55] Figure 55 is a schematic diagram showing EMR2 / CD33 multiplex ABE base editing. [Figure 56] Figure 56 shows exemplary EMR2 / CD33 multiplex ABE base editing conditions. [Figure 57] Figure 57 shows potential ABE guides at in silico off-target sites. [Figure 58A] Figures 58A and 58B show cell counts and cell viability for ABE editing of EMR2 and CD33, respectively. [Figure 58B] Figures 58A and 58B show cell counts and cell viability for ABE editing of EMR2 and CD33, respectively. [Figure 59A] Figures 59A and 59B show ABE EMR2 and CD33 DNA editing frequencies, respectively. [Figure 59B] Figures 59A and 59B show ABE EMR2 and CD33 DNA editing frequencies, respectively. [Figure 60A] 60A-60C show summaries of ABE EMR2 edit frequencies, edit results, and base edits, respectively. [Figure 60B] 60A-60C show summaries of ABE EMR2 edit frequencies, edit results, and base edits, respectively. [Figure 60C] 60A-60C show summaries of ABE EMR2 edit frequencies, edit results, and base edits, respectively. [Figure 61A] Figures 61A and 61B show the frequency and consequences of EMR2 off-target editing in CD97, respectively. [Figure 61B] Figures 61A and 61B show the frequency and consequences of EMR2 off-target editing in CD97, respectively. [Figure 62A] Figures 62A-62C show a summary of ABE CD33 editing frequency, editing results, and base editing, respectively. [Figure 62B] Figures 62A-62C show a summary of ABE CD33 editing frequency, editing results, and base editing, respectively. [Figure 62C] Figures 62A-62C show a summary of ABE CD33 editing frequency, editing results, and base editing, respectively. [Figure 63] Figure 63 shows EMR2 surface protein expression. [Figure 64] Figure 64 shows EMR2 surface protein expression. [Figure 65] Figure 65 shows CD33 surface protein expression. [Figure 66] Figure 66 shows CD33 surface protein expression. [Figure 67] Figure 67 is a schematic diagram showing CBE single and quadroplex editing of CD33, CLL1, CD123, and EMR2. [Figure 68] Figure 68 is a schematic diagram showing the experimental design for delivering CBE to CD34+ cells to simultaneously target four loci. [Figure 69A] Figures 69A and 69B show cell viability and cell counts for CBE single and quadroplex editing for CD33, CLL1, CD123, and EMR2, respectively. These data demonstrate that quadroplex editing does not affect cell health. [Figure 69B] Figures 69A and 69B show cell viability and cell counts for CBE single and quadroplex editing for CD33, CLL1, CD123, and EMR2, respectively. These data demonstrate that quadroplex editing does not affect cell health. [Figure 70] Figure 70 shows the total editing efficiency of CBE single and quadroplex editing of CD33, CLL1, CD123, and EMR2. These data demonstrate that multiplex allelic deletion of myeloid antigens by base editing in human hematopoietic stem and progenitor cells (HSPCs) enables the potential for next-generation transplantation for the treatment of acute myeloid leukemia (AML). [Figure 71] Figure 71 shows the editing efficiencies of CBE single and quadroplex editing of CD33, CLL1, CD123, and EMR2. These data demonstrate that multiplex allelic deletion of myeloid antigens by base editing in human hematopoietic stem and progenitor cells (HSPCs) enables the potential for next-generation transplantation for the treatment of acute myeloid leukemia (AML). [Figure 72] Figure 72 is a schematic diagram showing ABE CD33 / CD123 / EMR2 triple KO. [Figure 73] Figure 73 shows exemplary electroporation conditions for ABE CD33 / CD123 / EMR2 triple KO. [Figure 74A]Figures 74A and 74B show DNA editing frequencies at days 2 and 5 after EP, respectively. These data show >80% editing for CD33 g16 and >90% editing for CD123 g18, EMR2 sDex13, and EMR2 sDex19 at day 5 after EP. Similar editing was observed for CD123 g18, EMR2 sDex13, and EMR2 sDex19 in the single and triplex EP conditions. A slight decrease (approximately 5% decrease) in editing of CD33 g16 was also observed in triplex compared to single EP. Across all guides and conditions, higher editing was observed at day 5 compared to day 2. No off-target editing was observed in CD97 for EMR2 sDex13 and EMR2 sDex19. [Figure 74B] Figures 74A and 74B show DNA editing frequencies at days 2 and 5 after EP, respectively. These data show >80% editing for CD33 g16 and >90% editing for CD123 g18, EMR2 sDex13, and EMR2 sDex19 at day 5 after EP. Similar editing was observed for CD123 g18, EMR2 sDex13, and EMR2 sDex19 in the single and triplex EP conditions. A slight decrease (approximately 5% decrease) in editing of CD33 g16 was also observed in triplex compared to single EP. Across all guides and conditions, higher editing was observed at day 5 compared to day 2. No off-target editing was observed in CD97 for EMR2 sDex13 and EMR2 sDex19. [Figure 75A] Figures 75A and 75B show DNA editing frequencies at 2 and 5 days after EP, respectively. These data indicate that the majority of edits for all guides in single and triplex conditions result in splice site disruption. EMR2 sDex13 shows approximately 4% INDEL formation at 2 and 5 days after EP. [Figure 75B]Figures 75A and 75B show DNA editing frequencies at 2 and 5 days after EP, respectively. These data indicate that the majority of edits for all guides in single and triplex conditions result in splice site disruption. EMR2 sDex13 shows approximately 4% INDEL formation at 2 and 5 days after EP. [Figure 76A] Figures 76A and 76B are schematic diagrams outlining the detailed substitution rates in the CD33sg16 and CD123sg18 groups, respectively. [Figure 76B] Figures 76A and 76B are schematic diagrams outlining the detailed substitution rates in the CD33sg16 and CD123sg18 groups, respectively. [Figure 77A] Figures 77A and 77B are schematic diagrams outlining the detailed substitution rates in the EMR2sg13 and EMR2sg19 groups, respectively. [Figure 77B] Figures 77A and 77B are schematic diagrams outlining the detailed substitution rates in the EMR2sg13 and EMR2sg19 groups, respectively. [Figure 78A-1] 78A-78C are schematic diagrams showing flow cytometry gating strategies. [Figure 78A-2] 78A-78C are schematic diagrams showing flow cytometry gating strategies. [Figure 78B-1] 78A-78C are schematic diagrams showing flow cytometry gating strategies. [Figure 78B-2] 78A-78C are schematic diagrams showing flow cytometry gating strategies. [Figure 78C] 78A-78C are schematic diagrams showing flow cytometry gating strategies. [Figure 79A] Figures 79A and 79B show EMR2 surface protein expression and total gMFI, respectively. [Figure 79B] Figures 79A and 79B show EMR2 surface protein expression and total gMFI, respectively. [Figure 80]Figures 80 and 81 show CD33 surface protein expression and total gMFI, respectively. [Figure 81] Figures 80 and 81 show CD33 surface protein expression and total gMFI, respectively. [Figure 82A] Figures 82A and 82B show CD123 surface protein expression and total gMFI, respectively. [Figure 82B] Figures 82A and 82B show CD123 surface protein expression and total gMFI, respectively. [Figure 83A] Figures 83A and 83B show CD33, CD123, and EMR2 surface protein expression, and triple KO surface expression, respectively. [Figure 83B] Figures 83A and 83B show CD33, CD123, and EMR2 surface protein expression, and triple KO surface expression, respectively. [Figure 84A] Figures 84A and 84B show CD33, CD123, and EMR2 DNA editing and triple KO surface protein analysis, respectively. [Figure 84B] Figures 84A and 84B show CD33, CD123, and EMR2 DNA editing and triple KO surface protein analysis, respectively. [Figure 85A] Figure 85A shows a CLL-1 ABE guide with SpCas9 NGG PAM. [Figure 85B] Figure 85B shows a CLL-1 ABE guide with an incomplete PAM (NG). [Figure 85C] Figure 85C shows a CLL-1 ABE guide with an incomplete PAM (NRG). [Figure 85D] Figure 85D shows a CLL-1 ABE guide with a Cpf1 TTTN PAM. [Figure 85E] Figure 85E is a schematic diagram outlining the CLL-1 gene. [Figure 86] Figure 86 shows the ABE / CBE g17 alignment to Siglec-6, predicting that g17 likely targets and disrupts both Siglec-3 and Siglec-6. [Figure 87] Figure 87 shows that Siglec-6 surface expression is reduced after editing with CD33g17. DETAILED DESCRIPTION OF THE INVENTION

[0047] definition As used herein with respect to gRNA interactions with a target domain, the term "binding" refers to a gRNA molecule and a target domain forming a complex. The complex may include two strands forming a double-stranded structure or three or more strands forming a multi-stranded complex. Binding may constitute a step in a more extensive process, such as cleavage of the target domain by a Cas endonuclease. In some embodiments, the gRNA binds to the target domain with perfect complementarity; in other embodiments, the gRNA binds to the target domain with partial complementarity, e.g., with one or more mismatches. In some embodiments, when the gRNA binds to the target domain, the entire targeting domain of the gRNA base pairs with the targeting domain. In other embodiments, only a portion of the targeting domain and / or only a portion of the targeting domain base pairs with the other. In one embodiment, the interaction is sufficient to mediate a targeting domain-mediated cleavage event.

[0048] As used herein, the term "Cas9 molecule" refers to a molecule or polypeptide that can interact with a gRNA and, in coordination with the gRNA, target or localize to a site containing a target domain. Cas9 molecules include naturally occurring Cas9 molecules and engineered, altered, or modified Cas9 molecules that differ, for example, by at least one amino acid residue from a naturally occurring Cas9 molecule.

[0049] The terms "gRNA" and "guide RNA" are used interchangeably throughout and refer to a nucleic acid that facilitates specific targeting or homing of a gRNA / Cas9 molecule complex to a target nucleic acid. A gRNA, sometimes referred to herein as an sgRNA, can be unimolecular (having a single RNA molecule) or modular (comprising more than one, typically two separate RNA molecules). A gRNA may bind to a target domain in the genome of a host cell. A gRNA may include a targeting domain that may be partially or fully complementary to the target domain. A gRNA may also include a "scaffold sequence" (e.g., a tracrRNA sequence) that recruits a Cas9 molecule to a target domain bound to the gRNA sequence (e.g., by the targeting domain of the gRNA sequence). A scaffold sequence may include at least one stem-loop structure and recruit an endonuclease. Exemplary scaffold sequences can be found, for example, in Jinek, et al. Science (2012) 337(6096):816-821, Ran, et al. Nature Protocols (2013) 8:2281-2308, PCT Publication No. WO2014 / 093694, and PCT Publication No. WO2013 / 176772.

[0050] The term "mutation," as used herein, refers to a genetic change (e.g., an insertion, deletion, inversion, or substitution) in a nucleic acid compared to a reference sequence, e.g., the corresponding sequence of a cell that does not have such a mutation, or the corresponding wild-type nucleic acid sequence. In some embodiments provided herein, a mutation in a gene encoding a lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) results in loss of expression of the lineage-specific cell surface antigen in cells that carry the mutation. In some embodiments, the mutation to the gene detargets the protein produced by the gene. In some embodiments, the detargeted lineage-specific cell surface antigen protein is not bound, or is bound at a lower level, by an agent that targets the lineage-specific cell surface antigen. In some embodiments, mutations in genes encoding lineage-specific cell surface antigens (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) result in the expression of variant forms of the lineage-specific cell surface antigens that are not bound by immunotherapeutic agents targeting the lineage-specific cell surface antigens, or that are bound at significantly lower levels than the non-mutated lineage-specific cell surface antigen forms encoded by the genes.In some embodiments, cells harboring genomic mutations in the lineage-specific cell surface antigen genes provided herein (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) can be treated with immunotherapeutics targeting the lineage-specific cell surface antigen, such as an anti-CD33 antibody or chimeric antigen receptor (CAR), an anti-CLL-1 antibody or chimeric antigen receptor (CAR), an anti-CD123 antibody or chimeric antigen receptor (CAR), an anti-CD19 antibody, or an anti-CD20 antibody. or chimeric antigen receptor (CAR), anti-CD30 antibody or chimeric antigen receptor (CAR), anti-CD5 antibody or chimeric antigen receptor (CAR), anti-CD6 antibody or chimeric antigen receptor (CAR), anti-CD7 antibody or chimeric antigen receptor (CAR), anti-CD34 antibody or chimeric antigen receptor (CAR), anti-CD38 antibody or chimeric antigen receptor (CAR), and / or anti-BCMA antibody or chimeric antigen receptor (CAR). In some embodiments, cells with genomic mutations in the lineage-specific cell surface antigen genes provided herein (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) are not bound, or are bound at significantly lower levels, by immunotherapeutics that target lineage-specific cell surface antigens, e.g., antibodies or chimeric antigen receptors (CARs) that target CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2).

[0051] In some embodiments, cells with genomic mutations in cell surface antigen genes provided herein are not bound, or are bound at significantly lower levels, by immunotherapeutics that target cell surface antigens, e.g., antibodies or chimeric antigen receptors (CARs). In some embodiments, the immunotherapeutics, e.g., antibodies or chimeric antigen receptors (CARs), target cell surface antigens and are selected from the group consisting of CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CD13, CD14, CD15, CD16a, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42, CD43, CD44, CD45, CD46, CD47, CD48, CD49, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60, CD61, CD62, CD63, CD64, CD65, CD66, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD76, CD77, CD78, CD79, CD80, CD81, CD82, CD83, CD84, CD85, CD86, CD87, CD88, CD89, CD90, CD81, CD82, CD83, 4, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d , CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, C D53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65s, CD66a, CD66b, CD66c, C D66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, C D85a, CD85b, CD85c, CD85d, CD85e, CD85f, CD85g, CD85h, CD85i, CD85j, CD85k, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112,CD113、CD114、CD115、CD116、CD117、CD118、CD119、CD120a、CD120b、CD121a、CD121b、CD122、CD123、CD124、CD125、CD126、CD127、CD128a、CD128b、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CD146、CD147、CD148、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CD156c、CD157、CD158a、CD158b1、CD158b2、CD158c、CD158d、CD158e1、CD158e2、CD158f、CD158g、CD158h、CD158i、CD158j、CD158k、CD159a、CD159c、CD160、CD161、CD162、CD163、CD164、CD165、CD166、CD167a、CD167b、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CD198、CD199、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210a、CD210b、CD212、CD213a1、CD213a2、CD215、CD217、CD218a、CD218b、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD236、CD238、CD239、CD240CE、CD240D、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CD248、CD249、CD252、CD253、CD254、CD256, CD257, CD258, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD27 1, CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD2 86, CD288, CD289, CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300 c, CD300d, CD300e, CD300f, CD300g, CD301, CD302, CD303, CD304, CD305, CD306, CD307a, CD307b, CD3 07c, CD307d, CD307e, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD320, CD321, CD32 2, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD3 38, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, or any combination thereof. See also examples of lineage-specific cell surface antigens from the BD Biosciences Human CD Marker Chart, https: / / www.bdbiosciences.com / content / dam / bdb / campaigns / reagent-education / BD_Reagents_CDMarkerHuman_Poster.pdf (incorporated by reference in its entirety).

[0052] The "targeting domain" of a gRNA is complementary to a "targeting domain" on a target nucleic acid. The strand of a target nucleic acid that contains a nucleotide sequence complementary to the core domain of a gRNA is referred to herein as the "complementary strand" of the target nucleic acid. The targeting domain mediates targeting of the gRNA-bound RNA-guided nuclease to the target site. Guidance on the selection of a targeting domain can be found, for example, in Fu Y et al., Nat Biotechnol 2014 (doi:10.1038 / nbt.2808) and Sternberg SH et al., Nature 2014 (doi:10.1038 / naturel3011).

[0053] The term "base editing" refers to genome editing techniques that involve the use of base editors, e.g., nuclease-damaged gene editing enzymes or partial nuclease-damaged gene editing enzymes (e.g., RNA-guided CRISPR / Cas proteins) fused to deaminase that target and deaminate specific nucleobases, e.g., cytosine or adenosine nucleobases of C or A nucleotides, resulting in a C to T nucleotide change or an A to G nucleotide change via cellular mismatch repair mechanisms. See, e.g., Komor et al. Nature (2016) 533:420-424; Rees et al. Nat. Rev. Genet. (2018) 19(12):770-788; Anzalone et al. Nat. Biotechnol. (2020) 38:824-844.

[0054] The terms "target domain," "target site," or "target sequence" refer to a sequence within a nucleic acid molecule (e.g., a DNA molecule) that is deaminated by a base editor as described herein. In some embodiments, the target sequence is a polynucleotide (e.g., a double-stranded DNA molecule), and the polynucleotide comprises a coding strand and a complementary strand. The meanings of "coding strand" and "complementary strand" are those common to the terms in the art. In some embodiments, the target sequence is a sequence in a mammalian genome. In some embodiments, the target sequence is a sequence in a human genome. The term "target codon" refers to an amino acid codon that is edited by a base editor and converted to a different codon via deamination of a nucleic acid base. In some embodiments, the target codon is edited in the coding strand. In some embodiments, the target codon is edited in the complementary strand.

[0055] The term "surface antigen" or "cell surface antigen" refers to an antigen on the surface of a cell that is extracellularly accessible during at least one cell cycle or developmental stage of the cell, and includes antigens that are extracellularly accessible during all stages of the cell cycle. In this context, "extracellularly accessible" refers to an antigen that can be bound by an agent, such as an antibody, that is provided extracellularly without requiring permeabilization of the cell membrane. As used herein, the term "cell surface antigen" can include proteins, peptides, sugars, lipids, or other moieties that are displayed on the surface of cells, such as on the surface of hematopoietic stem and progenitor cells (HSPCs).

[0056] The term "antigen" refers to a portion of a macromolecule (e.g., a polypeptide) that is specifically recognized by a component of the immune system, e.g., an antibody or antigen-binding portion thereof. As used herein, the term "antigen" encompasses any molecule that elicits an immune response. This immune response may involve either antibody production or activation of specific immunocompetent cells, or both. Thus, one of skill in the art will understand that any nucleic acid comprising a nucleotide sequence that encodes a protein or portion thereof that elicits an immune response encodes an "antigen" as that term is used herein. Cell surface antigens include, but are not limited to, cell surface molecules such as proteins, peptides, sugars, lipids, or other moieties on the cell surface.

[0057] Exemplary cell surface antigens include, but are not limited to, CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CD13, CD14, CD15, CD16a, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28, CD29, CD30, CD31, CD32, CD33, CD34, CD 35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD4 9b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, CD60c, CD61, CD62E, CD62L, CD62P, CD63, CD 64, CD65s, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79a, CD79b, CD80, CD81, C D82, CD83, CD84, CD85a, CD85b, CD85c, CD85d, CD85e, CD85f, CD85g, CD85h, CD85i, CD85j, CD85k, CD86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108, CD109, CD110, CD111, CD112, CD113, CD114, C D115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD122, CD123, CD124, CD125, CD126, CD127, CD128a, CD128b, CD129, CD130, CD131,CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CD146、CD147、CD148、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CD156c、CD157、CD158a、CD158b1、CD158b2、CD158c、CD158d、CD158e1、CD158e2、CD158f、CD158g、CD158h、CD158i、CD158j、CD158k、CD159a、CD159c、CD160、CD161、CD162、CD163、CD164、CD165、CD166、CD167a、CD167b、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CD198、CD199、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210a、CD210b、CD212、CD213a1、CD213a2、CD215、CD217、CD218a、CD218b、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD236、CD238、CD239、CD240CE、CD240D、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CD248、CD249、CD252、CD253、CD254、CD256、CD257、CD258、CD261、CD262、CD263、CD264、CD265、CD266、CD267、CD268、CD269、CD270、CD271、CD272、CD273、CD274、CD275、CD276、CD277、CD278、CD279、CD280、CD281, CD282, CD283, CD284, CD286, CD288, CD289, CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD300d, CD300e, CD300f , CD300g, CD301, CD302, CD303, CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD309, CD312, CD314, CD315, CD316, CD317, CD318, CD319, CD32 CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, CD371, or any combination thereof. See also examples of lineage-specific cell surface antigens from the BD Biosciences Human CD Marker Chart, https: / / www.bdbiosciences.com / content / dam / bdb / campaigns / reagent-education / BD_Reagents_CDMarkerHuman_Poster.pdf.

[0058] The term "exon" refers to a nucleic acid sequence that comprises the coding sequence of a gene. A gene typically contains two or more exons separated by an intron between them.

[0059] The term "intron" refers to a nucleic acid sequence adjacent to the coding sequence of a gene. The term "intron" encompasses non-coding sequences located within precursor mRNA (pre-mRNA) transcripts that are typically excised before nuclear transport. Splicing of pre-mRNA requires a sequence motif in the intron and is mediated by a ribonucleoprotein complex called the spliceosome. Introns typically contain a 5' donor splice site and a 3' acceptor splice site, usually with GU and AG dinucleotides at each intron end and a branch point located within the intron. At the 5' end, the DNA nucleotides can be GT (GU in pre-mRNA), and at the 3' end, they can be "AG." These nucleotides are part of the splice site. In some embodiments, introns are spliced ​​or removed from the RNA or mRNA sequence in which they reside. During splicing, the branch point nucleotide initiates nucleophilic attack on the 5' donor splice site. The free end of the upstream intron then initiates a second nucleophilic attack on the 3' acceptor splice site, releasing the intron as an RNA lariat and covalently linking the two exons. Introns are typically removed by the major spliceosome, a large ribonucleoprotein (RNP) complex found primarily in the nuclei of eukaryotic cells. The spliceosome is assembled from small nuclear RNAs (snRNAs) and numerous proteins. Base pairing of snRNAs with the intron and with each other, as well as protein-protein and protein-RNA interactions of splicing factors, positions the splice site for splicing.

[0060] The term "splice donor site" refers to a nucleic acid sequence or domain on the 5' end of an intron. In one embodiment, a splice donor site marks the start of an intron and / or the boundary of an intron with the immediately preceding coding sequence (e.g., an exon).

