Military genome editing in primary bone marrow cells

Abstract

Provided herein are, inter alia, compositions, methods, and systems for efficient genetic manipulation of bone marrow cells without the use of viral vectors. Strategies for gene disruption in primary bone marrow cells (e.g., of human and mouse origin) using electroporation-based delivery of Cas / ribonucleoprotein (RNP) are also provided. The methods provided herein, including embodiments thereof, can provide near-population-level gene knockout of single and multiple targets in a range of cell types without selection or enrichment. Cell fitness and response to immune stimuli may be unaffected by the gene editing process.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims the interests of U.S. Provisional Application No. 62 / 983,568 filed on 28 February 2020 and U.S. Provisional Application No. 63 / 010,476 filed on 15 April 2020, each of which is incorporated herein by reference in whole for all purposes. A table or computer reference in the "arrangement list" Program listing appendix submitted as an ASCII file

[0002] The sequence listing contained in the 5,790-byte machine-format IBM-PC, MS-Windows operating system file 048893-530001WO_ST25, created on February 23, 2021, is incorporated herein by reference. [Background technology]

[0003] background Myeloid cells play crucial and diverse roles in mammalian physiology, including tissue development and repair, natural defense against pathogens, and the generation of adaptive immunity. Macrophages and dendritic cells, in particular, are therapeutic targets for many diseases, including cancer. However, few approaches for gene editing of these cell types have been developed, likely due to their susceptibility to exogenous genetic material or virus-based manipulation.

[0004] Myelocytes constitute the innate immune system, providing the first line of defense against pathogens while simultaneously generating the inflammation necessary for optimal adaptive immunity. Myelocyte subsets include granulocytes, macrophages, monocytes, and dendritic cells. These cells are crucial components of the tissue microenvironment, acting as effectors to directly kill pathogens or infected cells, as phagocytic cells to remove dead cells or pathogens, as professional antigen-presenting cells (APCs) to drive adaptive immunity, and finally as microenvironment modifiers by generating inflammatory or reparative factors such as cytokines. Strategies targeting myelocytes have emerged as relevant for promoting antimicrobial or antitumor immunity, leading to biological research or modification of these cell types necessary to advance immunomodulatory therapies. Nevertheless, current research on innate immunity is largely limited to transformed myelocyte cell lines, myelocytes derived from Cas9-knock-in mouse models, or other engineered mouse gene models (e.g., knockout (KO), inducible cell deletion, reporter). [Overview of the project]

[0005] overview This disclosure generally relates to strategies for gene perturbation in primary myeloid cells of human and mouse origin, for example. Using transfection techniques (e.g., electroporation / nucleofection) for delivering ribonucleoprotein complexes (RNPs) containing guide RNA non-covalently associated with recombinant Cas (e.g., Cas9), near-population-level gene knockout of single and multiple targets can be achieved in various cell types without requiring selection or enrichment of genetically modified cells. The cytocompatibility and response to immune stimulation of cells modified according to the methods described herein are not significantly affected by the gene editing process. Such advances enable species-wide pathway discovery and drug target validation in the field of innate immunity.

[0006] Transfection, for example, delivery of the Cas-RNP complex to nongranulocytic cells (i.e., monocytes, macrophages, and dendritic cells) via nucleofection as described herein, can result in knockout of at least approximately 90% of one or more target genes without requiring further selection of genetically modified cells. This can be achieved in both differentiated primary myeloid cell populations and newly isolated cells. For example, a method is provided herein that enables rapid loss-of-function gene assessment in donor cell populations without substantially affecting normal cellular function, including responsiveness to stimuli.

[0007] In one embodiment, a method for genetic modification of myeloid cells is provided herein, comprising transfecting myeloid cells with a gene editing reagent targeting a gene site of interest, wherein the myeloid cells are not transduced by a viral vector. In embodiments, gene editing reagents suitable for use in the method herein may include an RNP comprising a guide RNA non-covalently contacted with a Cas protein, such as the Cas9 protein.

[0008] In one embodiment, a method for genetically modifying multiple myeloid cells is provided herein, comprising the step of transfecting multiple myeloid cells with a gene editing reagent that targets a site of interest, wherein the myeloid cells are not transduced by a viral vector or viral delivery system.

[0009] In one embodiment, the Specified Information provides a method for genetically modifying a plurality of bone marrow cells, comprising transfecting the bone marrow cells with a gene editing reagent, wherein the bone marrow cells are not transfected with a viral vector and the method does not include a selection or enrichment step after bone marrow cell transfection.

[0010] In one embodiment, a system for genetically modifying bone marrow cells in the absence of a viral vector is provided herein. In an embodiment, the system includes an electroporation system. In an embodiment, the system includes a nucleofection system. In an embodiment, the system includes a chamber compatible with an electroporation or nucleofection system, a plurality of bone marrow cells in the chamber in a culture medium compatible with electroporation / nucleofection, and at least one gene editing system designed to target at least one site of interest in the genome of the bone marrow cells.

[0011] In one embodiment, a method for treating a disease treatable by bone marrow cells provided herein (including embodiments thereof) is provided herein. The method comprises providing genetically modified bone marrow cells that have not been transformed with a virus, wherein the bone marrow cells have not been transfected with a gene editing reagent, and administering the bone marrow cells to a patient in need of bone marrow cells.

[0012] In one embodiment, genetically modified bone marrow cells produced by a method provided herein, including embodiments thereof, are provided herein.

[0013] In one embodiment, an assay for drug discovery is provided herein, which includes screening the effects of one or more compounds on genetically modified bone marrow cells (including embodiments thereof) provided herein.

[0014] In one embodiment, a method for target validation of a compound is provided herein, which includes contacting genetically modified bone marrow cells provided herein, including embodiments thereof, with the compound and monitoring the effect on the cells.

[0015] In one embodiment, a composition comprising a plurality of bone marrow cells in contact with a gene editing reagent, a transfection buffer, and an electroporation enhancer, which does not include a viral vector, is provided herein. [Brief explanation of the drawing]

[0016] Figures 1A-1E show efficient gene editing in mouse monocytes, macrophages, and dendritic cells obtained from bone marrow. (Figure 1A) Workflow for screening eGFP crRNA / Cas9-RNP-mediated knockout in mouse monocytes. Representative FACS plots show a gating strategy for identifying eGFP KO, F4 / 80+ macrophages after 5 days of culture in M-CSF. (Figure 1B) Heatmap showing the relative effect of nucleofection conditions on cell viability and eGFP knockout. White boxes indicate best conditions (buffer P3, program CM-137). (Figure 1C) eGFP KO efficiency after nucleofection with single or pooled crRNA. NTC: untargeted control crRNA-Cas9-RNP. (Figure 1D) Workflow for CD45 crRNAXT / Cas9-RNP-mediated KO in mouse BMDC cultures. The upper FACS plot (right side) shows gating strategies for identifying macrophage, pDC, CD24+DC, and Sirpα+DC cells. The lower FACS plot shows representative gating strategies using NTC crRNAXT / Cas9 RNP as a control for determining CD45-negative cells in each cell population. (Figure 1E) CD45 KO efficiency measured by FACS (experimental workflow and gating strategies shown in Figure 1D) in BM-derived CD24+, Sirpα+, pDC, and macrophage cells nucleofected with crRNAXT-targeting NTC or CD45-loaded IDT V3 Cas9-RNP using either P3, CM137 (upper graph) or P3, EN138 (lower graph). Data are presented as mean + / -SD and collected from two independent experiments.

[0017] Figures 2A-2G show population-level gene editing in human monocyte-derived dendritic cells and macrophages. (Figure 2A) Workflow of β2-microglobulin (B2M) gRNA / Cas9-RNP-mediated knockout in human monocyte-derived dendritic cell and macrophage cultures. Representative FACS plots show gating strategies for determining B2M-negative cells in each cell population using Cas9-RNP carrying NTC gRNA. (Figure 2B) B2M knockout efficiency in monocyte-derived DC cells (left bar graph) and macrophage cells (right bar graph) that underwent nucleofection with different B2M-targeting sequences in either crRNA (B2M cr1, B2M cr2) or crRNAXT (B2M crXT1, B2M crXT2) format, or with a non-targeted control (NTC) complexed with IDT V3 Cas9 (dark bar) or Thermo Fisher TruCut V2 Cas9 (light bar). Data are from a single experiment. (Figure 2C) B2M knockout efficiency in monocyte-derived macrophages nucleofected with IDT V3 Cas9-RNP carrying two different crRNAXTs (crXT1, crXT2) targeting B2M or NTC (NTC crXT). Cas9-RNPs were added individually or in combination. Each Cas9-RNP was labeled with 1X or 2X to indicate the relative molar amount nucleofected in the cells. (Figure 2D) Same as (Figure 2B), but with sgRNAs (B2M sg1, sg2) instead of crRNA or crRNAXTs loaded onto the gRNA / Cas9-RNP. Data are from one experiment. (Figure 2E) B2M knockout efficiency in monocyte-derived macrophages of IDT V3 Cas9-RNP, which is loaded with NTC sgRNA or B2M sgRNA2 and complexed in an sgRNA:Cas9 molar ratio of 2:1 or 3:1. Data is from one experiment. (Figures 2F-2G) Dose-response curves of B2M knockout efficiency in monocyte-derived macrophages. Cells were nucleofected with IDT V3 Cas9-RNP loaded with two different sgRNAs targeting B2M (Figure 2F shows data for sg2, and Figure 2G shows data for sg4) at the indicated amounts.Cas9-RNP was complexed and delivered with and without 4 μM of IDT "Electroporation Enhancer". The data are from one experiment. For (Figs. 2B–2D), cells without nucleofection (Nuc−), or cells nucleofected with NTC crRNA, crRNAXT or sgRNA / Cas9-RNP were used as controls. Buffer P3, CM-137 conditions were used for all Cas9-RNP deliveries.

[0018] Figures 3A-3E illustrate efficient CRISPR / Cas9 deletion of Toll-like receptor 7 in mouse BMDCs and MAVS in human monocyte-derived dendritic cells. (Figure 3A) Top panel: Percentage of TLR7-negative cells from BM-derived CD24+, Sirpα+ DCs, pDCs, and macrophages electroporated with NTCs or IDT V3 Cas9-RNP carrying two different TLR7 sgRNAs (sg1, sg2). TLR7-negative cells were assayed by intracellular FACS. Bottom panel: Histograms show the maximum TLR7% for each BM-derived cell subset electroporated with NTCs, Cas9-RNP carrying TLR7 sg1, TLR7 sg2, or isotype-stained Cas9-RNP. Data are expressed as mean + / - SD. (Figure 3B) Cytokine levels measured by Luminex in the supernatant from BMDC cultures (complex cell type) in (Figure 3A) after 17 hours of stimulation with a mock or 800 ng / ml TLR7 agonist R848. Data are expressed as mean + / -SD and are from one experiment out of three technically replicated experiments. (Figure 3C) Surface CD80 levels (gMFI) of each BMDC cell population in (Figure 3A) after 17 hours of stimulation with a mock or 800 ng / ml TLR7 agonist R848. Data are from one experiment out of three technically replicated experiments. Data are expressed as mean + / -SD and are from one experiment out of three technically replicated experiments. (Figure 3D) TIDE analysis (sg1, sg2) of genomic DNA from monocyte-derived dendritic cells electroporated with IDT V3 Cas9-RNP carrying two different sgRNAs against MAVS. ICE represents % indel, and KO represents % knockout. (Figure 3E) Cytokine levels measured by Luminex from the supernatant of monocyte-derived dendritic cells in (Figure 3D) after overnight stimulation with mock or RIG-I agonist, 3P-dsRNA. Data are expressed as mean + / - SD and are from one experiment out of three technically replicated experiments.

[0019] Figures 4A-4B show that single or compound deletions of MYD88, TRIF, and STING in mouse BMDMs affect TLR and cytosolic sensing of microbial ligands. (Figure 4A) Representative Western blot showing single, double, and triple gene knockdown by sgRNA / Cas9-RNP in mouse BMDMs. (Figure 4B) Cytokine measurements (ELISA) of IFNβ and TNF in cell culture supernatants after stimulation with the indicated ligands for 18 h. Data are mean + / - SD (n = 3) and represent two independent experiments.

[0020] Figures 5A-5D show the screening of an optimal Cas9-RNP electroporation protocol for KO in mouse monocytes and BMDMs. (Figure 5A) Ranking of nucleofection conditions (left graph) and viability (right graph) of crRNA / Cas9-mediated eGFP KO in monocyte-derived macrophages after 5 days of culture. Gray arrow: buffer P3, program CM-137. White arrow: buffer P5, program CM-150. Black arrow: buffer P5, program CA-137. Black bar: nucleofection control without crRNA / Cas9. (Figure 5B) Workflow for generation of mouse BMDMs and screening of crRNA / Cas9-mediated Itgam / CD11b KO. (Figure 5C) Representative histogram showing mean fluorescence intensity (MFI) of CD11b for the indicated nucleofection conditions after 5 days of culture in BMDM medium. (Figure 5D) Heat map showing the relative effects of nucleofection conditions on cell viability and CD11b MFI. White box indicates the best condition (buffer P3, program CM-137).

[0021] Figures 6A–6D show the screening of the optimal Cas9-RNP nucleofection protocol for KO in mouse BMDCs. (Figure 6A) Workflow of screening of initial 80 conditions (5 buffers, 15 electroporation programs, and 5 no-nucleofection (Nuc-free) controls) to optimize nucleofection parameters for efficient KO of CD45 by electroporation of IDT V3 containing CD45 crRNAXT / Cas9-RNP in mouse BMDCs. Representative FACS plots showing the same gating strategy for identifying macrophages, pDCs, CD24+DCs and Sirpα+DCs, as well as CD45 KO efficiency, as shown in Figure 1D. (Figure 6B) Data from the initial optimization screen are shown in four heatmaps reporting cell abundance and CD45 KO efficiency in CD24+ (left panel) and Sirpα+ (right panel) DCs. White boxes indicate the five conditions that showed the highest KO efficiency while maintaining acceptable cell abundance. Data are from one experiment. (Figure 6C) Confirmation of deletion efficiency of the top 5 conditions from initial optimization using IDT V3 containing CD45 crRNAXT / Cas9-RNP compared with IDT V3 containing NTC crRNAXT / Cas9-RNP and a control without nucleofection (NN). Data are from one experiment. (Figure 6D) Relative CD80 levels measured by FACS in BM-derived CD24+DCs, Sirpα+DCs, pDCs and macrophages that are either unnucleofected (Nuc-free) or nucleofected with NTC crRNAXT / Cas9-RNP (NTC crXT) using one of the following combinations: P3, CM137 or P3, EN138. Data are presented as mean + / - SEM and collected from two independent experiments.

[0022] Figures 7A-7H show supporting data for population-level gene disruption in human monocyte-derived dendritic cells and macrophages. (Figure 7A) B2M KO efficiency in monocyte-derived macrophages nucleofected with four different crRNAXTs (crXT1, crXT2, crXT3, crXT4) targeting B2M and complexed with IDT V3 Cas9. Data is from one experiment. (Figure 7B) B2M KO efficiency in monocyte-derived macrophages electroporated with two different crRNAXTs (crXT1, crXT2) targeting B2M and complexed with IDT V3 Cas9. Data from three different donors are shown. (Figure 7C) B2M KO efficiency (n=3) in monocyte-derived macrophages nucleofected with specified crRNAs targeting B2M or an untargeted control (NTC) and complexed with IDT V3 Cas9. (Figure 7D) B2M KO efficiency in monocyte-derived macrophages complexed with IDT V3 Cas9 after nucleofection with a single sgRNA (sg2). Data are from three different donors with three technical replicates and are shown as mean + / -SD. (Figure 7E) B2M KO efficiency in monocyte-derived macrophages nucleofected with IDT V3 containing Cas9-RNP carrying two different sgRNAs (sg1, sg2) targeting B2M or a non-targeting control sgRNA (NTCsg). Cas9-RNP 1021 was added individually or in combination. Each Cas9-RNP was labeled 1× or 2× to indicate the relative molar amount nucleofected in the cells. Data are mean + / -SD (n=3). (Figure 7F) B2M knockout efficiency in monocyte-derived macrophages of IDT V3 Cas9-RNP, which carries NTC sgRNA or B2M sgRNA2 and is complexed with an sgRNA:Cas9 molar ratio of 2:1 or 3:1. (Figures 7G-7H) Cell viability in monocyte-derived macrophages from samples in Figures 2E and 2F. Figure 7G shows data for sg2, and Figure 7H shows data for sg4.

[0023] Figures 8A–8D show supporting data for CRISPR / Cas9 deletion of MAVS and PKR in human monocyte macrophages and dendritic cells. (Figure 8A) MAVS KO efficiency determined by intracellular FACS staining in monocyte-derived dendritic cells complexed with IDT V3 Cas9 after electroporation of two different sgRNAs (sg1, sg2) against MAVS. Data are from one experiment out of three technically replicated experiments and are shown as mean + / -SD (Figure 8B) Sanger sequencing traces used to determine the TIDE value in Figure 3D. Underlined nucleotides in the control sample (electroporated NTCsg) indicate sgRNA targeting sequences. T1 and T2 represent technical replicates. (Figure 8C) Workflow of PKR sgRNA / Cas9-RNP-mediated KO in human monocyte-derived macrophages followed by stimulation with PKR activator Poly I:C or mock. (Figure 8D) Western blots of cell lysates from PKR sgRNA / Cas9-RNP electroperforated human monocyte-derived macrophages, with or without stimulation by PKR activator Poly I:C. Blots were performed for total PKR, phosphorylated eIF2α, total eIF2α, or β-tubulin as a loading control.

[0024] Figures 9A–9F show supporting data for disruption of single or multiple genes in mouse BMDCs and BMDMs to study TLR signaling. (Figure 9A) Percentage of TLR7-negative cells (sg1, sg2) from BM-derived CD24+, Sirpα+ DCs, pDCs and macrophages nucleofected with NTCs or IDT V3 Cas9-RNP carrying two different TLR7-specific sgRNAs. Top panel: Buffer P3, programmed CM-137; bottom panel: Buffer P3, programmed EN-138. TLR7-negative cells were assayed by intracellular FACS. Data are expressed as mean + / -SD. (Figure 9B) Cytokine levels measured by Luminex in the supernatant from BMDC cultures (complex cell type) of (Figure A) after 17 hours of stimulation with mock or 800 ng / ml TLR7 agonist R848. Data are expressed as mean + / -SD and are from one experiment out of three technically replicated experiments. (Figure 9C) Representative Western blot showing MyD88 or TRIF knockdown by sgRNA-Cas9-RNP in mouse BMDM. (Figure 9D) Assessment of gene editing efficiency by Sanger sequencing 5 days after electroporation. Data are mean + / - SEM (n=3). (Figure 9E) ELISA measurement of IFNβ levels in cell culture medium of BMDM treated as described above, 24 hours after electroporation and 5 days after electroporation. (Figure 9F) ELISA measurement of TNF in cell culture supernatant after 18 hours of stimulation with a specified ligand. Data in Figures 9E and 9F are mean + / - SD (n=3) and are representative of three independent experiments.

[0025] Figures 10A-10C show supporting data for single and multiple gene disruption in human monocyte-derived macrophages. (Figure 10A) Histograms showing B2M, CD14, and CD81 knockout in monocyte-derived macrophages, measured by flow cytometry. (Figure 10B) Quantification of gene deletion. Data are mean + / - SD (n=3) and representative of three independent donors. (Figure 10C) Assessment of gene editing efficiency by Sanger sequencing 7 days after nucleofection. Data are mean + / - SEM (n=3).

[0026] Figures 11A-11H show supporting data for the analysis of activation markers, cytokine release, and phagocytosis in human monocyte-derived macrophages after RNP nucleofection. (Figure 11A) Cell surface levels of indicated phenotypic markers measured by flow cytometry 5 days after nucleofection. (Figure 11B) Efficiency of B2M-KO after nucleofection by two unique sgRNAs:Cas9 RNP. (Figure 11C) Comparison of cell surface levels of CD80 and CD86 measured by flow cytometry 5 days after nucleofection. (Figure 11D) ELISA measurement of TNF levels in cell culture medium of monocyte-derived macrophages after nucleofection. (Figure 11E) Quantification of living monocyte-derived macrophages using imaging of living cell nuclei. Micrographs show representative images of cultured cells. (Figures 11F, 11G) Quantification of particulate phagocytosis dynamics (Figure 11F, myelin-pHrodo; Figure 11G, beads-pHrodo). The microscopic images show representative images of phagocytosis after nucleofection, taken at 5 hours. The graphs in Figures 11A-11C show the average fluorescence intensity. The graphs in Figures 11F-11G show the pHrodo signal intensity measured every hour after incubation with the indicated particulate cargo. (Figure 11H) Quantification of the phagocytosis index measured as the area under the curve of the data in (Figure 11F; top three bars) and (Figure 11G; bottom three bars) over 5 hours of imaging. The data in Figures 11C-11H are mean + / -SD (n=3).

[0027] Figures 12A-12F show the analysis of the effects of nucleofection on Tlr7 editing efficiency and BMDC phenotype. (Figure 12A) Representative histogram of TLR7 flow cytometry after nucleofection with the indicated RNP complex using buffer P3 and program CM-137. Quantification of TLR7-KO shown in Figure 3A. (Figure 12B) Frequency of the indicated myeloid cell subset 12 days after nucleofection and Flt3 ligand-mediated BMDC differentiation. (Figure 12C) Relative abundance of BMDCs cultured in (Figure 12B). (Figure 12D) Assessment of cell surface levels of the indicated phenotypic and activation markers on BMDCs cultured as in (Figure 12B). (Figures 12E, 12F) Quantification of CD8+ T cell / OT-I (Figure 12E) or CD4+ T cell / OT-II (Figure 12F) proliferation after 3 days of co-culture with BMDCs pulsed with ovalbumin (OVA) at indicated concentrations after nucleofection. Histograms show proliferation of OT-I or OT-II cells as measured by CFSE dilution. The data in Figures 12B-12F are mean + / - SD (n=3) and represent two independent experiments. [Modes for carrying out the invention]

[0028] Detailed explanation After reading this description, it will become clear to those skilled in the art how to implement the disclosure in various alternative embodiments and applications. However, not all of the various embodiments of the invention are described herein. It will be understood that the embodiments presented herein are presented only as examples and are not limiting. Therefore, this detailed description of various alternative embodiments should not be construed as limiting the scope or breadth of the disclosure as described herein.

[0029] Before disclosing and describing this technology, it should be understood that the embodiments described below are not limited to specific compositions, methods for preparing such compositions, or uses thereof, and are therefore naturally subject to change. It should also be understood that the terminology used herein is intended solely to describe specific embodiments and is not intended to limit them.