[0061] The term "splice acceptor site" refers to a nucleic acid sequence or domain on the 3' end of an intron. In some embodiments, the splice acceptor site marks the start of an intron and its boundary in the following coding sequence (e.g., an exon). In some embodiments, the splice acceptor site comprises an intron branch point. In some embodiments, the intron branch point is the point where the 5' end of an intron is joined during the splicing process. In some embodiments, the splice acceptor sequence and the intron branch site are adjacent to each other. In some embodiments, the splice acceptor sequence and the intron branch site may be separated, for example, the branch site may be further 5' from the splice acceptor sequence.

[0062] The term "splice branch point" refers to a nucleotide in an intron that is involved in splicing by facilitating the formation of a branched RNA lariat.

[0063] The term "splice site" refers to a nucleic acid sequence or domain that is located at either the 5' or 3' end of an intron as described herein.

[0064] The term "splice site mutation" refers to a genetic mutation that inserts, deletes, or alters one or more nucleotides at a specific site where splicing occurs during the processing of precursor messenger RNA into mature messenger RNA. The splicing process itself is controlled, at least in part, by splice donor and splice acceptor sequences surrounding each exon. Mutations in these sequences can lead, for example, to the retention of large segments of intronic DNA by the mRNA, or to entire exons being spliced ​​out of the mRNA. Such changes can potentially result in the production of non-functional proteins.

[0065] Nucleases / Gene Editing Enzymes In some embodiments, the cells (e.g., HSCs or HPCs) described herein are generated using nucleases described herein. Exemplary nucleases include CRISPR / Cas molecules (also referred to as CRISPR / Cas nucleases, Cas nucleases, e.g., Cas9), TALENs, ZFNs, and meganucleases. In some embodiments, the nucleases are used in combination with lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) gRNAs described herein (e.g., according to Tables 1-19). Some aspects of the present disclosure provide compositions and methods for generating the genetically engineered cells described herein, e.g., genetically engineered cells that contain modifications in their genome that result in loss of expression of a lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)), or expression of a variant form of a lineage-specific cell surface antigen that is not recognized by immunotherapeutic agents that target the lineage-specific cell surface antigen. Such compositions and methods provided herein include, but are not limited to, suitable strategies and approaches for genetically engineering cells, for example, by using nucleases, such as CRISPR / Cas nucleases, and suitable RNAs that can bind to such nucleases and target them to suitable target sites within the genome of the cells, resulting in genomic modifications that result in loss of expression of lineage-specific cell surface antigens, or expression of variant forms of lineage-specific cell surface antigens that are not recognized by immunotherapeutic agents targeting lineage-specific cell surface antigens (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)).

[0066] In some embodiments, for example, the engineered cells described herein (e.g., engineered hematopoietic cells such as engineered hematopoietic stem or progenitor cells or engineered immune effector cells) are generated via genome editing techniques, including any technique capable of introducing targeted changes, also referred to as "editing," into the genome of a cell using a nuclease, such as any of the nucleases described herein.

[0067] One exemplary suitable genome editing technique is "cellular editing," which involves the use of nucleases, e.g., RNA-RNA-guided nucleases such as CRISPR / Cas nucleases, to introduce targeted single- or double-stranded DNA breaks into the genome of a cell, which triggers cellular repair mechanisms such as, for example, non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ, sometimes referred to as "alternative NHEJ" or "alt-NHEJ"), or homology-directed repair (HDR), which typically results in an altered nucleic acid sequence at or immediately proximal to the site of the nuclease cut (e.g., via a nucleotide or nucleotide sequence insertion, deletion, inversion, or substitution). See Yeh et al. Nat. Cell. Biol. (2019) 21:1468-1478, e.g., Hsu et al. Cell (2014) 157:1262-1278, Jasin et al. DNA Repair (2016) 44:6-16, Sfeir et al. Trends Biochem. Sci. (2015) 40:701-714.

[0068] Another exemplary suitable genome editing technique is "base editing," which involves the use of base editors, e.g., nuclease-damaging gene editing enzymes or partial nuclease-damaging enzymes (e.g., RNA-guided CRISPR / Cas proteins) fused to deaminases that target and deaminate specific nucleobases, e.g., cytosine or adenosine nucleobases of C or A nucleotides, resulting in a C to T nucleotide change or an A to G nucleotide change via cellular mismatch repair mechanisms. See, e.g., Komor et al. Nature (2016) 533:420-424; Rees et al. Nat. Rev. Genet. (2018) 19(12):770-788; Anzalone et al. Nat. Biotechnol. (2020) 38:824-844.

[0069] Yet another exemplary genome editing technique involves "primed editing," which involves the introduction of new genetic information, e.g., modified nucleotide sequences, into specifically targeted genomic sites using catalytically impaired or partially catalytically impaired nucleases (e.g., RNA-guided nucleases, e.g., CRISPR / Cas nucleases) fused to engineered reverse transcriptase (RT) domains. The Cas / RT fusion also contains a nucleic acid sequence encoding the desired edit and is targeted to the target site within the genome by a guide RNA, which can function as a primer for the RT. See, e.g., Anzalone et al. Nature (2019) 576(7785):149-157.

[0070] Cas9 molecule In some embodiments, the use of genome editing techniques is characterized by the use of suitable RNA-guided nucleases, which in some embodiments can be catalytically impaired or partially catalytically impaired, for example, for base editing or prime editing. Examples of suitable RNA-guided nucleases include CRISPR / Cas nucleases such as Cas9, or other Cas nucleases such as Cas12a / Cpf1.

[0071] In some embodiments, a lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) gRNA described herein is complexed with a Cas9 molecule. A variety of Cas9 molecules can be used. In some embodiments, a Cas9 molecule is selected with the desired PAM specificity to target the gRNA / Cas9 molecule complex to a target domain in the lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)). In some embodiments, engineering the cell comprises introducing one or more (e.g., 1, 2, 3, or more) Cas9 molecules into the cell.

[0072] In some embodiments, the CD33 gRNA described herein is complexed with a Cas9 molecule. Various Cas9 molecules can be used. In some embodiments, a Cas9 molecule with desired PAM specificity is selected to target the gRNA / Cas9 molecule complex to a target domain in CD33. In some embodiments, genetically engineering the cell includes introducing one or more (e.g., 1, 2, 3 or more) Cas9 molecules into the cell.

[0073] In some embodiments, the CLL-1 gRNA described herein is complexed with a Cas9 molecule. Various Cas9 molecules can be used. In some embodiments, a Cas9 molecule with the desired PAM specificity is selected to target the gRNA / Cas9 molecule complex to a target domain in CLL-1. In some embodiments, genetically engineering the cell includes introducing one or more (e.g., 1, 2, 3 or more) Cas9 molecules into the cell.

[0074] In some embodiments, the CD123 gRNA described herein is complexed with a Cas9 molecule. Various Cas9 molecules can be used. In some embodiments, a Cas9 molecule with desired PAM specificity is selected to target the gRNA / Cas9 molecule complex to a target domain in CD123. In some embodiments, genetically engineering the cell includes introducing one or more (e.g., 1, 2, 3 or more) Cas9 molecules into the cell.

[0075] In some embodiments, the CD327 (Siglec-6) gRNA described herein is complexed with a Cas9 molecule. Various Cas9 molecules can be used. In some embodiments, a Cas9 molecule with the desired PAM specificity is selected to target the gRNA / Cas9 molecule complex to a target domain in CD327 (Siglec-6). In some embodiments, genetically engineering the cell includes introducing one or more (e.g., 1, 2, 3 or more) Cas9 molecules into the cell.

[0076] In some embodiments, the CD312 (EMR2) gRNA described herein is complexed with a Cas9 molecule. Various Cas9 molecules can be used. In some embodiments, a Cas9 molecule with the desired PAM specificity is selected to target the gRNA / Cas9 molecule complex to a target domain in CD312 (EMR2). In some embodiments, genetically engineering the cell includes introducing one or more (e.g., 1, 2, 3 or more) Cas9 molecules into the cell.

[0077] Cas9 molecules of various species can be used in the methods and compositions described herein. In some embodiments, the Cas9 molecule is from or derived from Streptococcus pyogenes (SpCas9), Staphylococcus aureus (SaCas9), or Streptococcus thermophilus (StCas9). Additional suitable Cas9 molecules include Staphylococcus aureus, Neisseria meningitidis (NmCas9), Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces, Cycliphilus denitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni(CjCas9), Campylobacter lari, Candidatus puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, gamma proteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, Ilyobacter polytropus, Kingellakingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis spp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria spp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella Suitable Cas9 nucleases include those derived from or derived from the genus Cas9, such as Cas9 of the genus Cas9, ...

[0078] In some embodiments, the Cas9 molecule is a naturally occurring Cas9 molecule. In some embodiments, the Cas9 molecule is an engineered, altered, or modified Cas9 molecule that differs, e.g., by at least one amino acid residue, from a reference sequence, e.g., the most similar naturally occurring Cas9 molecule, or a sequence in Table 50 of PCT Publication No. WO2015 / 157070, which is incorporated by reference herein in its entirety. In some embodiments, the Cas9 molecule comprises Cpfl or a fragment or variant thereof.

[0079] Naturally occurring Cas9 molecules typically comprise two lobes: a recognition (REC) lobe and a nuclease (NUC) lobe, each of which further comprises domains described, for example, in PCT Publication No. WO2015 / 157070, e.g., at Figures 9A-9B therein (which application is incorporated herein by reference in its entirety).

[0080] The REC lobe contains an arginine-rich bridge helix (BH), a REC1 domain, and a REC2 domain. The REC lobe is thought to be a Cas9-specific functional domain. The BH domain is a long alpha helix and arginine-rich region, encompassing amino acids 60-93 of the S. pyogenes Cas9 sequence. The REC1 domain is involved in recognizing repeat:anti-repeat duplexes, for example, of gRNAs or tracrRNAs. The REC1 domain contains two REC1 motifs at amino acids 94-179 and 308-717 of the S. pyogenes Cas9 sequence. These two REC1 domains are separated by the REC2 domain in the linear primary structure, but assemble in the tertiary structure to form the REC1 domain. The REC2 domain, or a portion thereof, may also play a role in recognizing repeat:anti-repeat duplexes. The REC2 domain encompasses amino acids 180-307 of the S. pyogenes Cas9 sequence.

[0081] The NUC lobe contains a RuvC domain (also referred to herein as a RuvC-like domain), an HNH domain (also referred to herein as an HNH-like domain), and a PAM-interacting (PI) domain. The RuvC domain shares structural similarity with members of the retroviral integrase superfamily and cleaves single strands, e.g., non-complementary strands, of target nucleic acid molecules. The RuvC domain is assembled from three separate RuvC motifs (RuvC I, RuvC II, and RuvC III, often referred to in the art as the RuvCI domain, or the N-terminal RuvC domain, RuvC II domain, and RuvC III domain) at amino acids 1-59, 718-769, and 909-1098 of the S. pyogenes Cas9 sequence, respectively. Similar to the REC1 domain, the three RuvC motifs are linearly separated by other domains in the primary structure, but in the tertiary structure, the three RuvC motifs assemble to form the RuvC domain. The HNH domain shares structural similarity with HNH endonucleases and cleaves a single strand, e.g., the complementary strand, of a target nucleic acid molecule. The HNH domain is located between the RuvC II-III motifs and includes amino acids 775-908 of the S. pyogenes Cas9 sequence. The PI domain interacts with the PAM of the target nucleic acid molecule and includes amino acids 1099-1368 of the S. pyogenes Cas9 sequence.

[0082] Crystal structures have been determined for the naturally occurring bacterial Cas9 nuclease (Jinek et al., Science, 343(6176):1247997, 2014) and S. pyogenes Cas9 with guide RNAs (e.g., synthetic fusions of crRNA and tracrRNA) (Nishimasu et al., Cell, 156:935-949, 2014, and Anders et al., Nature, 2014, doi:10.1038 / naturel3579).

[0083] In some embodiments, the Cas9 molecules described herein have nuclease activity, e.g., double-strand cleavage activity, at or directly proximal to the target site. In some embodiments, the Cas9 molecule is modified to inactivate one of the endonuclease's catalytic residues. In some embodiments, the Cas9 molecule is a nickase and generates a single-strand cleavage. See, e.g., Dabrowska et al. Frontiers in Neuroscience (2018) 12(75). It has been shown that one or more mutations in the enzyme's RuvC and HNH catalytic domains can improve Cas9 efficiency. See, e.g., Sarai et al. Currently Pharma. Biotechnol. (2017) 18(13). In some embodiments, the Cas9 molecule is fused to a second domain, e.g., a domain that modifies DNA or chromatin, e.g., a deaminase or demethylase domain. In some such embodiments, the Cas9 molecule is modified to eliminate its endonuclease activity.

[0084] In some embodiments, a Cas nuclease described herein (e.g., a Cas9 molecule or a Cas / gRNA complex) is administered together with a template for homology-directed repair (HDR). In some embodiments, a Cas9 molecule described herein is administered without an HDR template.

[0085] In some embodiments, Cas9 molecules are used that are modified to enhance the specificity of the enzyme (e.g., to reduce off-target effects and maintain robust on-target cleavage). In some embodiments, the Cas9 molecule is an enhanced specificity Cas9 variant (e.g., eSPCas9). See, e.g., Slaymaker et al. Science (2016) 351(6268):84-88. In some embodiments, the Cas9 molecule is a high-fidelity Cas9 variant (e.g., SpCas9-HF1). See, e.g., Kleinstiver et al. Nature (2016) 529:490-495.

[0086] Various Cas9 molecules are known in the art and are available from a variety of sources, and can be engineered / modified to modulate one or more activities or specificities of the enzyme. In some embodiments, the Cas9 molecule is engineered / modified to recognize one or more PAM sequences. In some embodiments, the Cas9 molecule is engineered / modified to recognize one or more PAM sequences that are different from the PAM sequences that the Cas9 molecule recognizes without engineering / modification. In some embodiments, the Cas9 molecule is engineered / modified to reduce off-target activity of the enzyme.

[0087] In some embodiments, the nucleotide sequence encoding the Cas9 molecule is further modified to alter the specificity of the endonuclease activity (e.g., to reduce off-target cleavage, decrease intracellular endonuclease activity or duration, increase homology-guided recombination, or reduce non-homologous end joining). See, e.g., Komor et al. Cell (2017) 168:20-36. In some embodiments, the nucleotide sequence encoding the Cas9 molecule is modified to alter the PAM recognition of the endonuclease. For example, the Cas9 molecule SpCas9 recognizes the PAM sequence NGG, while truncated variants of SpCas9 containing one or more modifications of the endonuclease (e.g., VQR SpCas9, EQR SpCas9, VRER SpCas9) can recognize the PAM sequences NGA, NGAG, and NGCG. The PAM recognition of a modified Cas9 molecule is considered "imperfect" if the Cas9 molecule recognizes more potential PAM sequences than an unmodified Cas9 molecule. For example, the Cas9 molecule SaCas9 recognizes the PAM sequence NNGRRT, while an imperfect variant of SaCas9 containing one or more modifications (e.g., KKH SaCas9) may recognize the PAM sequence NNNRRT. In one example, the Cas9 molecule FnCas9 recognizes the PAM sequence NNG, while an imperfect variant of FnCas9 containing one or more endonuclease modifications (e.g., RHA FnCas9) may recognize the PAM sequence YG. In one example, a Cas9 molecule containing the Cpf1 endonuclease substitution mutations S542R and K607R recognizes the PAM sequence TYCV. In one example, a Cas9 molecule containing the Cpf1 endonuclease substitution mutations S542R, K607R, and N552R recognizes the PAM sequence TATV. See, for example, Gao et al. Nat. Biotechnol. (2017) 35(8):789-792.

[0088] In some embodiments, more than one Cas9 molecule (e.g., two, three, or more) are used. In some embodiments, at least one of the Cas9 molecules is a Cas9 enzyme. In some embodiments, at least one of the Cas molecules is a Cpfl enzyme. In some embodiments, at least one of the Cas9 molecules is derived from Streptococcus pyogenes. In some embodiments, at least one of the Cas9 molecules is derived from Streptococcus pyogenes and at least one Cas9 molecule is derived from an organism that is not Streptococcus pyogenes.

[0089] In some embodiments, the Cas9 molecule is a base editor. In some embodiments, base editors are used to create genomic modifications that result in loss of expression of lineage-specific cell surface antigens (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)), or expression of variants of lineage-specific cell surface antigens that are not targeted by immunotherapy. Base editor endonucleases generally comprise a catalytically inactive Cas9 molecule fused to a functional domain, e.g., a deaminase domain. See, e.g., Eid et al. Biochem. J. (2018) 475(11):1955-1964; Rees et al. Nature Reviews Genetics (2018) 19:770-788. In some embodiments, a catalytically inactive Cas9 molecule is referred to as "death-Cas" or "dCas9." In some embodiments, the catalytically inactive Cas molecule has reduced activity and is, for example, a nickase (referred to as nCas). In some embodiments, the endonuclease comprises dCas9 fused to one or more uracil glycosylase inhibitor (UGI) domains. In some embodiments, the endonuclease comprises dCas9 fused to an adenine base editor (ABE), e.g., an ABE evolved from the RNA adenine deaminase TadA. In some embodiments, the endonuclease comprises dCas9 fused to a cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-triggered cytidine deaminase (AID)). In some embodiments, the catalytically inactive Cas9 molecule has reduced activity and is nCas9. In some embodiments, the catalytically inactive Cas9 molecule (dCas9) is fused to one or more uracil glycosylase inhibitor (UGI) domains. In some embodiments, the Cas9 molecule comprises an inactive Cas9 molecule (dCas9) fused to an adenine base editor (ABE), e.g., an ABE evolved from the RNA adenine deaminase TadA.In some embodiments, the Cas9 molecule comprises nCas9 fused to an adenine base editor (ABE), e.g., an ABE evolved from the RNA adenine deaminase TadA. In some embodiments, the Cas9 molecule comprises dCas9 fused to a cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-triggered cytidine deaminase (AID)). In some embodiments, the Cas9 molecule comprises nCas9 fused to a cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-triggered cytidine deaminase (AID)).

[0090] Examples of base editors include, but are not limited to, BE1, BE2, BE3, HF-BE3, BE4, BE4max, BE4-Gam, YE1-BE3, EE-BE3, YE2-BE3, YEE-CE3, VQR-BE3, VRER-BE3, SaBE3, SaBE4, SaBE4-Gam, Sa(KKH)-BE3, Target-AID, Target-AID-NG, xBE3, eA3A-BE3, BE-PLUS, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, and CRISPR-SKIP. Additional examples of base editors can be found, for example, in U.S. Publication No. 2018 / 0312825(A1), U.S. Publication No. 2018 / 0312828(A1), and PCT Publication No. WO2018 / 165629(A1), which are incorporated by reference in their entireties.

[0091] In some embodiments, the base editor is further modified to inhibit base excision repair at the target site and induce cellular mismatch repair. Any of the Cas9 molecules described herein can be fused to a Gam domain (bacteriophage Mu protein) to protect the Cas9 molecule from degradation and exonuclease activity. See, e.g., Eid et al. Biochem. J. (2018) 475(11):1955-1964.

[0092] In some embodiments, the Cas9 molecule belongs to the Class 2 Type V of Cas endonucleases. Class 2 Type V Cas endonucleases can be further classified as Type VA, Type VB, Type VC, and Type VU. See, e.g., Stella et al. Nature Structural & Molecular Biology (2017) 24:882-892. In some embodiments, the Cas molecule is a Type VA Cas endonuclease, such as Cpf1 (Cas12a) nuclease. In some embodiments, the Cas9 molecule is a Type VB Cas endonuclease, such as C2c1 endonuclease. See, e.g., Shmakov et al. Mol Cell (2015) 60:385-397. In some embodiments, the Cas molecule is MAD7™. Alternatively, or in addition, the Cas9 molecule is a Cpf1 nuclease or a variant thereof. As will be understood by those skilled in the art, Cpf1 nuclease may also be referred to as Cas12a. See, e.g., Strohkendl et al. Mol. Cell (2018) 71:1-9. In some embodiments, the compositions or methods described herein include, or the host cell expresses, a Cpf1 nuclease derived from Provetella spp. or Francisella spp., Acidaminococcus sp. (AsCpf1), Lachnospiraceae bacterium (LpCpf1), or Eubacterium rectale. In some embodiments, the nucleotide sequence encoding the Cpf1 nuclease may be codon optimized for expression in the host cell. In some embodiments, the nucleotide sequence encoding the Cpf1 endonuclease is further modified to alter the activity of the protein.

[0093] Both naturally occurring and modified variants of CRISPR / Cas nucleases are suitable for use according to aspects of the present disclosure. For example, dCas or nickase variants, Cas variants with altered PAM specificity, and Cas variants with improved nuclease activity are encompassed by some embodiments of the present disclosure. In some embodiments, catalytically inactive variants of Cas molecules (e.g., Cas9 or Cas12a) are used according to the methods described herein. A catalytically inactive variant of Cpf1 (Cas12a) may be referred to as dCas12a. As described herein, a catalytically inactive variant of Cpf1 may be fused to a functional domain to form a base editor. See, e.g., Rees et al. Nature Reviews Genetics (2018) 19:770-788. In some embodiments, the catalytically inactive Cas9 molecule is dCas9. In some embodiments, the endonuclease comprises dCas12a fused to one or more uracil glycosylase inhibitor (UGI) domains. In some embodiments, the Cas9 molecule comprises dCas12a fused to an adenine base editor (ABE), e.g., an ABE evolved from the RNA adenine deaminase TadA. In some embodiments, the Cas molecule comprises dCas12a fused to a cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-triggered cytidine deaminase (AID)).

[0094] Alternatively, or in addition, the Cas9 molecule is a Cas14 endonuclease or a variant thereof. Cas14 endonucleases are derived from archaea and tend to be small in size (e.g., 400-700 amino acids). Furthermore, Cas14 endonucleases do not require a PAM sequence. See, e.g., Harrington et al., Science (2018).

[0095] Any of the Cas9 molecules described herein may be modulated to control the level of expression and / or activity of the Cas9 molecule at a desired time point. For example, it may be advantageous to increase the level of expression and / or activity of the Cas9 molecule during a particular phase of the cell cycle. It has been demonstrated that the level of homology-directed repair is reduced during the G1 phase of the cell cycle; therefore, increasing the level of expression and / or activity of the Cas9 molecule during the S, G2, and / or M phases may increase homology-directed repair after Cas endonuclease editing. In some embodiments, the level of expression and / or activity of the Cas9 molecule is increased during the S, G2, and / or M phases of the cell cycle. In one example, the Cas9 molecule is fused to the N-terminal region of human geminin. See, e.g., Gutschner et al. Cell Rep. (2016) 14(6):1555-1566. In some embodiments, the level of expression and / or activity of the Cas9 molecule is reduced during the G1 phase. In one example, the Cas9 molecule is modified to reduce activity during the G1 phase. See, e.g., Lomova et al. Stem Cells (2018).

[0096] Alternatively, or in addition, any of the Cas9 molecules described herein may be fused to an epigenetic modifier (e.g., a chromatin-modifying enzyme, e.g., a DNA methylase, a histone deacetylase). See, e.g., Kungulovski et al. Trends Genet. (2016) 32(2):101-113. Cas9 molecules fused to epigenetic modifiers are referred to as "epi-effectors" and may enable transient and / or transient endonuclease activity. In some embodiments, the Cas9 molecule is dCas9 fused to a chromatin-modifying enzyme.

[0097] Base Editor In some embodiments, the cells or cell populations described herein are produced using base editing technology. As described above, base editing involves the use of a base editor, e.g., a nuclease-damaging gene editing enzyme or a partial nuclease-damaging enzyme (e.g., an RNA-guided CRISPR / Cas protein) fused to a deaminase that targets and deaminates specific nucleobases, e.g., cytosine or adenosine nucleobases of C or A nucleotides, resulting in a C to T nucleotide change or an A to G nucleotide change via cellular mismatch repair mechanisms. See, e.g., Komor et al. Nature (2016) 533:420-424; Rees et al. Nat. Rev. Genet. (2018) 19(12):770-788; Anzalone et al. Nat. Biotechnol. (2020) 38:824-844.

[0098] As described herein, base editing techniques can be used to achieve multiplex base editing. For example, in some embodiments, a method of multiplex base editing can include (i) providing a cell, and (ii) introducing into the cell one or more guide RNAs (gRNAs) targeting CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), (b) one or more gRNAs targeting CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), and (c) a base editor that binds to the one or more gRNAs, wherein the one or more gRNAs are configured to provide editing events within different target domains, thereby producing a genetically engineered cell, as described herein. In particular, multiplex base editing can be used to modify one or more target lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) genes. In particular, multiplex base editing can be used to modify multiple target lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) genes. Notably, multiplex base editing can be used without any risk of translocation. In certain embodiments, multiplex base editing can involve combining a base editor and a CRISPR nuclease, including, for example, a Cas9 or Cas12a nuclease, without any risk of translocation.

[0099] In some embodiments, the Cas9 molecule is a base editor. In some embodiments, a base editor is used to create a genomic modification that results in the loss of expression of a lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)), or the expression of a variant of a lineage-specific cell surface antigen that is not targeted by immunotherapy. In some embodiments, a base editor is used to create a genomic modification that results in the loss of expression of CD33, or the expression of a CD33 variant that is not targeted by immunotherapy. In some embodiments, a base editor is used to create a genomic modification that results in the loss of expression of CLL-1, or the expression of a CLL-1 variant that is not targeted by immunotherapy. In some embodiments, a base editor is used to create a genomic modification that results in the loss of expression of CD123, or the expression of a CD123 variant that is not targeted by immunotherapy. In some embodiments, base editors are used to create genomic modifications that result in the loss of expression of CD327 (Siglec-6) or the expression of a CD327 (Siglec-6) variant that is not targeted by immunotherapy. In some embodiments, base editors are used to create genomic modifications that result in the loss of expression of CD312 (EMR2) or the expression of a CD312 (EMR2) variant that is not targeted by immunotherapy.

[0100] In some embodiments, a base editor is used to create an editing event (e.g., create a genomic modification) that reduces the activity of a lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) in a cell. In some embodiments, a base editor is used to create an editing event (e.g., create a genomic modification) that reduces the expression level of a nucleic acid encoding a lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) in a cell.

[0101] In some embodiments, a base editor is used to create an editing event (e.g., create a genomic modification) that disables expression of a full-length lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) RNA in a cell. In some embodiments, a base editor is used to create an editing event (e.g., create a genomic modification) that disables expression of a full-length lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) protein in a cell.

[0102] In some embodiments, the cells express truncated forms of lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) RNA. In some embodiments, the cells express truncated forms of lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) protein.

[0103] In some embodiments, the truncated form of the lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) RNA is expressed at a level equal to or greater than the level of full-length lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) RNA in non-edited cells. In some embodiments, the truncated form of the lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) protein is expressed at a level equal to or greater than the level of the full-length lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) protein in a non-edited cell.

[0104] In some embodiments, the function or activity of a truncated form of a lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) RNA is impaired or abolished. In some embodiments, the function or activity of a truncated form of a lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) protein is impaired or abolished. In some embodiments, the function or activity of the truncated form of a lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) RNA that is impaired or abolished comprises binding to an antibody or chimeric antigen receptor (CAR).

[0105] Base editor endonucleases generally comprise a catalytically inactive Cas9 molecule fused to a functional domain, e.g., a deaminase domain. See, e.g., Eid et al. Biochem. J. (2018) 475(11):1955-1964; Rees et al. Nature Reviews Genetics (2018) 19:770-788. In some embodiments, the catalytically inactive Cas9 molecule is referred to as a "dead-Cas" or "dCas9." In some embodiments, the catalytically inactive Cas molecule has reduced activity, e.g., a nickase (referred to as nCas). In some embodiments, the endonuclease comprises dCas9 fused to one or more uracil glycosylase inhibitor (UGI) domains. In some embodiments, the endonuclease comprises dCas9 fused to an adenine base editor (ABE), e.g., an ABE evolved from the RNA adenine deaminase TadA. In some embodiments, the endonuclease comprises dCas9 fused to a cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-induced cytidine deaminase (AID)). In some embodiments, the catalytically inactive Cas9 molecule has reduced activity and is nCas9. In some embodiments, the catalytically inactive Cas9 molecule (dCas9) is fused to one or more uracil glycosylase inhibitor (UGI) domains. In some embodiments, the Cas9 molecule comprises an inactive Cas9 molecule (dCas9) fused to an adenine base editor (ABE), e.g., an ABE evolved from RNA adenine deaminase TadA. In some embodiments, the Cas9 molecule comprises nCas9 fused to an adenine base editor (ABE), e.g., an ABE evolved from RNA adenine deaminase TadA. In some embodiments, the Cas9 molecule comprises dCas9 fused to a cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-induced cytidine deaminase (AID)).In some embodiments, the Cas9 molecule comprises nCas9 fused to a cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-induced cytidine deaminase (AID)).

[0106] In some embodiments, the base editor is a cytosine base editor (CBE). In some embodiments, the CBE is CBE1, CBE2, CBE3, or CBE4. In some embodiments, the CBE is selected from the group consisting of nCas9-2xUGI, BE4-rAPOBEC1, BE4-rAPOBEC1 K34A H122A, BE4-PpAPOBEC1, BE4-PpAPOBEC1 R33A, BE4-PpAPOBEC1 H122A, BE4-RrA3F, BE4-AmAPOBEC1, and BE4-SsAPOBEC3B.

[0107] In some embodiments, the base editor is an adenine base editor (ABE). In some embodiments, the ABE is ABE1, ABE2, ABE3, ABE4, ABE5, ABE6, ABE7, or ABE8. In some embodiments, the ABE is selected from the group consisting of ABE7.10-m, ABE7.10-d, ABE8.8-m, ABE8.8-d, ABE8.13-m, ABE8.13-d, ABE8.17-m, ABE8.17-d, ABE8.20-m, and ABE8.20-d.

[0108] In some embodiments, base editors include, but are not limited to, BE1, BE2, BE3, HF-BE3, BE4, BE4max, BE4-Gam, YE1-BE3, EE-BE3, YE2-BE3, YEE-CE3, VQR-BE3, VRER-BE3, SaBE3, SaBE4, SaBE4-Gam, Sa(KKH)-BE3, Target-AID, Target-AID-NG, xBE3, eA3A-BE3, BE-PLUS, TAM, CRISPR-X, ABE7.9, ABE7.10, ABE7.10*, xABE, ABESa, VQR-ABE, VRER-ABE, Sa(KKH)-ABE, and CRISPR-SKIP.

[0109] Additional examples of base editors can be found, for example, in U.S. Publication No. 2018 / 0312825(A1), U.S. Publication No. 2018 / 0312828(A1), and PCT Publication No. WO2018 / 165629(A1), Yu et al. Nat Commun. (2020) 11(1):2052, and Gaudelli et al. Nat Biotechnol. (2020) 38(7):892-900, which are incorporated by reference herein in their entireties.

[0110] An exemplary abe8_20m sequence is provided below.

[0111] An exemplary ppabobec1-orf-r33a sequence is provided below.

[0112] An exemplary ppabobec1-orf-wt sequence is provided below.

[0113] An exemplary prtn_SzW8eqL7-abe8_20m sequence is provided below.

[0114] An exemplary prtn_ZJVPExXY-ppabobec1-r33a-protein sequence is provided below.

[0115] An exemplary prtn_ZyqE8AYc-ppabobec1-wt-protein sequence is provided below.

[0116] In some embodiments, the base editor is further modified to inhibit base excision repair at the target site and induce cellular mismatch repair. Any of the Cas9 molecules described herein can be fused to a Gam domain (bacteriophage Mu protein) to protect the Cas9 molecule from degradation and exonuclease activity. See, e.g., Eid et al. Biochem. J. (2018) 475(11):1955-1964.

[0117] In some embodiments, the Cas9 molecule belongs to the Class 2 Type V of Cas endonucleases. Class 2 Type V Cas endonucleases can be further classified as Type VA, Type VB, Type VC, and Type VU. See, e.g., Stella et al. Nature Structural & Molecular Biology (2017) 24:882-892. In some embodiments, the Cas molecule is a Type VA Cas endonuclease, such as Cpf1 (Cas12a) nuclease. In some embodiments, the Cas9 molecule is a Type VB Cas endonuclease, such as C2c1 endonuclease. See, e.g., Shmakov et al. Mol Cell (2015) 60:385-397. In some embodiments, the Cas molecule is MAD7™. Alternatively, or in addition, the Cas9 molecule is a Cpf1 nuclease or a variant thereof. As will be understood by those skilled in the art, Cpf1 nuclease may also be referred to as Cas12a. See, e.g., Strohkendl et al. Mol. Cell (2018) 71:1-9. In some embodiments, the compositions or methods described herein include, or the host cell expresses, a Cpf1 nuclease derived from Provetella spp. or Francisella spp., Acidaminococcus sp. (AsCpf1), Lachnospiraceae bacterium (LpCpf1), or Eubacterium rectale. In some embodiments, the nucleotide sequence encoding the Cpf1 nuclease may be codon optimized for expression in the host cell. In some embodiments, the nucleotide sequence encoding the Cpf1 endonuclease is further modified to alter the activity of the protein.

[0118] Both naturally occurring and modified variants of CRISPR / Cas nucleases are suitable for use according to aspects of the present disclosure. For example, dCas or nickase variants, Cas variants with altered PAM specificity, and Cas variants with improved nuclease activity are encompassed by some embodiments of the present disclosure. In some embodiments, catalytically inactive variants of Cas molecules (e.g., Cas9 or Cas12a) are used according to the methods described herein. A catalytically inactive variant of Cpf1 (Cas12a) may be referred to as dCas12a. As described herein, a catalytically inactive variant of Cpf1 may be fused to a functional domain to form a base editor. See, e.g., Rees et al. Nature Reviews Genetics (2018) 19:770-788. In some embodiments, the catalytically inactive Cas9 molecule is dCas9. In some embodiments, the endonuclease comprises dCas12a fused to one or more uracil glycosylase inhibitor (UGI) domains. In some embodiments, the Cas9 molecule comprises dCas12a fused to an adenine base editor (ABE), e.g., an ABE evolved from the RNA adenine deaminase TadA. In some embodiments, the Cas molecule comprises dCas12a fused to a cytidine deaminase enzyme (e.g., APOBEC deaminase, pmCDA1, activation-triggered cytidine deaminase (AID)).

[0119] Zinc finger nuclease In some embodiments, the cells or cell populations described herein are produced using zinc finger (ZFN) technology. In some embodiments, the ZFN recognizes a target domain described herein, e.g., a target domain in CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) described herein. Generally, zinc finger-mediated genome editing involves the use of zinc finger nucleases, which typically include a zinc finger DNA-binding domain and a nuclease domain. The zinc finger binding domain may be engineered to recognize and bind to any target domain of interest and may be designed to recognize, for example, a DNA sequence ranging from about 3 nucleotides to about 21 nucleotides in length, or from about 8 to about 19 nucleotides in length. The zinc finger binding domain typically includes at least three zinc finger recognition regions (e.g., zinc fingers).

[0120] Restriction endonucleases (restriction enzymes) capable of sequence-specific binding to DNA (at a recognition site) and cleaving the DNA at or near the binding site are known in the art and can be used to form ZFNs for use in genome editing. For example, type IIS restriction endonucleases cleave DNA at sites removed from the recognition site and have separable binding and cleavage domains. In one example, the DNA cleavage domain can be derived from FokI endonuclease.

[0121] TALEN In some embodiments, the cells or cell populations described herein are produced using TALEN technology. In some embodiments, TALENs recognize target domains described herein, such as those in CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2). Generally, TALENs are engineered restriction enzymes that can specifically bind to and cleave a desired target DNA molecule. TALENs typically contain a transcription activator-like effector (TALE) DNA-binding domain fused to a DNA cleavage domain. The DNA-binding domain may contain a highly conserved 33-34 amino acid sequence with a bifurcated two-amino acid RVD (repeated variable dipeptide motif) at positions 12 and 13. The RVD motif determines binding specificity to nucleic acid sequences and can be engineered to specifically bind to a desired DNA sequence. In one example, the DNA cleavage domain may be derived from FokI endonuclease. In some embodiments, the FokI domain functions as a dimer, using two constructs with unique DNA binding domains in the appropriate orientation and spacing to a site in the target genome.

[0122] TALEN specific to a target gene of interest can be used in cells to generate double-strand breaks (DSBs).If the repair mechanism improperly repairs the break through non-homologous end joining, mutations can be introduced at the break site.For example, improper repair can cause frameshift mutations.Alternatively, foreign DNA molecules with desired sequences can be introduced into cells together with TALEN.Depending on the sequence of foreign DNA and chromosomal sequence, this process can be used to correct defects or introduce DNA fragments into a target gene of interest, or introduce such defects into endogenous genes, thereby reducing the expression of the target gene.

[0123] Some exemplary, non-limiting embodiments of endonucleases and nuclease variants suitable for use in connection with the guide RNAs and genetic engineering methods provided herein are described above. Additional suitable nucleases and nuclease variants will be apparent to those skilled in the art based on this disclosure and knowledge in the art. The present disclosure is not limited in this regard.

[0124] gRNA sequence and configuration General gRNA configuration A gRNA can comprise several domains. In one embodiment, a single molecule, sgRNA, or chimeric gRNA comprises, for example, from 5' to 3': a targeting domain (complementary or partially complementary to a target nucleic acid sequence in a target gene, e.g., a lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) gene); a first complementarity domain, binding domain, a second complementarity domain (complementary to the first complementarity domain), the proximal domain, and Optionally, it includes a tail domain.

[0125] Each of these domains is now described in more detail.

[0126] The targeting domain may, for example, comprise a nucleotide sequence that is at least 80, 85, 90, or 95% complementary, e.g., fully complementary, to a target sequence on a target nucleic acid. The targeting domain is a portion of an RNA molecule and therefore typically comprises the base uracil (U), whereas any DNA encoding a gRNA molecule comprises the base thymine (T). Without wishing to be bound by theory, it is believed that the complementarity of the targeting domain to the target sequence contributes to the specificity of the interaction of the gRNA / Cas9 molecule complex with the target nucleic acid. In a targeting domain and target sequence pair, it is understood that the uracil base in the targeting domain pairs with the adenine base in the target sequence. In one embodiment, the targeting domain itself comprises, from 5' to 3', an optional secondary domain and a core domain. In one embodiment, the core domain is fully complementary to the target sequence. In one embodiment, the targeting domain is 5 to 50 nucleotides in length. The targeting domain may be 15-30 nucleotides in length, 15-25 nucleotides in length, 18-22 nucleotides in length, or 19-21 nucleotides in length. In some embodiments, the targeting domain is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In some embodiments, the targeting domain is 10-30 or 15-25 nucleotides in length. The targeting domain sequence may correspond exactly to the targeting domain sequence (i.e., without any mismatched nucleotides) or may contain one or more, but typically no more than four, mismatches. Because the targeting domain is part of an RNA molecule, a gRNA typically contains ribonucleotides, while a DNA-targeting domain will contain deoxyribonucleotides.

[0127] Thus, the targeting domain of the gRNA base-pairs (with full or partial complementarity) with the sequence of the double-stranded target site that is complementary to the sequence of the targeting domain, and thus with the strand that is complementary to the strand containing the PAM sequence. It will be understood that the targeting domain of the gRNA typically does not contain a PAM sequence. It will be further understood that the location of the PAM can be 5' or 3' of the targeting domain sequence, depending on the nuclease employed. For example, the PAM is typically 3' of the targeting domain sequence for Cas9 nucleases and 5' of the targeting domain sequence for Cas12a nucleases. For an illustration of the location of the PAM and the mechanism of gRNA binding to the target site, see, e.g., Figure 1 in Vanegas et al., Fungal Biol Biotechnol. 2019;6:6, incorporated herein by reference. For additional illustrations and explanations of the mechanism of gRNA targeting of RNA-guided nucleases to target sites, see Fu Y et al., Nat Biotechnol 2014 (doi:10.1038 / nbt.2808), and Sternberg SH et al., Nature 2014 (doi:10.1038 / naturel3011), both of which are incorporated herein by reference.

[0128] An exemplary illustration of a Cas9 target site comprising a 22-nucleotide targeting domain and an NGG PAM sequence, and a gRNA comprising a targeting domain that corresponds perfectly to the targeting domain (and thus base-pairs with perfect complementarity to the DNA strand complementary to the strand comprising the targeting domain and PAM), is provided below.

[0129] [ka]

[0130] An exemplary illustration of a Cas12a target site comprising a 22-nucleotide targeting domain and a TTN PAM sequence, and a gRNA comprising a targeting domain that corresponds perfectly to the targeting domain (and thus base-pairs with perfect complementarity to the DNA strand complementary to the strand comprising the targeting domain and PAM), is provided below. [ka]

[0131] In some embodiments, the Cas12a PAM sequence is 5'-TTTV-3'.

[0132] While not wishing to be bound by theory, it is believed that, at least in some embodiments, the length of the targeting domain and its complementarity with the target sequence contribute to the specificity of the interaction of the gRNA / Cas9 molecular complex with the target nucleic acid. In some embodiments, the targeting domain of a gRNA provided herein is 5-50 nucleotides in length. In some embodiments, the targeting domain is 15-25 nucleotides in length. In some embodiments, the targeting domain is 18-22 nucleotides in length. In some embodiments, the targeting domain is 19-21 nucleotides in length. In some embodiments, the targeting domain is 15 nucleotides in length. In some embodiments, the targeting domain is 16 nucleotides in length. In some embodiments, the targeting domain is 17 nucleotides in length. In some embodiments, the targeting domain is 18 nucleotides in length. In some embodiments, the targeting domain is 19 nucleotides in length. In some embodiments, the targeting domain is 20 nucleotides in length. In some embodiments, the targeting domain is 21 nucleotides in length. In some embodiments, the targeting domain is 22 nucleotides in length. In some embodiments, the targeting domain is 23 nucleotides in length. In some embodiments, the targeting domain is 24 nucleotides in length. In some embodiments, the targeting domain is 25 nucleotides in length. In some embodiments, the targeting domain fully corresponds to a targeting domain sequence provided herein or a portion thereof, without mismatches. In some embodiments, the targeting domain of a gRNA provided herein contains one mismatch to a targeting domain sequence provided herein. In some embodiments, the targeting domain contains two mismatches to a targeting domain sequence. In some embodiments, the targeting domain contains three mismatches to a targeting domain sequence.

[0133] In some embodiments, the targeting domain comprises a core domain and a secondary targeting domain, e.g., as described in PCT Publication No. WO2015 / 157070, which is incorporated by reference in its entirety. In some embodiments, the core domain comprises about 8 to about 13 nucleotides from the 3' end of the targeting domain (e.g., the 3'-most 8 to 13 nucleotides of the targeting domain). In one embodiment, the secondary domain is positioned 5' to the core domain. In many embodiments, the core domain has exact complementarity (perfect correspondence) with the corresponding region or portion thereof of the target sequence. In other embodiments, the core domain may include one or more nucleotides that are not complementary (mismatch) to the corresponding nucleotides of the targeting domain sequence.

[0134] The first complementarity domain is complementary to the second complementarity domain and, in one embodiment, has sufficient complementarity to the second complementarity domain to form a double-stranded region under at least some physiological conditions. In one embodiment, the first complementarity domain is 5 to 30 nucleotides in length. In one embodiment, the first complementarity domain comprises three subdomains, in the 5' to 3' direction: a 5' subdomain, a central subdomain, and a 3' subdomain. In one embodiment, the 5' subdomain is 4 to 9 nucleotides in length, e.g., 4, 5, 6, 7, 8, or 9 nucleotides in length. In one embodiment, the central subdomain is 1, 2, or 3 nucleotides in length, e.g., 1 nucleotide in length. In one embodiment, the 3' subdomain is 3 to 25 nucleotides in length, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. The first complementarity domain shares homology with or can be derived from a naturally occurring first complementarity domain. In one embodiment, it has at least 50% homology with a S. pyogenes, S. aureus, or S. thermophilus first complementarity domain.