[0030] Detailed explanations divided into various sections for the convenience of the reader, and disclosures found in any section, may be combined with those in other sections. Titles or subtitles may be used herein for the convenience of the reader and are not intended to affect the scope of this disclosure. definition

[0031] Unless otherwise defined, all technical and scientific terms used herein have the meanings generally understood by those skilled in the art in the field to which this invention pertains. The following references provide general definitions of many of the terms used herein: Singleton et al., Dictionary of Microbiology and Molecular Biology (2nd ed. 1994); The Cambridge Dictionary of Science and Technology (Walker ed., 1988); The Glossary of Genetics, 5th Ed., R. Rieger et al. (eds.), Springer Verlag (1991); and Hale & Marham, The Harper Collins Dictionary of Biology (1991). Where used herein, the following terms have the meanings described herein unless otherwise specified.

[0032] The use of singular indefinite or definite articles (e.g., “a,” “an,” “the,” etc.) in this disclosure and the following claims follows the conventional patent practice of meaning “at least one” unless the context makes it clear that in a particular case the term is intended to mean specifically one in that particular case. Similarly, the term “comprising” is open-ended and does not exclude additional items, features, components, etc. References identified herein are expressly incorporated herein by reference in their entirety unless otherwise specified.

[0033] "Optional" or "optionally" means that the event or situation described below may or may not occur, and that the description includes both the cases in which the event or situation occurs and the cases in which it does not.

[0034] The term "approximately" when used before a numerical specification that includes a range, such as temperature, time, quantity, or concentration, indicates an approximate value that may vary by (+) or (-) 10%, 5%, 1%, or any sub-range or sub-value between them. Preferably, the term "approximately" means that the value may vary by + / - 10%.

[0035] Where used herein, the terms “comprising” or “comprises” are intended to mean that a composition and method includes the elements described but does not exclude others. “Consisting essentially of,” where used to define a composition and method, means excluding other elements that are essentially important to the combination for the purposes described. Thus, a composition consisting essentially of the elements defined herein does not exclude other materials or processes that do not substantially affect the basic and novel features of the claimed invention. “Consisting of” means excluding other components and elements that are more than trace amounts of substantial method processes. Embodiments defined by each of these transitional terms are within the scope of this disclosure.

[0036] As used herein, the terms “control” or “control experiment” are used in their simple, ordinary sense, referring to an experiment in which the subject or reagents of the experiment are treated similarly to those of a parallel experiment, except for the omission of experimental procedures, reagents, or variables. In some cases, a control is used as a criterion for comparison when evaluating the effect of an experiment. In embodiments, the control is a measurement of gene expression in the absence of the compounds described herein (including embodiments and examples). In embodiments, the control is a measurement of protein activity in the absence of the compounds described herein (including embodiments and examples).

[0037] A "control" sample or value refers to a sample that serves as a reference for comparison with a test sample, usually a known reference. For example, a test sample may be taken from test conditions, for example, in the presence of the test compound, and compared to a sample from known conditions, for example, in the absence of the test compound (negative control) or in the presence of a known compound (positive control). A control can also represent the mean value collected from a large number of tests or results. A person skilled in the art will recognize that controls can be designed for the evaluation of any number of parameters. For example, controls can be devised to compare therapeutic benefits based on pharmacological data (e.g., half-life) or therapeutic means (e.g., comparison of side effects). A person skilled in the art will understand which controls are valuable in a given situation and which data can be analyzed based on comparison with control values. Controls are also useful for determining the significance of data. For example, if the value of a particular parameter varies greatly in the control, the variability of the test sample may not be considered significant.

[0038] Where used herein, the terms “nucleic acid,” “nucleic acid molecule,” “nucleic acid oligomer,” “oligonucleotide,” “nucleic acid sequence,” “nucleic acid fragment,” and “polynucleotide” are interchangeable and are intended to include, but not be limited to, polymeric forms of covalently linked nucleotides having various lengths, such as deoxyribonucleotides or ribonucleotides, or their analogs, derivatives, or modifications. Different polynucleotides may have different three-dimensional structures and may perform a variety of known or unknown functions. Non-exclusive examples of polynucleotides include genes, gene fragments, exons, introns, intergenic DNA (including, but not limited to, heterochromatic DNA), messenger RNA (mRNA), transfer RNA, ribosomal RNA, ribozymes, cDNA, sgRNA, guide RNA, tracrRNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA sequences, isolated RNA sequences, PCR products, nucleic acid probes, and primers. Polynucleotides useful in the methods of this disclosure may include natural nucleic acid sequences and their variants, artificial nucleic acid sequences, or combinations of such sequences.

[0039] "Nucleic acid" refers to nucleotides (e.g., deoxyribonucleotides or ribonucleotides) and their polymers or complements, or nucleosides (e.g., deoxyribonucleosides or ribonucleosides), in single-stranded, double-stranded, or multi-stranded form. In embodiments, "nucleic acid" does not include nucleosides. The terms "polynucleotide," "oligonucleotide," and "oligo" refer to linear sequences of nucleotides in their usual and customary sense. The term "nucleoside" refers to glycosylamines containing nucleic acid bases and pentoses (ribose or deoxyribose) in their usual and customary sense. Non-limiting examples of nucleosides include cytidine, uridine, adenosine, guanosine, thymidine, and inosine. The term "nucleotide" refers to a single unit of a polynucleotide, i.e., a monomer, in its usual and customary sense. A nucleotide may be a ribonucleotide, a deoxyribonucleotide, or a modification thereof. Examples of polynucleotides intended herein include single-stranded and double-stranded DNA, single-stranded and double-stranded RNA, and hybrid molecules having mixtures of single-stranded and double-stranded DNA and RNA. Examples of nucleic acids intended herein, such as polynucleotides, include any type of RNA, such as mRNA, siRNA, miRNA, and guide RNA, and any type of DNA, such as genomic DNA, plasmid DNA, and minicircle DNA, and any fragments thereof. In the context of polynucleotides, the term “double-stranded” refers to double-strandedness in the usual and customary sense. Nucleic acids can be linear or branched. For example, a nucleic acid can be a linear chain of nucleotides, or it can be branched, for example, such that the nucleic acid contains one or more arms or branches of nucleotides. Optionally, branched nucleic acids can be repeatedly branched to form higher-order structures such as dendrimers.

[0040] Nucleic acids, for example, nucleic acids having a phosphorothioate skeleton, may contain one or more reactive moieties. As used herein, the term reactive moiety includes any group that can react with another molecule, such as a nucleic acid or polypeptide, via covalent, non-covalent, or other interactions. For example, a nucleic acid may contain an amino acid-reactive moiety that reacts with an amino acid on a protein or polypeptide via covalent, non-covalent, or other interactions.

[0041] This term also encompasses nucleic acids containing known nucleotide analogues or modified backbone residues or linkages, which are synthetic, native, and unnatural, possess similar binding properties to the reference nucleic acid, and are metabolized in similar manner to the reference nucleotide. Examples of such analogues include, but are not limited to, phosphoramidates, phosphorodiamidates, phosphorothioates (also known as phosphothioates having a double-bonded sulfur that substitutes oxygen in the phosphate), phosphorodithioates, phosphonocarboxylic acids, phosphonocarboxylates, phosphonoacetic acids, phosphonoformic acids, methylphosphonates, boronphosphonates, or phosphodiester derivatives containing O-methylphosphoamidite linkages (see Eckstein, Oligonucleotides and Analogues: A Practice Approach, Oxford University Press), as well as modifications to nucleotide bases, such as 5-methylcytidine or pseudouridine; and peptide nucleic acid skeletons and linkages. Other nucleic acid analogs include those with a positive skeleton; nonionic skeletons, modified sugars, and non-ribose skeletons (e.g., phosphorodiamidate morpholino oligos or locked nucleic acids (LNAs) known in the art), including those described in U.S. Patents 5,235,033 and 5,034,506, and Chapters 6 and 7 of ASC Symposium Series 580, CARBOHYDRATE MODIFICATIONS IN ANTISENSE RESEARCH, Sanghui & Cook, eds. Nucleic acids containing one or more carbocyclic sugars are also included within one definition of nucleic acid. Modification of the ribose-phosphate skeleton may be performed for various reasons, for example, to increase the stability and half-life of such molecules in a physiological environment or as probes on biochips. Mixtures of native nucleic acids and analogs can be prepared, or mixtures of different nucleic acid analogs, as well as mixtures of native nucleic acids and analogs. In embodiments, the internucleotide bonds in DNA are phosphodiesters, phosphodiester derivatives, or a combination of both.

[0042] Polynucleotides typically consist of a specific sequence of four nucleotide bases: adenine (A); cytosine (C); guanine (G); and thymine (T) (or uracil (U) for thymine (T) if the polynucleotide is RNA). Therefore, the term "polynucleotide sequence" can be either an alphabetical representation of a polynucleotide molecule or the term can apply to the polynucleotide molecule itself. This alphabetical representation can be entered into a database in a computer with a central processing unit and used in bioinformatics applications such as functional genomics and homology searches. Polynucleotides may optionally contain one or more non-standard nucleotides, nucleotide analogs, and / or modified nucleotides.

[0043] As used herein, the term “complement” refers to a nucleotide (e.g., RNA or DNA) or sequence of nucleotides that can base-pair with a complementary nucleotide or sequence of nucleotides. As described herein and as commonly known in the art, the complementary (matching) nucleotide of adenosine is thymidine, and the complementary (matching) nucleotide of guanosine is cytosine. Thus, a complement may include a sequence of nucleotides that base-pair with the corresponding complementary nucleotide of a second nucleic acid sequence. The nucleotides of the complement may partially or completely match the nucleotides of the second nucleic acid sequence. If the nucleotides of the complement completely match each nucleotide of the second nucleic acid sequence, the complement forms a base-pair with each nucleotide of the second nucleic acid sequence. If the nucleotides of the complement partially match the nucleotides of the second nucleic acid sequence, only some of the nucleotides of the complement form a base-pair with the nucleotides of the second nucleic acid sequence.

[0044] As described herein, sequence complementarity may be partial, where only a portion of the nucleic acid matches according to base pairing, or it may be complete, where all of the nucleic acid matches according to base pairing. Thus, two complementary sequences may have a certain percentage of identical nucleotides (i.e., about 60% identity across a particular region, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity).

[0045] As used herein, the term “gene” is used according to its simple, ordinary meaning, referring to a segment of DNA involved in protein production, which includes the regions before and after the coding region (leaders and trailers) as well as the intervening sequences (introns) between individual coding segments (exons). Leaders, trailers, and introns contain regulatory elements necessary during gene transcription and translation. Furthermore, a “protein gene product” is a protein expressed from a particular gene.

[0046] The term "amino acid" refers to natural and synthetic amino acids, as well as amino acid analogs and amino acid mimetic compounds that function in a similar manner to natural amino acids. Natural amino acids are those encoded by the genetic code, as well as naturally modified natural amino acids, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same base chemical structure as natural amino acids, i.e., hydrogen, an amino group, and an α-carbon bonded to an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs have a modified R group (e.g., norleucine) or a modified peptide backbone, but retain the same base chemical structure as natural amino acids. Amino acid mimetic compounds refer to compounds that have a structure different from the general or chemical structure of an amino acid, but function in a similar manner to a natural amino acid. The terms "unnatural amino acid" and "non-natural amino acid" refer to amino acid analogs, synthetic amino acids, and amino acid mimetic compounds that are not found in nature.

[0047] Amino acids may be referred herein by their generally known three-letter or one-letter notation as recommended by the IUPAC-IUB Biochemical Nomenclature Committee. Similarly, nucleotides may be referred to by their generally accepted single-letter codes.

[0048] The terms “polypeptide,” “peptide,” and “protein” are used interchangeably herein to refer to polymers of amino acid residues, which may be compounded with portions that do not consist of amino acids. This term applies to amino acid polymers, in which one or more amino acid residues are artificial chemical mimics of corresponding native amino acids, as well as to native and non-native amino acid polymers. A “fusion protein” refers to a chimeric protein that encodes two or more distinct protein sequences that are recombinantly expressed as a single portion.

[0049] With respect to amino acid sequences, those skilled in the art will recognize that individual substitutions, deletions, or additions to nucleic acids, peptides, polypeptides, or protein sequences, which modify, add, or delete a single amino acid or a small proportion of amino acids in the encoded sequence, are “conservatively modified variants” in which the modification results in the substitution of an amino acid with a chemically similar amino acid. Conservative substitution tables that provide functionally similar amino acids are well known in the art. Such conservatively modified variants are added to, and not excluded from, the polymorphic variants, interspecific homologs, and alleles of this disclosure.

[0050] The following eight groups each contain amino acids that are conserved substitutions with each other: (1) alanine (A), glycine (G); (2) aspartic acid (D), glutamic acid (E); (3) asparagine (N), glutamine (Q); (4) arginine (R), lysine (K); (5) isoleucine (I), leucine (L), methionine (M), valine (V); (6) phenylalanine (F), tyrosine (Y), tryptophan (W); (7) serine (S), threonine (T); and (8) cysteine ​​(C), methionine (M) (see, for example, Creighton, Proteins (1984)).

[0051] The "percentage of sequence identity" is determined by comparing two optimally aligned sequences on a comparison window, where some polynucleotide or polypeptide sequences within the comparison window may show additions or deletions (i.e., gaps) compared to a reference sequence (without additions or deletions) for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions in which identical nucleic acid bases or amino acid residues appear in both sequences, dividing the number of matching positions by the total number of positions in the comparison window, and multiplying the result by 100 to obtain the percentage of sequence identity.

[0052] In the context of two or more nucleic acid or polypeptide sequences, the terms “identical” or “percent “identical” refer to two or more sequences or subsequences that are identical or have a specified percentage of the same amino acid residues or nucleotides (i.e., approximately 60% identity, preferably 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher identity, when compared and aligned to the maximum correspondence across the comparison window or specified region). In such cases, such sequences are said to be “substantially identical.” This definition may also refer to or apply to complements of test sequences. This definition also includes sequences with deletions and / or additions, as well as sequences with substitutions. As described below, preferred algorithms can take gaps and the like into account. Preferably, identity exists over a region of at least about 25 amino acids or nucleotides in length, or more preferably over a region of 50 to 100 amino acids or nucleotides in length.

[0053] The "position" of an amino acid or nucleotide base is indicated by a number that sequentially identifies each amino acid (or nucleotide base) in the reference sequence based on its position relative to the N-terminus (or 5'-terminus). Due to deletions, insertions, cleavages, fusions, etc., which must be considered when determining the optimal alignment, the numbers of amino acid residues in a test sequence, which are generally determined by simply counting from the N-terminus, are not necessarily the same as the numbers of their corresponding positions in the reference sequence. For example, if a mutant has a deletion relative to the aligned reference sequence, there is no amino acid in the mutant that corresponds to the position of the deletion site in the reference sequence. If an insertion exists in the aligned reference sequence, that insertion does not correspond to a numbered amino acid position in the reference sequence. In the case of cleavage or fusion, there may be stretches of amino acids in the reference or aligned sequence that do not correspond to any amino acid in the corresponding sequence.

[0054] The terms "numbered by reference" or "corresponding to," when used in relation to the numbering of a given amino acid or polynucleotide sequence, refer to the numbering of residues in a particular reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.

[0055] For the specific proteins described herein, the named protein includes either a native form that maintains the protein's activity, a native or engineered variant, or a homolog (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the activity compared to the native protein). In embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity across the entire sequence or a portion of the sequence (e.g., a contiguous portion of 50, 100, 150, or 200 amino acids) compared to the native form. Protein activity may be, for example, enzymatic activity or editing activity.

[0056] As used herein, the terms “virus” or “viral particle” are used according to their simple, ordinary meanings within the context of viral transduction. Viral vector transduction can be used to insert or modify genes in mammalian cells. It is frequently used as a tool in basic research and is actively studied as a potential means of gene therapy. However, viral transduction may be inefficient or have low variability.

[0057] As used herein, terms such as “genetic modification,” “gene alteration,” “gene editing,” “genome editing,” and “genome engineering” refer to a type of genetic engineering in which DNA is inserted, deleted, modified, or replaced at one or more designated locations within the genome of a cell. Unlike earlier genetic engineering techniques that randomly insert genetic material into the host genome, genome editing targets insertion at site-specific locations. One key step in gene editing is to create a double-strand break at a specific point in a gene or genome. Examples of gene editing tools that achieve this step include, but are not limited to, zinc finger nucleases (ZFNs), effector nuclease-like transcription activators (TALENs), meganucleases, and clustered, regularly arranged short palindromic sequence repeat systems (CRISPR / Cas).

[0058] As used herein, the terms “gene knockout” or “KO” refer to a genetic technique in which one of the genes of an organism is rendered completely or partially inoperable. Knockouts can be heterozygous and homozygous KOs. In the former, only one of two gene copies (alleles) is knocked out, while in the latter, both are knocked out. In embodiments, near-complete loss of target gene expression at the population level can be achieved, which reduces the need for a selection process.

[0059] As used herein, the term “loss of function” refers to a mutation in a gene, or a deletion of part or all of a gene, resulting in a loss of function of a gene product or protein encoded by that gene. In embodiments, loss of function means reducing or inhibiting the activity of a gene product or protein by 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, or 10% compared to the activity of the gene product or protein in the absence of the mutation or gene deletion. In embodiments, loss of function means reducing or inhibiting the activity of a gene product or gene by 1.5, 2, 3, 4, 5, 10, or more compared to the activity of the gene product or gene in the absence of the mutation or gene deletion.

[0060] As used herein, the term “gene editing reagent” refers to the components required for a gene editing tool and may include enzymes, riboproteins, solutions, cofactors, etc. For example, a gene editing reagent may include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and one or more components required for clustered and regularly arranged short palindromic repeat system (CRISPR / Cas) gene editing.

[0061] As used herein, the terms “CRISPR” or “clustered, regularly arranged short palindromic sequence repeats” are used according to their simple, ordinary meaning and refer to genetic elements used by bacteria as a type of acquired immunity to protect against viruses. CRISPRs consist of short sequences derived from viral genomes and incorporated into bacterial genomes. Cas (CRISPR-associated protein) processes these sequences and cleaves matching viral DNA sequences. Thus, CRISPR sequences serve as guides for Cas to recognize and cleave DNA that is at least partially complementary to the CRISPR sequence. By introducing plasmids containing the Cas gene and specifically constructed CRISPRs into eukaryotic cells, the eukaryotic genome can be cleaved at any desired location.

[0062] As used herein, the terms “Cas9” or “CRISPR-related protein 9” are used in their simple, ordinary sense and refer to an enzyme that uses a CRISPR sequence as a guide to recognize and cleave a specific strand of DNA that is at least partially complementary to the CRISPR sequence. The Cas9 enzyme, along with the CRISPR sequence, forms the basis of a technology known as CRISPR-Cas9, which can be used to edit genes in living organisms. This editing process has a wide variety of applications, including basic biological research, the development of biotechnology products, and the treatment of diseases.

[0063] As used herein, “CRISPR-related protein 9,” “Cas9,” “Csn1,” or “Cas9 protein” includes either a recombinant or native form of Cas9 endonuclease or its variant or homolog (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to Cas9) that maintains Cas9 endonuclease enzyme activity. In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a contiguous portion of 50, 100, 150, or 200 amino acids) compared to the native Cas9 protein. In some embodiments, the Cas9 protein is substantially identical to the protein identified by UniProt reference number Q99ZW2 or a variant or homolog substantially identical thereto. In one embodiment, the Cas9 protein has at least 75% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In another embodiment, the Cas9 protein has at least 80% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In another embodiment, the Cas9 protein has at least 85% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In another embodiment, the Cas9 protein has at least 90% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2. In yet another embodiment, the Cas9 protein has at least 95% sequence identity with the amino acid sequence of the protein identified by UniProt reference number Q99ZW2.

[0064] As used herein, “CRISPR-related endonuclease Cas12a,” “Cas12a,” “Cas12,” or “Cas12 protein” includes either a recombinant or native form of Cas12 endonuclease or its variant or homolog (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to Cas12) that maintains Cas12 endonuclease enzyme activity. In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a contiguous portion of 50, 100, 150, or 200 amino acids) compared to the native Cas12 protein. In one embodiment, the Cas12 protein is substantially identical to the protein identified by UniProt reference number A0Q7Q2, or to a variant or homolog having substantial identity therewith.

[0065] As used herein, “CRISPR-related endoribonuclease 13a,” “Cas13a,” “Cas13,” or “Cas13 protein” includes either a recombinant or native form of Cas13 endoribonuclease or its variant or homolog (e.g., activity within 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% compared to Cas13) that maintains Cas13 endoribonuclease enzyme activity. In some embodiments, the variant or homolog has at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a contiguous portion of 50, 100, 150, or 200 amino acids) compared to the native Cas13 protein. In one embodiment, the Cas13 protein is substantially identical to the protein identified by UniProt reference number P0DPB8, or to a variant or homolog having substantial identity therewith.

[0066] As used herein, “Cascade” refers to a complex of Cas proteins associated with an RNA sequence containing a CRISPR sequence. For example, a cascade may contain one or more Cas proteins (e.g., Cas9) that can cleave target DNA as directed by the CRISPR sequence. In other examples, a cascade complex may present a CRISPR RNA and recruit a Cas protein (e.g., Cas3) to cleave target DNA.

[0067] As used herein, “Argonaut endonucleases,” “Argonaut,” “Protein Argonaut-2,” or “Argonaut protein” include Argonaut endonucleases, or recombinant or native forms of its variants or homologs, that maintain Argonaut endonucleases enzyme activity (e.g., within the range of at least 50%, 80%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of activity compared to Argonaut). In some embodiments, the variants or homologs have at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% amino acid sequence identity over the entire sequence or a portion of the sequence (e.g., a contiguous portion of 50, 100, 150, or 200 amino acids) compared to the native Argonaut protein. In one embodiment, the Argonaut protein is substantially identical to the protein identified by UniProt reference number Q9UKV8, or to a variant or homolog having substantial identity therewith.

[0068] As used herein, "TALEN" or "transcription activator-like effector nuclease" refers to restriction enzymes produced by binding a DNA-binding domain (e.g., a TAL effector DNA-binding domain) to a nuclease (e.g., FokI). TALENs typically contain a native DNA-binding domain comprising multiple modules, referred to as TAL or TALE. Therefore, a TAL containing two variable residues confers DNA-binding specificity.