[0135] The sequences and arrangements of the above domains are described in more detail in PCT Publication No. WO2015 / 157070, which is incorporated by reference in its entirety, including pages 88-112 thereof.

[0136] The linking domain serves to link the first complementary domain to the second complementary domain of the unimolecular gRNA. The linking domain can link the first and second complementary domains covalently or non-covalently. In one embodiment, the linkage is a covalent bond. In one embodiment, the linking domain is or includes a covalent bond interposed between the first complementary domain and the second complementary domain. In some embodiments, the linking domain comprises one or more, e.g., 2, 3, 4, 5, 6, 7, 8, 9, or 10, nucleotides. In some embodiments, the linking domain comprises at least one non-nucleotide bond, e.g., as disclosed in PCT Publication No. WO2018 / 126176, the entire contents of which are incorporated herein by reference.

[0137] The second complementarity domain is at least partially complementary to the first complementarity domain and, in one embodiment, has sufficient complementarity to the second complementarity domain to form a double-stranded region under at least some physiological conditions. In one embodiment, the second complementarity domain can include a sequence that lacks complementarity with the first complementarity domain, e.g., a sequence that creates a loop from the double-stranded region. In one embodiment, the second complementarity domain is 5 to 27 nucleotides in length. In one embodiment, the second complementarity domain is longer than the first complementarity domain. In one embodiment, the complementarity domain is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In one embodiment, the second complementarity domain comprises three subdomains, in a 5' to 3' direction: a 5' subdomain, a central subdomain, and a 3' subdomain. In one embodiment, the 5' subdomain is 3 to 25 nucleotides in length, e.g., 4 to 22, 4 to 18, or 4 to 10, or 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25 nucleotides in length. In one embodiment, the central subdomain is 1, 2, 3, 4, or 5 nucleotides in length, e.g., 3 nucleotides in length. In one embodiment, the 3' subdomain is 4 to 9 nucleotides in length, e.g., 4, 5, 6, 7, 8, or 9 nucleotides in length. In one embodiment, the 5' and 3' subdomains of the first complementarity domain are complementary, eg, perfectly complementary, to the 3' and 5' subdomains of the second complementarity domain, respectively.

[0138] In one embodiment, the proximal domain is 5-20 nucleotides in length. In one embodiment, the proximal domain shares homology with or can be derived from a naturally occurring proximal domain. In one embodiment, it has at least 50% homology with a S. pyogenes, S. aureus, or S. thermophilus proximal domain.

[0139] A wide range of tail domains are suitable for use in gRNAs. In one embodiment, the tail domain is 0 (absent), 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 nucleotides in length. In some embodiments, the tail domain nucleotides are derived from or share homology with sequences from the 5' end of a naturally occurring tail domain. In some embodiments, the tail domains comprise sequences that are complementary to each other and form a double-stranded region under at least some physiological conditions. In some embodiments, the tail domain is absent or is 1-50 nucleotides in length. In some embodiments, the tail domain can share homology with or be derived from a naturally occurring proximal tail domain. In some embodiments, it has at least 50% homology to a S. pyogenes, S. aureus, or S. thermophilus tail domain. In one embodiment, the tail domain includes nucleotides at the 3' end that are relevant for in vitro or in vivo transcription methods.

[0140] In some embodiments, the modular gRNA comprises: For example, from 5' to 3', a targeting domain (complementary to a target nucleic acid in a lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) gene), and a first strand comprising a first complementarity domain; Preferably from 5' to 3', optionally, a 5' extension domain; a second complementarity domain, the proximal domain, and and optionally a second strand comprising a tail domain.

[0141] In some embodiments, the gRNA is chemically modified. In some embodiments, any of the gRNAs provided herein comprises one or more chemically modified nucleotides. Chemical modifications of gRNAs have been previously described, and suitable chemical modifications include any modifications that are beneficial to gRNA function and do not measurably increase any undesirable properties of a given gRNA, such as off-target effects. Suitable chemical modifications include, for example, those that render the gRNA less susceptible to endonuclease or exonuclease catalytic activity, including, but not limited to, that the gRNA may comprise one or more modifications selected from phosphorothioate backbone modifications, 2'-O-Me modified sugars (e.g., at one or both of the 3' and 5' ends), 2'F modified sugars, replacement of the ribose sugar with the bicyclic nucleotide-cEt, 3'thioPACE (MSP), or any combination thereof. Additional suitable gRNA modifications will be apparent to those of skill in the art based on the present disclosure, and include, but are not limited to, those described in, for example, Rahdar et al. PNAS December 22, 2015 112(51)E7110-E7117 and Hendel et al., Nat Biotechnol. 2015) Sep;33(9):985-989, each of which is incorporated herein by reference in its entirety. In some embodiments, the gRNAs described herein comprise one or more 2'-O-methyl-3'-phosphorothioate nucleotides, e.g., at least 2, 3, 4, 5, or 6 2'-O-methyl-3'-phosphorothioate nucleotides. In some embodiments, the gRNAs described herein comprise modified nucleotides (e.g., 2'-O-methyl-3'-phosphorothioate nucleotides) at the three terminal positions and at the 5' terminus and / or at the three terminal positions and at the 3' terminus. In some embodiments, the gRNA may comprise one or more modified nucleotides, e.g., as described in PCT Publication Nos. WO2017 / 214460, WO2016 / 089433, and WO2016 / 164356, which are incorporated by reference in their entireties.

[0142] In some embodiments, the gRNAs described herein are chemically modified. For example, the gRNA may contain one or more 2'-O-modified nucleotides, e.g., 2'-O-methyl nucleotides. In some embodiments, the gRNA contains 2'-O-modified nucleotides, e.g., 2'-O-methyl nucleotides, at the 5' end of the gRNA. In some embodiments, the gRNA contains 2'-O-modified nucleotides, e.g., 2'-O-methyl nucleotides, at the 3' end of the gRNA. In some embodiments, the gRNA contains 2'-O-modified nucleotides, e.g., 2'-O-methyl nucleotides, at both the 5' and 3' ends of the gRNA. In some embodiments, the gRNA is 2'-O-modified, e.g., at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA. In some embodiments, the gRNA is 2'-O modified, e.g., the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA are 2'-O-methyl modified. In some embodiments, the gRNA is 2'-O modified, e.g., the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA are 2'-O-methyl modified. In some embodiments, the gRNA is 2'-O modified, e.g., the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA are 2'-O-methyl modified. In some embodiments, the nucleotide at the 3' end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3' end of the gRNA does not have a chemically modified sugar.In some embodiments, the gRNA is 2'-O modified, for example, at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA. In some embodiments, the 2'-O-methyl nucleotide comprises a phosphate linkage to the adjacent nucleotide. In some embodiments, the 2'-O-methyl nucleotide comprises a phosphorothioate linkage to the adjacent nucleotide. In some embodiments, the 2'-O-methyl nucleotide comprises a thioPACE linkage to the adjacent nucleotide.

[0143] In some embodiments, the gRNA may comprise one or more 2'-O-modified and 3'-phosphorus modified nucleotides, such as 2'-O-methyl 3' phosphorothioate nucleotides. In some embodiments, the gRNA comprises a 2'-O-modified and 3'-phosphorus modified nucleotide at the 5' end of the gRNA, such as 2'-O-methyl 3' phosphorothioate nucleotides. In some embodiments, the gRNA comprises a 2'-O-modified and 3'-phosphorus modified nucleotide at the 3' end of the gRNA, such as 2'-O-methyl 3' phosphorothioate nucleotides. In some embodiments, the gRNA comprises a 2'-O-modified and 3'-phosphorus modified nucleotide at the 5' and 3' ends of the gRNA, such as 2'-O-methyl 3' phosphorothioate nucleotides. In some embodiments, the gRNA comprises a backbone in which one or more non-bridging oxygen atoms are replaced with a sulfur atom. In some embodiments, the gRNA is 2'-O modified and 3' phosphorus modified, for example, 2'-O-methyl 3' phosphorothioate modified at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA. In some embodiments, the gRNA is 2'-O modified and 3' phosphorus modified, for example, 2'-O-methyl 3' phosphorothioate modified at the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA. In some embodiments, the gRNA is 2'-O modified and 3' phosphorus modified, e.g., 2'-O-methyl 3' phosphorothioate modified at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA. In some embodiments, the gRNA is 2'-O modified and 3' phosphorus modified, e.g., 2'-O-methyl 3' phosphorothioate modified at the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA.In some embodiments, the nucleotide at the 3' end of the gRNA is not chemically modified. In some embodiments, the nucleotide at the 3' end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2'-O-modified and 3' phosphorus-modified, for example, 2'-O-methyl 3' phosphorothioate-modified at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA.

[0144] In some embodiments, the gRNA may comprise one or more 2'-O modifications and 3'-phosphorus modifications, such as 2'-O-methyl 3' thio PACE nucleotides. In some embodiments, the gRNA comprises a 2'-O modification and a 3'-phosphorus modification, such as 2'-O-methyl 3' thio PACE nucleotides, at the 5' end of the gRNA. In some embodiments, the gRNA comprises a 2'-O modification and a 3'-phosphorus modification, such as 2'-O-methyl 3' thio PACE nucleotides, at the 3' end of the gRNA. In some embodiments, the gRNA comprises a 2'-O modification and a 3'-phosphorus modification, such as 2'-O-methyl 3' thio PACE nucleotides, at the 5' and 3' ends of the gRNA. In some embodiments, the gRNA comprises a backbone in which one or more non-bridging oxygen atoms are replaced with a sulfur atom and one or more non-bridging oxygen atoms are replaced with an acetate group. In some embodiments, the gRNA is 2'-O modified and 3' phosphorus modified, for example, 2'-O-methyl 3' thio PACE modified at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA. In some embodiments, the gRNA is 2'-O modified and 3' phosphorus modified, for example, 2'-O-methyl 3' thio PACE modified at the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA. In some embodiments, the gRNA is 2'-O modified and 3' phosphorus modified, for example, 2'-O-methyl 3' thio PACE modified at the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA. In some embodiments, the gRNA is 2'-O modified and 3' phosphorus modified, e.g., 2'-O-methyl 3' thio PACE modified at the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA. In some embodiments, the nucleotide at the 3' end of the gRNA is not chemically modified.In some embodiments, the nucleotide at the 3' end of the gRNA does not have a chemically modified sugar. In some embodiments, the gRNA is 2'-O modified and 3' phosphorus modified, for example, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA are 2'-O-methyl 3' thio PACE modified.

[0145] In some embodiments, the gRNA comprises a chemically modified backbone. In some embodiments, the gRNA comprises a phosphorothioate linkage. In some embodiments, one or more non-bridging oxygen atoms are replaced with a sulfur atom. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each comprise a phosphorothioate linkage. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each comprise a phosphorothioate linkage.

[0146] In some embodiments, the gRNA comprises a thioPACE bond. In some embodiments, the gRNA comprises a backbone in which one or more non-bridging oxygen atoms are replaced with a sulfur atom and one or more non-bridging oxygen atoms are replaced with an acetate group. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, and the third nucleotide from the 5' end of the gRNA each comprise a thioPACE bond. In some embodiments, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each comprise a thioPACE bond. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end of the gRNA, the nucleotide at the 3' end of the gRNA, the second nucleotide from the 3' end of the gRNA, and the third nucleotide from the 3' end of the gRNA each comprise a thioPACE bond. In some embodiments, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each comprise a ThioPACE bond. In some embodiments, the nucleotide at the 5' end of the gRNA, the second nucleotide from the 5' end of the gRNA, the third nucleotide from the 5' end, the second nucleotide from the 3' end of the gRNA, the third nucleotide from the 3' end of the gRNA, and the fourth nucleotide from the 3' end of the gRNA each comprise a ThioPACE bond.

[0147] Some exemplary, non-limiting embodiments of suitable modifications, e.g., chemical modifications, for use in connection with the guide RNAs and genetic engineering methods provided herein are described above. Additional suitable modifications, e.g., chemical modifications, will be apparent to those skilled in the art based on this disclosure and knowledge in the art, including, but not limited to, those described in Hendel, A. et al., Nature Biotech., 2015, Vol. 33, No. 9, PCT Publication Nos. WO2017 / 214460, WO2016 / 089433, and WO2016 / 164356 (each of which is incorporated herein by reference in its entirety).

[0148] Lineage-specific cell surface antigens targeted by the gRNAs provided herein (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) can be delivered to cells in any suitable manner. For example, various suitable methods have been described for delivery of CRISPR / Cas systems, including ribonucleoproteins (RNPs) containing gRNAs linked to RNA-guided nucleases; exemplary suitable methods include, but are not limited to, electroporation of RNPs into cells, electroporation of mRNA encoding the Cas nuclease and gRNA into cells, various protein or nucleic acid transfection methods, and delivery of encoding RNA or DNA via viral vectors, such as retroviral (e.g., lentiviral) vectors. Any suitable delivery method is encompassed by the present disclosure, which is not limited in this respect.

[0149] gRNA targeting CD33 (Siglec-3) The present disclosure provides several useful gRNAs capable of targeting endonucleases to human CD33. In some embodiments, the gRNA is a gRNA disclosed in any of PCT Publication Nos. WO2017 / 066760, WO2020 / 047164, WO2020 / 150478, and WO2020 / 237217, WO2019 / 046285, WO2018 / 160768, or Borot et al. PNAS June 11, 2019 116(24)11978-11987, each of which is incorporated herein by reference in its entirety. Tables 1-3 and Table A below illustrate target domains within human endogenous CD33 that can be bound by the gRNAs described herein. [Table 1] [Table 2] [Table 3-1] [Table 4] [Table 5] [Table 15]

[0150] gRNA targeting CLL-1 The present disclosure provides several useful gRNAs capable of targeting endonucleases to human CLL-1. In some embodiments, the gRNAs capable of targeting endonucleases to human CLL-1 are gRNAs disclosed in either PCT Publication No. WO2020 / 047164 or WO2021 / 041971, each of which is incorporated herein by reference in its entirety. Table 1 below illustrates target domains within human endogenous CLL-1 that can be bound by the gRNAs described herein. Table 6 Table 7 Table 16 Table 17-1 Table 17-2 Table 17-3 Table 8 Table 9 Table 10 Table 11-1 Table 11-2 Table 11-3 Table 11-4 Table 11-5 Table 11-6

[0151] A representative CLL-1 (NM_138337.6) cDNA sequence is provided below as SEQ ID NO: 31. Underlining, bolding, or italics indicate regions complementary to gRNA A, B, C, D, E, F, G, H, I, J, or O2 (or their reverse complements). Bolding and italics are used where there is overlap between two or more such regions. [ka] (SEQ ID NO: 600)

[0152] Additional CLL-1 isoform (ENST00000355690.8) cDNAs are provided as follows:

[0153] An additional CLL-1 isoform (NM_001207010.2) cDNA is provided as follows:

[0154] An additional CLL-1 isoform (NM_001300730.2) cDNA is provided as follows:

[0155] An additional CLL-1 isoform (NM_201623.4) cDNA is provided as follows:

[0156] gRNA targeting CD123 The present disclosure provides a number of useful gRNAs capable of targeting endonucleases to human CD123. In some embodiments, the gRNA capable of targeting endonucleases to human CD33 is a gRNA disclosed in either PCT Publication No. WO2020 / 047164 or WO2021 / 041977, each of which is incorporated herein by reference in its entirety. Table 1 below illustrates target domains within human endogenous CD123 that can be bound by the gRNAs described herein. [Table 12] [Table 13] [Table 14-1] [Table 14-2] [Table 14-3] [Table 14-4] [Table 14-5] [Table 14-6] [Table 14-7] [Table 14-8] [Table 14-9] [Table 14-10] [Table 14-11]

[0157] A representative CD123 (NM_001267713.1) cDNA sequence is provided below as SEQ ID NO: 31. Underlined or bold text indicates regions complementary to gRNA A, B, C, D, E, F, G, H, I, J, P3, or S3 (or their reverse complements). Bold text is used when there is overlap between two such regions. [ka] (SEQ ID NO: 700)

[0158] An additional CD123 isoform (NM_002183.4) cDNA is provided as follows: [ka] (SEQ ID NO: 701)

[0159] The underlined region indicates the region complementary to gRNA D1 (or its reverse complement).

[0160] gRNA targeting CD327 (Siglec-6) The present disclosure provides a number of useful gRNAs that can target endonucleases to human CD327, also known as Siglec-6. In some embodiments, targeting domain sequences that can be targeted by suitable gRNAs, which may include equivalent RNA targeting domain sequences (comprising RNA nucleotides instead of DNA nucleotides), include about 16 to about 30 nucleotides of a human CD327 isoform, for example, having the nucleic acid and amino acid sequence of CD327 (ENSG00000105492) isoforms ENST00000425629.8, ENST00000346477.7, ENST00000359982.8, ENST00000343300.8, ENST00000436458.5, ENST00000391797.3, ENST00000474054.1, ENST00000496422.5, or ENST00000489837.1.

[0161] gRNA targeting CD312 (EMR2) The present disclosure provides numerous useful gRNAs capable of targeting endonucleases to human EMR2, also known as CD312. Table 18 below illustrates target domains within human endogenous EMR2 that can be bound by the gRNAs described herein. In some embodiments, target domain sequences that can be targeted by suitable gRNAs, which may include equivalent RNA targeting domain sequences (comprising RNA nucleotides instead of DNA nucleotides), include human CD312 (EMR2) isoforms of about 16 to about 30 nucleotides, such as the nucleic acid and amino acid sequences of the full-length EMR2 (ENSG00000127507) isoform ENST00000315576.8 (Table 19). [Table 18] [Table 19-1] [Table 19-2] [Table 19-3] Table 19-4 Table 19-5 Table 19-6 Table 19-7 Table 19-8 Table 19-9 Table 19-10 Table 19-11 Table 19-12 Table 19-13 Table 19-14 Table 19-15 Table 19-16 Table 19-17 Table 19-18 Table 19-19 [Table 19-20] [Table 19-21] [Table 19-22]

[0162] Dual and multiple gRNA compositions and uses thereof In some embodiments, a gRNA described herein (e.g., a gRNA in Tables 1-19) can be used in combination with one or more gRNAs, e.g., to target a nuclease to one or more sites within a genome. In some embodiments, multiple gRNAs described herein (e.g., two or more gRNAs in Tables 1-19) can be used in combination, e.g., to target a nuclease to multiple sites within a genome. In some embodiments, a gRNA described herein (e.g., a gRNA in Tables 1-19) can be used in combination with a second gRNA, e.g., to target a nuclease to two sites within a genome. In some embodiments, a gRNA described herein (e.g., a gRNA in Tables 1-19) can be used in combination with a third gRNA, e.g., to target a nuclease to three sites within a genome. In some embodiments, a gRNA described herein (e.g., a gRNA in Tables 1-19) can be used in combination with a fourth gRNA, e.g., to target a nuclease to four sites within a genome. In some embodiments, a gRNA described herein (e.g., a gRNA in Tables 1-19) can be used in combination with a fifth gRNA, e.g., to target a nuclease to five sites within a genome. In some embodiments, a gRNA described herein (e.g., a gRNA in Tables 1-19) can be used in combination with a sixth gRNA, e.g., to target a nuclease to six sites within a genome. In some embodiments, a gRNA described herein (e.g., a gRNA in Tables 1-19) can be used in combination with a seventh gRNA, e.g., to target a nuclease to seven sites within a genome. In some embodiments, a gRNA described herein (e.g., a gRNA in Tables 1-19) can be used in combination with an eighth gRNA, e.g., to target a nuclease to eight sites within a genome. In some embodiments, a gRNA described herein (e.g., a gRNA in Tables 1-19) can be used in combination with a ninth gRNA, e.g., to target a nuclease to nine sites within a genome.In some embodiments, a gRNA described herein (e.g., a gRNA in Tables 1-19) can be used in combination with a tenth gRNA, e.g., to target a nuclease to ten sites within a genome. In some embodiments, a gRNA described herein (e.g., a gRNA in Tables 1-19) can be used in combination with more than ten gRNAs, e.g., to target a nuclease to more than ten sites within a genome.

[0163] For example, in some embodiments, it is desirable to produce hematopoietic cells that are deficient for a first lineage-specific cell surface antigen (e.g., a lineage-specific cell surface antigen, e.g., CD33, CLL-1, CD123, CD19, CD30, CD5, CD6, CD7, CD34, CD38, or BCMA) and a second lineage-specific cell surface antigen (e.g., a lineage-specific cell surface antigen, e.g., CD33, CLL-1, CD123, CD19, CD30, CD5, CD6, CD7, CD34, CD38, or BCMA), such that the cells are resistant to two agents, one that targets the first lineage-specific cell surface antigen and one that targets the second lineage-specific cell surface antigen. In some embodiments, it is desirable to contact cells with two or more different gRNAs that target different regions of a lineage-specific cell surface antigen (e.g., a lineage-specific cell surface antigen, e.g., CD33, CLL-1, CD123, CD19, CD30, CD5, CD6, CD7, CD34, CD38, or BCMA) to make two or more cleavages and create a deletion between the two cleavage sites.

[0164] Thus, the present disclosure provides various combinations of gRNAs and associated CRISPR systems, as well as cells produced by genome editing methods using such combinations of gRNAs and associated CRISPR systems. In some embodiments, a first lineage-specific cell surface antigen gRNA binds to a different nuclease than a second gRNA. For example, in some embodiments, a first lineage-specific cell surface antigen gRNA may bind to Cas9, and a second gRNA may bind to Cas12a, or vice versa.

[0165] Accordingly, the present disclosure provides various combinations of gRNAs and associated base editing systems, as well as cells produced by genome editing methods using such combinations of gRNAs and associated base editing systems.

[0166] In some embodiments, two or more (e.g., three, four, or more) gRNAs described herein are mixed. In some embodiments, each gRNA is in a separate container. In some embodiments, a kit described herein (e.g., a kit including one or more gRNAs according to Tables 1-19) also includes a Cas9 molecule or a nucleic acid encoding a Cas9 molecule.

[0167] In some embodiments, it may be desirable to contact cells with two or more different gRNAs that target different sites on CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), e.g., to effect multiple chemical changes to nucleobases. In some embodiments, the first and second gRNAs are gRNAs according to Tables 1-19 or variants thereof.

[0168] In some embodiments, it may be desirable to contact cells with two or more different gRNAs that target different sites on CD33, e.g., to effect multiple chemical changes to the nucleobases. In some embodiments, the first and second gRNAs are gRNAs according to Tables 1-19 or variants thereof.

[0169] In some embodiments, it may be desirable to contact cells with two or more different gRNAs that target different sites on CLL-1, e.g., to effect multiple chemical changes to the nucleobases. In some embodiments, the first and second gRNAs are gRNAs according to Tables 1-19 or variants thereof.

[0170] In some embodiments, it may be desirable to contact cells with two or more different gRNAs that target different sites on CD123, e.g., to effect multiple chemical changes to the nucleobases. In some embodiments, the first and second gRNAs are gRNAs according to Tables 1-19 or variants thereof.

[0171] In some embodiments, the first gRNA is a CD33 gRNA described herein. The second gRNA targets a lineage-specific cell surface antigen selected from BCMA, CD19, CD20, CD30, ROR1, B7H6, B7H3, CD23, CD33, CD38, C-type lectin-like molecule-1 (CLL-1), CS1, IL-5, L1-CAM, PSCA, PSMA, CD138, CD133, CD70, CD7, CD13, NKG2D, NKG2D ligand, CLEC12A, CD11, CD123, CD56, CD30, CD34, CD14, CD66b, CD41, CD61, CD62, CD235a, CD146, CD326, LMP2, CD22, CD52, CD10, CD3 / TCR, CD79 / BCR, and CD26. In certain embodiments, the second gRNA is a CLL-1 or CD123 gRNA.

[0172] In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA according to Tables 1-5 or a variant thereof), and the second gRNA is a gene encoding ... They target lineage-specific cell surface antigens associated with specific types of cancer, such as TCR (T-cell lymphoma and leukemia), CD79 / B-cell receptor (BCR) (B-cell lymphoma and leukemia), CD26 (epithelial and lymphoid malignancies), human leukocyte antigen (HLA)-DR, HLA-DP, HLA-DQ (lymphoid malignancies), RCAS1 (gynecologic cancers, cholangiocarcinoma, and pancreatic ductal adenocarcinoma), and prostate-specific membrane antigen.

[0173] In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA or variant thereof according to Tables 1-5), and the second gRNA targets a lineage-specific cell surface antigen selected from CD7, CD13, CD19, CD22, CD20, CD25, CD32, CD38, CD44, CD45, CD47, CD56, CD96, CD117, CD123, CD135, CD174, CLL-1, folate receptor beta, IL1RAP, MUC1, NKG2D / NKG2DL, TIM-3, or WT1.

[0174] In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA according to Tables 1-5 or a variant thereof) and the second gRNA is a gRNA encoding any of CD1a, CD1b, CD1c, CD1d, CD1e, CD2, CD3, CD3d, CD3e, CD3g, CD4, CD5, CD6, CD7, CD8a, CD8b, CD9, CD10, CD11a, CD11b, CD11c, CD11d, CDw12, CD13, CD14, CD15, CD16, CD16b, CD17, CD18, CD19, CD20, CD21, CD22, CD23, CD24, CD25, CD26, CD27, CD28 , CD29, CD30, CD31, CD32a, CD32b, CD32c, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59 , CD60a, CD61, CD62E, CD62L, CD62P, CD63, CD64a, CD65, CD65s, CD66a, CD66b, CD66c, CD66F, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75 S, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85A, CD85C, CD85D, CD85E, CD85F, CD85G, CD85H, CD85I, CD85J, CD85K, CD86, CD87, CD88, CD 89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD99R, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD107b, CD108 , CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD121a, CD121b, CD122, CD123,CD124、CD125、CD126、CD127、CD129、CD130、CD131、CD132、CD133、CD134、CD 135、CD136、CD137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD1 4、CDw145、CD146、CD147、CD148、CD150、CD152、CD152、CD153、CD154、CD155 、CD156a、CD156b、CD156c、CD157、CD158b1、CD158b2、CD158d、CD158e1 / e2、C D158f、CD158g、CD158h、CD158i、CD158j、CD158k、CD159a、CD159c、CD160、C D161、CD163、CD164、CD165、CD166、CD167a、CD168、CD169、CD170、CD171、CD1 72a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、C D179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CD191、CD 192、CD193、CD194、CD195、CD196、CD197、CDw198、CDw199、CD200、CD201、CD 202b、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210a、CDw210b、 CD212, CD213a1, CD213a2, CD215, CD217, CD218a, CD218b, CD220, CD221, CD222, CD223, CD224, CD225, CD226, CD227, CD228, CD229, CD230, CD231, CD232 CD233, CD234, CD235a, CD235b, CD236, CD236R, CD238, CD239, CD240, CD241, CD242, CD243, CD244, CD245, CD246, CD247, CD248, CD249, CD252, CD253, C D254, CD256, CD257, CD258, CD261, CD262, CD263, CD264, CD265, CD266, CD267, CD268, CD269, CD270, CD272, CD273, CD274, CD275, CD276, CD277CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD286, CD288, CD289, CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a , CD300c, CD300e, CD301, CD302, CD303, CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD309, CD312, CD314, CD315, CD316, CD317 , targeting a lineage-specific cell surface antigen selected from CD318, CD319, CD320, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD359, CD360, CD361, CD362, or CD363.

[0175] In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA according to Tables 1-5 or a variant thereof), and the second gRNA is a CD34 gRNA, e.g., a gRNA according to Tables 1-5 or a variant thereof, or a variant thereof. , CD30, CD31, CD32, CD33, CD34, CD35, CD36, CD37, CD38, CD39, CD40, CD41, CD42a, CD42b, CD42c, CD42d, CD43, CD44, CD45, CD45RA, CD45RB, CD45RC, CD45RO, CD46, CD47, CD48, CD49a, CD49b, CD49c, CD49d, CD49e, CD49f, CD50, CD51, CD52, CD53, CD54, CD55, CD56, CD57, CD58, CD59, CD60a, CD60b, C D60c, CD61, CD62E, CD62L, CD62P, CD63, CD64, CD65s, CD66a, CD66b, CD66c, CD66d, CD66e, CD66f, CD67, CD68, CD69, CD70, CD71, CD72, CD73, CD74, CD75, CD75s, CD77, CD79a, CD79b, CD80, CD81, CD82, CD83, CD84, CD85a, CD85b, CD85c, CD85d, CD85e, CD85f, CD85g, CD85h, CD85i, CD85j, CD85k, CD 86, CD87, CD88, CD89, CD90, CD91, CD92, CD93, CD94, CD95, CD96, CD97, CD98, CD99, CD100, CD101, CD102, CD103, CD104, CD105, CD106, CD107a, CD10 7b, CD108, CD109, CD110, CD111, CD112, CD113, CD114, CD115, CD116, CD117, CD118, CD119, CD120a, CD120b, CD121a, CD121b, CD122, CD123, CD124,CD125、CD126、CD127、CD128a、CD128b、CD129、CD130、CD131、CD132、CD133、CD134、CD135、CD136、CD137、CD138、CD139、CD140a、CD140b、CD141、CD142、CD143、CD144、CD146、CD147、CD148、CD150、CD151、CD152、CD153、CD154、CD155、CD156a、CD156b、CD156c、CD157、CD158a、CD158b1、CD158b2、CD158c、CD158d、CD158e1、CD158e2、CD158f、CD158g、CD158h、CD158i、CD158j、CD158k、CD159a、CD159c、CD160、CD161、CD162、CD163、CD164、CD165、CD166、CD167a、CD167b、CD168、CD169、CD170、CD171、CD172a、CD172b、CD172g、CD173、CD174、CD175、CD175s、CD176、CD177、CD178、CD179a、CD179b、CD180、CD181、CD182、CD183、CD184、CD185、CD186、CD191、CD192、CD193、CD194、CD195、CD196、CD197、CD198、CD199、CD200、CD201、CD202b、CD203c、CD204、CD205、CD206、CD207、CD208、CD209、CD210a、CD210b、CD212、CD213a1、CD213a2、CD215、CD217、CD218a、CD218b、CD220、CD221、CD222、CD223、CD224、CD225、CD226、CD227、CD228、CD229、CD230、CD231、CD232、CD233、CD234、CD235a、CD235b、CD236、CD238、CD239、CD240CE、CD240D、CD241、CD242、CD243、CD244、CD245、CD246、CD247、CD248、CD249、CD252、CD253、CD254、CD256、CD257、CD258、CD261、CD262、CD263、CD264、CD265、CD266、CD267、CD268、CD269、CD270、CD271、CD272, CD273, CD274, CD275, CD276, CD277, CD278, CD279, CD280, CD281, CD282, CD283, CD284, CD286, CD288, CD289, CD290, CD292, CDw293, CD294, CD295, CD296, CD297, CD298, CD299, CD300a, CD300c, CD300d, CD300e, CD300f, CD300g, CD301, CD302, CD303, CD304, CD305, CD306, CD307a, CD307b, CD307c, CD307d, CD307e, CD309, CD312, CD314, CD315, CD 316, CD317, CD318, CD319, CD320, CD321, CD322, CD324, CD325, CD326, CD327, CD328, CD329, CD331, CD332, CD333, CD334, CD335, CD336, CD337, CD338, CD339, CD340, CD344, CD349, CD350, CD351, CD352, CD353, CD354, CD355, CD357, CD358, CD360, CD361, CD362, CD363, CD364, CD365, CD366, CD367, CD368, CD369, CD370, and CD371.

[0176] In some embodiments, the second gRNA is a gRNA disclosed in any of PCT Publication Nos. WO2017 / 066760, WO2019 / 046285, WO2018 / 160768, or Borot et al. PNAS (2019) 116(24):11978-11987, each of which is incorporated herein by reference in its entirety.

[0177] In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA according to Tables 1-5 or a variant thereof), and the second gRNA is a gRNA selected from the group consisting of CD19; CD123; CD22; CD30; CD171; CS-1 (CD2 subset 1, also referred to as CRACC, SLAMF7, CD319, and 19A24); C-type lectin-like molecule-1 (CLECL1); epidermal growth factor receptor variant III (EGFRvIII); ganglioside G2 (CD2); ganglioside GD3 (aNeu5Ac(2-8)aNeu5Ac(2-3)bDGalp(1-4)bDGlep(1-1)Cer); TNF receptor family member B cell maturation (BCMA), Tn antigen ((Tn Ag) or (GalNAc.alpha.-Ser / Thr)); prostate-specific membrane antigen (PSMA); receptor tyrosine kinase-like orphan receptor 1 (ROR1); Fms-like tyrosine kinase 3 (FLT3); tumor-associated glycoprotein 72 (TAG72); CD38; CD44v6; carcinoembryonic antigen (CEA); epithelial cell adhesion molecule (EPCAM); B7H3 (CD276); KIT (CD117); interleukin-13 receptor subunit alpha-2 (IL-13Ra2 or CD213A2); mesothelin; interleukin-11 receptor alpha (IL-11Ra); prostate stem cell antigen (PSCA); proteolytic enzyme serine 21 (testisin or PRSS21); vascular endothelial growth factor receptor 2 (VEGFR2); Lewis (Y ) antigen; CD24; platelet-derived growth factor receptor beta (PDGFR-beta); stage-specific embryonic antigen-4 (SSEA-4); CD20; folate receptor alpha; receptor tyrosine-protein kinase ERBB2 (Her2 / neu); mucin 1, cell surface associated (MUC1); epidermal growth factor receptor (EGFR); neural cell adhesion molecule (NCAM); prostase; prostatic acid phosphatase (PAP); elongation factor 2 mutated (ELF2M); ephrin B2; fibroblast activation protein alpha (FAP); insulin-like growth factor I receptor (IGF-I receptor), carbonic anhydrase IX (CAIX), proteasome (prosome, macropain) subunit, beta type 9 (LMP2); glycoprotein 100 (gp100);Oncogene fusion protein consisting of breakpoint cluster region (BCR) and Abelson murine leukemia viral oncogene homolog 1 (Abl) (bcr-abl); tyrosinase; ephrin type A receptor 2 (EphA2); fucosyl-GM1; sialyl Lewis adhesion molecule (sLe); ganglioside GM3 (aNeu5Ac(2-3)bDGalp(1-4)bDGlcp(1-1)Cer); transglutaminase 5 (TGS5); high molecular weight melanoma-associated antigen (HMWMAA); o-acetyl-GD2 ganglioside (OAcGD2); folate receptor beta ; tumor vascular endothelial marker 1 (TEM1 / CD248); tumor vascular endothelial marker 7-related (TEM7R); claudin 6 (CLDN6); thyroid-stimulating hormone receptor (TSHR); G protein-coupled receptor class C group 5, member D (GPRC5D); chromosome X open reading frame 61 (CXORF61); CD97; CD179a; anaplastic lymphoma kinase (ALK); polysialic acid; placenta-specific 1 (PLAC1); hexasaccharide moiety of globoH glycoceramide (GloboH); mammary differentiation antigen (NY-BR-1); uroplakin 2 (UPK2); hepatitis A virus cellular receptor 1 (HAVCR1); adrenergic receptor beta 3 (ADRB3); pannexin 3 (PANX3); G protein-coupled receptor 20 (GPR20); lymphocyte antigen 6 complex; locus K9 (LY6K); taste receptor 51E2 (OR51E2); TCR gamma alternative reading frame protein (TARP); Wilms tumor protein (WT1); cancer / testis antigen 1 (NY-ESO-1); cancer / testis antigen 2 (LAGE-1a); melanoma-associated antigen 1 (MAGE-A1), ETS translocation located on chromosome 12p variant gene 6 (ETV6-AML); sperm protein 17 (SPA17); X antigen family, member 1A (XAGE1); angiopoietin-binding cell surface receptor 2 (Tie2); melanoma cancer testis antigen-1 (MAD-CT-1); melanoma cancer testis antigen-2 (MAD-CT-2); Fos-related antigen 1; tumor protein p53 (p53); p53 mutant; prostein; survivin; telomerase; prostate carcinoma tumor antigen-1 (PCTA-1 or galectin-8), melanoma antigen recognized by T cells 1 (MelanA or MART1);Rat sarcoma (Ras) mutant; human telomerase reverse transcriptase (hTERT); sarcoma translocation breakpoint; melanoma inhibitor of apoptosis (ML-1AP); ERG (transmembrane protease, serine 2 (TMPRSS2) ETS fusion gene); N-acetylglucosaminyltransferase V (NA17); paired box protein Pax-3 (PAX3); androgen receptor; cyclin B1; v-myc avian myelocytomatosis viral oncogene neuroblastoma-derived homolog (MYCN); Ras homolog family member C (RhoC); tyrosinase-related protein 2 (TRP-2); cytochrome P450 1B1 (CYP1B1); CCCTC-binding factor (zinc finger protein)-like (BORIS or related regulator of imprinted sites), squamous cell carcinoma antigen recognized by T cells 3 (SART3); paired box protein Pax-5 (PAX5); proacrosin-binding protein sp32 (OY-TES1); lymphocyte-specific protein tyrosine kinase (LCK); A-kinase anchoring protein 4 (AKAP-4); synovial sarcoma, X-breakpoint 2 (SSX2); receptor for advanced glycation end products (RAGE-1); renal ubiquitous 1 (RU1); renal ubiquitous 2 (RU2); legumain; human papillomavirus E6 (HPV E6); human papillomavirus E7 (HPV E7); intestinal carboxylesterase; heat shock protein 70-2 mutated (mutated) hsp70-2); CD79a; CD79b; CD72; leukocyte-associated immunoglobulin-like receptor 1 (LAIR1); Fc fragment of IgA receptor (FCAR or CD89); leukocyte immunoglobulin-like receptor subfamily A member 2 (LILRA2); CD300 molecule-like family member f (CD300LF); C-type lectin domain family 12 member A (CLEC12A); bone marrow stromal cell antigen 2 (BST2); EGF-like module containing mucin-like hormone receptor-like 2 (EMR2), lymphocyte antigen 75 (LY75); glypican-3 (GPC3); Fc receptor-like 5 (FCRL5); and immunoglobulin lambda-like polypeptide 1 (IGLL1).

[0178] In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA according to Tables 1-5 or a variant thereof), and the second gRNA targets a lineage-specific cell surface antigen selected from CD11a, CD18, CD19, CD20, CD31, CD33, CD34, CD44, CD45, CD47, CD51, CD58, CD59, CD63, CD97, CD99, CD100, CD102, CD123, CD127, CD133, CD135, CD157, CD172b, CD217, CD300a, CD305, CD317, CD321, and CLL-1.

[0179] In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA or variant thereof according to Tables 1-5), and the second gRNA targets a lineage-specific cell surface antigen selected from CD123, CLL-1, CD38, CD135 (FLT3), CD56 (NCAM1), CD117 (c-KIT), FRβ (FOLR2), CD47, CD82, TNFRSF1B (CD120B), CD191, CD96, PTPRJ (CD148), CD70, LILRB2 (CD85D), CD25 (IL2R alpha), CD44, CD96, NKG2D ligand, CD45, CD7, CD15, CD19, CD20, CD22, CD37, and CD82.

[0180] In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA according to Tables 1-5 or a variant thereof) and the second gRNA is a gRNA selected from the group consisting of CD7, CD11a, CD15, CD18, CD19, CD20, CD22, CD25, CD31, CD34, CD37, CD38, CD44, CD45, CD47, CD51, CD56, CD58, CD59, CD63, CD70, CD82, CD85D, CD96, CD 97, CD99, CD100, CD102, CD117, CD120B, CD123, CD127, CD133, CD135, CD148, CD157, CD172b, CD191, CD217, CD300a, CD305, CD317, CD321, CLL-1, FRβ (FOLR2), or NKG2D ligand.

[0181] In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA according to Tables 1-5 or a variant thereof) and the second gRNA targets CLL-1.

[0182] In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA according to Tables 1-5 or a variant thereof) and the second gRNA targets CD123.

[0183] In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA according to Tables 1-5 or a variant thereof) and the second gRNA comprises a sequence in Tables 1-19.

[0184] In some embodiments, the first gRNA is a CD33 gRNA comprising a targeting domain, wherein the targeting domain comprises a sequence in Tables 1-5, and the second gRNA comprises a targeting domain corresponding to a sequence in Tables 1-19.

[0185] In some embodiments, the first gRNA is a CD33 gRNA described herein (e.g., a gRNA according to Tables 1-s or a variant thereof), and the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more gRNAs comprise a sequence in Tables 1-19.

[0186] In some embodiments, the first gRNA is a CD33 gRNA comprising a targeting domain, wherein the targeting domain comprises a sequence in Tables 1-5, and the third, fourth, fifth, sixth, seventh, eighth, ninth, tenth, or more gRNAs comprise targeting domains corresponding to sequences in Tables 1-19.

[0187] In some embodiments, the second gRNA is a gRNA disclosed in any of WO2017 / 066760, WO2019 / 046285, WO / 2018 / 160768, or Borot et al. PNAS June 11, 2019 116(24)11978-11987.

[0188] Cells containing two or more chemical changes to nucleic acid bases In some embodiments, the engineered cells described herein comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemical alterations to nucleobases. In some embodiments, the engineered cells described herein comprise two or more mutations (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more). In some embodiments, the engineered cells described herein comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemical alterations to nucleobases in CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2). In some embodiments, the engineered cells described herein comprise two or more (e.g., 2, 3, 4, 5, 6, 7, 8, 9, 10, or more) chemical alterations to a nucleobase, wherein a first chemical alteration to a nucleobase is in CD33 and a second chemical alteration to a nucleobase is in a second lineage-specific cell surface antigen. Such cells may, in some embodiments, be resistant to two antigens: an anti-CD33 agent and an agent targeting the second lineage-specific cell surface antigen. In some embodiments, such cells can be produced using two or more gRNAs described herein, e.g., a gRNA in Tables 1-19 and a second gRNA. In some embodiments, such cells can be produced using two or more gRNAs described herein, e.g., a gRNA in Tables 1-19 and a second gRNA. In some embodiments, the cells can be produced using, for example, ZFNs or TALENs. The present disclosure also provides populations comprising the cells described herein.

[0189] In some embodiments, the second chemical alteration to the nucleobase is in a gene encoding a lineage-specific cell surface antigen, such as those listed in the preceding section. In some embodiments, the second mutation is at a site listed in Tables 1-19.

[0190] Typically, mutations effected by the methods and compositions provided herein, e.g., mutations in target genes such as CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), and / or any other target genes mentioned in this disclosure, result in a loss of function of the gene product encoded by the target gene, e.g., in the case of mutations in the CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes, result in a loss of function of the CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) protein. In some embodiments, the loss of function is a reduction in the level of expression of the gene product, e.g., a reduction to a lower level of expression or complete abolition of expression of the gene product. In some embodiments, the mutation results in expression of a non-functional variant of the gene product. A gene product characterized by an altered amino acid sequence that renders the gene product non-functional, e.g., in the case of mutations that create premature stop codons in the coding sequence, truncated gene products, or in the case of mutations that create nonsense or missense mutations. In some embodiments, the function of the gene product is to bind or recognize a binding partner. In some embodiments, the reduction in expression of a gene product, e.g., the gene product of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), the gene product of a second lineage-specific cell surface antigen, or both gene products, is 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 5% or less, 2% or less, or 1% or less of the level in wild-type or unengineered counterpart cells.

[0191] In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the copies of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) in a population of cells generated by the methods and / or using the compositions provided herein have a mutation. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the copies of a second lineage-specific cell surface antigen in a population of cells have a mutation. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the copies of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) and the second lineage-specific cell surface antigen in the population of cells have a mutation. In some embodiments, the population comprises one or more wild-type cells. In some embodiments, the population comprises one or more cells comprising a wild-type copy of one of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2). In some embodiments, the population comprises one or more cells comprising a wild-type copy of one of the second lineage-specific cell surface antigens.