[0069] As used herein, the term “donor DNA” refers to single-stranded or double-stranded DNA that can be inserted into the genome of a cell (e.g., a bone marrow cell) using a gene editing method (e.g., CRISPR). For example, donor DNA may have homology arms that are homologous to the region of the gene into which the donor DNA is inserted. For example, donor DNA may form a complex with a Cas protein. In an example, cells can be transfected with a gene editing reagent and donor DNA.

[0070] As used herein, the term "crRNA" refers to CRISPR RNA, which is a short guide RNA containing a unique single repeat spacer unit. In bacterial cells, crRNA interferes with invading congeneral foreign genomes by targeting foreign DNA. Thus, short, mature crRNAs are a crucial component in the interference phase of the immune pathway. crRNAs contain a nucleotide sequence that is at least partially complementary to the target DNA. Therefore, crRNAs direct target sequence recognition and enable specificity for the CRISPR gene editing mechanism. In embodiments, crRNAs may be provided as pre-crRNAs. Pre-crRNAs can form complexes with regions at least partially complementary to tracrRNAs, thereby forming an RNA double helix. Pre-crRNAs can be cleaved by ribonucleases (e.g., RNase III), thus yielding a crRNA / tracrRNA hybrid. This hybrid acts as a guide for the endonuclease Cas9, which cleaves the invading nucleic acid.

[0071] As used herein, the terms “tracrRNA” or “trans-activated crRNA” refer to a small transcoding RNA. TracrRNA is at least partially complementary to crRNA and forms base pairs with crRNA, thus forming an RNA double helix. In some embodiments, tracrRNA forms an RNA double helix with pre-crRNA. TracrRNA can non-covalently associate with Cas (e.g., Cas9) and thereby function as a binding scaffold for Cas. In some embodiments, recognition and binding of tracrRNA by Cas results in the formation of a Cas9 / tracrRNA / crRNA complex.

[0072] The “guide RNA” or “gRNA” provided herein refers to an RNA sequence that hybridizes with a target sequence and has sufficient complementarity to a target polynucleotide sequence to direct sequence-specific binding of the CRISPR complex to the target sequence. For example, a gRNA can direct Cas to a target polynucleotide. In embodiments, the gRNA includes crRNA and tracrRNA. For example, the gRNA may include crRNA and tracrRNA hybridized by base pairing. Thus, in embodiments, the two RNAs may be separately encoded by crRNA and tracrRNA as two RNA molecules that form an RNA / RNA complex due to complementary base pairing between crRNA and tracrRNA. In embodiments, the degree of complementarity between the guide RNA sequence and its corresponding target sequence is about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or greater, when optimally aligned using an appropriate alignment algorithm. In some embodiments, the degree of complementarity between the guide RNA sequence and its corresponding target sequence is at least about 50%, 60%, 75%, 80%, 85%, 90%, 95%, 97.5%, and 99% when optimally aligned using an appropriate alignment algorithm.

[0073] The terms "sgRNA," "single guide RNA," and "single guide RNA sequence" are used interchangeably and refer to RNA sequences containing both crRNA and tracrRNA sequences. For example, sgRNA may be a single RNA sequence containing both crRNA and tracrRNA. For example, sgRNA may be a fusion sequence containing both crRNA and tracrRNA. In some embodiments, sgRNA is synthesized in vitro. In some embodiments, sgRNA is prepared in vivo from a DNA sequence encoding sgRNA.

[0074] In embodiments, the methods provided herein are used in combination with a type II CRISPR system to generate single-strand and / or double-strand breaks in the host genome. In certain embodiments, a nuclease, such as Cas9 nuclease, is guided to a target site by a guide RNA (e.g., crRNA hybridized to tracrRNA). The guide RNA and the nuclease form a colocalization complex on DNA, on which the nuclease induces cleavage of the target DNA. In exemplary embodiments where the nuclease is Cas9, Cas9 generates a blunt-end double-strand break 3 bp upstream of a protospacer-adjacent motif (PAM) in the target genome via a process mediated by two catalytic domains in the protein.

[0075] Non-limiting examples of CIRSPR enzymes include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12, Cas13, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2, Csa5, Csn2, Examples include Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs, or their mutants. In embodiments, the CRISPR enzyme is the Cas9 enzyme. In embodiments, the Cas9 enzyme is Cas9 from S. pneumoniae, S. pyogenes, or S. thermophilus, or mutants derived from these organisms. In embodiments, the CRISPR enzyme is codon-optimized for expression in eukaryotic cells. In one embodiment, the CRISPR enzyme directs the cleavage of one or two strands at the location of the target sequence. In another embodiment, the CRISPR enzyme lacks DNA strand cleavage activity.

[0076] As used herein, “zinc finger” is a polypeptide structural motif folded around a bound zinc cation. In embodiments, the polypeptide of the zinc finger is X3-Cys-X 2~4 -Cys-X 12 -His-X 3~5 -Has a sequence of the form His-X4, where X is any amino acid (for example, X 2-4 (This represents an oligopeptide with a length of 2-4 amino acids). Therefore, as used herein, "zinc finger nuclease" refers to a nuclease that contains a zinc finger motif and a domain capable of inducing the cleavage of target DNA.

[0077] Non-limiting examples of methods for homologous recombination and gene editing using various nuclease systems can be found, for example, in U.S. Patent No. 8,945,839, International Publication No. WO 2013 / 163394 of International PCT Application, and U.S. Patent Application No. 2016 / 0060657, U.S. Patent Application No. 2012 / 0192298 A1, and U.S. Patent Application No. 2007 / 0042462 (each of which is incorporated herein by reference in its entirety). These and any other known methods for homologous recombination can be used with the plasmid vectors provided herein.

[0078] As used herein, the term "myeloid cell" is used according to its plain and ordinary meaning and refers to any cell derived from and including myeloid stem cells. In embodiments, the myeloid stem cells are derived from hematopoietic stem cells. Myeloid cells are progenitor cells of different types of cells. They give rise to many different types of blood cells including monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, megakaryocytes, and platelets.

[0079] As used herein, the term "monocyte" or "monocyte cell" is used according to its plain and ordinary meaning and refers to a type of white blood cell or leukocyte. They are the largest type of white blood cell and can differentiate into macrophages and myeloid dendritic cells. As part of the vertebrate innate immune system, monocytes also influence the process of adaptive immunity. There are at least three types of monocytes present in human blood: 1) Classical monocytes may be characterized by high expression of the CD14 cell surface receptor (CD14 ++ CD16 - monocytes); 2) Non-classical monocytes show low expression of CD14 and additional co-expression of the CD16 receptor (CD14 + [[ID=1,4]]CD16 ++ monocytes); and 3) Intermediate monocytes with high expression of CD14 and low expression of CD16 (CD14 ++ CD16 + monocytes).

[0080] As used herein, the term “macrophage” is used in its simple, ordinary sense, referring to a type of white blood cell in the immune system that engulfs and digests cellular debris, foreign substances, microorganisms, cancer cells, and other substances that do not have types of proteins specific to healthy somatic cells on their surface, in a process called phagocytosis. Beyond increasing inflammation and stimulating the immune system, macrophages also play an important anti-inflammatory role, and can reduce immune responses through the release of cytokines.

[0081] As used herein, the term “dendritic cell” is used according to its simple, ordinary meaning and refers to antigen-presenting cells (also known as accessory cells) of the mammalian immune system. Their primary function is to process antigenic material and present it on the cell surface to T cells of the immune system. They act as messengers between the innate and adaptive immune systems. The most common division of dendritic cells is “myeloid” versus “plasmacytoid dendritic cells” (lymphoid system). In embodiments herein, dendritic cells are myeloid dendritic cells.

[0082] As used herein, the terms “electroporation,” “electrical permeability,” and “electrotransfer” are used according to their simple, ordinary meanings and refer to a technique in which an electric field is applied to a cell to increase the permeability of the cell membrane, thereby enabling the introduction of chemicals, drugs, proteins, or nucleic acids, or combinations thereof, into the cell. The cell must then be handled carefully until it has the opportunity to divide. This process is about 10 times more effective than chemical transformation. Therefore, the term “electroporation enhancer” refers to a compound or composition that improves the delivery of chemicals, drug compounds, proteins, or nucleic acids to cells, improves the efficiency of genetic modification of cells, and / or increases the level of cell viability after transfection. In embodiments, the electroporation enhancer improves the delivery of chemicals, drug compounds, proteins, or nucleic acids to cells. In embodiments, the electroporation enhancer increases the efficiency of genetic modification in cells. In embodiments, the electroporation enhancer increases the efficiency of genetic modification in cells compared to the efficiency of genetic modification in the absence of the electroporation enhancer. In embodiments, the electroporation enhancer increases the level of cell viability after transfection. In one embodiment, the electroporation enhancer increases the level of cell viability after transfection compared to the cell viability after transfection in the absence of the electroporation enhancer.

[0083] As used herein, the term “transfection” is used in its simple, ordinary sense and refers to the process of intentionally introducing naked or purified nucleic acids into eukaryotic cells. In examples, “transfection” may refer to other methods and cell types, but other terms are often preferred. For example, the term “transformation” is typically used to describe the transfer of nonviral DNA in bacteria and non-animal eukaryotic cells, including plant cells. In animal cells, transfection is the preferred term. For example, the term “transduction” is often used to describe the introduction of virus-mediated genes into eukaryotic cells.

[0084] A "transfection reagent" can be any compound and / or composition that increases the uptake of one or more nucleic acids into one or more target cells.

[0085] As used herein, the term “contact” is used in its simple, ordinary sense and refers to a process that allows at least two different species (e.g., a chemical compound including a biomolecule or a cell) to become sufficiently proximal to react, interact, or physically come into contact. However, naturally, the reaction products obtained can be produced from the reaction between the added reagents, or directly from intermediates from one or more of the added reagents that can be produced in the reaction mixture.

[0086] The term "modulate" is used according to its simple, ordinary meaning, referring to the act of changing or altering one or more properties. "Modification" refers to the process of changing or altering one or more properties. For example, when applied to the effect of a modulator on a target gene, modulation is the modification of a means by which gene expression or gene activity is altered, either by increasing or decreasing it.

[0087] As used herein, the term “abnormal” means different from normal. When used to describe enzyme activity, abnormal means activity that is greater or less than the mean of a normal control or normal non-disease control sample. Abnormal activity may refer to a disease-causing amount of activity, and restoring the abnormal activity to a normal or non-disease-related amount (e.g., by using the methods described herein) results in a reduction of the disease or one or more disease symptoms.

[0088] The term "expression" includes, but is not limited to, any steps involved in the production of a polypeptide, including transcription, post-transcriptional modification, translation, post-translational modification, and secretion. Expression can be detected using conventional techniques for detecting proteins (e.g., ELISA, Western blotting, flow cytometry, immunofluorescence, immunohistochemistry, etc.).

[0089] The term "recombinant," when used, for example, in relation to cells, nucleic acids, proteins, or vectors, indicates that the cell, nucleic acid, protein, or vector has been modified by the introduction of a heterologous nucleic acid or protein or by the alteration of a native nucleic acid or protein, or that the cell originates from such a modified cell. For example, a recombinant cell will express a gene not found in the cell's native (non-recombinant) form, or a native gene that is otherwise abnormally expressed, lowly expressed, or not expressed at all. Transgenic cells and plants typically express heterologous genes or coding sequences as a result of a recombination method.

[0090] When the term "heterogeneous" is used in reference to a portion of a nucleic acid, it indicates that the nucleic acid contains two or more subsequences that are not inherently related to each other. For example, a nucleic acid typically has two or more sequences from unrelated genes that are typically recombinantly produced and arranged to create a new functional nucleic acid, e.g., a promoter from one source and a coding region from another. Similarly, a heterogeneous protein refers to a protein that contains two or more subsequences that are not inherently related to each other in nature (e.g., a fusion protein).

[0091] "Patient" or "subject requiring it" means an organism suffering from or susceptible to a disease or condition that can be treated by administration of a composition or pharmaceutical composition as provided herein. Non-limiting examples include humans, other mammals, cattle, rats, mice, dogs, monkeys, goats, sheep, deer, and other non-mammals. In some embodiments, the patient is human.

[0092] As used herein, the term “administration” means a route suitable for cell therapy. Examples include intravenous (IV), intramuscular (IM), intrathecal (lumbar puncture), or interarterial (IA) administration. Parenteral administration includes, for example, intravenous, intramuscular, intraarterial, intradermal, subcutaneous, intraperitoneal, intraperitoneal, and intracranial administration. Other modes of delivery include, but are not limited to, the use of liposomal formulations and intravenous infusion. In embodiments, administration does not include the administration of active agents other than those listed. In embodiments, administration includes co-administration with another agent. “Co-administration” means that the compositions described herein are administered simultaneously with, immediately before, or immediately after, one or more additional therapeutic treatments, such as cancer therapy, such as chemotherapy, hormone therapy, radiotherapy, or immunotherapy. The compounds of the present invention may be administered alone or co-administered to a patient. Co-administration means that the compounds may be administered individually or in combination (multiple compounds) simultaneously or sequentially. Therefore, the preparation can be combined with other active substances as needed (for example, to reduce metabolic degradation).

[0093] "Pharmacopoeia-acceptable excipients" and "pharmacopoeia-acceptable carriers" refer to substances that can be included in the compositions of the Disclosure to assist in the administration and absorption of active agents to a subject without causing significant adverse toxic effects to the patient. Non-limiting examples of pharmacopoeia-acceptable excipients include water, NaCl, ordinary saline, lactated Ringer's solution, ordinary sucrose, ordinary glucose, binders, fillers, disintegrants, lubricants, coatings, sweeteners, flavorings, salt solutions (such as Ringer's solution), alcohols, oils, gelatin, carbohydrates such as lactose, amylose or starch, fatty acid esters, hydroxymethylcellulose, polyvinylpyrrolidine, and colorants. Such preparations may be sterilized and, if necessary, mixed with auxiliary agents such as lubricants, preservatives, stabilizers, wetting agents, emulsifiers, salts to affect osmotic pressure, buffers, colorants, and / or aromatic substances that do not react adversely with the compounds of the Disclosure. Those skilled in the art will recognize that other pharmaceutical excipients may be useful in the Disclosure.

[0094] The term "leukemia" broadly refers to progressive malignant diseases of the hematopoietic organs, generally characterized by abnormal proliferation and development of white blood cells and their precursors in the blood and bone marrow. Leukemia is generally clinically classified based on (1) the duration and characteristics of the acute or chronic disease; (2) the type of cells involved; bone marrow (myeloid), lymphoid (lymphoid), or monocytic; and (3) an increase or absence of a number of abnormal cells, whether hematological leukemic or non-leukemic (subleukemic). Leukemias that can be treated with the compounds or methods provided herein include, for example, acute myeloid leukemia, chronic myeloid leukemia, acute nonlymphocytic leukemia, chronic lymphocytic leukemia, acute granulocytic leukemia, chronic granulocytic leukemia, acute promyelocytic leukemia, adult T-cell leukemia, nonleukemic leukemia, leukocyte leukemia, basophilic leukemia, blastic leukemia, bovine leukemia, chronic myeloid leukemia, cutaneous leukemia, embryonic leukemia, eosinophilic leukemia, Gross leukemia, hairy cell leukemia, and hemoblastic leukemia. Leukemia, hemocytoblastic leukemia, histiocytic leukemia, stem cell leukemia, acute monocytic leukemia, leukopenic leukemia, lymphatic leukemia, lymphoblastic leukemia, lymphocytic leukemia, lymphogenous leukemia, lymphoid leukemia, lymphosarcoma cell leukemia, mast cell leukemia, megakaryocytic leukemia, micromyeloblastic leukemia, monocytic leukemia, myeloblastic leukemia, myeloid leukemia Examples include granulocytic leukemia, myelomonocytic leukemia, Naegeri's leukemia, plasma cell leukemia, multiple myeloma, plasma cell leukemia, promyelocytic leukemia, Ryder cell leukemia, Schilling's leukemia, stem cell leukemia, subleukemia, or anaplastic cell leukemia.

[0095] It will be understood that the examples and embodiments described herein are for illustrative purposes only, and that various modifications or changes are suggested in light of them and should be included in the spirit and scope of this application and in the claims. All publications, patents and patent applications referenced herein are incorporated herein by reference in their entirety for all purposes. Methods for genetic modification

[0096] A method for genetically modifying bone marrow cells without using a viral transduction method for delivering gene editing reagents to cells is provided herein. The applicant has remarkably discovered a method that overcomes problems associated with previous delivery methods, including the inefficient or variably efficient delivery of compounds (e.g., oligonucleotides, proteins, etc.). The method provided herein includes embodiments thereof for consistently modifying bone marrow cells at a substantially population level. Thus, the cells may not require enrichment or selection steps after delivery of the gene editing reagents to the cells. Accordingly, in one embodiment, a method for genetically modifying bone marrow cells is provided herein, comprising transfecting the bone marrow cells with a gene editing reagent targeting a site of interest, wherein the bone marrow cells are not transduced by a viral vector.

[0097] In embodiments of the gene modification methods provided herein, the bone marrow cells are primary bone marrow cells. In embodiments of the gene modification methods provided herein, the bone marrow cells are passaged bone marrow cells.

[0098] In embodiments of the gene modification methods provided herein, the bone marrow cells are monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, or megakaryocytes. In embodiments, the bone marrow cells are monocytes. In embodiments, the bone marrow cells are macrophages. In embodiments, the bone marrow cells are dendritic cells. In embodiments, the bone marrow cells are neutrophils. In embodiments, the bone marrow cells are basophils. In embodiments, the bone marrow cells are eosinophils. In embodiments, the bone marrow cells are erythrocytes. In embodiments, the myoform cells are megakaryocytes. In various embodiments, the bone marrow cells are promyelocytes, promonocytes, myeloid stem cells, proerythroblasts, or promegakaryocytes. In embodiments, the bone marrow cells are promyelocytes. In embodiments, the bone marrow cells are promonocytes. In embodiments, the bone marrow cells are myeloid stem cells. In embodiments, the bone marrow cells are proerythroblasts. In embodiments, the bone marrow cells are promegakaryocytes.

[0099] In embodiments of the gene modification methods provided herein, transfection of cells is performed via electroporation using an electroporation system. In embodiments of the gene modification methods provided herein, transfection of cells is performed via nucleofection using a nucleofection system. As used herein, “nucleofection” refers to an electroporation-based transfection method used to deliver nucleic acids (e.g., DNA) to cells. For example, nucleofection enables the delivery of DNA to the cytoplasm or nucleus of bone marrow cells. In embodiments, the nucleofection system is one of several Nucleofector® systems provided by Lonza (Basel, Switzerland). In embodiments, transfection of cells is performed via nucleofection.

[0100] In embodiments, the transfection process is achieved using a chemical transfection system, such as a polymer-based transfection reagent. Various polymer-based transfection reagents are known to those skilled in the art. In embodiments, the transfection reagent is a polymer-based transfection reagent. Suitable transfection reagents include, but are not limited to, one or more compounds and / or compositions comprising cationic polymers such as polyethyleneimine (PEI), polymers of positively charged amino acids such as polylysine and polyarginine, positively charged dendrimers and crushed dendrimers, polymers, cationic β-cyclodextrin (CD-polymer), DEAE-dextran, TURBOFECT® transfection reagent (available from ThermoFisher Scientific), Xfect transfection reagent (available from Takara Bio USA), and Sigma Universal Transfection Reagent (available from Sigma-Aldrich). In embodiments, the transfection reagent is a cationic polymer-based transfection reagent, such as polyglycolic acid (PGA), POLYMER In Vivo Transfection Reagent (available from Altogen Biosystems), and polyethyleneimine (PEI).

[0101] In embodiments of the gene modification methods provided herein, cells are transfected using a lipid-based transfection system. In embodiments, the reagent for introducing macromolecules into cells may include one or more lipids, which may be cationic lipids and / or neutral lipids. Examples of lipids include, but are not limited to, N-[1-(2,3-dioleyloxy)propyl]-N,N,N-trimethylammonium chloride (DOTMA), dioleoylphosphatidylcholine (DOPE), 1,2-bis(oleoyloxy)-3-(4'-trimethylammonio)propane (DOTAP), dihydrooxy-dimyristylspermine tetrahydrochloride (DHDMS), hydroxy-dimyristylspermine tetrahydrochloride (HDMS), 1,2-dioleoyl-3-(4'-trimethylammonio)butanoyl-sn-glycerol (DOTB), 1,2-dioleoyl-3-succinyl-sn-glycerolcholine ester (DOSC), and cholesteryl (4' -Trimethylammonio)butanoate (ChoTB), cetyltrimethylammonium bromide (CTAB), 1,2-dioleoyl-3-dimethyl-hydroxyethylammonium bromide (DORI), 1,2-dioleyloxypropyl-3-dimethyl-hydroxyethylammonium bromide (DOME), 1,2-dimyristyloxypropyl-3-dimethylhydroxyethylammonium bromide (DMRIE), O,O'-didodecyl-N-[p(2-trimethylammonioethyloxy)benzoyl]-N,N,N-trimethylammonium chloride, complexed with one or more lipids (e.g., 5-carboxyspermylglycine dioctadecylamide (DOGS), N,N I ,N II ,N III -tetramethyl-N,N I ,N II ,N III-tet-Lapalmitylspermine (TM-TPS) and dipalmitoylphosphatidylethanolamine 5-carboxyspermylamide (DPPES)), lipopolylysine (polylysine complexed with DOPE), TRIS (Tris(hydroxymethyl)aminomethane, tromethamine) complex fatty acids (TFAs) and / or peptides, e.g., trilysyl-alanyl-TRIS mono, di- and tripalmitate, (3B-[N-(N',N'-dimethylaminoethane)-carbamoyl]cholesterol (DCChol), N-(α-trimethylammonioacetyl)-didodecyl-D-glutamate chloride (TMAG), dimethyldioctadecylammonium Examples include umbromide (DDAB), 2,3-dioleyloxy-N-[2(spermine-carboxamide)ethyl]-N,N-dimethyl-1-propanamine-inium trifluoroacetate (DOSPA), and combinations thereof. Those skilled in the art will understand that certain combinations of the above lipids, such as cationic lipids, have been shown to be particularly suitable for the introduction of nucleic acids, proteins, ribonucleoproteins, etc., into cells. In embodiments, the transfection reagent is a cationic lipid transfection reagent. An example of a cationic lipid transfection reagent suitable for the introduction of nucleic acids into cells is Life, Carlsbad, California. Examples include a 3:1 (w / w) combination of DOSPA and DOPE available from Life Technologies Corporation under the trade name LIPOFECTAMINE®, a 1:1 (w / w) combination of DOTMA and DOPE available from Life Technologies under the trade name LIPOFECTIN®, a 1:1 (M / M) combination of DIVIRIE and cholesterol available from Life Technologies Corporation (Carlsbad, California) under the trade name DIVIRIE-C reagent, a 1:1.5 (M / M) combination of TM-TPS and DOPE available from Life Technologies, and CRISPRMAX® available from Life Technologies Corporation (Carlsbad, California).Other commercially available cationic lipid transfection reagents include, but are not limited to, TRANSFAST® (available from Promega Corporation), LYOVEC® (available from InvivoGen), DOTAP Liposome Transfection Reagent (available from Roche), TRANSIT® Transfection Reagent (available from Mirus), and GENEJUICE® Transfection Reagent (EMD Millipore). Further transfection reagents that may be used herein include VIAFECT® Transfection Reagent, FUGENE® 6 Transfection Reagent, and FUGENE® HD Transfection Reagent (each available from Promega Corporation), and TRANSFECTIN® Lipid Reagent (available from BioRad Laboratories, Inc.).