[0192] cell Some aspects of the present disclosure provide genetically engineered cells that contain modifications in their genomes that result in loss of expression of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), or expression of variant forms of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) that are not recognized by immunotherapeutic agents targeting CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2). Such modifications can be introduced via base editing events. Such base editing events can include, but are not limited to, chemical changes to nucleic acid bases. In certain embodiments, editing events can include deamination of cytosine. In some embodiments, editing events can include deamination of adenine. In certain embodiments, the editing event may comprise a nucleobase transition. In certain embodiments, the editing event may comprise a nucleobase transversion. In certain embodiments, the editing event may comprise converting a cytosine-guanine (CG) base pair to a thymine-adenine (TA) base pair within the target nucleic acid molecule. In certain embodiments, the editing event may comprise converting a thymine-adenine (TA) base pair to a cytosine-guanine (CG) base pair within the target nucleic acid molecule. In certain embodiments, the editing event may comprise introducing a premature stop codon into the target nucleic acid molecule. In certain embodiments, the editing event may comprise introducing a splice site into the target nucleic acid molecule. In certain embodiments, the editing event may comprise destroying a splice site within the target nucleic acid molecule.Accordingly, some aspects of the present disclosure provide genetically engineered cells that contain multiple modifications in their genome that result in loss of expression of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), or expression of variant forms of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) that are not recognized by immunotherapeutic agents that target CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2).

[0193] For example, some aspects of the present disclosure provide novel cells having modifications (e.g., stop codons or mutant splice sites) in, for example, endogenous CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes. In particular, provided herein are cell populations comprising a plurality of genetically engineered hematopoietic stem or progenitor cells, wherein at least a portion of the cells have: (i) an edited CD33 (Siglec-3) gene; (ii) an edited CLL-1 gene; (iii) an edited CD123 gene; (iv) an edited CD327 (Siglec-6) gene; (v) an edited CD312 (EMR2) gene; (vi) an edited CD33 (Siglec-3) gene and an edited CLL-1 gene; (vii) an edited CD33 (Siglec-3) gene and an edited CD123 gene; (viii) an edited CD33 (Siglec-3) gene and an edited CD327 (Siglec-6) gene; or (ix) an edited CD33 (Siglec-3) gene and an edited CD312 (EMR2) gene. genes, (x) an edited CD33 (Siglec-3) gene, an edited CLL-1 gene, and an edited CD123 gene, (xi) an edited CD33 (Siglec-3) gene, an edited CLL-1 gene, an edited CD123 gene, and an edited CD327 (Siglec-6) gene, (xii) an edited CD33 (Siglec-3) gene, an edited CLL-1 gene, an edited CD123 gene, an edited CD327 (Siglec-6) gene, and an edited CD312 (EMR2) gene, or (xiii) an edited CD33 (Siglec-3) gene, an edited CLL-1 gene, an edited CD123 gene, an edited CD327 (Siglec-6) gene, and / or an edited CD312 (EMR2) gene. In some embodiments, cells (e.g., HSCs or HPCs) with CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) modifications are generated using nucleases and / or gRNAs described herein.In some embodiments, cells (e.g., HSCs or HPCs) having modifications of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) and a second lineage-specific cell surface antigen are generated using nucleases and / or gRNAs described herein. In some embodiments, the modifications in the cell's genome are mutations in the genomic sequence encoding CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2). In some embodiments, the modifications are affected via genome editing, e.g., using Cas nucleases and gRNAs targeting the CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) target sites provided herein, or including targeting domain sequences provided herein. It is understood that the cells may be generated by contacting the cells themselves with the nuclease and / or gRNA, or the cells may be daughter cells of cells that have been contacted with the nuclease and / or gRNA. In some embodiments, the cells (e.g., HSCs) described herein are capable of reconstituting the hematopoietic system of a subject. In some embodiments, the cells (e.g., HSCs) described herein are capable of one or more (e.g., all) of engrafting in a human subject, producing myeloid cells, and producing lymphoid cells.

[0194] While the compositions, methods, strategies, and treatment modalities provided herein may be applied to any cell or cell type, some exemplary cells and cell types that are particularly suitable for genomic modification in the CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) genes in accordance with aspects of the present disclosure are described in more detail herein. Those skilled in the art will recognize, however, that the provision of such examples is for purposes of illustrating some specific embodiments, and that additional suitable cells and cell types will be apparent to those skilled in the art based on the present disclosure, which is not limited in this respect.

[0195] In some embodiments, the cells described herein are human cells that have mutations in exons of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2).

[0196] In some embodiments, the cells described herein are human cells with mutations in introns of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2).

[0197] In some embodiments, the cells described herein are human cells that have a mutation in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, and / or exon 7 of CD33.

[0198] In some embodiments, the cells described herein are human cells having a mutation in exon 1, exon 2, exon 3, exon 4, exon 5, exon 6, exon 7, exon 8, exon 9, exon 10, exon 11, exon 12, exon 13, exon 14, exon 15, exon 16, exon 17, exon 18, exon 19, exon 20, and / or exon 21 of CD312 (EMR2).

[0199] In some embodiments, the cells described herein are human cells with a mutation in exon 2 of CLL-1 and / or CD123. In some embodiments, the cells described herein are human cells with a mutation in exon 4 of CLL-1 and / or CD123 5. In some embodiments, the cells described herein are human cells with a mutation in exon 6 of CD123.

[0200] In some embodiments, the populations of cells described herein include hematopoietic stem cells (HSCs), hematopoietic progenitor cells (HPCs), or both (HSPCs). In some embodiments, the cells are CD34+. In some embodiments, the cells are hematopoietic cells. In some embodiments, the cells are hematopoietic stem cells. In some embodiments, the cells are hematopoietic progenitor cells. In some embodiments, the cells are immune effector cells. In some embodiments, the cells are lymphocytes. In some embodiments, the cells are T lymphocytes. In some embodiments, the cells are NK cells. In some embodiments, the cells are stem cells. In some embodiments, the stem cells are embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells, or tissue-specific stem cells.

[0201] In some embodiments, the cells comprise only one genetic modification. In some embodiments, the cells are genetically modified only at the CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) loci. In some embodiments, the cells are genetically modified at a second locus. In some embodiments, the cells do not comprise a transgenic protein, e.g., do not comprise a CAR.

[0202] Some aspects of the present disclosure provide genetically engineered hematopoietic cells that contain modifications in their genome that result in loss of expression of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), or expression of variant forms of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) that are not recognized by immunotherapeutic agents targeting CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2). In some embodiments, the modified cells described herein are substantially free of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) protein. In some embodiments, the modified cells described herein are substantially free of wild-type CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) proteins, but comprise mutant CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) proteins. In some embodiments, the mutant CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) proteins are not bound by agents that target CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) for therapeutic purposes. In some embodiments, the genetically engineered cells comprising the modification in their genome result in reduced cell surface expression of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), and / or reduced binding by immunotherapeutic agents targeting CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), e.g., compared to hematopoietic cells (e.g., HSCs) of the same cell type but that do not comprise the genomic modification.

[0203] In some embodiments, the cell is a hematopoietic cell, e.g., a hematopoietic stem cell, a hematopoietic progenitor cell (HPC), a hematopoietic stem or progenitor cell. Hematopoietic stem cells (HSCs) are cells characterized by pluripotency, self-renewal, and / or the ability to generate and / or reconstitute all lineages of the hematopoietic system, including both myeloid and lymphoid progenitor cells, which further give rise to myeloid cells (e.g., monocytes, macrophages, neutrophils, basophils, dendritic cells, erythrocytes, platelets, etc.) and lymphoid cells (e.g., T cells, B cells, NK cells), respectively. HSCs are characterized by the expression of one or more cell surface markers, e.g., CD34 (e.g., CD34+), and the absence of cell surface markers associated with lineage commitment, which can be used to identify and / or isolate HSCs. In some embodiments, the genetically engineered cells described herein (e.g., genetically engineered HSCs) typically do not express, express reduced amounts of, or express variant cell surface markers that are not recognized by immunotherapeutic agents that target cell surface markers, but are still capable of self-renewal and generating and / or reconstituting all lineages of the hematopoietic system.

[0204] In some embodiments, the cell populations described herein comprise a plurality of hematopoietic stem cells; in some embodiments, the cell populations described herein comprise a plurality of hematopoietic progenitor cells; in some embodiments, the cell populations described herein comprise a plurality of hematopoietic stem cells and a plurality of hematopoietic progenitor cells.

[0205] In some embodiments, the genetically engineered cells provided herein comprise one or more genomic modifications in addition to two or more genomic modifications, e.g., a genomic modification that results in loss of expression of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), or expression of a variant form of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) that is not recognized by immunotherapeutic agents targeting CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2).

[0206] In some embodiments, the genetically engineered cells provided herein comprise a genomic modification that results in loss of expression of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), or expression of a variant form of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) that is not recognized by an immunotherapeutic agent that targets CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), and further comprise an expression construct encoding a chimeric antigen receptor, e.g., in the form of an expression construct encoding a CAR integrated into the genome of the cell. In some embodiments, the CAR comprises a binding domain, e.g., an antibody fragment, that binds to CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2).

[0207] Some aspects of the present disclosure provide engineered immune effector cells that contain modifications in their genome that result in loss of expression of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), or expression of variant forms of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) that are not recognized by immunotherapeutics targeting CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2). In some embodiments, the immune effector cells are lymphocytes. In some embodiments, the immune effector cells are T lymphocytes. In some embodiments, the T lymphocytes are alpha / beta T lymphocytes. In some embodiments, the T lymphocytes are gamma / delta T lymphocytes. In some embodiments, the immune effector cells are natural killer T (NKT) cells. In some embodiments, the immune effector cells are natural killer (NK) cells. In some embodiments, the immune effector cells do not express an endogenous transgene, e.g., a transgenic protein. In some embodiments, the immune effector cells express a chimeric antigen receptor (CAR). In some embodiments, the immune effector cells express a CAR that targets CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2). In some embodiments, the immune effector cells do not express a CAR that targets CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2).

[0208] In some embodiments, the genetically engineered cells provided herein do not substantially express CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) proteins, e.g., do not express CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) proteins, which can be measured by a suitable method, such as, for example, an immunostaining method. In some embodiments, the genetically engineered cells provided herein do not substantially express wild-type CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) proteins, but do express mutant CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) protein variants, e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)-targeting immunotherapeutics, e.g., CAR-T cell therapeutics, or anti-CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) antibodies, antibody fragments, or antibody-drug conjugates (ADCs).

[0209] In some embodiments, the HSCs are obtained from a subject, such as a human subject. Methods for obtaining HSCs are described, for example, in PCT / US2016 / 057339, which is incorporated herein by reference in its entirety. In some embodiments, the HSCs are peripheral blood HSCs. In some embodiments, the mammalian subject is a non-human primate, rodent (e.g., mouse or rat), cow, pig, horse, or livestock. In some embodiments, the HSCs are obtained from a human subject, such as a human subject with a hematopoietic malignancy. In some embodiments, the HSCs are obtained from a healthy donor. In some embodiments, the HSCs are obtained from a subject to whom immune cells expressing a chimeric receptor will subsequently be administered. HSCs administered to the same subject from whom the cells were obtained are referred to as autologous cells, whereas HSCs obtained from a subject other than the subject to whom the cells will be administered are referred to as allogeneic cells.

[0210] In some embodiments, the population of engineered cells is a heterogeneous population of cells, e.g., a heterogeneous population of engineered cells comprising different CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) mutations. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the copies of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) in the population of engineered cells have a mutation. In some embodiments, at least 40%, at least 50%, at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95% of the copies of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) in the population of engineered cells have a mutation affected by the genome editing approaches described herein, e.g., by a CRISPR / Cas system using the gRNAs provided herein. As an example, a population can include a plurality of different CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) mutations, each of the plurality of mutations contributing to a proportion of copies of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) in the population of cells that have the mutation.

[0211] In some embodiments, the expression of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) on the genetically engineered hematopoietic cells is compared to the expression of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) on naturally occurring hematopoietic cells (e.g., wild-type counterparts). In some embodiments, the genetic engineering results in a reduction in the expression levels of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) by at least 50%, at least 60%, at least 70%, at least 80%, 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%, or at least 99% compared to the expression of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) on naturally occurring hematopoietic cells (e.g., wild-type counterparts). For example, in some embodiments, the genetically engineered hematopoietic cells express less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) compared to naturally occurring hematopoietic cells (e.g., wild-type counterparts).

[0212] In some embodiments, the genetic engineering results in a reduction in the expression levels of wild-type CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) by at least 50%, at least 60%, at least 70%, at least 80%, 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%, or at least 99% compared to the expression levels of wild-type CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) on naturally occurring hematopoietic cells (e.g., wild-type counterparts). That is, in some embodiments, the genetically engineered hematopoietic cells express less than 20%, less than 19%, less than 18%, less than 17%, less than 16%, less than 15%, less than 14%, less than 13%, less than 12%, less than 11%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, or less than 1% of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) compared to naturally occurring hematopoietic cells (e.g., wild-type counterparts).

[0213] In some embodiments, the genetic engineering results in a reduction in the expression level of a wild-type lineage-specific cell surface antigen (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)) by at least 50%, at least 60%, at least 70%, at least 80%, 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%, or at least 99% compared to a suitable control (e.g., a cell or plurality of cells). In some embodiments, a suitable control comprises the level of the wild-type lineage-specific cell surface antigen measured or predicted in multiple unengineered cells from the same subject. In some embodiments, a suitable control comprises the level of the wild-type lineage-specific cell surface antigen measured or predicted in multiple cells from a healthy subject. In some embodiments, a suitable control comprises the level of wild-type lineage-specific cell surface antigen measured or expected in a population of cells from a pool of healthy individuals (e.g., 10, 20, 50, or 100 individuals). In some embodiments, a suitable control comprises the level of wild-type lineage-specific cell surface antigen measured or expected in a subject in need of a treatment described herein, e.g., anti-CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) therapy, e.g., the subject has cancer and cells of the cancer express CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2).

[0214] In some embodiments, the methods of genetically engineering cells described herein include providing a wild-type cell, e.g., a wild-type hematopoietic stem or progenitor cell. In some embodiments, the wild-type cell is an unedited cell that contains (e.g., expresses) two functional copies of genes encoding CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2).

[0215] In some embodiments, the cells used in the methods are naturally occurring or non-engineered cells.

[0216] In some embodiments, wild-type cells express CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), or give rise to further differentiated cells that express CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) at levels comparable to (or within 90%-110%, 80%-120%, 70%-130%, 60%-140%, or 50%-150% of) a cell line expressing CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2).

[0217] In some embodiments, wild-type cells give rise to further differentiated cells that bind CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) (e.g., anti-CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) antibodies) or bind such antibodies at a level comparable to (or within 90%-110%, 80%-120%, 70%-130%, 60%-140%, or 50%-150%) the binding of the antibodies to cell lines expressing CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2). Antibody binding may be measured, for example, by flow cytometry or immunohistochemistry.

[0218] Methods of production, treatment, and administration The present disclosure provides, among other things, compositions and methods for multiplex base editing and generating genetically engineered cells.

[0219] Multiplex engineering is a strategy and method in which multiple gene targets are engineered in the same cell in the same manufacturing process.Multiplex engineering allows for the removal or modification of two or more separate genes, and therefore allows for the combined or sequential use of targeted therapies directed at two or more separate targets, which may be particularly useful for preventing escape mechanisms involving tumor cells that downregulate target expression.

[0220] In particular, the present disclosure provides methods for multiplex base editing, which involves converting specific DNA bases to alternative bases at targeted genomic loci. As such, base editing does not require cleavage, reducing the risk of translocation errors. The methods provided herein can be used, for example, to efficiently knock out the expression of multiple genomic targets, such as cell surface targets, from hematopoietic stem cells (HSCs) and / or hematopoietic stem and progenitor cells (HSPCs) using a single base editing step.

[0221] In particular, the present disclosure provides methods for multiplex base editing in which hematopoietic stem cells (HSCs) and / or hematopoietic stem and progenitor cells (HSPCs) are genetically modified to remove surface targets, and these cells are then provided to a patient as a hematopoietic stem cell transplant (HSCT). Once these cells engraft in the bone marrow, the patient's healthy cells can be protected from the negative on-target, off-tumor effects of targeted immunotherapy, for example, because they no longer express the surface targets, leaving only cancer cells exposed. Thus, in some embodiments, the present disclosure provides compositions and methods for targeted therapy to selectively destroy cancer cells while sparing healthy cells, among other things. As a result, the engineered cells described herein can be designed to limit the on-target toxicity associated with these targeted therapies, thereby enhancing their utility and broadening their applicability. In certain embodiments, the engineered cells can be administered in combination with targeted therapeutic agents, such as chimeric antigen receptor ("CAR")-T therapies, bispecific antibodies, and antibody-drug conjugates designed to target cell surface proteins.

[0222] Without wishing to be bound by theory, a multiplex approach may offer advantages in at least two areas. First, in the context of cancer, target expression may vary among tumor cells from the same patient, a phenomenon known as tumor heterogeneity. Applying a therapy such as multispecific CAR-T may reduce this concern. Second, it is theoretically possible for tumor cells to downregulate target expression to avoid death, a phenomenon known as tumor escape. Again, simultaneously pursuing multiple targets may reduce the effectiveness of tumor escape mechanisms.

[0223] In some embodiments, an effective number of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) modified cells described herein are administered to a subject in combination with an anti-CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) therapy, e.g., an anti-CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) cancer therapy.

[0224] In some embodiments, an effective number of cells comprising a modified CD33 and a modified second lineage-specific cell surface antigen are administered in combination with an anti-CD33 therapy, e.g., an anti-CD33 cancer therapy. In some embodiments, the anti-CD33 therapy comprises immune cells expressing an antibody, a bispecific T cell engager, an ADC, or a CAR.

[0225] In some embodiments, an effective number of cells comprising the modified CLL-1 and the modified second lineage-specific cell surface antigen are administered in combination with an anti-CLL-1 therapy, e.g., an anti-CLL-1 cancer therapy. In some embodiments, the anti-CLL-1 therapy comprises an antibody, a bispecific T cell engager, an ADC, or an immune cell expressing a CAR.

[0226] In some embodiments, an effective number of cells comprising a modified CD123 and a modified second lineage-specific cell surface antigen are administered in combination with an anti-CD123 therapy, e.g., an anti-CD123 cancer therapy. In some embodiments, the anti-CD123 therapy comprises immune cells expressing an antibody, a bispecific T cell engager, an ADC, or a CAR.

[0227] In some embodiments, an effective number of cells comprising a modified CD327 (Siglec-6) and a modified second lineage-specific cell surface antigen are administered in combination with an anti-CD327 (Siglec-6) therapy, e.g., an anti-CD327 (Siglec-6) cancer therapy. In some embodiments, the anti-CD327 (Siglec-6) therapy comprises immune cells expressing an antibody, a bispecific T cell engager, an ADC, or a CAR.

[0228] In some embodiments, an effective number of cells comprising a modified CD312 (EMR2) and a modified second lineage-specific cell surface antigen are administered in combination with an anti-CD312 (EMR2) therapy, e.g., an anti-CD312 (EMR2) cancer therapy. In some embodiments, the anti-CD312 (EMR2) therapy comprises immune cells expressing an antibody, a bispecific T cell engager, an ADC, or a CAR.

[0229] In some embodiments, the number of genetically engineered cells provided herein administered to a subject in need thereof is greater than or equal to 10 6 ~10 11 However, amounts below or above this exemplary range are also within the scope of the present disclosure. For example, in some embodiments, the number of genetically engineered cells provided herein, e.g., HSCs, HPCs, or immune effector cells, administered to a subject in need thereof is about 10 6 , about 10 7 , about 10 8 , about 10 9 , about 10 10 , or about 10 11In some embodiments, the number of genetically engineered cells provided herein administered to a subject in need thereof is 10 6 ~10 9 Within the range of 10 6 ~10 8 Within the range of 10 7 ~10 9 Within the range of about 10 7 ~10 10 Within the range of 10 8 ~10 10 Within the range of 10 9 ~10 11 is within the range.

[0230] It is understood that when agents (e.g., CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) modified cells and anti-CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) therapy) are administered in combination, the agents can be administered simultaneously or at different times close in time. Furthermore, the treatments can be mixed or in separate volumes. For example, in some embodiments, administration in combination includes administration during the same course of treatment, e.g., with an anti-CD33 therapy, and the subject can be administered an effective number of CD33-modified cells simultaneously with the anti-CD33 therapy or sequentially, e.g., before, during, or after treatment. In some embodiments, administration in combination includes administration during the same course of therapy, e.g., during a course of therapy with an anti-CLL-1 therapy, where the subject may be administered an effective number of CLL-1 modified cells simultaneously with or sequentially, e.g., before, during, or after, the anti-CLL-1 therapy. In some embodiments, administration in combination includes administration during the same course of therapy, e.g., during a course of therapy with an anti-CD123 therapy, where the subject may be administered an effective number of CD123 modified cells simultaneously with or sequentially, e.g., before, during, or after, the anti-CD123 therapy. In some embodiments, administration in combination includes administration during the same course of therapy, e.g., during a course of therapy with an anti-CD327 (Siglec-6) therapy, where the subject may be administered an effective number of CD327 (Siglec-6) modified cells simultaneously with or sequentially, e.g., before, during, or after, the anti-CD327 (Siglec-6) therapy. In some embodiments, administration in combination includes administration during the same course of treatment, e.g., during a course of treatment with anti-CD312 (EMR2) therapy, and the subject can be administered an effective number of CD312 (EMR2)-modified cells simultaneously with the anti-CD312 (EMR2) therapy or sequentially, e.g., before, during, or after treatment.

[0231] In some embodiments, agents targeting CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) described herein are immune cells that express a chimeric receptor, which comprises an antigen-binding fragment (e.g., a single-chain antibody) that can bind to CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2). The immune cells can be, for example, T cells (e.g., CD4+ or CD8+ T cells) or NK cells.

[0232] A chimeric antigen receptor (CAR) can comprise a recombinant polypeptide comprising at least an extracellular antigen-binding domain, a transmembrane domain, and a cytoplasmic signaling domain comprising a functional signaling domain, e.g., derived from a stimulatory molecule. In some embodiments, the cytoplasmic signaling domain further comprises one or more functional signaling domains derived from at least one costimulatory molecule, e.g., 4-1BB (i.e., CD137), CD27, and / or CD28, or fragments of these molecules. The extracellular antigen-binding domain of the CAR can comprise a CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)-binding antibody fragment. The antibody fragment can comprise one or more CDRs, a variable region (or portion thereof), a constant region (or portion thereof), or any combination of the foregoing.

[0233] Exemplary CD33 CAR constructs can be found, for example, in PCT Publication No. WO2019 / 178382, which is incorporated herein by reference in its entirety.

[0234] Exemplary CLL-1 CAR constructs are found, for example, in PCT Application No. PCT / CN2014 / 082602 and U.S. Publication No. 2016 / 0051651(A1), which are incorporated by reference herein in their entireties.

[0235] The amino acid and nucleic acid sequences of exemplary heavy and light chain variable regions of anti-human CLL-1 antibodies are provided below. The CDR sequences are shown in bold in the amino acid sequences. Anti-CLL-1 heavy chain variable region amino acid sequence (SEQ ID NO: 3032) [ka] Anti-CLL-1 light chain variable region amino acid sequence (SEQ ID NO: 3033) [ka]

[0236] Additional anti-CLL-1 sequences can be found, for example, in US Pat. No. 8,536,310, which is incorporated herein by reference in its entirety.

[0237] Anti-CLL-1 antibody binding fragments for use in constructing CLL-1-targeting agents described herein may comprise the same heavy and / or light chain CDR regions as those of SEQ ID NO: 3032 and SEQ ID NO: 3033. Such antibodies may contain amino acid residue variations in one or more of the framework regions. In some examples, an anti-CLL-1 antibody fragment may comprise a heavy chain variable region that shares at least 70% sequence identity (e.g., 75%, 80%, 85%, 90%, 95%, or more) with SEQ ID NO: 3032 and / or a light chain variable region that shares at least 70% sequence identity (e.g., 75%, 80%, 85%, 90%, 95%, or more) with SEQ ID NO: 3033.

[0238] The amino acid and nucleic acid sequences of exemplary heavy and light chain variable regions of anti-human CD123 antibodies are provided below. The CDR sequences are shown in bold in the amino acid sequences. Amino acid sequence of anti-CD123 heavy chain variable region (SEQ ID NO: 2032) [ka] Amino acid sequence of anti-CD123 light chain variable region (SEQ ID NO: 2033) [ka]

[0239] Additional CD123 sequences can be found, for example, in PCT Publication No. WO2015 / 140268(A1), which is incorporated by reference herein in its entirety.

[0240] Anti-CD123 antibody binding fragments for use in constructing CD123-targeting agents described herein may comprise the same heavy and / or light chain CDR regions as those of SEQ ID NO:2032 and SEQ ID NO:2033. Such antibodies may contain amino acid residue variations in one or more of the framework regions. In some examples, an anti-CD123 antibody fragment may comprise a heavy chain variable region that shares at least 70% sequence identity (e.g., 75%, 80%, 85%, 90%, 95%, or more) with SEQ ID NO:2032 and / or may comprise a light chain variable region that shares at least 70% sequence identity (e.g., 75%, 80%, 85%, 90%, 95%, or more) with SEQ ID NO:2033.

[0241] Exemplary chimeric receptor component sequences are provided in Table 3 below. [Table 3-2]

[0242] In some embodiments, the CAR comprises a 4-1BB costimulatory domain (e.g., as shown in Table 3), a CD8α transmembrane domain and a portion of the extracellular domain of CD8α (e.g., as shown in Table 3), and a CD3ζ cytoplasmic signaling domain (e.g., as shown in Table 3).

[0243] A typical number of cells, e.g., immune cells or hematopoietic cells, administered to a mammal (e.g., a human) may be, for example, in the range of 1 million to 100 billion cells, although amounts below or above this exemplary range are also within the scope of the present disclosure.

[0244] In some embodiments, an agent targeting CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) is an antibody-drug conjugate (ADC). An ADC can be a molecule comprising an antibody or antigen-binding fragment thereof conjugated to a toxin or drug molecule. Binding of the antibody or fragment thereof to the corresponding antigen enables delivery of the toxin or drug molecule to cells (e.g., target cells) that display the antigen on their cell surface, thereby resulting in the death of the target cell.

[0245] Suitable antibodies and antibody fragments that bind to CLL-1 will be apparent to those of skill in the art. In some embodiments, the antigen-binding fragment of the antibody-drug conjugate has the same heavy chain CDRs as the heavy chain variable region provided by SEQ ID NO: 3032 and the same light chain CDRs as the light chain variable region provided by SEQ ID NO: 3033. In some embodiments, the antigen-binding fragment of the antibody-drug conjugate has the same heavy chain variable region as provided by SEQ ID NO: 3032 and the same light chain variable region as provided by SEQ ID NO: 3033.

[0246] Suitable antibodies and antibody fragments that bind to CD123 will be apparent to those of skill in the art. In some embodiments, the antigen-binding fragment of the antibody-drug conjugate has the same heavy chain CDRs as the heavy chain variable region provided by SEQ ID NO: 2032 and the same light chain CDRs as the light chain variable region provided by SEQ ID NO: 2033. In some embodiments, the antigen-binding fragment of the antibody-drug conjugate has the same heavy chain variable region as provided by SEQ ID NO: 2032 and the same light chain variable region as provided by SEQ ID NO: 2033.

[0247] Toxins or drugs suitable for use in antibody-drug conjugates are known in the art and will be apparent to those skilled in the art (see, e.g., Peters et al. Biosci. Rep. (2015) 35(4):e00225, Beck et al. Nature Reviews Drug Discovery (2017) 16:315-337, Marin-Acevedo et al. J. Hematol. Oncol. (2018) 11:8, Elgundi et al. Advanced Drug Delivery Reviews (2017) 122:2-19).

[0248] In some embodiments, the antibody-drug conjugate may further comprise a linker (e.g., a peptide linker such as a cleavable linker) attaching the antibody and the drug molecule.

[0249] Examples of antibody-drug conjugates include, but are not limited to, brentuximab vedotin, glentuximab vedotin / CDX-011, depatuximab mafodotin / ABT-414, PSMA ADC, polatuzumab vedotin / RG7596 / DCDS4501A, denintuzumab mafodotin / SGN-CD19A, AGS-16C3F, CDX-014, RG7841 / DLYE5953A, RG7882 / DMUC406A, RG7986 / DCDS0780A, SGN-LIV1A, enfortumab vedotin / ASG-22ME, AG-15ME, AGS67E, and tetrodotoxin. Risotuzumab vedotin / ABBV-399, ABBV-221, ABBV-085, GSK-2857916, tisotuzumab vedotin / HuMax-TF-ADC, HuMax-Axl-ADC, pinatuzumab vedotin / RG7593 / DCDT2980S, rifatuzumab vedotin / RG7599 / DNIB0600A, indusatuzumab vedotin / MLN-0264 / TAK-2 64, bundled tuzumab vedotin / RG7450 / DSTP3086S, sofituzumab vedotin / RG7458 / DMUC5754A, RG7600 / DMOT4039A, RG7336 / DEDN6526A, ME1547, PF-06263507 / ADC5T4, trastuzumab emtansine / T-DM1, mirvetuximab sorafutansine / IMGN853, coltuximab Bravtansine / SAR3419, naratuximab emtansine / IMGN529, indatuximab emtansine / BT-062, anetumab emtansine / BAY94-9343, SAR408701, SAR428926, AMG224, PCA062, HKT288, LY3076226, SAR566658, lorvotuzumab mertansine / IMGN901, cantuzumab Mertansine / SB-408075, cantuzumab lavtansine / IMGN242, laprituximab emtansine / IMGN289, IMGN388, bivatuzumab mertansine, AVE9633, BIIB015, MLN2704, AMG172, AMG595, LOP628, vadatuximab butarilin / SGN-CD123A, SGN-CD70A, SGN-CD19B, SGN-CD123A, SGN-CD352A, rovalpituzumab tesirin / SC16LD6.5, SC-002, SC-003, ADCT-301 / HuMax-TAC-PBD, ADCT-402, MEDI3726 / ADC-401, IMGN779, IMGN632, gemtuzumab ozogamicin, inotuzumab ozogamicin / CMC-544, PF-06647263, CMD-193, CMB-401, trastuzumab duocarmazine / SYD985, BMS-936561 / MDX-1203, sacituzumab govitecan / IMMU-132, labetuzumab govitecan / IMMU-130, DS-8 201a, U3-1402, milatuzumab doxorubicin / IMMU-110 / hLL1-DOX, BMS-986148, RC48-ADC / hertuzumab-vc-MMAE, PF-06647020, PF-06650808, PF-06664178 / RN927C, rupartuzumab amadotin / BAY1129980, aprtuzumab ixadotin / BAY1187982, ARX788, AGS62P1, XMT-1522, AbGn-107, MEDI4276, and DSTA4637S / RG7861. In one example, the antibody-drug conjugate is gemtuzumab ozogamicin.

[0250] In some embodiments, binding of an antibody-drug conjugate to an epitope of a cell-surface lineage-specific protein can induce internalization of the antibody-drug conjugate, resulting in the release of the drug (or toxin) intracellularly. In some embodiments, binding of an antibody-drug conjugate to an epitope of a cell-surface lineage-specific protein can induce internalization of the toxin or drug, thereby enabling the toxin or drug to kill cells expressing the lineage-specific protein (target cells). In some embodiments, binding of an antibody-drug conjugate to an epitope of a cell-surface lineage-specific protein can induce internalization of the toxin or drug, thereby enabling the toxin or drug to modulate the activity of cells expressing the lineage-specific protein (target cells). The type of toxin or drug used in the antibody-drug conjugates described herein is not limited to any particular type.

[0251] CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2)-associated diseases and / or disorders The present disclosure provides, inter alia, compositions and methods for treating diseases associated with cells expressing CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), or conditions associated with cells expressing CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2). In some embodiments, diseases associated with expression of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), or conditions associated with cells expressing CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), include, for example, proliferative disorders such as cancer or malignancies (e.g., hematopoietic malignancies), or precancerous conditions such as myelodysplastic syndromes or preleukemias. In some embodiments, diseases associated with expression of CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), or conditions associated with cells expressing CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2), include, for example, myeloproliferative neoplasms (MPNs). In some embodiments, the present disclosure provides, inter alia, compositions and methods for use as or in combination with conditioning targets, or for the treatment of various immune disorders, e.g., based on expression profiles.

[0252] In some embodiments, the hematopoietic malignancy or blood disorder is associated with CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) expression. Hematopoietic malignancies are described as malignant disorders involving hematopoietic cells (e.g., blood cells, including progenitor cells and stem cells). Examples of hematopoietic malignancies include, but are not limited to, Hodgkin's lymphoma, non-Hodgkin's lymphoma, leukemia, or multiple myeloma. Exemplary leukemias include, but are not limited to, acute myeloid leukemia, acute lymphocytic leukemia, chronic myelogenous leukemia, acute lymphoblastic leukemia or chronic lymphoblastic leukemia, and chronic lymphocytic leukemia.

[0253] In some embodiments, cells involved in hematopoietic malignancies are resistant to conventional or standard therapeutic agents used to treat the malignancy. For example, the cells (e.g., cancer cells) may be resistant to chemotherapeutic agents and / or CAR T cells used to treat the malignancy.

[0254] In some embodiments, the leukemia is acute myeloid leukemia (AML). AML is characterized by a heterogeneous, clonal neoplastic disease originating from transformed cells that gradually acquire profound genetic alterations that disrupt key differentiation and proliferation control pathways. (Dohner et al., NEJM, (2015) 373:1136). Without wishing to be bound by theory, in some embodiments, CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) are expressed on myeloid leukemia cells and normal myeloid and monocytic precursors, and are believed to be promising targets for AML therapy.

[0255] In some cases, a subject may initially respond to a therapy (e.g., for a hematopoietic malignancy) and subsequently experience a relapse. Any of the methods or populations of genetically engineered hematopoietic cells described herein may be used to reduce or prevent recurrence of a hematopoietic malignancy. Alternatively, or in addition, any of the methods described herein may include administering any of the populations of genetically engineered hematopoietic cells described herein and immunotherapeutic agents (e.g., cytotoxic agents) that target cells associated with a hematopoietic malignancy, and further administering one or more additional immunotherapeutic agents upon recurrence of the hematopoietic malignancy. In some embodiments, the subject has had or is prone to recurrence of a hematopoietic malignancy (e.g., AML) after administration of one or more prior therapies. In some embodiments, the methods described herein reduce the subject's risk of recurrence or the severity of recurrence.

[0256] In some embodiments, the hematopoietic malignancy or blood disorder associated with CD33 (Siglec-3), CLL-1, CD123, CD327 (Siglec-6), and / or CD312 (EMR2) is a precancerous condition, such as myelodysplasia, myelodysplastic syndrome, or preleukemia. Myelodysplastic syndrome (MDS) is a hematological condition characterized by unregulated and ineffective hematopoiesis or blood production. Thus, the number and quality of blood-forming cells are irreversibly reduced. Some MDS patients develop severe anemia, while others are asymptomatic. Classification schemes for MDS are known in the art, including criteria specifying the proportions or frequencies of specific blood cell types, such as myeloblasts, monocytes, and erythrocyte precursors. MDS includes refractory anemia, refractory anemia with ringed sideroblasts, refractory anemia with excess blasts, refractory anemia with excess transformed blasts, and chronic myelomonocytic leukemia (CML). In some embodiments, MDS can progress to acute myeloid leukemia (AML). [Example]

[0257] Example 1: Evaluation of CD33 / CLL-1 multiplex editing using a base editor A base editing strategy was devised to evaluate base editors and guide RNA combinations for efficient singleplex and multiplex editing of CD33 and CLL-1. See Figures 1A-1C. Both cytosine base editors (CBEs) and adenine base editors (ABEs) were tested with guide RNAs targeting either CD33 or CLL-1. See Figures 2A-2C and 3A-3E. The design and prioritization of base editor guides is outlined in Figures 2A-2C and 3A-3E, which describe various guides designed for use with various base editors (BEs), which can be used to achieve gene knockout (KO) using base editors through the introduction of premature stop codons or splice site disruptions. Different combinations of the guides described in Figure 2B and Tables 1, 2, and 6-8 with the BEs disclosed in Figure 3 were evaluated in more detail.

[0258] Guide RNAs were electroporated into target cells, e.g., immobilized human CD34+ hematopoietic cells, along with mRNAs encoding the respective base editors. The encoding mRNAs were chemically modified to improve expression of the encoded base editors, e.g., using 5-methoxyuridine or N1-methylpseudouridine modifications (Figure 3B). CD34+ cells were obtained from two different donors, and guide RNAs and BE-encoding mRNAs were electroporated into the cells. The cells were analyzed at different time points after electroporation, e.g., 48 and 120 hours. Analyses included cell viability, cell counting, and target protein expression (e.g., CD33 and CLL-1). Genomic DNA (gDNA) was obtained from the edited cell populations, e.g., via DNA sequencing, to analyze genome editing.

[0259] Figure 3D shows high on-target CD33 base editing in HSPCs using guides 7, 8, and 17 combined with a CBE containing an R33A substitution. The observed editing efficiency was higher when N1-methylpseudouridine-modified mRNA was administered to cells compared with 5-methoxyuridine-modified mRNA, and delivery of 9 micrograms of mRNA compared with 6 micrograms also resulted in improved editing efficiency. Editing efficiency was observed to improve over time, with higher observed editing efficiency at 120 hours compared with 48 hours after electroporation. Sequence analysis revealed a subset of cells containing an unintended C to G conversion.

[0260] Figure 3E shows high on-target CLL-1 base editing in HSPCs using guides 3, 4, and 1 combined with a CBE containing an R33A substitution. The observed editing efficiency was higher when N1-methylpseudouridine-modified mRNA was administered to cells compared with 5-methoxyuridine-modified mRNA, and delivery of 9 micrograms of mRNA compared with 6 micrograms also resulted in improved editing efficiency. Editing efficiency was observed to improve over time, with higher observed editing efficiency at 120 hours compared with 48 hours after electroporation. Sequence analysis revealed a subset of cells containing unintended C to G conversions.

[0261] Different CBEs, such as the "WT" and "R33A" variants, were evaluated for improved base editing efficiency and lack of unintended base conversions. Additionally, ABE editing strategies were also evaluated. Figure 4 discloses various combinations that were evaluated. Figure 4A shows the experimental design. Figure 4B shows the specific BE and guide combinations tested. Figure 4C shows that cytosine / adenine base editing of the CD33E1 splice site using different guide RNAs efficiently disrupts CD33 expression. Insertion indicates the results for guide RNA 17. EP: electroporation. Without wishing to be bound by theory, it is contemplated that some of the edits disclosed herein, for example, when the CD33E1 splice donor site is disrupted by a CBE / ABE in combination with guide sg17, result in efficient CD33 knockout via a nonsense-mediated decay mechanism. See Figure 4D. Figure 4E shows that cytosine base editing of CLL-1 using various guide RNAs efficiently disrupts protein expression. The insertion shows the results for guide RNA 3.

[0262] The base editing efficiency, measured as the percentage of cells showing target protein (CD33 or CLL-1) knockout, of different CBEs combined with different guides is summarized in Figure 4F. These data show that the exemplary CD33 BE and guide combination can achieve CD33 protein loss in HSPCs or greater than 60%, and the exemplary CLL-1 BE and guide combination can achieve CLL-1 protein loss in HPSCs or greater than 60%.

[0263] Further characterization of the gene editing efficiency of various BE and guide RNA combinations was performed. See Figure 5 for a summary of the results.

[0264] Example 2: Multiplex base editing of CD33 and CLL-1 Multiplex editing of CD33 and CLL-1 was performed using different CD33 guide RNAs (sg7, sg8, or sg17) in combination with CLL-1 guide RNA sg3. Figure 6A shows the experimental design. Figure 6B shows the groups in this study, which included dose-titrated multiplex base editing using three top CBE CD33 guides combined with the top CLL-1 guide. Figure 6C shows efficient knockout of both CD33 and CLL-1 in immobilized human CD34+ hPSCs. CD33 expression was present in less than 10% of cells in the edited hPSC cell population, and CLL-1 expression was present in less than 20% of cells in the edited hPSC cell population. Figure 6D shows FACS data for edited cell populations using various guide combinations. Figure 6E shows CD33 and CLL-1 protein knockout data in hPSCs for different ratios of guides used in the experiment. FACS data for CD33g8 and CLL-1g3 are shown on the right. An exemplary off-target analysis of CD33sg8 is shown in the lower left, demonstrating the desired off-target profile. FACS analysis showed that approximately 80% of the multiplex-edited cells lacked CD33 and CLL-1 surface protein expression (Figure 6F). The multiplex-edited cells (CD33KO / CLL-1KO) were subjected to a colony formation assay, and the data are shown in Figure 7, demonstrating that base-edited cells did not affect colony formation and differentiation potential. [Table 20]

[0265] Example 3: Complexing and co-delivering cytosine base editors and AsCpf1 As shown in Figure 8A, multiplexing using co-delivery of a cytosine base editor (CBE) and Cpf1 nuclease allows for single delivery without the risk of translocations because the base editor does not create double-strand breaks. These data show >50% editing at both loci when the base editor and Cpf1 are delivered together.

[0266] As shown in Figure 8B, when CBE and AsCpf1 are delivered simultaneously in CD34 cells, viability and cell proliferation are not affected.

[0267] Example 4: Simultaneous multiplexed base editing engineering protocol in HSCs using exemplary CD33 and CLL-1 cytosine base editor (CBE) guides Multiplex editing of CD33 and CLL-1 was performed using different CD33 guide RNAs (sg7, sg8, or sg17) in combination with CLL-1 guide RNA sg3. Figure 9A shows the experimental design. After in silico guide design, mCD34+ cells were thawed for culture on day 1. After 48 hours, CD33 and CLL-1 guides were introduced into mCD34+ cells for screening. After 24 hours, cell counts and cell viability were assessed. After 48 hours, cell counts and cell viability were assessed again, and cells were harvested. gDNA was purified and editing readouts were obtained via NGS. Furthermore, bone marrow in vitro differentiation was set up, and after 6 days, cells were evaluated by flow cytometry for protein knockout (KO) readouts. Figure 9B shows data from day 6 after bone marrow in vitro differentiation. Combination of base editors (BE) with CD33g8 and CLL-1g3 demonstrated 80% dual surface protein KO. Figure 9C shows that no balanced translocations were detected in the multiplexed base-edited samples, as determined by the RhampSeq assay. These data demonstrate the feasibility and success achievable with simultaneous multiplexed base editing. Notably, this experiment achieved approximately 80% CD33 / CLL-1 double KO cells and 0% translocations; i.e., the RhampSeq assay was unable to detect any translocations. Notably, there was no effect on cell viability or cell proliferation.

[0268] Example 5: Multiplex base editing in human hematopoietic stem and progenitor cells (HSPCs) enables efficient removal of multiple surface antigens for acute myeloid leukemia (AML) immunotherapy Multiplex base editing of CD34+ hematopoietic stem and progenitor cells (HSPCs) derived from healthy donors was performed using different CD33 and CLL-1 guide RNAs. Figure 10 shows the experimental design. A CBE4 base editing guide screen for CD33 and CLL-1 was performed, generating highly efficient edits in CD34+ HSPCs. Figure 11A shows the on-target base editing efficiency of three CBE gene knockouts (KOs) using three different CBE4 mRNA-encoding constructs to induce single guides (sg7, sg8, or sg17) compared to the Cas9-induced indel frequency at the CD33 locus. Figure 11B shows the on-target base editing efficiency of two CBE gene knockouts (KOs) using three different CBE4 mRNA-encoding constructs to induce single guides (sg3, sg4) compared to the Cas9-induced indel frequency at the CLL-1 locus. Figure 12 shows that efficient base editing of CD33 and CLL-1 abolishes CD33 and CLL-1 protein surface expression. Figures 13A and 13B show that multiplex base editing of the CD33 and CLL-1 loci demonstrates efficient on-target editing and CD33 and CLL-1 protein surface expression knockout. The right panel of Figure 13B shows an 80% true double KO population for edited CD33+CLL-1 (i.e., CD33-CLL-1- cells). Figure 14 shows that the multilineage potential of double-edited CD34+ HSPCs was maintained after multiplex base editing. Figures 15A and 15B show that bone marrow in vitro differentiation demonstrated editing persistence in monocytes and protein KO expression of CD33 and CLL-1 multiplex-edited cells. Figures 16A and 16B show that no translocations were detected in the CD33+CLL-1 multiplex-base-edited sample.