[0102] In embodiments of the gene modification methods provided herein, cells are not subjected to a selection step and / or enrichment step after gene modification. In embodiments, cells are not subjected to a selection step after gene modification. The selection step may be positive or negative selection for a desired phenotype. For example, selection may be based on antibiotic resistance. In embodiments, cells are not subjected to an enrichment step after transfection. In embodiments, the enrichment step is a process of enriching or expanding the cells of interest or a population of cells obtained from the selection step. In embodiments of the gene modification methods provided herein, cells are not subjected to both a selection step and an enrichment step after transfection, thus significantly improving efficiency and reducing the time required to obtain gene-modified cells.

[0103] In embodiments, the methods described herein include gene modification methods. In embodiments, the gene modification methods provided herein include gene editing. In embodiments, the gene editing methods provided herein include nucleases. In embodiments, the methods for gene modification provided herein include nucleases for gene editing, such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), meganucleases, and clustered and regularly arranged short palindromic sequence repeat systems (CRISPR / CAS), as well as their variants readily identifiable by those skilled in the art. In embodiments, the gene modification methods provided herein include the CRISPR-CAS system.

[0104] In embodiments, the gene editing method provided herein includes a gene editing reagent. In embodiments, the gene editing reagent includes an RNA guide nuclease. In embodiments, the RNA guide nuclease is a CRISPR-Cas system. In embodiments, the CRISPR-Cas system includes Cas9 or a Cas9 variant. In embodiments, the CRISPR-Cas system includes Cas9. In embodiments, the CRISPR-Cas system includes a Cas9 variant. In embodiments, the CRISPR-Cas system includes Cas12, Cascade, Cas13, or variants of each thereof. In embodiments, the CRISPR-Cas system includes Cas12. In embodiments, the CRISPR-Cas system includes Cascade. In embodiments, the CRISPR-Cas system includes Cas13. In embodiments, the CRISPR-Cas system includes a Cas12 variant. In embodiments, the CRISPR-Cas system includes Cascade, and one or more members of the Cascade complex are variants. In embodiments, the CRISPR-Cas system includes a Cas13 variant.

[0105] In embodiments of the gene editing methods provided herein, the gene editing reagent comprises a CRISPR-Cas system comprising a Cas protein, guide RNA, and optionally donor DNA. In embodiments, the Cas protein and guide RNA are non-covalently associated. In embodiments, the Cas protein, guide RNA, and donor DNA are non-covalently associated.

[0106] In some embodiments, the gene editing reagent comprises a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, or an algonaut endonuclease. In some embodiments, the gene editing reagent comprises a transcription activator-like effector nuclease (TALEN). In some embodiments, the gene editing reagent comprises a zinc finger nuclease. In some embodiments, the gene editing reagent comprises an algonaut endonuclease.

[0107] In embodiments of the gene modification methods provided herein using electroporation / nucleofection, cells can be brought into contact with an electroporation enhancer during transfection. In embodiments, the electroporation enhancer is a carrier DNA, single-stranded DNA, a combination of single-stranded and double-stranded DNA, a polymer additive, and / or oligonucleotides. In embodiments, the electroporation enhancer is single-stranded DNA. In embodiments, the electroporation enhancer is a combination of single-stranded and double-stranded DNA. In embodiments, the electroporation enhancer is a polymer additive. In embodiments, the electroporation enhancer is an oligonucleotide. In embodiments, the oligonucleotide is a TLR antagonist such as A151. In embodiments, the carrier DNA is a single-stranded DNA oligonucleotide. In embodiments, the carrier DNA is a double-stranded DNA oligonucleotide. In embodiments, the carrier DNA is non-homologous to the human, mouse, and / or rat genome. In embodiments, the carrier DNA is non-homologous to the human genome. In embodiments, the carrier DNA is non-homologous to the mouse genome. In embodiments, the carrier DNA is non-homologous to the rat genome. In embodiments, electroporation is enhanced by the use of one or more JAK2 inhibitors, for example, when Cas mRNA is delivered to the cells.

[0108] In embodiments of the gene modification methods provided herein, myeloid cells are differentiated before electroporation. In embodiments, myeloid cells are differentiated into dendritic cells. In embodiments, myeloid cells are differentiated into macrophages. In embodiments, newly isolated human monocytes are electroporated and then differentiated into macrophages. Methods for differentiating myeloid cells are well known in the art. See, for example, Harada, Y., et al. Cytokine-based high log-scale expansion of functional human dendritic cells from cord-blood CD34-positive cells. Sci Rep 1, 174 (2011); Ohradanova-Repic A, et al. Differentiation of human monocytes and derived subsets of macrophages and dendritic cells by the HLDA10 monoclonal antibody panel. Clin Transl Immunology. 2016; 5(1):e55 (each of which is incorporated in whole by reference).

[0109] In embodiments of the gene modification methods provided herein, bone marrow cells are not differentiated prior to electroporation. In embodiments, total bone marrow cells (e.g., mouse) are electroporated and then differentiated into bone marrow-derived dendritic cells (BMDCs).

[0110] Myelocytes can be activated by exposure to various factors, including viruses. However, activation of myelocytes during, for example, genetic engineering of cells reduces their usefulness in downstream applications. In embodiments of the genetic engineering methods provided herein, myelocytes are not activated before or during genetic engineering. In embodiments of the genetic engineering methods provided herein, myelocytes are not activated before genetic engineering. In embodiments of the genetic engineering methods provided herein, myelocytes are moderately activated during genetic engineering. In embodiments, mild activation may include slight or minimal activation. For example, the absence or reduced expression of a particular marker may indicate that myelocytes or multiple myelocytes are not activated, are slightly activated, or are not significantly activated. For example, CD64, CD169, or HLA-DR expression may be reduced or absent in myelocytes or multiple myelocytes.

[0111] In embodiments of the gene modification methods provided herein, two or more different crRNAs complementary to the target site are introduced into bone marrow cells. In embodiments of the gene modification methods provided herein, two different crRNAs complementary to the target site are introduced into bone marrow cells. In embodiments of the gene modification methods provided herein, three different crRNAs complementary to the target site are introduced into bone marrow cells. In embodiments of the gene modification methods provided herein, four different crRNAs complementary to the target site are introduced into bone marrow cells. In embodiments of the gene modification methods provided herein, five different crRNAs complementary to the target site are introduced into bone marrow cells. In embodiments of the gene modification methods provided herein, six different crRNAs complementary to the target site are introduced into bone marrow cells. In embodiments of the gene modification methods provided herein, seven different crRNAs complementary to the target site are introduced into bone marrow cells. In embodiments of the gene modification methods provided herein, eight different crRNAs complementary to the target site are introduced into bone marrow cells.

[0112] In one embodiment, introducing crRNA into bone marrow cells includes transfecting the cells with crRNA. In another embodiment, contacting crRNA with tracrRNA. In yet another embodiment, contacting crRNA includes annealing crRNA to tracrRNA. In yet another embodiment, crRNA is a single guide RNA (sgRNA).

[0113] In embodiments of the gene modification methods provided herein, multiple target sites are targeted. In embodiments of the gene modification methods provided herein, two target sites are targeted. In embodiments of the gene modification methods provided herein, three target sites are targeted. In embodiments of the gene modification methods provided herein, four target sites are targeted. In embodiments of the gene modification methods provided herein, five target sites are targeted. In embodiments of the gene modification methods provided herein, two or more different crRNAs targeting each target site are introduced into bone marrow cells.

[0114] In embodiments of the gene modification methods provided herein, the bone marrow cells are mammalian cells. In embodiments, the mammalian bone marrow cells are selected from cattle, rat, mouse, dog, monkey, goat, sheep, deer, or other mammalian bone marrow cells. In embodiments, the mammalian bone marrow cells are bovine bone marrow cells. In embodiments, the mammalian bone marrow cells are rat bone marrow cells. In embodiments, the mammalian bone marrow cells are mouse bone marrow cells. In embodiments, the mammalian bone marrow cells are dog bone marrow cells. In embodiments, the mammalian bone marrow cells are monkey bone marrow cells. In embodiments, the mammalian bone marrow cells are goat bone marrow cells. In embodiments, the mammalian bone marrow cells are sheep bone marrow cells. In embodiments, the mammalian bone marrow cells are cow bone marrow cells. In embodiments, the mammalian bone marrow cells are cow bone marrow cells. In embodiments, the mammalian bone marrow cells are deer bone marrow cells. In embodiments, the bone marrow cells are non-mammalian bone marrow cells. In embodiments, the bone marrow cells are human bone marrow cells.

[0115] In one embodiment, a method for genetically modifying a plurality of myeloid cells is provided herein, comprising the step of transfecting the plurality of myeloid cells in the presence of a gene editing reagent that targets a site of interest, wherein the myeloid cells are not transfected by a viral vector. In embodiments of the genetic modification method provided herein, the plurality of myeloid cells are cultured myeloid cells. In embodiments of the genetic modification method provided herein, the plurality of myeloid cells are transfected via electroporation. In embodiments of the genetic modification method provided herein, the plurality of myeloid cells are transfected via lipid-based transfection.

[0116] In embodiments of the gene modification methods provided herein, multiple myeloid cells are not activated before or during gene modification. In embodiments, at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% of the myeloid cells in multiple myeloid cells are not activated. In embodiments, at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the myeloid cells in multiple myeloid cells are not activated. In embodiments, at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the myeloid cells in multiple myeloid cells are not activated. For example, the absence or reduced expression of a particular marker may indicate that myeloid cells or multiple myeloid cells are not activated, are slightly activated, or are not significantly activated. For example, CD64, CD169, or HLA-DR expression may be reduced or absent in bone marrow cells or multiple bone marrow cells.

[0117] With respect to the methods provided herein, including embodiments thereof, gene knockout of single and multiple targets at a substantially population level can be achieved in various cell types without requiring selection or enrichment of genetically modified cells. As used herein, “substantially population level” refers to substantially all cells in a given cell population. Thus, in embodiments, substantially population level refers to at least 70% of the population of myeloid cells. Thus, in embodiments, substantially population level refers to at least 75% of the population of myeloid cells. Thus, in embodiments, substantially population level refers to at least 80% of the population of myeloid cells. Thus, in embodiments, substantially population level refers to at least 85% of the population of myeloid cells. Thus, in embodiments, substantially population level refers to at least 90% of the population of myeloid cells. Thus, in embodiments, substantially population level refers to at least 95% of the population of myeloid cells. In embodiments, the approximate population level is at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the myeloid cell population. In embodiments, the approximate population level is at least 90%, 95%, 96%, 97%, 98%, 99%, or 100% of the myeloid cell population.

[0118] Therefore, in embodiments of the gene modification methods provided herein, the target site is modified in at least 70% of multiple bone marrow cells. In embodiments of the gene modification methods provided herein, the target site is modified in at least 80% of multiple bone marrow cells. In embodiments of the gene modification methods provided herein, the target site is modified in at least 85% of multiple bone marrow cells. In embodiments of the gene modification methods provided herein, the target site is modified in at least 90% of multiple bone marrow cells. In embodiments of the gene modification methods provided herein, the target site is modified in at least 95% of multiple bone marrow cells. In the embodiment, the target site is modified in at least 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of a plurality of myeloid cells.

[0119] In embodiments of the gene modification methods provided herein, a plurality of bone marrow cells include dendritic cells (DCs), and the site of interest is modified in at least 50% of the DCs. In embodiments of the gene modification methods provided herein, a plurality of bone marrow cells include dendritic cells (DCs), and the site of interest is modified in at least 60% of the DCs. In embodiments of the gene modification methods provided herein, a plurality of bone marrow cells include dendritic cells (DCs), and the site of interest is modified in at least 70% of the DCs. In embodiments of the gene modification methods provided herein, a plurality of bone marrow cells include dendritic cells (DCs), and the site of interest is modified in at least 80% of the DCs. In embodiments of the gene modification methods provided herein, a plurality of bone marrow cells include dendritic cells (DCs), and the site of interest is modified in at least 90% of the DCs. In embodiments of the gene modification methods provided herein, a plurality of bone marrow cells include dendritic cells (DCs), and the site of interest is modified in at least 95% of the DCs. In the embodiment, the multiple bone marrow cells include dendritic cells (DCs), and the site of interest is modified in at least 55%, 60%, 70%, 75%, 80%, 85%, 90%, or 95% of the DCs. In the embodiment, the plurality of bone marrow cells include dendritic cells (DCs), and the site of interest is modified in at least 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the DCs.

[0120] The methods described herein can be used to simultaneously modify a single gene or multiple genes in bone marrow cells. One gene, two genes, three genes, or more than three genes may be simultaneously modified in bone marrow cells. Using the methods provided herein, including its embodiments, up to 20 genes can be simultaneously modified in bone marrow cells.

[0121] In embodiments of the gene modification methods provided herein, the viability of multiple bone marrow cells after electroporation is at least 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In embodiments of the gene modification methods provided herein, the viability of multiple bone marrow cells after electroporation is at least 80%. In embodiments of the gene modification methods provided herein, the viability of multiple bone marrow cells after electroporation is at least 85%. In embodiments of the gene modification methods provided herein, the viability of multiple bone marrow cells after electroporation is at least 90%. In embodiments of the gene modification methods provided herein, the viability of multiple bone marrow cells after electroporation is at least 95%. Survival rates can be measured by any technique known to those skilled in the art, including but not limited to cell lysis, caspase, functional, genomic, proteomics, and / or flow cytometry assays.

[0122] In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease. In embodiments, the RNA guide nuclease is a CRISPR system. In embodiments of the methods provided herein, the CRISPR system may include Cas1, Cas1B, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csn1 and Csx12), Cas10, Cas12, Cas13, Csy1, Csy2, Csy3, Cse1, Cse2, Csc1, Csc2 Examples include Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmr1, Cmr3, Cmr4, Cmr5, Cmr6, Csb1, Csb2, Csb3, Csx17, Csx14, Csx10, Csx16, CsaX, Csx3, Csx1, Csx15, Csf1, Csf2, Csf3, Csf4, their homologs, or their variants. In embodiments, the RNA guide nuclease is a CRISPR-Cas system. In embodiments, the CRISPR-Cas system includes Cas9, Cas12, Cascade, Cas13, or each of their variants. In embodiments, the CRISPR-Cas system includes Cas9 or a Cas9 variant. In embodiments, the CRISPR-Cas system includes Cas12, Cascade, Cas13, or each of their variants. In embodiments of the gene modification methods provided herein, the gene editing reagent includes a CRISPR-Cas system containing Cas9. In embodiments of the gene modification methods provided herein, the gene editing reagent includes a CRISPR-Cas system containing a Cas9 variant. In embodiments of the gene modification methods provided herein, the gene editing reagent includes a CRISPR-Cas system containing Cas12 or its variants. In embodiments of the gene modification methods provided herein, the gene editing reagent includes a CRISPR-Cas system containing Cascade or its variants. In embodiments of the gene modification methods provided herein, the gene editing reagent includes Cas3.In embodiments of the gene modification methods provided herein, the gene editing reagent comprises a CRISPR-Cas system containing Cas13 or a variant thereof.

[0123] In the methods provided herein, in embodiments, the gene editing reagent comprises a CRISPR-Cas system comprising a Cas protein, guide RNA, and optionally donor DNA. In embodiments, the Cas protein and guide RNA are non-covalently associated. In embodiments, the Cas protein, guide RNA, and donor DNA are non-covalently associated.

[0124] In embodiments of the gene modification methods provided herein, the gene editing reagent comprises a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, or an algonaut endonuclease. In embodiments, the gene editing reagent comprises a TALEN. In embodiments, the gene editing reagent comprises a zinc finger nuclease. In embodiments, the gene editing reagent comprises an algonaut endonuclease.

[0125] In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease. In embodiments, the RNA guide nuclease comprises a guide RNA and a ribonucleoprotein (RNP), wherein the ratio of the guide RNA to the ribonucleoprotein (RNP) is approximately 100:1 to approximately 1:100 or less. In embodiments of the genetic modification methods provided herein, the gene editing reagent includes an RNA guide nuclease and a ribonucleoprotein (RNP), where the guide RNA to RNP ratio is approximately 100:1 or less, approximately 90:1 or less, approximately 80:1 or less, approximately 70:1 or less, approximately 60:1 or less, approximately 50:1 or less, approximately 40:1 or less, approximately 30:1 or less, approximately 20:1 or less, approximately 10:1 or less, approximately 1:10 or less, approximately 1:20 or less, approximately 1:30 or less, approximately 1:40 or less, approximately 1:50 or less, approximately 1:60 or less, approximately 1:70 or less, approximately 1:80 or less, approximately 1:90 or less, or approximately 1:100. In embodiments of the genetic modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and a ribonucleoprotein (RNP), where the guide RNA to RNP ratio is approximately 100:1 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and ribonucleoprotein (RNP), with a guide RNA to RNP ratio of about 90:1 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and ribonucleoprotein (RNP), with a guide RNA to RNP ratio of about 80:1 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and ribonucleoprotein (RNP), with a guide RNA to RNP ratio of about 70:1 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and ribonucleoprotein (RNP), with a guide RNA to RNP ratio of about 60:1 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and a ribonucleoprotein (RNP), wherein the ratio of guide RNA to RNP is approximately 50:1 or less.In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and ribonucleoprotein (RNP), with a guide RNA to RNP ratio of about 40:1 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and ribonucleoprotein (RNP), with a guide RNA to RNP ratio of about 30:1 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and ribonucleoprotein (RNP), with a guide RNA to RNP ratio of about 20:1 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and ribonucleoprotein (RNP), with a guide RNA to RNP ratio of about 10:1 or less.

[0126] In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and a ribonucleoprotein (RNP), with a guide RNA to RNP ratio of approximately 1:10 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and a ribonucleoprotein (RNP), with a guide RNA to RNP ratio of approximately 1:20 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and a ribonucleoprotein (RNP), with a guide RNA to RNP ratio of approximately 1:30 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and a ribonucleoprotein (RNP), with a guide RNA to RNP ratio of approximately 1:40 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and a ribonucleoprotein (RNP), with a guide RNA to RNP ratio of about 1:50 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and a ribonucleoprotein (RNP), with a guide RNA to RNP ratio of about 1:60 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and a ribonucleoprotein (RNP), with a guide RNA to RNP ratio of about 1:70 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and a ribonucleoprotein (RNP), with a guide RNA to RNP ratio of about 1:80 or less, or about 1:90 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and a ribonucleoprotein (RNP), wherein the guide RNA to RNP ratio is approximately 1:100 or less. In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and a ribonucleoprotein (RNP), wherein the guide RNA to RNP ratio is approximately 3:1 or less.In embodiments of the gene modification methods provided herein, the gene editing reagent comprises an RNA guide nuclease and a ribonucleoprotein (RNP), wherein the ratio of guide RNA to RNP is approximately 2:1 or less.

[0127] In embodiments of the gene modification methods provided herein, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% of the transfected cells are administered to a patient in need without cell selection or concentration. In embodiments, at least 70% of the transfected cells are administered to a patient in need without cell selection or concentration. In embodiments, at least 75% of the transfected cells are administered to a patient in need without cell selection or concentration. In embodiments, at least 80% of the transfected cells are administered to a patient in need without cell selection or concentration. In one embodiment, at least 85% of the transfected cells are administered to patients in need without cell selection or concentration. In another embodiment, at least 90% of the transfected cells are administered to patients in need without cell selection or concentration. In yet another embodiment, at least 95% of the transfected cells are administered to patients in need without cell selection or concentration.

[0128] In embodiments of the gene modification methods provided herein, at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or at least 99% of the transfected cells are used in subsequent reactions without cell selection or concentration. In embodiments of the gene modification methods provided herein, at least 70% of the transfected cells are used in subsequent reactions without cell selection or concentration. In embodiments of the gene modification methods provided herein, at least 75% of the transfected cells are used in subsequent reactions without cell selection or concentration. In embodiments of the gene modification methods provided herein, at least 80% of the transfected cells are used in subsequent reactions without cell selection or concentration. In embodiments of the gene modification methods provided herein, at least 85% of the transfected cells are used in subsequent reactions without cell selection or concentration. In embodiments of the gene modification methods provided herein, at least 90% of the transfected cells are used in subsequent reactions without cell selection or concentration. In embodiments of the gene modification methods provided herein, at least 95% of the transfected cells are used in subsequent reactions without cell selection or concentration.

[0129] One embodiment provides a method for genetically modifying a plurality of myeloid cells, comprising transfecting myeloid cells with a gene editing reagent, wherein the myeloid cells are not transfected with a viral vector and the method does not involve a selection or enrichment step after myeloid cell transfection. In one embodiment, the cells are transfected via electroporation. In another embodiment, the cells are transfected via lipid-based transfection.

[0130] In the methods provided herein, including those embodiments, the site of interest is modified in at least 70% of a plurality of myeloid cells. In embodiments, the site of interest is modified in at least 80% of a plurality of myeloid cells. In embodiments, the site of interest is modified in at least 85% of a plurality of myeloid cells. In embodiments, the site of interest is modified in at least 90% of a plurality of myeloid cells. In embodiments, the plurality of myeloid cells include dendritic cells (DCs), and the site of interest is modified in at least 50% of the DCs. In embodiments, the survival rate of the plurality of myeloid cells after electroporation is at least 50%. In embodiments, the survival rate of the plurality of myeloid cells after electroporation is at least 60%. In embodiments, the survival rate of the plurality of myeloid cells after electroporation is at least 70%. In embodiments, the survival rate of the plurality of myeloid cells after electroporation is at least 80%. In embodiments, the survival rate of the plurality of myeloid cells after electroporation is at least 90%.

[0131] In an embodiment, the gene editing reagent comprises an RNA guide nuclease. In an embodiment, the RNA guide nuclease is a CRISPR-Cas system. In an embodiment, the CRISPR-Cas system comprises Cas9 or a Cas9 variant. In an embodiment, the CRISPR-Cas system comprises Cas12, Cascade, Cas13 or variants thereof. In an embodiment, the gene editing reagent comprises a CRISPR-Cas system comprising a Cas protein, guide RNA, and optionally donor DNA. In an embodiment, the Cas protein and guide RNA are non-covalently associated. In an embodiment, the Cas protein, guide RNA, and donor DNA are non-covalently associated. In an embodiment, the gene editing reagent comprises a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, or an algonaut endonuclease. Treatment method

[0132] In one embodiment, a method for treating a disease treatable with bone marrow cells is provided herein. The method comprises providing genetically modified bone marrow cells that have not been transformed with a virus, wherein the bone marrow cells have not been transfected with a gene editing reagent, and administering the bone marrow cells to a patient in need of bone marrow cells.

[0133] In embodiments, diseases treatable with bone marrow cells include cancer or diseases characterized by abnormal or dysfunctional bone marrow cells. In several embodiments, cancer is chronic myeloid leukemia. In embodiments, cancer is acute myeloid leukemia. In embodiments, diseases are treatable using bone marrow cells. In embodiments, diseases are autoimmune conditions, neuropathologies, diseases involving immunomodulatory components with bone marrow cells, myelodysplastic syndromes, or myeloproliferative disorders. In embodiments, diseases are autoimmune conditions. In embodiments, autoimmune conditions are systemic lupus erythematosus, rheumatoid arthritis, inflammatory bowel disease, or multiple sclerosis. In embodiments, autoimmune conditions are systemic lupus erythematosus. In embodiments, autoimmune pathology is inflammatory bowel disease. In several embodiments, autoimmune pathology is multiple sclerosis. In embodiments, diseases are neuropathologies. In embodiments, diseases include immunomodulatory components with bone marrow cells. In embodiments, diseases are myelodysplastic syndromes. In some embodiments, the disease is a myeloproliferative disorder.

[0134] In embodiments, a method for treating a disease treatable with bone marrow cells includes administering genetically modified bone marrow cells provided herein, including those embodiments. In embodiments, bone marrow cells are transfected with gene editing reagents according to any embodiment or model provided herein. In embodiments, bone marrow cells are genetically modified to mutate and replace disease-causing genes. In embodiments, bone marrow cells are genetically modified to mutate disease-causing genes. In embodiments, bone marrow cells are genetically modified to replace disease-causing genes. In multiple embodiments, bone marrow cells are genetically modified for cell therapy.

[0135] In one embodiment, the bone marrow cells are primary bone marrow cells. In another embodiment, the bone marrow cells are cultured bone marrow cells.

[0136] In an embodiment, the gene editing reagent comprises an RNA guide nuclease. In an embodiment, the RNA guide nuclease is a CRISPR-Cas system. In an embodiment, the CRISPR-Cas system comprises Cas9 or a Cas9 variant. In an embodiment, the CRISPR-Cas system comprises Cas9. In an embodiment, the CRISPR-Cas system comprises a Cas9 variant. In an embodiment, the CRISPR-Cas system comprises Cas12, Cascade, Cas13, or variants of each thereof. In an embodiment, the CRISPR-Cas system comprises Cas12. In an embodiment, the CRISPR-Cas system comprises Cascade. In an embodiment, the CRISPR-Cas system comprises Cas13. In an embodiment, the CRISPR-Cas system comprises a Cas12 variant. In an embodiment, the CRISPR-Cas system comprises Cascade, and one or more components of the Cascade complex are variants. In an embodiment, the CRISPR-Cas system comprises a Cas13 variant.

[0137] In the methods provided herein, in embodiments, the gene editing reagent comprises a CRISPR-Cas system comprising a Cas protein, guide RNA, and optionally donor DNA. In embodiments, the Cas protein and guide RNA are non-covalently associated. In embodiments, the Cas protein, guide RNA, and donor DNA are non-covalently associated.

[0138] In the embodiments provided herein, the gene editing reagent comprises a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, or an algonaut endonuclease. In the embodiments, the gene editing reagent comprises a transcription activator-like effector nuclease (TALEN). In the embodiments, the gene editing reagent comprises a zinc finger nuclease. In the embodiments, the gene editing reagent comprises an algonaut endonuclease.

[0139] In the embodiments of the methods provided herein, the administration of genetically modified bone marrow to a patient in need includes intravenous, intramuscular, intra-arterial, or intrathecal administration. In embodiments, administration includes intravenous administration. In embodiments, administration includes intramuscular administration. In embodiments, administration includes intra-arterial administration. In embodiments, administration includes intrathecal administration. In embodiments, administration includes intravenous, parenteral, intraperitoneal, intramuscular, intrafocal, or subcutaneous administration. In embodiments, administration includes intravenous administration. In embodiments, administration includes parenteral administration. In embodiments, administration includes intraperitoneal administration. In embodiments, administration includes intramuscular administration. In embodiments, administration includes intrafocal administration. In embodiments, administration includes subcutaneous administration. system

[0140] A system for efficiently generating genetically modified bone marrow cells is provided herein. The cells can be genetically modified to contain a mutant gene or a knockout gene. The system provided herein produces population-level gene knockouts or gene mutants in bone marrow cells. Thus, in one embodiment, a system for genetically modifying bone marrow cells in the absence of a viral vector is provided herein. The system comprises a chamber compatible with a transfection system, a plurality of bone marrow cells in the chamber in a culture medium compatible with electroporation, and at least one gene editing system designed to target at least one site of interest in the genome of the bone marrow cells.

[0141] In one embodiment, the system for genetically modifying bone marrow cells in the absence of a viral vector provided herein includes a chamber that fits into a transfection system. In another embodiment, the transfection system includes an electroporation device.

[0142] In some embodiments, a system for genetically modifying bone marrow cells in the absence of a viral vector provided herein comprises a plurality of bone marrow cells in a chamber adapted to a transfection system. In some embodiments, the plurality of bone marrow cells are in a culture medium adapted to electroporation. In some embodiments, the culture medium comprises nutrients, growth factors, and / or antibiotics. In some embodiments, the culture medium comprises DMEM High Glucose, fetal bovine serum, GlutaMAX (Gibco), and penicillin / streptomycin. In some embodiments, the bone marrow cells are primary bone marrow cells or cultured bone marrow cells. In some embodiments, the bone marrow cells are primary bone marrow cells. In some embodiments, the bone marrow cells are cultured bone marrow cells. In some embodiments, the bone marrow cells are monocytes, macrophages, or dendritic cells. In some embodiments, the bone marrow cells are monocytes. In some embodiments, the bone marrow cells are macrophages. In some embodiments, the bone marrow cells are dendritic cells.

[0143] In some embodiments, the system for genetically modifying bone marrow cells in the absence of a viral vector provided herein includes bone marrow cells that are not subjected to a selection step and / or enrichment step after transfection.

[0144] In embodiments, the system for genetically modifying bone marrow cells in the absence of a viral vector provided herein comprises a gene editing system comprising a gene editing reagent. In embodiments, the gene editing reagent comprises an RNA guide nuclease. In embodiments, the RNA guide nuclease is a CRISPR-Cas system. In embodiments, the CRISPR-Cas system comprises Cas9 or a Cas9 variant. In embodiments, the CRISPR-Cas system comprises Cas12, Cascade, Cas13 or variants thereof. In embodiments, the gene editing reagent comprises a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, or an algonaut endonuclease.

[0145] In embodiments, a system for genetically modifying bone marrow cells in the absence of a viral vector provided herein includes contacting the cells with an electroporation enhancer during transfection. In embodiments, the electroporation enhancer is a carrier DNA. In embodiments, the electroporation enhancer is a carrier DNA, single-stranded DNA, a combination of single-stranded and double-stranded DNA, a polymer additive, and / or an oligonucleotide. In embodiments, the electroporation enhancer is single-stranded DNA. In embodiments, the electroporation enhancer is a combination of single-stranded and double-stranded DNA. In embodiments, the electroporation enhancer is a polymer additive. In embodiments, the electroporation enhancer is an oligonucleotide. In embodiments, the oligonucleotide is a TLR antagonist such as A151. In embodiments, the carrier DNA is a single-stranded DNA oligonucleotide. In embodiments, electroporation is enhanced by the use of one or more JAK2 inhibitors, for example, when Cas mRNA is delivered to the cells.

[0146] In the embodiments of the systems provided herein, bone marrow cells are not activated before or during genetic modification. In the embodiments, bone marrow cells are not activated before genetic modification. In the embodiments, bone marrow cells are not activated during genetic modification. Composition and its use

[0147] In one embodiment, a composition comprising a plurality of bone marrow cells in contact with a gene editing reagent, a transfection buffer, and an electroporation enhancer, wherein the composition is free of a viral vector, is provided herein. In an embodiment, the composition provided herein comprises bone marrow cells according to any embodiment or model described herein. In an embodiment, the composition provided herein comprises a gene editing reagent according to any embodiment or model described herein.

[0148] In embodiments, the compositions provided herein include a transfection buffer. In embodiments, the transfection buffer may include a salt, a divalent cation, and a buffering agent. In embodiments, examples of salts include NaCl, KCl, and sodium succinate. In embodiments, examples of divalent cations include magnesium and calcium. In embodiments, examples of buffering agents include sodium phosphate and HEPES. In embodiments, the transfection buffer may include other agents, such as mannitol and sodium lactobionate.

[0149] In embodiments, the compositions provided herein include an electroporation enhancer. In embodiments, the electroporation enhancer is a carrier DNA, single-stranded DNA, a combination of single-stranded and double-stranded DNA, a polymer additive, and / or an oligonucleotide. In embodiments, the electroporation enhancer includes carrier DNA. In embodiments, the carrier DNA is a single-stranded DNA oligonucleotide. In embodiments, the electroporation enhancer is single-stranded DNA. In embodiments, the electroporation enhancer is a combination of single-stranded and double-stranded DNA. In embodiments, the electroporation enhancer is a polymer additive. In embodiments, the electroporation enhancer is an oligonucleotide. In embodiments, the oligonucleotide is a TLR antagonist. In several embodiments, the TLR antagonist is A151. In embodiments, electroporation is enhanced by the use of one or more JAK2 inhibitors, for example, when Cas mRNA is delivered to cells.

[0150] In the embodiments of the compositions provided herein, the bone marrow cells are cultured bone marrow cells. In the embodiments of the compositions provided herein, the plurality of bone marrow cells are a plurality of monocytes, macrophages, or dendritic cells. In the embodiments, the plurality of bone marrow cells are a plurality of monocyte cells. In the embodiments, the plurality of bone marrow cells are a plurality of macrophage cells. In the embodiments, the plurality of bone marrow cells are a plurality of dendritic cells.

[0151] In an embodiment, the gene editing reagent comprises an RNA guide nuclease. In an embodiment, the RNA guide nuclease is a CRISPR-Cas system. In an embodiment, the CRISPR-Cas system comprises Cas9 or a Cas9 variant. In an embodiment, the CRISPR-Cas system comprises Cas9. In an embodiment, the CRISPR-Cas system comprises a Cas9 variant. In an embodiment, the CRISPR-Cas system comprises Cas12, Cascade, Cas13, or variants of each thereof. In an embodiment, the CRISPR-Cas system comprises Cas12. In an embodiment, the CRISPR-Cas system comprises Cascade. In an embodiment, the CRISPR-Cas system comprises Cas13. In an embodiment, the CRISPR-Cas system comprises a Cas12 variant. In an embodiment, the CRISPR-Cas system comprises a Cascade variant. In an embodiment, the CRISPR-Cas system comprises a Cas13 variant.

[0152] In the embodiments of the compositions provided herein, the gene editing reagent comprises a CRISPR-Cas system comprising a Cas protein, guide RNA, and optionally donor DNA. In the embodiments, the Cas protein and guide RNA are associated non-covalently. In the embodiments, the Cas protein, guide RNA, and donor DNA are associated non-covalently.

[0153] In embodiments of the compositions provided herein, the gene editing reagent comprises a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, or an algonaut endonuclease.

[0154] In one embodiment, the RNA guide nuclease comprises guide RNA and ribonucleoprotein (RNP), with a guide RNA to RNP ratio between 100:1 and 1:100. In another embodiment, the RNA guide nuclease comprises guide RNA and ribonucleoprotein (RNP), with a guide RNA pair ratio of approximately 3:1 or less. In yet another embodiment, the RNA guide nuclease comprises guide RNA and ribonucleoprotein (RNP), with a guide RNA to ribonucleoprotein (RNP) ratio of approximately 2:1 or less. In yet another embodiment, the RNA guide nuclease comprises guide RNA and ribonucleoprotein (RNP), with a guide RNA to ribonucleoprotein (RNP) ratio of approximately 3:1. In yet another embodiment, the RNA guide nuclease comprises guide RNA and ribonucleoprotein (RNP), with a guide RNA to ribonucleoprotein (RNP) ratio of approximately 2:1. In embodiments, the electroporation enhancer is selected from carrier DNA, single-stranded DNA, a combination of single-stranded and double-stranded DNA, a polymer additive, and an oligonucleotide. In embodiments, the carrier DNA is a single-stranded DNA oligonucleotide.

[0155] The genetically modified cells provided herein are considered effective in treating diseases characterized by abnormal or dysfunctional bone marrow cells (e.g., chronic myeloid leukemia, acute myeloid leukemia, etc.). Accordingly, one embodiment provides genetically modified bone marrow cells provided herein, including embodiments thereof. Another embodiment provides genetically modified bone marrow cells provided herein, including embodiments thereof comprising pharmaceutically acceptable excipients.

[0156] Genetically modified cells can be further used in methods for discovering and validating drugs for treating diseases characterized by abnormal or dysfunctional bone marrow cells. Thus, in one embodiment, an assay for drug discovery is provided, comprising screening the effects of one or more compounds on bone marrow cells (including embodiments thereof) provided herein. In another embodiment, a method for target validation of compounds is provided, comprising contacting bone marrow cells (including embodiments thereof) provided herein with the compound and monitoring the effect on the cells. Embodiment

[0157] Embodiment 1: A method for genetic modification of bone marrow cells, comprising transfecting the bone marrow cells with a gene editing reagent that targets a gene site of interest, wherein the bone marrow cells are not transduced by a viral vector.

[0158] Embodiment 2: The method according to Embodiment 1, wherein the bone marrow cells are primary bone marrow cells.

[0159] Embodiment 3: The method according to Embodiment 1 or 2, wherein the bone marrow cells are monocytes, macrophages, neutrophils, basophils, eosinophils, erythrocytes, dendritic cells, or megakaryocytes.

[0160] Embodiment 4: The method according to any one of Embodiments 1 to 3, wherein the cells are transfected via electroporation.

[0161] Embodiment 5: The method according to any one of Embodiments 1 to 4, wherein the cells are transfected via nucleofection.

[0162] Embodiment 6: The method according to any one of Embodiments 1 to 5, wherein the cells are transfected via lipid-based transfection or polymer-based transfection.

[0163] Embodiment 7: The method according to Embodiment 6, wherein the cells are transfected via lipid-based transfection.

[0164] Embodiment 8: The method according to any one of Embodiments 1 to 7, wherein the cells are not subjected to a selection step and / or a concentration step after transfection.

[0165] Embodiment 9: The method according to any one of Embodiments 1 to 8, wherein the gene editing reagent comprises an RNA guide nuclease.

[0166] Embodiment 10: The method according to Embodiment 9, wherein the RNA guide nuclease is a CRISPR-Cas system.

[0167] Embodiment 11: The method according to Embodiment 10, wherein the CRISPR-Cas system includes Cas9 or a Cas9 variant.

[0168] Embodiment 12: The method according to Embodiment 10, wherein the CRISPR-Cas system comprises Cas12, Cascade, Cas13, or each of their variants.

[0169] Embodiment 13: The method according to any one of Embodiments 1 to 12, wherein the gene editing reagent comprises a CRISPR-Cas system comprising a Cas protein, guide RNA, and optionally donor DNA.

[0170] Embodiment 14: The method according to Embodiment 13, wherein the Cas protein and the guide RNA are associated non-covalently.

[0171] Embodiment 15: The method according to Embodiment 13 or 14, wherein the Cas protein, the guide RNA, and the donor DNA are associated non-covalently.

[0172] Embodiment 16: The method according to any one of Embodiments 1 to 8, wherein the gene editing reagent comprises a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, or an algonautendonuclease.

[0173] Embodiment 17: The method according to any one of Embodiments 1 to 16, wherein the cells are brought into contact with an electroporation enhancer during transfection.

[0174] Embodiment 18: The method according to Embodiment 17, wherein the electroporation enhancer is selected from carrier DNA, single-stranded DNA, a combination of single-stranded and double-stranded DNA, a polymer additive, and an oligonucleotide.

[0175] Embodiment 19: The method according to Embodiment 18, wherein the carrier DNA is a single-stranded DNA oligonucleotide.

[0176] Embodiment 20: The method according to Embodiment 18 or 19, wherein the carrier DNA is non-homologous to the genomes of humans, mice, and / or rats.

[0177] Embodiment 21: The method according to any one of Embodiments 1 to 20, wherein the bone marrow cells are differentiated before electroporation.

[0178] Embodiment 22: The method according to Embodiment 21, wherein the bone marrow cells differentiate into dendritic cells.

[0179] Embodiment 23: The method according to Embodiment 21, wherein the bone marrow cells differentiate into macrophages.

[0180] Embodiment 24: The method according to any one of Embodiments 1 to 23, wherein the bone marrow cells are not activated before or during the genetic modification.

[0181] Embodiment 25: The method according to any one of Embodiments 1 to 24, wherein two or more different crisprRNAs (crRNAs) targeting the desired site are introduced into the bone marrow cells.

[0182] Embodiment 26: The method according to Embodiment 25, wherein introducing the crRNA into the bone marrow cells is equivalent to transfecting the cells with the crRNA.

[0183] Embodiment 27: The method according to Embodiment 25 or 26, wherein the crRNA is brought into contact with tracrRNA.

[0184] Embodiment 28: The method according to Embodiment 27, wherein the crRNA is annealed to the tracrRNA.

[0185] Embodiment 29: The method according to Embodiment 25 or 26, wherein the crRNA is a single guide RNA (sgRNA).

[0186] Embodiment 30: The method according to any one of Embodiments 1 to 29, wherein multiple target gene sites are targeted.

[0187] Embodiment 31: The method according to Embodiment 30, wherein two or more different crRNAs for each target site are introduced into the bone marrow cells.

[0188] Embodiment 32: The method according to any one of Embodiments 1 to 31, wherein the bone marrow cells are human cells.

[0189] Embodiment 33: A method for genetically modifying a plurality of myeloid cells, comprising transfecting the plurality of myeloid cells in the presence of a gene editing reagent targeting a site of interest, wherein the myeloid cells are not transduced by a viral vector.

[0190] Embodiment 34: The method according to Embodiment 33, wherein the bone marrow cells are cultured bone marrow cells.

[0191] Embodiment 35: The method according to Embodiment 33 or 34, wherein the cells are transfected via electroporation.

[0192] Embodiment 36: The method according to any one of Embodiments 33 to 35, wherein the cells are transfected via lipid-based transfection.

[0193] Embodiment 37: The method according to any one of Embodiments 33 to 36, wherein the target site is modified in at least 70% of the plurality of bone marrow cells.

[0194] Embodiment 38: The method according to Embodiment 37, wherein the target site is modified in at least 80% of the plurality of bone marrow cells.

[0195] Embodiment 39: The method according to Embodiment 37 or 38, wherein the target site is modified in at least 85% of the plurality of bone marrow cells.

[0196] Embodiment 40: The method according to any one of Embodiments 37 to 39, wherein the target site is modified in at least 90% of the plurality of bone marrow cells.

[0197] Embodiment 41: The method according to any one of Embodiments 33 to 40, wherein the plurality of bone marrow cells include dendritic cells (DCs), and the site of interest is modified in at least 50% of the DCs.

[0198] Embodiment 42: The method according to any one of Embodiments 33 to 41, wherein the viability of the plurality of bone marrow cells after electroporation is at least 80%.

[0199] Embodiment 43: The method according to Embodiment 42, wherein the viability of the plurality of bone marrow cells after electroporation is at least 90%.

[0200] Embodiment 44: The method according to any one of Embodiments 33 to 43, wherein the gene editing reagent comprises an RNA guide nuclease.

[0201] Embodiment 45: The method according to Embodiment 44, wherein the RNA guide nuclease is a CRISPR-Cas system.

[0202] Embodiment 46: The method according to Embodiment 45, wherein the CRISPR-Cas system includes Cas9 or a Cas9 variant.

[0203] Embodiment 47: The method according to Embodiment 45, wherein the CRISPR-Cas system includes Cas12, Cascade, Cas13, or each of their variants.

[0204] Embodiment 48: The method according to any one of Embodiments 33 to 47, wherein the gene editing reagent comprises a CRISPR-Cas system comprising a Cas protein, guide RNA, and optionally donor DNA.

[0205] Embodiment 49: The method according to Embodiment 48, wherein the Cas protein and the guide RNA are associated non-covalently.

[0206] Embodiment 50: The method according to Embodiment 48 or 49, wherein the Cas protein, the guide RNA, and the donor DNA are associated non-covalently.

[0207] Embodiment 51: The method according to any one of Embodiments 33 to 43, wherein the gene editing reagent comprises a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, or an algonautendonuclease.

[0208] Embodiment 52: The method according to Embodiments 9-13 or 44-47, wherein the RNA guide nuclease comprises guide RNA and ribonucleoprotein (RNP), and the ratio of guide RNA to RNP is between 100:1 and 1:100.

[0209] Embodiment 53: The method according to Embodiment 52, wherein the guide RNA to RNP ratio is approximately 3:1 or less.

[0210] Embodiment 54: The method according to Embodiment 52, wherein the ratio of guide RNA to ribonucleoprotein (RNP) is approximately 2:1 or less.

[0211] Embodiment 55: The method according to Embodiment 52, wherein the guide RNA to ribonucleoprotein (RNP) ratio is approximately 3:1.

[0212] Embodiment 56: The method according to Embodiment 52, wherein the ratio of guide RNA to ribonucleoprotein (RNP) is approximately 2:1.

[0213] Embodiment 57: The method according to any one of Embodiments 1 to 56, wherein at least 70% of the transfected cells are administered to a patient in need of the transfected cells without selection or concentration of the cells.

[0214] Embodiment 58: The method according to any one of Embodiments 1 to 57, wherein at least 70% of the transfected cells are used in subsequent reactions without selection or concentration of the cells.

[0215] Embodiment 59: A method for genetically modifying a plurality of bone marrow cells, comprising transfecting the bone marrow cells with a gene editing reagent, wherein the bone marrow cells are not transfected with a viral vector, and the method does not include a selection step or a concentration step after bone marrow cell transfection.

[0216] Embodiment 60: The method according to Embodiment 59, wherein the cells are transfected via electroporation.

[0217] Embodiment 61: The method according to Embodiment 59, wherein the cells are transfected via lipid-based transfection.

[0218] Embodiment 62: The method according to any one of Embodiments 59 to 61, wherein the target site is modified in at least 70% of the plurality of bone marrow cells.

[0219] Embodiment 63: The method according to Embodiment 62, wherein the target site is modified in at least 80% of the plurality of bone marrow cells.

[0220] Embodiment 64: The method according to Embodiment 62 or 63, wherein the target site is modified in at least 85% of the plurality of bone marrow cells.

[0221] Embodiment 65: The method according to any one of Embodiments 62 to 64, wherein the target site is modified in at least 90% of the plurality of bone marrow cells.

[0222] Embodiment 66: The method according to any one of Embodiments 59 to 61, wherein the plurality of bone marrow cells include dendritic cells (DCs), and the site of interest is modified in at least 50% of the DCs.

[0223] Embodiment 67: The method according to any one of Embodiments 59 to 66, wherein the viability of the plurality of bone marrow cells after electroporation is at least 80%.

[0224] Embodiment 68: The method according to Embodiment 67, wherein the viability of the plurality of bone marrow cells after electroporation is at least 90%.

[0225] Embodiment 69: The method according to any one of Embodiments 59 to 68, wherein the gene editing reagent comprises an RNA guide nuclease.

[0226] Embodiment 70: The method according to Embodiment 69, wherein the RNA guide nuclease is a CRISPR-Cas system.

[0227] Embodiment 71: The method according to Embodiment 70, wherein the CRISPR-Cas system includes Cas9 or a Cas9 variant.

[0228] Embodiment 72: The method according to Embodiment 70, wherein the CRISPR-Cas system comprises Cas12, Cascade, Cas13, or each of their variants.

[0229] Embodiment 73: The method according to any one of Embodiments 59 to 72, wherein the gene editing reagent comprises a CRISPR-Cas system comprising a Cas protein, guide RNA, and optionally donor DNA.

[0230] Embodiment 74: The method according to Embodiment 73, wherein the Cas protein and the guide RNA are associated non-covalently.

[0231] Embodiment 75: The method according to Embodiment 73 or 74, wherein the Cas protein, the guide RNA, and the donor DNA are associated non-covalently.

[0232] Embodiment 76: The method according to any one of Embodiments 59 to 68, wherein the gene editing reagent comprises a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, or an algonaut endonuclease.

[0233] Embodiment 77: A system for genetically modifying bone marrow cells in the absence of a viral vector, comprising: a chamber adapted to a transfection system; a plurality of bone marrow cells in the chamber in a culture medium adapted to electroporation; and at least one gene editing system designed to target at least one site of interest in the genome of the bone marrow cells.

[0234] Embodiment 78: The system according to Embodiment 77, further comprising a transfection system.

[0235] Embodiment 79: The system according to Embodiment 77 or 78, wherein the transfection system includes an electroporation apparatus.

[0236] Embodiment 80: The system according to any one of Embodiments 77 to 79, wherein the bone marrow cells are primary bone marrow cells.

[0237] Embodiment 81: The system according to any one of Embodiments 77 to 79, wherein the bone marrow cells are cultured bone marrow cells.

[0238] Embodiment 82: The system according to any one of Embodiments 77 to 81, wherein the bone marrow cells are monocytes, macrophages, or dendritic cells.

[0239] Embodiment 83: The system according to any one of Embodiments 77 to 82, wherein the cells are not subjected to a selection step and / or a concentration step after transfection.

[0240] Embodiment 84: The system according to any one of Embodiments 77 to 83, wherein the gene editing system includes a gene editing reagent.

[0241] Embodiment 85: The system according to Embodiment 84, wherein the gene editing reagent comprises an RNA guide nuclease.

[0242] Embodiment 86: The system according to Embodiment 85, wherein the RNA guide nuclease is a CRISPR-Cas system.

[0243] Embodiment 87: The system according to Embodiment 85, wherein the CRISPR-Cas system includes Cas9 or a Cas9 variant.

[0244] Embodiment 88: The system according to Embodiment 86, wherein the CRISPR-Cas system includes Cas12, Cascade, Cas13, or each of their variants.

[0245] Embodiment 89: The method according to any one of Embodiments 84 to 88, wherein the gene editing reagent comprises a CRISPR-Cas system comprising a Cas protein, guide RNA, and optionally donor DNA.

[0246] Embodiment 90: The method according to Embodiment 89, wherein the Cas protein and the guide RNA are associated non-covalently.

[0247] Embodiment 91: The method according to Embodiment 89 or 90, wherein the Cas protein, the guide RNA, and the donor DNA are associated non-covalently.

[0248] Embodiment 92: The system according to Embodiment 84, wherein the gene editing reagent comprises a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, or an algonaut endonuclease.

[0249] Embodiment 93: The system according to any one of Embodiments 77 to 92, wherein the cells are brought into contact with an electroporation enhancer during transfection.

[0250] Embodiment 94: The system according to Embodiment 93, wherein the electroporation enhancer is selected from carrier DNA, single-stranded DNA, a combination of single-stranded and double-stranded DNA, polymer additives, and oligonucleotides.

[0251] Embodiment 95: The system according to Embodiment 94, wherein the carrier DNA is a single-stranded DNA oligonucleotide.

[0252] Embodiment 96: The system according to any one of embodiments 77 to 95, wherein the bone marrow cells are not activated before or during genetic modification.

[0253] Embodiment 97: A method for treating a disease treatable with bone marrow cells, comprising providing genetically modified bone marrow cells that have not been transfected with a virus, wherein the bone marrow cells have not been transfected with a gene editing reagent, and administering the bone marrow cells to a patient in need of the bone marrow cells.

[0254] Embodiment 98: The method according to Embodiment 97, wherein the bone marrow cells are primary bone marrow cells.

[0255] Embodiment 99: The method according to Embodiment 97, wherein the bone marrow cells are cultured bone marrow cells.

[0256] Embodiment 100: The method according to any one of Embodiments 97 to 99, wherein the gene editing reagent comprises an RNA guide nuclease.

[0257] Embodiment 101: The method according to Embodiment 100, wherein the RNA guide nuclease is a CRISPR-Cas system.

[0258] Embodiment 102: The method according to Embodiment 101, wherein the CRISPR-Cas system includes Cas9 or a Cas9 variant.

[0259] Embodiment 103: The method according to Embodiment 101, wherein the CRISPR-Cas system comprises Cas12, Cascade, Cas13, or each of their variants.

[0260] Embodiment 104: The method according to any one of Embodiments 97 to 103, wherein the gene editing reagent comprises a CRISPR-Cas system comprising a Cas protein, guide RNA, and optionally donor DNA.

[0261] Embodiment 105: The method according to Embodiment 104, wherein the Cas protein and the guide RNA are associated non-covalently.

[0262] Embodiment 106: The method according to Embodiment 104 or 105, wherein the Cas protein, the guide RNA, and the donor DNA are associated non-covalently.

[0263] Embodiment 107: The method according to any one of Embodiments 97 to 99, wherein the gene editing reagent comprises a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, or an algonautendonuclease.

[0264] Embodiment 108: The method according to any one of Embodiments 97 to 107, wherein the administration includes oral, intravenous, parenteral, intraperitoneal, intramuscular, intrafocal, or subcutaneous administration.

[0265] Embodiment 109: A composition comprising a plurality of bone marrow cells, a gene editing reagent, a transfection buffer, and an electroporation enhancer, wherein the composition does not include a viral vector.

[0266] Embodiment 110: The composition according to Embodiment 109, wherein the bone marrow cells are cultured bone marrow cells.

[0267] Embodiment 111: The composition according to Embodiment 109 or 110, wherein the plurality of bone marrow cells are a plurality of monocytes, macrophages, or dendritic cells.

[0268] Embodiment 112: The composition according to any one of Embodiments 109 to 111, wherein the gene editing reagent comprises an RNA guide nuclease.

[0269] Embodiment 113: The composition according to Embodiment 112, wherein the RNA guide nuclease is a CRISPR-Cas system.

[0270] Embodiment 114: The composition according to Embodiment 113, wherein the CRISPR-Cas system comprises Cas9 or a Cas9 variant.

[0271] Embodiment 115: The composition according to Embodiment 113, wherein the CRISPR-Cas system comprises Cas12, Cascade, Cas13, or each of their variants.

[0272] Embodiment 116: The composition according to any one of Embodiments 109 to 115, wherein the gene editing reagent comprises a CRISPR-Cas system comprising a Cas protein, guide RNA, and optionally donor DNA.

[0273] Embodiment 117: The composition according to Embodiment 116, wherein the Cas protein and the guide RNA are associated non-covalently.

[0274] Embodiment 118: The composition according to Embodiment 116 or 117, wherein the Cas protein, the guide RNA, and the donor DNA are associated non-covalently.

[0275] Embodiment 119: The composition according to any one of Embodiments 109 to 112, wherein the gene editing reagent comprises a transcription activator-like effector nuclease (TALEN), a zinc finger nuclease, or an argonautendonuclease.

[0276] Embodiment 120: The composition according to any one of Embodiments 112 to 115, wherein the RNA guide nuclease comprises guide RNA and ribonucleoprotein (RNP), and the ratio of guide RNA to RNP is between 100:1 and 1:100.

[0277] Embodiment 121: The composition according to Embodiments 112 to 115, wherein the RNA guide nuclease comprises guide RNA and ribonucleoprotein (RNP), and the ratio of guide RNA pairs is about 3:1 or less.

[0278] Embodiment 122: The composition according to Embodiments 112 to 115, wherein the RNA guide nuclease comprises guide RNA and ribonucleoprotein (RNP), and the ratio of guide RNA to ribonucleoprotein (RNP) is about 2:1 or less.

[0279] Embodiment 123: The composition according to Embodiments 112 to 115, wherein the RNA guide nuclease comprises guide RNA and ribonucleoprotein (RNP), and the ratio of guide RNA to ribonucleoprotein (RNP) is approximately 3:1.

[0280] Embodiment 124: The composition according to Embodiments 112 to 115, wherein the RNA guide nuclease comprises guide RNA and ribonucleoprotein (RNP), and the ratio of guide RNA to ribonucleoprotein (RNP) is approximately 2:1.

[0281] Embodiment 125: The composition according to any one of Embodiments 109 to 124, wherein the electroporation enhancer is selected from carrier DNA, single-stranded DNA, a combination of single-stranded DNA and double-stranded DNA, a polymer additive, and an oligonucleotide.

[0282] Embodiment 126: The composition according to Embodiment 125, wherein the carrier DNA is a single-stranded DNA oligonucleotide.

[0283] Embodiment 127: A genetically modified bone marrow cell produced by the method according to any one of Claims 1 to 76.

[0284] Embodiment 128: An assay for drug discovery, comprising screening for the effect of one or more compounds on the bone marrow cells of Embodiment 127.

[0285] Embodiment 129: A method for target verification of a compound, comprising contacting the bone marrow cells of Embodiment 127 with the compound and monitoring the effect on the cells.

Example

[0286] Those skilled in the art will understand that the description of the preparation and use of the cells or compositions described herein is for illustrative purposes only and that the present disclosure is not limited by this illustration. Example 1: Efficient Genomic Engineering in Mouse Bone Marrow-Derived Macrophages and Dendritic Cells

[0287] It should be noted that for item , it seems there is an incorrect "照" in the original Chinese. I translated it as is based on the provided text. If this is a misprint, please correct it in the original text for a more accurate translation.)Macrophages are key sensors of infection and tissue damage, possessing the ability to promote inflammation, cellular and humoral immunity, or tissue regeneration and potential fibrosis (Wynn and Vannella, 2016). Mouse bone marrow-derived macrophages (BMDMs) are induced from hematopoietic progenitor cells by culturing whole bone marrow in the presence of CSF1 / M-CSF, generating a large number of these cells for experimental use. BMDMs have proven highly useful in studying macrophage function and are widely used to understand innate immune signaling. We performed a flow cytometry (FACS)-based screening of 80 conditions in mouse BMDMs, using integrin CD11b (encoded by Itgam) as a model target locus (Figure 5A-C) to identify the optimal electroporation-based conditions for gene disruption.

[0288] Five different buffers and 15 electroporation conditions were compared to identify which protocol provided high efficiency of gene editing while maintaining cell viability. BMDM cells were differentiated in M-CSF for 5 days and then used with 500,000 cells for each electroporation condition. Two different crRNAs were designed to target the CD11b coding sequence and synthesized using Integrated DNA Technologies (IDT). These guides were then annealed to tracrRNA and complexed with Streptococcus pyogenes Cas9 (SpCas9 (hereinafter referred to as Cas9)) protein (IDT Cas9 V2) to improve the probability of gene deletion. A 3:1 molar ratio of gRNA:Cas9 was used as a starting condition, as previously reported for lymphocytes (Seki and Rutz, 2018) (60 pmol or 10 μg of Cas9 per reaction). After electroporation, BMDM cells were cultured in M-CSF for an additional 5 days. Viability and CD11b deletion efficiency were compared by flow cytometry (FACS, Figure 5B, Figure 5C). While most electroporation conditions proved toxic (Figure 5B), we identified several conditions that preserved cell viability while inducing CD11b disruption (Figure 5C). Of these, the top three conditions (Figure 5C, Figure 5D) revealed equivalent loss of CD11b expression with buffer P3, and program CM137 maintained the highest level of cell viability (white box, Figure 5D).

[0289] Next, using monocytes isolated from the femoral bone marrow of eGFP transgenic mice, a similar study was conducted using different crRNA-tracrRNA sequences targeting egfp (Chen et al., 2017) to assess whether population-level gene deletion could be obtained from a subset of primary bone marrow cells (Figure 1A). The ability to screen for loss of intracellular markers such as eGFP eliminated receptor internalization as a potential confounding factor in the previous assay. After Cas9 / RNP electroporation, monocytes were cultured in M-CSF for 5 days to generate monocyte-derived macrophages. Consistent with the findings for CD11b disruption, buffer P3, programmed CM137 again resulted in maximum loss of target (eGFP) expression while maintaining cell viability (ranking shown in white boxes, Figure 1B, and Figure 5D). Next, these conditions were used to confirm the benefits of pooling multiple guide RNAs for enhancing target gene disruption. For example, egfp-targeting sequences g1 and g3 were delivered as a pool for monocyte eGFP screening. As shown in Figure 1C, individual gRNAs targeting egfp resulted in varying degrees of gene loss, but pooling g3 with either g1 or g2 resulted in the greatest eGFP loss. Combining all three gRNAs did not improve eGFP loss beyond the most effective pairing. Therefore, it was concluded that pooling two different crRNA-tracrRNAs targeting the same gene provides optimal gene knockout in mouse BMDM or monocyte-derived macrophages.

[0290] Dendritic cells (DCs) play a crucial role in directing T cell responses through the process of antigen presentation (review by Merad et al., 2013). Therefore, we conducted experiments to extend the protocol to genetically modified DCs. Bone marrow-derived dendritic cells (BMDCs) serve as a model for investigating the fundamental mechanisms of DC biology, and more broadly, engineered DCs could constitute important material for future cell-based vaccines. Traditional methods for differentiating bone marrow cells into DCs involve supplementation with GM-CSF (Cornel et al., 2018; Gundry et al., 2016). However, this does not produce the different physiologically relevant subsets found in vivo. Therefore, we used a more recently developed protocol using Flt3 ligand supplementation to differentiate bone marrow cells into at least three DC cell types found in vivo: plasmacytoid DCs (pDCs, B220+) and two conventional DC (cDC) cell types (Sirpα+ and CD24+) (Naik et al., 2005). Furthermore, this differentiation method enables the production of a large number of BMDCs, which would otherwise be rare in vivo. While both plasmid transfection and viral transduction methods have been developed for primary DC gene delivery, aside from variable and incomplete delivery across target cell populations, these methods require the use of biologically harmful substances and / or can introduce potential cytotoxicity or differentiation artifacts (Bowles et al., 2011).

[0291] To adapt the protocol for DC gene editing, the initial delivery of Cas9 RNP was evaluated. Following the BMDM and monocyte workflows, primary cell optimization screening was performed in mouse BMDCs using the same five buffer and 15 electroporation program combinations (Figure 6A). During these studies, an improved guide RNA chemistry for crRNA called crRNAXT became available from IDT. XT-modified crRNA was expected to improve RNP stability and therefore knockout efficiency. Therefore, a single CD45-specific targeting sequence with this modification was used for BMDC optimization screening. Bone marrow cells were isolated, and 2 million cells per condition were evaluated, which was determined to be the minimum number required for viability after electroporation. A gRNA:Cas9 molar ratio of 3:1 was maintained, but half the amount was used compared to BMDM and monocyte screening to increase the dynamic range for measuring improved knockout efficiency. Therefore, 30 pmol or 5 μg of Cas9 was used. After RNP electroporation, cells were cultured with Flt3 ligand (Figure 6A) for 12 days. cDCs were isolated by FACS, and cell surface CD45 expression was quantified by antibody staining (Figure 6A). Similar to macrophage screening, substantial variability in both knockout efficiency and viability was observed for both CD24+DC and Sirpα+DC populations across the 80 conditions tested, with CD24+DCs exhibiting lower cell viability across most conditions compared to Sirpα+DCs (Figure 6A).

[0292] Next, five conditions were selected that yielded substantial knockouts and acceptable survival rates: buffer P3 with program CM-137, DS130, EN-138, buffer P4 with program DS-130, and buffer P5 with program CM137 (white boxes, Figure 5D; Figure 6B). These conditions were tested at higher Cas9 / RNP concentrations used in macrophage screening, revealing that the combination of buffer P3 and program CM-137 (P3, CM137) yielded the highest knockout for CD24+DCs (74.5% KO), Sirpα+DCs (93% KO), and macrophages (72.5% KO) (Figure 1E). However, this combination produced a relatively low knockout efficiency within pDCs (21.5%). Instead, the results showed that buffer P3 containing program EN-138 (P3, EN-138) was most effective against the pDC subset (71% KO). Importantly, this condition was not as broadly effective as buffer P3 with program CM-137, but it resulted in over 50% KO across all DC cell types tested. Consistent with these results, when tested with cells from different mouse donors, P3, CM-137 showed high KO efficiency (>80%) in CD24+ DCs, Sirpα+ DCs, and macrophages, but not in pDCs, while P3, EN-138 resulted in higher KO efficiency (>50%) in pDCs, but slightly lower and more variable KO efficiency across the other three cell types (Figure 1E). Therefore, it was concluded that P3, CM-137 is the best condition for achieving KO in all DCs except the pDC subtype, and P3, EN-138 provides the highest KO efficiency.

[0293] Since maintaining a normal cellular state may be important as part of the gene editing process, the expression levels of the co-stimulatory protein and the myeloid cell activation marker CD80 were tested 12 days after electroporation using two conditions selected by the inventors. The results showed that, with the exception of Sirpα+DC, CD80 expression was similar between the negative control and cells electroporated using either condition, suggesting that Cas9 / RNP delivery by these methods does not induce widespread activation in mouse myeloid populations (Figure 6C). Example 2: Population-level gene disruption in human monocyte-derived dendritic cells and macrophages.

[0294] There are few available methods for effective non-viral genetic modification of primary human macrophages and dendritic cells, which limits direct phenotypic analysis of interspecies-shared genes, study of human-specific genes, or potentially, ex vivo editing of these populations for direct therapeutic benefits. Encouraged by the results from mouse monocyte-derived macrophages and dendritic cells (Example 1), we attempted similar conditions for Cas9 / RNP delivery in human cells. Furthermore, we sought to expand our findings from mouse cells by comparing updated Cas9 / RNP technology platforms in a human context. This included Cas9 protein (supplied from different sources) and guide RNA variants.

[0295] To initiate human bone marrow cell editing and β2-microglobulin (B2M) optimization, we targeted the widely expressed constant region of the human MHCI complex. This allows for the assessment of gene deletions across multiple donors, regardless of genetic background. Several RNP variants, each carrying two different guide sequences (referred to as B2M gRNA1 or 2), containing either Integrated DNA Technologies' "V3" Cas9 protein or ThermoFisher, Inc.'s "V2," were generated using standard or XT guide chemistry alone or in combination with each reaction. All RNPs were delivered to monocytes obtained from peripheral blood mononuclear cells (PBMCs) using buffer P3, CM-137 (Figure 2A), a generalizable condition identified in mouse assays. After electroporation of various B2M-specific Cas9 / guide RNA RNP variants, monocytes were differentiated into either macrophages (cultured for approximately 5 days in growth medium supplemented with M-CSF) or dendritic cells (cultured for approximately 7 days in growth medium supplemented with GM-CSF and IL-4). The results showed that specific B2M gRNA sequences exhibited different knockout efficiencies regardless of guide chemistry, with gRNA2 cleaving more efficiently than gRNA1, and knockout generally higher in the macrophage subset. Individual guide efficacy was close to a 90% population width knockout, but the Cas9 RNP pool was able to induce nearly complete knockout (over 90%) in both cell types (Figure 2B). As with individual guides, the standard and XT pool yielded similar results in these assays. When comparing the performance of Cas9 protein variants across all test conditions, negligible differences were observed. Example 3: Cas9 RNPs exhibit additiveity when two guides target the same gene.

[0296] Across all mouse or human myeloid cell types tested throughout the initial optimization process, a clear additive effect on gene disruption was routinely observed by combining multiple unique guide RNAs for the same target, regardless of guide chemistry. For example, this was observed in human cells for B2M-specific gRNA sequences 1 and 2, as well as a second set of B2M guides (sequences 3 and 4, Figure 7A), and this effect was independent of the human donor (Figure 7B). Therefore, this relationship was investigated in more detail.

[0297] Guide RNA pools were prepared by mixing equal amounts of RNPs loaded with specific guides prior to electroporation, effectively generating 2× stocks compared to individual guide preparations. While multiple targeting events may increase the probability of frameshift indels or local chromosomal rearrangements and improve KO efficiency, the additive effect observed by the inventors with multiple guides may still be attributable to the increased Cas9 / RNP loading introduced during the electroporation process. To test whether RNP loading is a determinant, the delivery of 1x and 2x RNPs loaded with individual XT guides targeting B2M in human cells was compared. As shown in Figure 2C, no significant difference in KO efficiency was observed for either guide 1 (1x: 32.5% or 2x: 31.3%) or guide 2 (1x: 82.5% or 2x: 86%), suggesting that the amount of Cas9 protein was not limiting under the 1x condition.

[0298] Separately, the inventors sought to evaluate whether guide RNAs act independently or synergistically when pooled. Here, the inventors mixed equal amounts of B2M-specific XT guide Cas9-RNP with untargeted guide (NTC crXT)-Cas9-RNP. The addition of NTC guide RNA did not affect the gene editing efficiency in either crRNA format (Figure 2C, Figure 7C). These results demonstrate that total RNP concentration is not a significant limiting factor in these assays, and that comparative evaluation of individual guide RNAs can reveal very strong candidates that function efficiently in isolation. Example 4: Fully synthesized sgRNA results in optimal gene disruption in primary bone marrow cells.

[0299] Two-part guide RNAs offer a cost-effective and easily produced solution for gene editing purposes. Recently, fully synthetic single guide RNAs (sgRNAs) have been developed, which ligate both crRNA and tracrRNA into a single unit (Jinek et al., 2012). Synthetic sgRNAs allow for chemical modifications to enhance function and / or stability, avoiding the need for guide annealing before RNP complex formation (Hendel et al., 2015; Kim et al., 2018; Ryan et al., 2018; Wienert et al., 2018). Due to their improved stability or function compared to two-part guides, we observed a slight but overall enhanced KO efficiency in both monocyte-derived macrophages and dendritic cells with B2M guides 1 and 2 formatted as sgRNAs (Figure 2D). This improvement was consistent across multiple donors (Figure 7D). Consistent with our findings using the crRNA format, we observed that the amount of Cas9 protein did not affect the gene knockout efficiency when combined with B2M-specific sgRNA (Figure 7E), nor did the presence of untargeted (NTC) sgRNA. Pooling two sgRNAs targeting the same gene slightly improved the knockout efficiency, as observed with the crRNA format (Figure 7E).

[0300] The increased activity of sgRNA was deemed to allow the use of sgRNA at a lower gRNA:Cas9 ratio compared to the 3:1 ratio that the inventors found to be optimal for crRNA and crRNAXT. In effect, this would help reduce the costs associated with the use of synthetic sgRNA. Therefore, the inventors tested a 2:1 sgRNA:Cas9 ratio (B2M sgRNA2:IDTV3 Cas9) and found that this yielded comparable knockout efficiency compared to the 3:1 ratio (Figure 7F). Encouraged by the maintenance of activity at reduced guide concentrations, the inventors evaluated the minimum amount of sgRNA:Cas9 RNP required for effective target disruption. Here, the inventors performed titration experiments comparing a less active (sgRNA 2) sgRNA with a more active (sgRNA 4) sgRNA targeting B2M, while decreasing the amount of RNP in 2-fold increments. As shown for the low-activity guide (B2M sgRNA2), when the RNP volume was halved from 4 μL (180 pmol gRNA, 60 pmol Cas9) to 2 μL (90 pmol gRNA, 30 pmol Cas9), the KO efficiency decreased by 36.5% (Figure 2E). The inventors also performed the same titration curve in the presence of an "electroporation enhancer" (single-stranded DNA carrier, IDT, 4 μM), and found that while this did not have a discernible effect on the KO efficiency at 4 μL, the enhancer reduced the sgRNA 2-RNP amount to 2 μL without substantially losing KO efficiency (4 μL: 96.2%, 2 μL: 92.9%). Importantly, the enhancer did not affect cell viability (Figures 7G, ​​7H). To the inventors' surprise, the activity curve of the more effective sgRNA showed a significant shift, and RNP retained nearly full activity at a dose of 0.125 μl in the presence of the enhancer, or retained approximately 16 times less protein than required for efficiency comparable to the less potent guide RNA (Figure 2F). In summary, the inventors conclude that a 2:1 sgRNA:Cas9 molar ratio in the presence of a 4 μM enhancer is optimal, but the minimum amount of RNP required to achieve maximum knockout efficiency should be tested for each guide, as a substantial reduction in the amount of effective RNP may be achievable.

[0301] The effects of nucleofection on innate cell activation and cytokine production were assessed. Measurement of myeloid phenotypic markers on monocyte-derived macrophages from two independent donors revealed comparable cell surface levels of CD14, DC-SIGN, HLA-DR, CD69, and CD11c across all experimental conditions (Figure 11A). CD11b expression increased after nucleofection (Figure 11A). Importantly, the efficiency of gene deletion did not correlate with the expression levels of any of the phenotypic markers tested (Figure 11B). The inventors also compared the effects of single or pooled sgRNAs on the expression of costimulatory proteins and cytokines in monocyte-derived macrophages. Elevated levels of the costimulatory protein CD86, rather than CD80, were observed at nucleofection, and these were independent of gRNA levels (Figure 11C). Although secreted TNF levels remained low after nucleofection (Figure 11D), type I interferon (IFNβ) levels were undetectable under all conditions (data not shown).

[0302] Finally, the effect of nucleofection on the phagocytic activity of monocyte-derived macrophages was characterized using live-cell imaging. Macrophages were co-incubated with particulate cargo of various sizes (myelin debris) or beads of a specified diameter labeled with a pH-sensitive fluorescent dye (pHrodo-red) and imaged periodically over 5 hours. This allowed for the quantification of cargo uptake and delivery of macrophages to the lysosomal (degradable) compartment. Considering that cell density significantly affects these measurements, the number of viable cells was determined immediately after nucleofection to ensure equal cell counts under each experimental condition. Comparable cell densities were confirmed among non-nucleofection (No Nuc), untargeted control (NTC sg), and B2M-KO (B2Msg) monocyte-derived macrophages based on the number of viable cells at the end of the assay (Figure 11E). The inventors observed that the phagocytic rates of myelin (Figure 11F) and beads (Figure 11G) were equal. The inventors generated a phagocytic index from time-series data (AUC over 5 hours), which showed comparable levels of phagocytosis by monocyte-derived macrophages in each treatment group (Figure 11H). The inventors conclude that nucleofection yields measurable differences for specific phenotypic and activation markers, but does not broadly induce phenotypic changes in monocyte-derived macrophages. These observations also motivate researchers to use non-nucleofection and non-targeted gRNAs as standardized controls in experimental design.

[0303] Similar to the two-part guide, B2M gRNA1 and gRNA2 sequences in sgRNA format were tested against either gRNA individually to determine whether there is additiveity between sgRNA-supported RNPs for the same target (Figure 7E). Similar to the two-part guide, the sgRNA pool yielded higher KO efficiency compared to either sgRNA alone (at equivalent concentrations). Also, similar to the crRNAXT conditions, the combination of NTC sgRNA RNP and B2M sgRNA RNP (complexed separately) reduced the KO efficiency of the B2M RNP, indicating that guide competition is a generalizable feature of Cas9 RNP use (Figure 7E). Example 5: Efficient CRISPR / Cas9 deletion of Toll-like receptor 7 in mouse BMDC

[0304] After determining the optimal conditions for genetic manipulation of primary myeloid cell types, we then determined whether these protocols could be used to study inflammatory responses. The general enhancement observed with sgRNAs compared to crRNA variants prompted us to compare sgRNA gene editing efficiencies between two previously identified optimal Cas9-RNP delivery conditions for Flt3 ligand-cultured BMDCs (buffer P3, program CM-137 vs. buffer P3, program EN-138 as shown in Figure 1E). We focused on Toll-like receptor 7 (TLR7), a microorganism-associated pattern recognition receptor highly expressed on pDCs. Two TLR7-specific sgRNAs with different efficiencies were nucleofectioned into mouse myeloid cells, and these were differentiated with Flt3 ligands for 12 days. Using buffer P3 and program CM-137, we observed that individual sgRNAs resulted in an efficient reduction of TLR7 in all myeloid cell subsets in a manner dependent on the intrinsic efficiency of each sgRNA, with the exception of CD24+ DCs lacking TLR7, which were detectable even before nucleofection, as previously shown (Naik et al., 2005) (Figure 3A, CM-137; histogram in Figure 12A). In contrast, program EN-138 showed a reduced efficiency of TLR7 deletion in pDCs and macrophages (Figure 3A, EN-138). To quantify the cellular response to TLR7 activation, we determined the levels of several cytokines in the supernatant. Tlr7 knockout (KO) reduced the levels of IFNα, IL-12p40, TNF, and IL-6 in the supernatant in response to stimulation with the TLR 7 agonist R848 compared to non-nucleofection and non-targeted control nucleofection samples (Figure 3B). This demonstrates that the protocol developed by our inventors can efficiently generate Tlr7 knockout while maintaining normal cell physiology, thereby neutralizing downstream cytokine responses in BMDCs.

[0305] To broadly assess the impact of sgRNA nucleofection on cell differentiation and function, bone marrow cell phenotypes, activation, and function were compared to non-targeting sgRNA (NTC) after nucleofection. The relative abundance of DC subsets was not affected by nucleofection, but the inventors observed a modest (about 5%) increase in macrophage abundance when compared to non-nucleofected cells (Figure 12B). A decrease in overall cell yield was observed after nucleofection, likely as a result of nucleofection immediately after whole bone marrow on day 0 (Figure 12C). Comparing the expression of activation and maturation markers revealed equivalent levels of MHC I and MHC II across all cell subsets, but levels of CD40 on pDCs and CD80 on SIRPα+ DCs were elevated (Figure 12D). Finally, the inventors compared MHC I- and MHC II-dependent antigen presentation. After nucleofection and differentiation as described above, BMDCs were pulsed with various concentrations of chicken ovalbumin (OVA) and co-cultured with antigen-specific CD8+ T cells (OT-I) or CD4+ T cells (OT-II). T cell proliferation was measured using flow cytometry after 3 days. OT-I proliferation was equivalent between non-nucleofected and nucleofected conditions (Figure 12E). Nucleofection enhanced OT-II proliferation at all concentrations of OVA (Figure 12F). These changes are mostly moderate but need to be considered when examining specific bone marrow cell subsets purified from a mixed culture system of Flt3 ligand-driven differentiation. Cumulatively, the inventors recommend using buffer P3 and program CM-137 for optimal gene editing when using single-guide RNA (sgRNA) chemistry. Example 6: Efficient single and multiple deletions of Ticam1 / TRIF and MyD88 in mouse bone marrow-derived macrophages

[0306] Next, the ability of this protocol to generate single or multiple gene knockouts in BMDM was tested under TLR signaling conditions. Macrophages engage with Toll-like receptors (TLRs) for antimicrobial immunity using cytoplasmic adapters MYD 88 or TRIF (encoded by Ticam1). Bacterial cell wall antigen LPS activates TLR4 and signals via both adapters, while double-stranded RNA engages TLR3 and signals only via TRIF (Gay et al., 2014). Single or double knockouts were generated by pooling two sgRNAs per target gene (Figure 9C). Sanger sequencing analysis of individual guide RNAs revealed that Myd88 sgRNA1 showed the highest editing efficiency, while both Ticam1 / TRIF sgRNAs showed approximately 50% editing efficiency. These did not change under single or double knockout conditions (Figure 9D). Measurement of IFNβ secretion by BMDM was consistent across all conditions for 24 hours post-nucleofection, demonstrating an enhanced interferon response after Cas9-RNP delivery and thus the absence of generalized activation (Figure 9E). Engagement of TLR3 with PolyI:C (synthetic double-stranded RNA analog) induced IFNβ and TNF in the control (NTC) and Myd88, but not in those lacking Ticam1 / TRIF (i.e., Ticam1 / TRIF-KO or Myd88;Ticam1 / TRIF-dKO) (Figure 9E, F, PolyI:C treatment). Similarly, engagement of TLR4 with LPS revealed that Ticam1 / TRIF mutations were sufficient to eliminate IFNβ secretion (Figure 9E, LPS treatment), and that TNF secretion depended on a combination of MYD88 or TRIF signaling (Figure 9F, LPS treatment). Furthermore, our results confirm the successful generation of compound knockout BMDMs in physiologically relevant situations for innate antimicrobial immunity.

[0307] Finally, the generation of single and multiple gene knockouts in human donor-derived monocyte-derived macrophages was demonstrated. The inventors chose to delete B2M along with CD14 and CD81, enabling quantitative flow cytometry-based assessment of gene editing efficiency. Single sgRNAs targeting each gene were introduced into monocytes by nucleofection using buffer P3, CM-137 protocol, and then the cells were differentiated into macrophages using M-CSF. Cells were harvested 6 days post-nucleofection and gene knockouts were compared. Flow cytometry revealed nearly complete population-wide editing in single, double, or triple knockout samples (Figure 10A, representative histogram; Figure 10B, quantification of gene knockout). Editing efficiency was confirmed by ICE analysis (Synthego), which provides a measure of mutant allele frequency across sequenced PCR products derived from the target locus (Figure 10C). Therefore, highly functional individual gene-specific sgRNAs can be combined for one-step production of multiple population-wide knockouts in primary human myeloid cell types without requiring selective or stable gene-editing component expression.

[0308] Consideration

[0309] This study provides a rapid, efficient, and economical method for producing population-level gene knockouts in primary myeloid cells of human and mouse origin (summarized in Tables 1 and 2). The optimizations developed herein enable single and multiple gene knockouts (up to 3 genes at a time) with efficiencies exceeding 90%, thus eliminating the need for stepwise gene disruption and / or transgenic marker selection. The results reveal that combining pairs of gene-specific crRNAs provides an additive and optimal effect on knockout efficiency. Alternatively, individual chemosynthetic sgRNAs can be mixed and used for nearly complete compound gene disruption. This study also demonstrates that the addition of an IDT electroporation enhancer in association with human monocyte-derived cells can significantly increase the gene editing efficiency of suboptimal gRNA sequences and reduce the effective amount of active sgRNA. By increasing the repertoire of functional guide RNA sequences while simultaneously reducing the expected cost and material requirements associated with the generation of KO cell types, we have overcome separate barriers to high-throughput KO analysis in primary human and mouse myeloid cells. Given the observation that human monocytes upregulated activation markers after nucleofection, it is prudent to use non-electropermeable cells as controls when monitoring their activation status and examining congenital inflammatory phenotypes. Further comparison of markers revealed the lack of elevation after nucleofection, demonstrating that this protocol has, if any, limited effect on the activation status of human myeloid cells.

[0310] A comparison of guide RNA chemistry in BMDCs revealed that pDCs are particularly affected by crRNA vs. sgRNA, and CRISPR-KO was efficiently achieved in pDCs as well as in other cell types of BMDC cultures using crRNA vs. sgRNA. Since pDCs differentiate mainly from common lymphoid primordial cells (CLPs), which are a different lineage from myeloid primordial cells (Rodrigues et al., 2019), CLPs may be more efficiently targeted by sgRNA. This observation warrants further evaluation of CRISPR-Cas9 RNP-mediated gene editing in hematopoietic progenitor cells. It is also important to note that myeloid cells edited using our method were cultured under various cell adhesion conditions. For example, mouse monocyte-derived macrophages and BMDMs were maintained in low-adhesion tissue culture (TC) multiwell plates or non-TC treated petri dishes, respectively. BMDCs were differentiated in conventional TC treated multiwell plates. For evaluation of gene knockout and functional assays, BMDMs were first cultured on petri dishes and then transferred to TC treated multiwell plates. Across all adhesion conditions, the inventors observed comparable efficiency of gene knockout. Therefore, bone marrow cell adhesion does not substantially affect the efficiency of gene editing after nucleofection or cell viability in the optimized protocol generated herein.

[0311] The inventors recently described the usefulness of this method in necroptosis studies, analyzing the signaling nodes of TRIF-mediated cell death using Cas9-RNP delivery to mouse BMDM (Lim et al., 2019). Beyond this example of reverse genetics, the scalability of the inventors' findings is likely to enable functional screening in primary myeloid cells. This is particularly relevant to human immunology, given the known but not yet widely investigated phenotypic differences between human and mouse immune cells. The inventors' approach utilizes an arrayed platform of concentrated cr / sgRNA libraries to elucidate the desired phenotype in donor-derived human myeloid cells, which can then be evaluated in preclinical model systems such as mice. Furthermore, the ability to deliver substantial amounts of recombinant Cas9 enables pooled functional screening in myeloid cells derived from knockout or mutant mouse lines. This reduces the need to breed the desired genotype in Cas9-knock-in lines and therefore offers significant advantages in study design and economics. Finally, the absence of significant chronic immune cell activation observed when using our method suggests the possibility of in vivo evaluation via adoptive cell therapy after RNP-mediated gene knockout.

[0312] These findings represent a significant technological advancement in the study of myeloid cells, which are typically considered a difficult cell type for gene editing. Further adaptation of our methods for alternative gene regulation (e.g., via CRISPRi or CRISPRa) or SNP / reporter knock-in strategies will expand our ability to investigate and modify innate immunity in the relevant primary cell subset(s) of interest. [Table 1] [Table 2] Example 7: Materials and Methods

[0313] mouse

[0314] All mice used in this study were handled in accordance with the Genentech Institutional Animal Care and Use Committee (IACUC). All experiments were conducted according to protocols approved by the IACUC. Female eGFP transgenic mice were obtained from Jackson Laboratories (C57BL / 6-Tg(CAG-EGFP)1Osb / J, stock number 003291). Female wild-type C57BL / 6J mice were obtained from Jackson Laboratories (stock number 000664). All mice were 8–12 weeks old.

[0315] Human donor

[0316] Peripheral blood and PBMCs were collected from healthy donors participating in the Genentech blood donor program using written informed consent from the Western Institutional Review Board.

[0317] Human monocyte preparation

[0318] PBMCs were isolated using Buffycoat or LeucoPac from healthy donors, or using Sepmate tubes (StemCell Technologies catalog number 84540) and ACK lysis buffer (Fisher catalog number A1049201) according to the manufacturer's protocol. Human monocytes were isolated from PBMCs using a human monocyte isolation kit (Miltenyi #130-091-153) according to the manufacturer's protocol. The monocytes were halved and frozen in 10% DMSO and FBS for further use.

[0319] Monocyte-derived dendritic cell cultures and macrophage cell cultures

[0320] Monocyte-derived dendritic cells were cultured in RPMI (Rapid Microwave Oxygenation) supplemented with DC medium (10% FBS (Gibco), 2 mM l-alanyl-l-glutamine (GlutaMAX; Gibco), 55 μM β-mercaptoethanol (Gibco), 100 U / ml penicillin, 100 μg / ml streptomycin (Thermo Fisher 15140122), and cytokines GM-CSF 800 U / ml (Peprotech) and IL-4 500 U / ml (Peprotech) for 1 × 10⁶ cells. 6 Cells were cultured at a density of cells / ml. The medium was changed every 2-3 days by removing half the volume of medium from each well, spinning down the collected cells at 400×g for 5 minutes, then resuspending the spun cells in DC medium containing 2× cytokines, and replating the remaining cells. Monocyte-derived macrophages were cultured in Mac medium (DMEM high glucose supplemented with 10% FBS (Gibco catalog no. 10082-147), 2 mM l-alanyl-l-glutamine (GlutaMAX; Gibco), 100 U / ml penicillin, 100 μg / ml streptomycin (Thermo Fisher catalog no. 15140122), and M-CSF 100 ng / ml (Peprotech)). The medium was changed every 2-3 days by adding half the volume of medium containing 1× cytokines to each well.

[0321] Optimized human monocyte dendritic cell and macrophage cell knockout

[0322] gRNA selection. All gRNA sequences were selected using IDT's pre-designed guides or in-house algorithms, which are searchable on their websites.

[0323] Cell preparation. Cells were isolated as described above. Frozen monocytes were thawed in 6-well plates (Fisher catalog number CKS336) without treatment at 1e6 cells / ml in DC or Mac medium containing appropriate cytokines and cultured overnight.

[0324] gRNA preparation. To prepare each gRNA, Alt-R crRNA or crRNAXT (crRNA(XT)), Alt-sgRNA, and Alt-tracrRNA (catalog number 1072534; IDT) were reconstituted to 100 μM using Nuclease-Free Duplex Buffer (IDT). To prepare crRNA(XT)-tracrRNA double helix, the two oligonucleotides were mixed at equimolar concentrations in a sterile PCR tube (e.g., crRNA(XT) containing 5 μl Alt-R crRNA and 5 μl Alt-R tracrRNA). The oligonucleotides were annealed by heating at 95°C for 5 minutes, the mixture was cooled to room temperature, and hybridized in a PCR thermocycler (program: 95°C for 30 seconds, 95°C for 4.5 minutes, 25°C indefinitely) at room temperature for 15 minutes. The mixture was then placed on ice or frozen at -20°C until further use.

[0325] Pre-complexation of Cas9-RNP. Unless otherwise indicated, for each reaction, in sterile PCR strips or 1.5 ml tubes, annealed crRNA(XT)-tracrRNA double helix or sgRNA was mixed with Cas9 (IDT SpCas9''Alt-R® SpCas9 Nuclease V3'' or Thermo Spy Cas9''True Cut Cas9 Protein V2'') in a molar ratio of 3:1 for crRNA(XT) (3 μl crRNA(XT)-tracrRNA double helix + 2 μl Cas9 5 mg / ml) or 2:1 for sgRNA (2 μl sgRNA + 2 μl Cas9 5 mg / ml). Cas9-RNP was then incubated at room temperature for at least 20 minutes.

[0326] Nucleofection of the Cas9-RNP complex. Appropriate medium (DC or Mac) was preheated in a cell culture plate in a 37°C incubator for at least 30 minutes. Cells were harvested for nucleofection. For monocyte-derived DCs, cells were harvested from a 6-well plate and rotated at 400×g for 5 minutes. Then, more than 300 μl of 1×PBS was added to each well, harvested, and the remaining cells were harvested by rotating at 400×g for 5 minutes. Monocyte-derived macrophages were harvested similarly, except that Detachin (Genlantis catalog number T100100) was added after PBS harvesting, and the 6-well plate was incubated at 37°C for 5 minutes to release and harvest the attached cells. Then, for both DCs and Macs, the final cell pellet was washed twice with more than 5 ml of 1×PBS and counted. 1×10 per reaction 6Each cell was resuspended in 20 μl of P3 primary nucleofection solution (P3 Primary Cell 4D-Nucleofector X Kit, catalog number V4XP-3032). Then, 20 μl of the cell / P3 nucleofection solution mixture was added to each Cas9-RNP complex, and the mixture was gently pipetted up and down 3-5 times to avoid air bubbles. The cell-RNP mixture was then immediately loaded onto the supplied nucleofector cassette strip. The strip was inserted into a Lonza 4D-Nucleofector (4D-Nucleofector Core Unit: Lonza, AAF-1002B; 4D-Nucleofector X Unit: AAF-1002X) and electroporated under buffer P3 and CM-137 conditions. The cassette strip was removed, and 150-180 μl of preheated medium was immediately added to each cassette well. Next, the culture medium / cell-RNP mixture was pipetted into a suitable cell culture dish, and the cells were cultured for 5 days (macrophages) or 7 days (dendritic cells) as described above. The knockout efficiency was assayed by FACS.

[0327] Enhancer and Cas9-RNP titration analysis. Monocyte-derived macrophage cells were collected, and sgRNA-containing Cas9-RNP complexes were electroporated using the P3, CM-137 conditions described above, with the following modifications. The amount of Cas9-RNP complex was titrated from 4 μl (2 μl sgRNA + 2 μl Cas9 at 5 mg / ml) to 0.0019 μl at a 2-fold dilution. Under conditions including the electroporation enhancer, 1 μl (4 μM) of IDT accelerator (IDT catalog number 1075915) was added to the Cas9-RNP complex before electroporation during complex formation, and the same titration was performed.

[0328] FACS analysis of human monocytes, dendritic cells, and macrophages

[0329] Cells were harvested as described above for nucleofection and then stained with LIVE / DEAD Fixable Aqua Dead Cell dye (ThermoFisher catalog number L34957) in 50 μl 1× PBS at room temperature for 10 minutes. Cells were washed by adding 150 μl 1× PBS to each well, and then the cells were centrifuged at 1600 rpm for 3 minutes. The pelleted cells were resuspended and incubated with fluorophore-conjugated antibody in 50 μl FACS buffer at 4°C for at least 30 minutes. After washing the cells and fixing them with 2% PFA for 10 minutes, flow cytometry was performed. Cells were then washed with 1× FACS buffer and resuspended in 180 μl FACS buffer for analysis by flow cytometry. Monocyte-derived macrophages and dendritic cells were identified by the expression of CD64 or DC-SIGN, respectively. KOs were determined by gating for negatively stained cells using an untargeted control (see Figure 2A for gating, e.g.). Heat-sterilized cells and unstained cells were used as controls for viability and positive antibody staining. All samples were analyzed using the FACSymphony system (BD). MAVS knockout and functional analysis of dendritic cells

[0330] MAVS KO. Monocyte-derived dendritic MAVS KO cells were prepared as described above using sgRNAs targeting two different sequences. As a control, Cas9-RNPs carrying untargeted control (NTC) sgRNA were also electroporated into the cells. Cells were harvested and counted 6 days after nucleofection. 150K cells / well for each genotype were seeded in triplicate in 150 μl of DC medium (cytokine-free) under two conditions (mock and RIG-I agonist 5'-3p-dsRNA stimulation (Coch et al., 2013)). The remaining cells for each genotype (over 300,000 cells per sample) were split in half to create technical replicas, rotated at 2,000 rpm for 5 minutes, washed twice with 1×PBS, and then snap-frozen for gDNA preparation and TIDE analysis.

[0331] Cell stimulation. Seeded cells were stimulated by adding a RIG-I-targeting 5'-3p-dsRNA agonist. 5'-3p-dsRNA was delivered to cells using lipofectamine 2000 (Thermo Fisher catalog number 11668019) according to a standard protocol. Briefly, 0.1 μg of 5'-3p-dsRNA or 0.5 μl / well of lipofectamine 2000 was resuspended in 25 μl / well of OptiMEM and incubated at room temperature for 5 minutes. Then, the 5'-3p-dsRNA and lipofectamine suspensions were mixed together and incubated at room temperature for a further 20 minutes. Then, 50 μl of the combined RNA-lipofectamine mixture was added to each well for all three genotypes (NTC, MAVSsg1, MAVSsg2). For mock control, 50 μl of preheated DC medium was added to the cells. Cells were stimulated overnight, and the supernatant was collected. Cytokine levels (IFNα, IL-6, MIP-1α, TNF, RANTES, IL-1α) were measured using Luminex (Bio-Rad Laboratories). Cells were harvested using Detachin as described above and seeded into U-bottom TC-treated 96-well plates for intracellular FACS staining to determine MAVS knockout efficiency.

[0332] Intracellular FACS staining. FC blocks (5 μl / well) (Biolegend catalog no. 422301) and viable / dead fixable blue stain (0.5 μl / well) (ThermoFisher catalog no. L34961) were added to all wells in 50 μl of 1× PBS (except for unstained controls). Cells were then stained for MAVS using the eBioscience Intracellular Fixation & Permeabilization Buffer Set (ThermoFisher catalog no. 88-8824-00) according to the manufacturer's protocol. After staining, cells were resuspended in 180 μl of FACS buffer for analysis. Heat-sterilized cells and unstained cells were used as controls for viable and positive antibody staining. All samples were analyzed using the FACSymphony system (BD).

[0333] PKR knockout and functional analysis of monocyte-derived macrophages

[0334] PKR knockout and cell stimulation. Monocyte-derived macrophage PKR knockout cells were stimulated at a rate of 3 × 10⁶ per reaction. 6 Except for electroporation of individual cells, the cells were prepared using a single sgRNA (PKR sg) as described above. As a control, Cas9-RNP carrying NTC sgRNA was electroporated into cells. After electroporation, cells under all three conditions (no nucleofection control, NTC, and PKR sg) were placed in 1 × 10⁶ Mac medium supplemented with cytokines in a TC-treated 24-well plate (Plating schematic Figure 8C). 6 Cells were seeded at a concentration of 1 / ml. On day 5 after electroporation, one replica of each cell condition was stimulated with poly I:C (Invivogen, tlrl-pic). For poly I:C stimulation, 6 ug of poly I:C (2 μg / ml) was added to 500 μl of Opti-MEM. Then, 12 μl of TransIT (Mirus, MIR 2225) was added to 500 μl of Opti-MEM and incubated at room temperature for 5 minutes. Then, the TransIT mix was added to the poly I:C mix and incubated at room temperature for a further 20 minutes. The complex was then added to the cells.

[0335] Cells for Western blot analysis were stimulated for 6 hours and then collected using Detachin as described above. The cells were pelleted, washed twice with 1×PBS, and the pellet was resuspended in RIPA buffer supplemented with a protease inhibitor (cOmplete, Mini, EDTA-free protease inhibitor cocktail tablets, #4693159001). The lysates were clarified by spinning at 13,000 rpm at 4°C for 10 minutes, and the protein content was measured by BCA (Pierce, #23225). For each sample, 10 μl of protein was separated by SDS-PAGE, transferred to a nitrocellulose membrane, and blotted according to a standard protocol. Chemiluminescence was imaged using the BioRad ChemiDoc imaging system.

[0336] CRISPR KO BMDM for MYD88, TRIF, and STING deletions was analyzed by lysing cells in RIPA buffer as described above. SDS-PAGE was performed using a 4-12% gradient Bis-Tris gel (Novex), followed by transfer of proteins onto a PVDF membrane and standard downstream immunoblotting. Chemiluminescence was detected by enhanced chemiluminescence (Western lightning-plus ECL, Perkin Elmer).

[0337] Mouse BMDM culture

[0338] Bone marrow was collected from the tibia and femur of mice. Total bone marrow cells were cultured in BMDM culture medium [DMEM high glucose supplemented with 50 ng / mL recombinant mouse M-CSF (Genentech), 10% FBS (VWR), GlutaMAX (Gibco), and penicillin / streptomycin (Gibco)] in 150 mm non-TC treated petri dishes (VWR) in quantities of 0.5 × 10⁶. 6 Cells were seeded at a density of cells / mL in 20 mL volumes per dish. For 2 days, 20 mL of fresh BMDM culture medium was added without removing the medium. On day 4, all medium was removed and 20 mL of fresh BMDM medium was added. On day 5, cells were gently scraped from the dish using a rubber policeman and transferred to a 50 mL conical tube. The petri dish was washed once with 20 mL of 1 × PBS, and the cells were recovered by centrifugation. The cells were resuspended in 10 mL of 1 × PBS, counted, and then centrifuged again to resuspend in appropriate buffer or medium for downstream assays.

[0339] BMDM CD11b CRISPR KO screening

[0340] On day 5, BMDM was added to the nucleofection solution for primary cells (Primary Cell Optimized 96-well Nucleofector Kit, catalog number V4 SP-9096, Lonza) in 20 μl of nucleofection solution, with 5 × 10 per reaction. 5The cells were resuspended at cell density and mixed with Cas9-RNP containing Itgam-targeted gRNA. This mixture was electroporated using a Lonza 4D Nucleofector (4D-Nucleofector core unit: Lonza, AAF-1002B; 4D-Nucleofector X unit: AAF-1002X). Immediately after electroporation, cells were collected by adding approximately 180 μl of preheated BMDM culture medium to each well and gently washing the wells. Each reaction product was transferred to a single well in a 6-well TC treatment plate containing 2 mL of preheated BMDM culture medium. The cells were cultured for a further 5 days, with complete medium changes 2 and 4 days after electroporation. On day 5, the cells were collected by gently scraping with a rubber policeman. The collected cells were processed for flow cytometry. First, cells were stained with an Fc blocking reagent (CD16 / 32FcR block, BD Biosciences) at 4°C for 10 minutes, followed by staining with an antibody cocktail against CD11b (anti-CD11b-APC, CD45 (anti-CD45-FITC)). Cells were washed twice with flow cytometry buffer and resuspended in flow cytometry buffer containing a viability marker (propidium iodide, PI). Flow cytometry was performed, and each electroporation condition was compared using a BD Fortessa analyzer. Each condition was ranked using loss of cell surface CD11b, indicated by a decrease in its mean fluorescence intensity (MFI), along with maintenance of cell viability, indicated by the absence of a PI-positive signal.

[0341] Mouse monocyte culture and eGFP CRISPR KO screening

[0342] Bone marrow was collected from the tibia and femur of eGFP transgenic mice. Red blood cells were lysed with ACK lysis buffer. Monocytes were isolated using a negative selection kit (Miltenyi Biotec, 130-100-629). Cells were washed once with 1×PBS and 2×10⁶ cells were placed per well. 5The cells were resuspended in nucleofection solution for primary cells (Primary Cell Optimized 96-well Nucleofector Kit, catalog no. V4SP-9096, Lonza) at the cell density. The cells were electroporated as described above and immediately transferred to a 6-well TC treatment plate containing preheated BMDM culture medium. The cells were cultured for 5 days, with complete medium changes 2 and 4 days after electroporation. On day 5, monocyte-derived macrophages (Mo-Macs) were collected by gently scraping with a rubber policeman. The collected cells were processed for flow cytometry. First, the cells were stained in 1×PBS at 4°C for 20 minutes using Fc blocking reagent (CD16 / 32FcR block, BD Biosciences) in the presence of a fixable survival dye (eFluor 780). After washing the cells twice with flow cytometry buffer, they were stained for F4 / 80 (anti-F4 / 80-BV421). Cells were washed twice, and flow cytometry was performed to compare each electroporation condition using a BD Fortessa analyzer. Each condition was ranked using the loss of eGFP in the F4 / 80 positive population, along with the maintenance of cell viability, indicated by the absence of an APC-Cy7 positive signal.

[0343] Mouse BMDC CRISPR KO

[0344] Bone marrow (BM) cells were prepared, and red blood cells were lysed with ACK lysis buffer. BM cells were washed twice with 1×PBS and electroporated in appropriate primary nucleofection solutions (Primary Cell Optimization 4D-Nucleofector® X Kit, catalog no. V4XP-9096, P3 Primary Cell 4 D-Nucleofector X Kit, catalog no. V4XP-3032) using Lonza 4D Nucleofector (4D-Nucleofector core unit: Lonza, AAF-1002 B; 4D-Nucleofector X unit: AAF-1002X) as described above. Specifically, 2×10⁶ cells per reaction. 6BM cells were resuspended in 20 μl of primary nucleofection solution and mixed with Cas9-RNP containing targeted or NTC gRNA. The cell / Cas9-RNP mixture was then electroporated using an appropriate program. The electroporated cells were cultured for 12 days in round-bottom 96-well TC treatment plates in preheated RPMI medium supplemented with 10% FBS, L-glutamate, HEPES, antibiotics, and 2-ME and 100 ng / ml Flt3-ligand (Peprotech).

[0345] Flow cytometry analysis of mouse BMDM

[0346] Collected BMDCs were stained with a fixable viability dye in 1×PBS for 20 minutes. The centrifuged cell pellet was pre-incubated for 10 minutes in FACS buffer containing CD16 / 32 Fc blocks. The cells were further incubated with fluorophore-conjugated antibody in FACS buffer for 30 minutes. The cells were washed, fixed with 2% PFA for 20 minutes, and then flow cytometry was performed. All staining was performed on ice. Subsets of cells in the BMDC culture were gated as shown in Figure 1D, 6A. BD fix and perm kits were used as directed for intracellular staining of TLR7. All samples were analyzed using the FACS Fortessa system (BD).

[0347] TLR3, TLR4, and STING stimulation of mouse BMDM

[0348] CRISPR knockouts were performed on wild-type BMDM on day 5, as shown in Figure 4, by electroporating either a non-targeting sgRNA (NTC) or a pool of two sgRNAs targeting MYD88, TRIF (encoded by Ticam1), or STING (encoded by Tmem173), either individually or in combination. 5 × 10⁶ reactions per reaction. 6BMDM was used (buffer P3, program CM-137). Immediately after electroporation, the cells were transferred to a 10 cm non-TC treated petri dish containing 10 mL of preheated BMDM culture medium. The cells were cultured for a further 5 days, with medium changes on days 2 and 4 after electroporation. On day 5, the cells were gently scraped off and 0.5 × 10⁶ oz for stimulation. 6 Cells were re-seed in TC-treated multiwell plates at a density of cells / mL. BMDM cells were stimulated overnight (18 hours) with 100 ng / mL ultrapure LPS, 10 ug / mL PolyI:C LMW, or 5 ug / mL 2'3'-cGAMP (InvivoGen) to activate TLR4, TLR3, or STING, respectively. The culture medium was collected after treatment for TNF and IFNβ measurement by ELISA. Gene deletions were assessed by Western blotting.

[0349] Sequencing and Cas9 editing verification using TIDE (indel tracking using DEcomposition)

[0350] Primer design. DNA primers were generated for each gene locus whose editing efficiency was to be evaluated. Briefly, RefSeq searches for the target gene were performed using NCBI Gene. The FASTA genome sequence was searched for 2 and 3 base pairs from one end for the guide sequence and cleavage site after GG PAM. Starting 350 bp upstream of the PAM, a 700 bp sequence was selected with the cleavage site in the center. This was attached to primer 3. "350,2" was entered in the Target box, "500~600" in the Product Size Ranges box, and "2" in the CG Clamp box. "Pick Primer" was clicked, and the result containing the cleavage site in the center was selected. The primers were ordered from IDT.

[0351] Genomic DNA (gDNA). gDNA was collected from cells using QuickExtract solution: Approximately 30 μL of QuickExtract solution was added to each well of a 96-well plate or to 100 μL / well of cells in 24 wells, and incubated at room temperature for 1–5 minutes, ensuring cell detachment. Cells were lifted by repeated pipetting and transferred to PCR tubes. The samples were briefly vortexed and incubated at 65°C for 6 minutes, then at 98°C for 2 minutes. The gDNA templates were then used for PCR.

[0352] PCR. PCR was performed using Terra polymerase for 35 cycles with 1 μl of gDNA per 20 μl reaction. For each sgRNA target editing site, one test (edited gDNA) reaction and one control (unedited gDNA) reaction were performed. Setup: 10 μl 2× buffer, 1 μl gDNA, 1.2 μl primer mix (containing 1 μM F and 1 μM R validation primers), 0.4 μl Terra Polymerase, 7.4 μl H2O. 35 cycles were performed according to the Terra PCR protocol (98°C 2 min, [98°C 10 sec, 58°C 15 sec, 68°C 60 sec] × 36, 68°C 5 min). Product size of approximately 500 bp was confirmed using eGel (it is sufficient to perform only the control unedited reaction for each new primer pair). 2 μL was run on 2% gel for 14 minutes. The PCR reaction was purified using DNAClean (Zymo Research) or Qiagen columns.

[0353] Sequencing and TIDE. Clean PCR products were subjected to Sanger sequencing using an F primer 250 bp upstream of the target editing site. Sanger results were analyzed using TIDE or the ICE website: www.deskgen.com / landing / tide.html or ice.synthego.com / # / .

[0354] antibody

[0355] (Mouse)IA / IE-BV421,B220-BV605,F4 / 80-BV711,Sirpα-PE-Cy7,CD45-FITC(Biolegend),TLR7-PE,CD24-BUV395,CD80-BUV747,F4 / 80-BV421,CD11b-APC(BD)

[0356] eBioscience® Fixable Viability Dye eFluor® 780 (Thermofisher catalog number 65-0865-14)

[0357] Western blotting antibodies: TRIF (Genentech, catalog number 1-3-5), MYD 88 (Abcam, catalog number ab2064), STING (Cell Signaling Technologies D2P2F, catalog number 13647S), beta-actin (Cell Signaling Technologies catalog number 3700).

[0358] Human. CD64 mouse anti-human, APC (BD Biosciences 561189), FITC anti-human CD163 antibody (Biolegend 333617), PE anti-human β2-microglobulin antibody (Biolegend 316305), APC anti-human CD209 (DC-SIGN) antibody (Biolegend 330107), BV786 mouse anti-human CD80 (BD Biosciences 564159), Alexa Fluor 488 (ThermoFisher catalog number A-11008). LIVE / DEAD Fixable Aqua Dead Cell dye (ThermoFisher catalog number L34957). LIVE / DEAD (trademark) Fixable Blue Dead Cell Stain Kit, for UV excitation (ThermoFisher catalog number L34961).

[0359] Western blotting antibodies: Anti-MAVS antibody (Abcam ab31334), goat anti-rabbit IgG(H+L) cross-adsorbed secondary antibody (ThermoFisher, catalog number 31212), goat anti-mouse IgG(H+L) cross-adsorbed secondary antibody (ThermoFisher, catalog number 31164), PKR (Cell Signaling, CST12297), eIF2a-P (Cell Signaling, CST3398), eIF2α-total (Abcam, ab5369), β-tubulin (Santa Cruz, sc-5274)

[0360] ELISA

[0361] TNF and IFNβ secretion by mouse BMDM was measured using a standard ELISA kit (TNF: Invitrogen 88-7324-88; IFNβ: PBL assay Sci. Verikin 422400-1).

[0362] statistical analysis

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Claims

1. A method for genetic modification of bone marrow cells, comprising transfecting the bone marrow cells by electroporation using a CRIPSR-Cas system comprising an electroporation enhancer and a Cas protein and guide RNA targeting a gene site of interest, wherein the bone marrow cells are not transduced by a viral vector, and the electroporation enhancer is single-stranded DNA or a combination of single-stranded DNA and double-stranded DNA.

2. The method according to claim 1, wherein the bone marrow cells are primary bone marrow cells.

3. The method according to claim 1, wherein the bone marrow cells are monocytes, macrophages, neutrophils, basophils, eosinophils, dendritic cells, or megakaryocytes.

4. The method according to claim 1, wherein the cells are not subjected to a selection step and / or a concentration step after transfection.

5. The method according to claim 1, wherein the CRISPR-Cas system further comprises donor DNA.

6. The method according to claim 1, wherein the bone marrow cells are differentiated before electroporation.

7. The method according to claim 6, wherein the bone marrow cells differentiate into dendritic cells.

8. The method according to claim 1, wherein the bone marrow cells are not activated before or during the genetic modification.

9. The method according to claim 1, wherein the guide RNA comprises two or more different crispr RNAs (crRNAs) for the target site, and the two or more different crRNAs are introduced into the bone marrow cells during the transfection step.

10. The method according to claim 9, wherein the crRNA is a single guide RNA (sgRNA).

11. The method according to claim 1, wherein multiple target gene regions are targeted.

12. A method for genetically modifying a plurality of myeloid cells, comprising transfecting the plurality of myeloid cells by electroporation in the presence of a CRIPSR-Cas system comprising an electroporation enhancer and a Cas protein and guide RNA targeting a site of interest, wherein the myeloid cells are not transduced by a viral vector, and the electroporation enhancer is single-stranded DNA or a combination of single-stranded DNA and double-stranded DNA.

13. The method according to claim 12, wherein the target site is modified in at least 70% of the plurality of bone marrow cells.

14. The method according to claim 13, wherein the target site is modified in at least 80% of the plurality of bone marrow cells.

15. The method according to claim 13, wherein the target site is modified in at least 85% of the plurality of bone marrow cells.

16. The method according to claim 13, wherein the target site is modified in at least 90% of the plurality of bone marrow cells.

17. The method according to claim 12, wherein the plurality of bone marrow cells include dendritic cells (DCs), and the target site is modified in at least 50% of the DCs.

18. The method according to claim 12, wherein the survival rate of the plurality of bone marrow cells after electroporation is at least 80%.

19. The method according to claim 18, wherein the survival rate of the plurality of bone marrow cells after electroporation is at least 90%.

20. The method according to claim 1, wherein the ratio of guide RNA to Cas protein is between 100:1 and 1:

100.

21. The method according to claim 20, wherein the ratio of the guide RNA to the Cas protein is 3:1 or less.

22. The method according to claim 20, wherein the ratio of the guide RNA to the Cas protein is 2:1 or less.

23. The method according to claim 20, wherein the ratio of the guide RNA to the Cas protein is 3:

1.

24. The method according to claim 20, wherein the ratio of the guide RNA to the Cas protein is 2:

1.

25. The method according to claim 12, which does not include a selection or enrichment step after bone marrow cell transfection.

26. A system for genetically modifying bone marrow cells in the absence of a viral vector, comprising: a chamber compatible with a transfection system; a plurality of bone marrow cells in the chamber in a culture medium compatible with electroporation; an electroporation enhancer; and a CRIPSR-Cas system comprising at least one Cas protein and guide RNA designed to target at least one site of interest in the genome of the bone marrow cells, wherein the electroporation enhancer is single-stranded DNA or a combination of single-stranded DNA and double-stranded DNA.

27. A composition comprising a plurality of bone marrow cells, a CRIPSR-Cas system containing Cas protein and guide RNA, a transfection buffer, and an electroporation enhancer, wherein the composition does not contain a viral vector, and the electroporation enhancer is single-stranded DNA or a combination of single-stranded DNA and double-stranded DNA.

28. The method according to any one of claims 1 to 25, further comprising screening the effects of one or more compounds on transfected bone marrow cells for the purpose of drug discovery assays.

29. The method according to any one of claims 1 to 25, further comprising contacting transfected bone marrow cells with the compound for the purpose of target verification of the compound, and monitoring the effect on the transfected bone marrow cells.

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