[0269] These data demonstrate the feasibility and success achievable with simultaneous multiplexed base editing. In particular, these data demonstrate that simultaneous delivery of base editing guides can preserve the health, proliferation, and stemness of HSPCs, which may facilitate the processing and manufacturing of cells for therapeutic applications, such as for the treatment of AML. Furthermore, this multiplexed base editing experiment achieved high base editing efficiency, robust surface protein knockout, and no detection of balanced translocations in multiplexed base-edited cells was achieved. Thus, these data demonstrate that multiplexed base editing of one, two, or multiple surface targets in CD34+ HSPCs provides a valuable, safe, and effective alternative for engineering next-generation transplants to treat AML patients.

[0270] Example 6: Evaluation of CD33 / CD123 multiplex editing using adenine base editors (ABEs) CD34+ hematopoietic stem and progenitor cells (HSPCs) from a single healthy donor were thawed and cultured in maintenance medium (SFEM + Flt3, SCF, TPO). Two days after thawing, CBE or ABE editing was performed. For each condition, 8e5 cells were electroporated with 9µg of CBE or ABE mRNA and 4.7µM guide RNA. N1-methylpseudouridine-modified ABE 8.20mM mRNA was used. Additional control conditions were included. Guide control samples received non-targeting guide RNA with the ABE enzyme and were electroporated. Mock-electroporated samples received no enzyme or guide RNA but were electroporated. All conditions were then cultured in maintenance medium (SFEM + Flt3, SCF, TPO) for 5 days after electroporation. Cells for gDNA were harvested 5 days after electroporation, and next-generation sequencing (NGS) was performed to measure DNA editing with CBE and ABE guides. Flow cytometry was performed 5 days after electroporation to measure surface protein expression of CD33 in guided-edited and control samples. Figure 17A shows that co-delivery of an adenine base editor (ABE) and gRNAs targeting CD33 and CD123 (e.g., CD33g17 and CD123g18, respectively) enabled approximately 90% on-target editing efficiency in CD123. In this experiment using ABE, there was no detectable bystander editing; only on-target splice site disruption was intended for all graphed targets (Figure 17A). As used herein, "bystander editing" refers to editing that occurs within the editing window (i.e., the guide protospacer) at a nucleotide other than the targeted "A" of the ABE.

[0271] Figure 17B shows the off-target profile of adenine base editing using CD123g18. The CD123g18 off-target profile outlines potential targeting sites using in silico predicted homology to all sites in the human genome. This off-target prediction pipeline considers mismatches and gaps in the guide sequence and then assigns a relative off-target score (e.g., the lower the score, the lower the potential activity). These sites are mapped and recorded in the "Locus" column of the table. Of the five listed sites, the first is the on-target site (score 100), while the next four are very low (score 1), indicating more favorable off-target predictions for CD123g18.

[0272] Figure 17C shows a schematic illustrating the experimental design for multiplex editing of CD33 and CD123 performed using different CD33 and CD123 guide RNAs in combination with adenine base editors (ABEs).

[0273] Figures 18A-18C show data from a cytosine base editor (CBE) screening effort, in which each CD33 guide (e.g., CD33g7, CD33g8, and CD33g17) target was edited as a single target using a different modified CBE (5-methoxyuridine (5-mO), N1-methylpseudouridine (N1), or wild-type (WT)), and CD33 guide / CBE pairs were evaluated for on-target editing and protein knockout compared to CBE editing and protein knockout.

[0274] Figure 18A shows the on-target editing efficiency of single base-edited cells for CD33 compared to Cas9 control-edited cells. Base editing was performed using different CD33 guide RNAs (e.g., CD33g7, CD33g8, and CD33g17) in combination with an adenine base editor (ABE) or a cytosine base editor (CBE). These data show that the combination of an adenine base editor (ABE) and CD33g17 resulted in approximately 90% on-target editing efficiency 120 hours after electroporation of CD34+ HSPCs with 9 μg of 5-methoxyuridine-modified mRNA encoding the ABE.

[0275] Figure 18B shows the on-target editing efficiency of single base-edited cells against CD33 using different CD33 guide RNAs (e.g., CD33g7, CD33g8, and CD33g17) in combination with ABE or CBE. These data show that the combination of ABE and CD33g17 resulted in virtually all edits creating splicing-disrupting substitutions 120 hours after electroporation of CD34+ HSPCs with 5-methoxyuridine-modified mRNA encoding ABE.

[0276] Figure 18C shows CD33 surface protein expression in edited and unedited CD34+ HSPCs 120 hours after EP. These data show that the combination of ABE and CD33g17 resulted in a robust loss of CD33 surface protein expression compared to unedited (MockEP) 120 hours after electroporation of CD34+ HSPCs with 9 μg of 5-methoxyuridine-modified mRNA encoding ABE.

[0277] Example 7: ABE CD33g17 is included as a group in the first BE multiplex experiment CD34+ hematopoietic stem and progenitor cells (HSPCs) from a single healthy donor were thawed and cultured in maintenance medium (SFEM + Flt3, SCF, TPO). Two days after thawing, ABE editing was performed. For each ABE multiplex editing condition, 8e5 cells were electroporated with 9 μg of N1-methylpseudouridine ABE8.20m and 4.7 μM ABE CD33g17 guide RNA combined with a different ABE CD123 guide (e.g., sg17, sg18, and sg21) to determine whether N1-methylpseudouridine ABE8.20m could simultaneously silence CD33 and CD123 in CD34+ cells. Figure 19 shows the experimental design. This experiment demonstrates, for the first time, the use of ABE mRNA with N1-methylpseudouridine chemical modifications to increase mRNA stability in cells.

[0278] This experiment also included a Cas9 CD33g811 / CLL1g6 multiplex editing condition for comparison with base editing multiplex editing of CD33 and CLL1.

[0279] Cells were harvested 5 days after electroporation for gDNA and next-generation sequencing (NGS) was performed to measure ABE-guided DNA editing using standard amplicon sequencing protocols. Flow cytometry was performed 5 days after electroporation to measure surface protein expression of CD33 and CD123 in guide-edited and control samples using a cytometer.

[0280] As shown in Figures 17A and 20A, co-delivery of adenine base editors (ABEs) with gRNAs targeting CD33 and CD123 (e.g., CD33g17 and CD123g18, respectively) enables approximately 90% on-target editing efficiency in CD123. Figure 20B shows that multiplex deletion of myeloid antigens by base editing in human hematopoietic stem and progenitor cells (HSPCs) enables the possibility of next-generation transplantation for the treatment of acute myeloid leukemia (AML). Figure 20C shows that the frequency of splice site disruptions induced by ABEs consistently increased across different groups in the study.

[0281] Example 8: Viivs042: Multiplexed base editing in vivo study Figures 21A and 21B and 22A and 22B show the experimental design and conditions for assessing the persistence of editing and long-term reconstitution of simultaneously CBE CD33+CLL1 and ABE CD33+CD123 multiplex-edited CD34+ HSPCs in NSG mice.

[0282] Cells were thawed according to the HSPC thawing protocol and then rested in culture for 48 hours. For the ABE portion, ABE CD33g17 was then paired with each of the ABE CD123g18 fragments for multiplex editing. The two guides and ABE cargo were electroporated into the cells using the Maxcyte electroporation system. The cells were then cultured for 48 hours before being harvested for mouse administration. A portion of the cells remained in culture to assess the efficacy of protein knockout at 144 hours post-electroporation. Cells were harvested for gDNA analysis at 48 and 144 hours post-electroporation.

[0283] As shown in Figures 23A and 23B, high cell viability of approximately 90% cells and similar cell counts were achieved in both the BE singleplex and multiplex conditions. As shown in Figures 24A and 24B, base editing efficiencies in samples taken 48 hours after electroporation (EP) for administration showed the expected alleles, including those with stop codon gains and disrupted splice sites. High total editing was also observed in all samples at 48 hours after EP (administered cells), slightly increasing at 144 hours after EP (Figure 25). Figures 26A and 26B show colony-forming unit (CFU) results at 200 and 400 dilutions, respectively.

[0284] Example 9: 16-week BM data Bone marrow was harvested at 16 weeks, and cells were stained for specific surface proteins to assess chimerism, surface protein knockout, lineage reconstitution of stem and progenitor cells, CD123 knockout in the CD34+ subpopulation, and CLL1 knockout in the subpopulations of the CBE portion of this experiment. As shown in Figure 27, after 16 weeks of engraftment in the bone marrow (BM), no effect on chimerism was observed in the edited group. After 16 weeks of engraftment in the bone marrow (BM), highly efficient knockout of CD33 (Figure 28A), CLL-1 (Figure 28B), and CD123 (Figure 28C) was observed in the edited group using ABE. No effect was observed on lineage reconstitution in the edited group (Figures 29A-29H). Figure 29A shows total lineage reconstitution in the edited group. Figures 29B-29H show lineage reconstitution in edited populations across different cell types, including B lymphocytes (Figure 29B), T lymphocytes (Figure 29C), monocytes (Figure 29D), HSPCs (Figure 29E), granulocytes (Figure 29F), cDCs (Figure 29G), and pDCs (Figure 29H). After 16 weeks of engraftment in the bone marrow (BM), high levels of CD123 KO in myeloid subpopulations across different cell types were observed, including monocytes (Figure 30A), granulocytes (Figure 30B), mast / basophils (Figure 30C), cDCs (Figure 30D), and pDCs (Figure 30E). However, low levels of double KO in myeloid subpopulations were observed, due to low levels of CLL1 KO across different cell types, including monocytes (Figure 31A), granulocytes (Figure 31B), obese / basophils (Figure 31C), cDCs (Figure 31D), and pDCs (Figure 31E). As shown in Figure 32, on-target editing analysis in bone marrow material across different study groups confirms the persistence of editing. Furthermore, the stop codon frequency induced by CBE was consistently slightly decreased across different study groups (Figure 33), whereas the splice site disruption frequency induced by ABE was consistently increased across different study groups (Figure 34).

[0285] Example 10: CBE CD33 / CLL-1 scale-up optimization To determine whether editing efficiency could be increased, CD34+ cells were multiplex-edited using CBEs containing CD33g8+CLL1g3 at twice the dose used in Viivs042 (the initial base editing in vivo study). This experiment also included electroporation of 6M cells at twice the original Viivs042 dose, rather than the standard 12M cells, to determine whether different cell numbers affected editing efficiency. Figures 35-37 show the experimental design and conditions evaluated.

[0286] Figures 38A and 38B show cell counts and viability. Cell proliferation was slightly reduced in the 6M cell, double dose condition. Figures 39, 40A, and 40B show the flow gating strategy and results for CD33 and CLL-1. Figure 41 shows double knockout of CD33 and CLL-1.

[0287] Figure 42 shows that a double dose results in a higher frequency of alleles that result in premature stop codon formation in CBE CD33g8, while Figure 43 shows that a double dose results in a higher frequency of alleles that result in premature stop codon formation in CBE CLL1g3.

[0288] Example 11: CBE and ABE CD33 / EMR2 A single CD34+ donor was thawed and cultured in maintenance medium (SFEM + Flt3, SCF, TPO). Two days after thawing, ABE or CBE editing was performed. For each condition, 1e6 cells were electroporated with 9µg of either ABE (N1-MPU ABE 8.20µm mRNA) or CBE (WT PpABOBEC1 mRNA) mRNA and 4.7µM of guide RNA. Additional control conditions were included. Guide control samples received non-targeting guide RNA with Cas9, ABE, or CBE enzyme and were electroporated. Mock electroporated samples received no enzyme or guide RNA but were electroporated. Two lead Cas9 guide conditions for knockout comparison received EMR2 guide-329 and CD33 guide-811 and were electroporated with 15µg of SpCas9 enzyme and 15µg of Cas9 guide RNA. All conditions were then cultured in maintenance medium (SFEM + Flt3, SCF, TPO) for 6 days after electroporation. Cell counts and viability were measured at 1, 2, and 6 days after electroporation using a Nexcelom Cellometer and AOPI staining (1:2 dilution). Cells for gDNA were harvested at 2 and 6 days after electroporation, and rhAmpSeq (NGS) was performed to measure ABE, CBE, or Cas9-guided DNA editing. Flow cytometry was performed at 6 days after electroporation to measure surface protein expression of CD33 and EMR2 in guide-edited and control samples using a cytometer.

[0289] Figures 44 and 46 show the experimental design and conditions. The CD33 and EMR2 guide screen landscapes are shown in Figure 45. Figures 47A and 47B show cell viability and cell counts for CBE and ABE editing of EMR2 and CD33, respectively. Figures 48A and 48B show reduced surface expression of EMR2, respectively. These data show that the ABE EMR2 guide exhibits strong protein KO 6 days after EP. Figures 49A and 49B show that the EMR2 experimental conditions resulted in various levels of protein KO 6 days after EP, respectively. Figures 50A and 50B show reduced surface expression of CD33, respectively. These data show that the ABE CD33 guide exhibits strong protein KO 6 days after EP. Figures 51A and 51B show that the CD33 experimental conditions resulted in various levels of protein KO 6 days after EP, respectively. Figures 52A and 52B show total editing efficiency and base editing efficiency by ABE, respectively. ABE-guided screening in HSPCs demonstrated high editing at various sites in the CD33 and EMR2 loci, with low frequencies of bystander editing. All experimental conditions demonstrated good viability (90%) and cell proliferation compared to the MockEP control. Figure 53 shows the editing efficiency of the ABE CD33 gRNA. Figure 54 shows the editing efficiency of the ABE and CBE EMR2 gRNAs.

[0290] Example 12: EMR2 / CD33 multiplex ABE base editing One CD34+ donor was thawed and cultured in maintenance medium (SFEM + Flt3, SCF, TPO). ABE editing was performed two days after thawing. For each condition, 8e5 cells were electroporated with 9µg of ABE (N1-MPU ABE 8.20mM mRNA) mRNA and 4.7µM of guide RNA. For the multiplex condition receiving EMR2 ABE guide RNA and CD33 ABE guide RNA, 8e5 cells were electroporated with 9µg of ABE mRNA and 4.7µM of both guides. Additional control conditions were included. The guide control sample received a non-targeting guide RNA with the ABE enzyme and was electroporated. The mock electroporated sample received no enzyme or guide RNA, but was electroporated. The electroporated condition was not electroporated and was only cultured. All conditions were then cultured in maintenance medium (SFEM + Flt3, SCF, TPO) for 5 days after electroporation. Cell counts and viability were measured at 1, 2, and 5 days after electroporation using a Nexcelom Cellometer and AOPI staining (1:2 dilution). Cells were harvested for gDNA at 2 and 5 days after electroporation, and rhAmpSeq (NGS) was performed to measure ABE-guided DNA editing. Flow cytometry was performed at 2 and 5 days after electroporation to measure surface protein expression of CD33 and EMR2 in guide-edited and control samples using a cytometer. RNA pellets were also taken for processing for transcript expression at 2 and 6 days after electroporation.

[0291] Figures 55 and 56 show the experimental design and conditions. Figure 57 shows potential ABE guides at in silico off-target sites.

[0292] Figures 58A and 58B show cell counts and cell viability for ABE editing of EMR2 and CD33, respectively. Figures 59A and 59B show ABE EMR2 and CD33 DNA editing frequencies, respectively. Figures 60A-60C show a summary of ABE EMR2 editing frequencies, editing outcomes, and base edits, respectively. Figures 61A and 61B show the frequency and outcomes of EMR2 off-target editing in CD97, respectively. Figures 62A-62C show a summary of ABE CD33 editing frequencies, editing outcomes, and base edits, respectively. Figures 63 and 64 show EMR2 surface protein expression, and Figures 65 and 66 show CD33 surface protein expression.

[0293] Example 13: CBE Quadruplex Base Editing of CD33, CLL1, CD123, and EMR2 Quadraplex (i.e., simultaneous multiplex editing of four genomic targets) editing of CD34+ cells was performed using CBE and guide RNAs targeting four different genes: CD33, CLL1, CD123, and EMR2. Figure 67 shows the editing conditions (i.e., guide RNAs used) for the single-editing control and quadruplex editing Quad1, Quad2, Quad3, and Quad4 of gene targets CD33, CLL1, CD123, and EMR2. Figure 68 shows the experimental design of the quadruplex experiment. Briefly, immobilized CD34+ (mCD34+) cells obtained from a single healthy donor (donor number SD01000510) were electroporated with a wild-type (WT) cytosine base editor (CBE) mRNA construct (WT CBE-PpAPOBEC1 mRNA N1-methyl-pseudouridine) and four guide RNAs simultaneously targeting the CD33, CLL1, CD123, and EMR2 genes. Single-target electroporations with each of the CD33, CLL1, CD123, and EMR2 guide RNAs were also performed as controls. The CBE construct contained an N1-methyl-pseudouridine chemical modification to improve mRNA stability in cells. On day 1, mCD34+ cells were thawed for culture. After 48 hours, CD33, CLL1, CD123, and EMR2 guide RNAs were introduced into the mCD34+ cells. After 24 hours, cell counts and cell viability were assessed. After 48 hours, cell counts and cell viability were assessed again, and the cells were harvested. gDNA was purified, and editing readouts were obtained via next-generation sequencing. In each case, 9 micrograms of CBE was complexed with 4.7 μM of sgRNA. Cell pellets 120 hours after electroporation (EP) were prepared for sequencing of the obtained editing readouts via next-generation sequencing (NGS). Computational analysis of the sequencing results was used to determine knockout (KO) of CD33, CLL1, CD123, and EMR2 protein expression.Figures 69A and 69B show cell viability and cell proliferation (cells / mL) at 24, 48, and 120 hours after electroporation (EP), indicating that quadruplex editing did not significantly affect cell viability or proliferation, impacting cell health. Figure 70 shows the lev...

Claims

1. (a) an mRNA encoding a base editor; and (b) one or more guide RNAs (gRNAs) comprising a targeting domain; A mixture comprising: The one or more gRNAs may be any of the following (i) to (v): (i) at least one gRNA that binds to a target domain in the CD33 gene; (ii) at least one gRNA that binds to a target domain in the CLL-1 gene; (iii) at least one gRNA that binds to a target domain in the CD123 gene; (iv) at least one gRNA that binds to a target domain in the CD312 (EMR2) gene; and (v) Any combination of (i) to (iv) The mixture is selected from:

2. 2. The mixture of claim 1, wherein the mRNA encoding the base editor has been chemically modified to improve expression of the encoded base editor, and optionally the chemically modified mRNA comprises a 5-methoxyuridine modification or an N1-methylpseudouridine modification.

3. 2. The mixture of claim 1, wherein the base editor comprises a Cas protein fused to a cytidine deaminase or an adenine deaminase, wherein the Cas protein is a catalytically inactive Cas protein or a nickase.

4. 4. The mixture of any one of claims 1 to 3, wherein the base editor is a cytosine base editor (CBE), and the CBE is CBE1, CBE2, CBE3, CBE4, nCas9-2xUGI, BE4-rAPOBEC1, BE4-rAPOBEC1 K34A H122A, BE4-PpAPOBEC1, BE4-PpAPOBEC1 R33A, BE4-PpAPOBEC1 H122A, BE4-RrA3F, BE4-AmAPOBEC1, BE4-SsAPOBEC3B, or CBE-PpAPOBEC1 WT.

5. 4. The mixture of any one of claims 1 to 3, wherein the base editor comprises a Cas protein fused to one or more uracil glycosylase inhibitor (UGI) domains, and wherein the Cas protein is a catalytically inactive Cas protein or a nickase.

6. 4. The mixture of any one of claims 1 to 3, wherein the base editor is an adenine base editor (ABE), and the ABE is ABE1, ABE2, ABE3, ABE4, ABE5, ABE6, ABE7, ABE8, ABE7.10-m, ABE7.10-d, ABE8.8-m, ABE8.8-d, ABE8.13-m, ABE8.13-d, ABE8.17-m, ABE8.17-d, ABE8.20-m, or ABE8.20-d.

7. 3. The mixture of claim 1 or 2, wherein the base editor is a wild-type base editor.

8. 4. The mixture of claim 1, wherein at least one gRNA is a single guide RNA (sgRNA) and / or at least one gRNA comprising one or more chemical modifications.

9. 4. The mixture of claim 1, wherein the one or more gRNAs comprises two gRNAs, three gRNAs, or four gRNAs.

10. 1. A method for producing a genetically engineered cell, comprising:

4. A method comprising introducing the mixture of any one of claims 1 to 3 into a cell comprising a target domain targeted by one or more gRNAs, thereby producing a genetically engineered cell.

11. 11. The method of claim 10, wherein the genetically engineered cells have reduced CD33 gene expression, reduced CLL-1 gene expression, reduced CD123 gene expression, and / or reduced EMR2 gene expression compared to wild-type counterpart cells.

12. The method of claim 10, wherein the cell is a hematopoietic cell, a hematopoietic stem cell, or a hematopoietic progenitor cell.

13. A cell population comprising the mixture of any one of claims 1 to 3.

14. A pharmaceutical composition for treating hematopoietic malignancies in a subject, comprising the cell population of claim 13.

15. 15. The pharmaceutical composition of claim 14, administered to a subject in combination with one or more pharmaceutical agents, wherein the one or more pharmaceutical agents are: (a) an agent that targets CD33; (b) agents that target CLL-1; (c) an agent that targets CD123; and / or (d) Drugs targeting EMR2 The pharmaceutical composition comprising: