Epigenetic editing methods and systems for differentiating stem cells

Abstract

Provided are DNA-targeting systems, such as CRISPR-Cas / guide RNA (gRNA) systems, that bind to or target a target site in a gene or regulatory element thereof in a stem cell. In some embodiments, the provided DNA-targeting systems promote differentiation of stem cells into hematopoietic progenitor cells. Among provided embodiments are embodiments in which the provided DNA-targeting systems promote transcriptional activation of genes that promote hematopoietic progenitor cell differentiation. Also provided herein are methods and uses related to the provided compositions, for example in facilitating hematopoietic progenitor cell differentiation without the provision of extrinsic differentiation signals.

Description

EPIGENETIC EDITING METHODS AND SYSTEMS FOR DIFFERENTIATING STEM CELLSCross-Reference to Related Applications

[0001] This application claims priority from U.S. provisional application No. 63 / 581,966 filed September 11, 2023, U.S. provisional application No. 63 / 635,967 filed April 18, 2024, and U.S. provisional application No. 63 / 669,204 filed July 9, 2024, the contents of which are incorporated by reference in their entireties.Incorporation by Reference of Sequence Listing

[0002] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file entitled 224742003140SeqList. xml, created September 6, 2024, which is 511,787 bytes in size. The information in the electronic format of the Sequence Listing is incorporated by reference in its entirety.Field

[0003] The present disclosure relates in some aspects to DNA-targeting systems, such as CRISPR-Cas / guide RNA (gRNA) systems, that bind to or target a target site in a gene or regulatory element thereof in a stem cell. In some aspects, the provided DNA-targeting systems of the present disclosure promote differentiation of stem cells into hematopoietic progenitor cells. In particular, the present disclosure relates to the transcriptional activation of genes that promote hematopoietic progenitor cell differentiation. In some aspects, the present disclosure is directed to methods and uses related to the provided compositions, for example in facilitating hematopoietic progenitor cell differentiation without the provision of extrinsic differentiation signals.Background

[0004] The ability to modulate stem cell differentiation into hematopoietic progenitor cells is an important step for generating effector cells. However, current methods for differentiating stem cells into hematopoietic progenitor cells rely on multiple media changes, feeder cell coculture, and / or addition of extrinsic factor to the culture, all of which are laborious, costly,224742003140 difficult to reproduce and difficult to scale. Therefore, there is a need for new and improved methods to overcome these challenges. The present disclosure addresses these and other needs.Summary

[0005] Provided herein is a DNA-targeting system comprising one or more DNA-targeting modules, wherein the one or more DNA-targeting modules target one or more transcription factor genes for promoting differentiation of stem cells to HPCs, and wherein each of the one or more DNA-targeting modules comprises a fusion protein comprising: (a) a DNA-binding domain that binds to a target site for one of the one or more transcription factor genes; and (b) at least one transcriptional activation domain that increases transcription of the one or more transcription factor genes. Also provided herein is a DNA-targeting system comprising one or more DNA-targeting modules, wherein the one or more DNA-targeting modules target one or more transcription factor genes for promoting differentiation of stem cells to HPCs, and wherein each of the one or more DNA-targeting modules comprises a fusion protein comprising: (a) a DNA-binding domain that binds to a target site for one of the one or more transcription factor genes; and (b) at least one transcriptional activation domain that increases transcription of the one or more transcription factor genes, wherein the one or more transcription factor genes is at least three genes GATA3, GATA2 and LM02. Also provided herein is a DNA-targeting system comprising one or more DNA-targeting modules, wherein the one or more DNA-targeting modules target one or more transcription factor genes for promoting differentiation of stem cells to HPCs, and wherein each of the one or more DNA-targeting modules comprises a fusion protein comprising: (a) a DNA-binding domain that binds to a target site for one of the one or more transcription factor genes, wherein the target site for at least one of the one or more transcription factor genes is a target site for LM02, and wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,869,125; and (b) at least one transcriptional activation domain that increases transcription of the one or more transcription factor genes.

[0006] In some embodiments, the one or more transcription factor genes are selected from the group consisting of CEBPA, CEBPD, CFOS, BMI1, FOXQ1, GATA2, GATA3, GFI1, GFI1B, HEY2, HOXB9, LM02, LYL1, RUNX3, NFE2, SIRT1, SPI1, TALI and ZEB2.224742003140

[0007] In some embodiments, the one or more transcription factor genes are further selected from the group consisting of CEBPA, CEBPD, CFOS, BMI1, FOXQ1, GATA2, GATA3, GFI1, GFI1B, HEY2, HOXB9, LYL1, RUNX3, NFE2, SIRT1, SPI1, TALI and ZEB2.

[0008] Provided herein is a DNA-targeting system comprising one or more DNA-targeting modules for increasing transcription of one or more transcription factor genes, wherein each of the one or more DNA-targeting modules comprises a fusion protein comprising: (a) a DNA- binding domain that binds to a target site for one of the one or more transcription factor genes, wherein the one or more transcription factor genes are selected from the group consisting of CEBPA, CEBPD, CFOS, BMI1, FOXQ1, GATA2, GATA3, GFI1, GFI1B, HEY2, HOXB9, LM02, LYL1, RUNX3, NFE2, SIRT1, SPI1, TALI and ZEB2; and (b) at least one transcriptional activation domain that increases transcription of the one or more transcription factor genes.

[0009] In some of any embodiments, transient delivery of the DNA-targeting system to a stem cell promotes differentiation to a HPC. In some of any embodiments, transient delivery of the DNA-targeting system to a stem cell promotes differentiation to a CD34+ cell. In some of any embodiments, transient delivery of the DNA-targeting system to a stem cell promotes differentiation to a CD43+ cell. In some of any embodiments, transient delivery of the DNA- targeting system to a stem cell promotes differentiation to a CD34+ / CD43+ cell. In some of any embodiments, the stem cell is an induced pluripotent stem cell (iPSC).

[0010] In some of any embodiments, the one or more transcription factor genes are selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally where the one or more transcription factor genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB 2.

[0011] In some of any embodiments, the one or more DNA targeting modules is a plurality of DNA-targeting modules for increasing transcription of one or more transcription factor genes, wherein each DNA-targeting module targets a target site for one of the one or more genes. In some of any embodiments, the plurality of DNA-targeting modules is 2, 3, 4, 6, 5, 7, 8, or 9 DNA-targeting modules, each targeting one of the one or more transcription factor genes. In some of any embodiments, the plurality of DNA-targeting modules is 2 DNA-targeting modules, each targeting one of the one or more transcription factor genes. In some of any embodiments, the one or more transcription factor genes is two genes selected from the group consisting of224742003140BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the two genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the plurality of DNA- targeting modules is 3 DNA-targeting modules, each targeting one of the one or more transcription factor genes. In some of any embodiments, the one or more transcription factor genes is three genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the three genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the plurality of DNA-targeting modules is 4 DNA- targeting modules, each targeting one of the one or more transcription factor genes. In some of any embodiments, the one or more transcription factor genes is four genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the four genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the plurality of DNA-targeting modules is 5 DNA-targeting modules, each targeting one of the one or more transcription factor genes. In some of any embodiments, the one or more transcription factor genes is five genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the five genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the plurality of DNA-targeting modules is 6 DNA- targeting modules, each targeting one of the one or more transcription factor genes. In some of any embodiments, the one or more transcription factor genes is six genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the six genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the plurality of DNA-targeting modules is 7 DNA-targeting modules, each targeting one of the one or more transcription factor genes. In some of any embodiments, the one or more transcription factor genes is seven genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the seven genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the plurality of DNA-targeting modules is 8 DNA-targeting modules, each targeting one of the one or more transcription factor genes. In224742003140 some of any embodiments, the one or more transcription factor genes is eight genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the genes are BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the plurality of DNA-targeting modules is 9 DNA-targeting modules is 9 DNA-targeting modules, each targeting one of the one or more transcription factor genes. In some of any embodiments, the one or more transcription factor genes is nine genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2.

[0012] In some of any embodiments, the target site for each of the one or more transcription factor genes is in the gene or a regulatory DNA element thereof. In some embodiments, the regulatory DNA element is an enhancer or a promoter of the gene. In some embodiments, the regulatory DNA element is a promoter of the gene. In some of any embodiments, the target site for each of the one or more transcription factor genes is within 1000 base pairs of the transcription start site (TSS) of the gene. In some of any embodiments, the target site for each of the one or more transcription factor genes is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene. In some of any embodiments, the target site for each of the one or more transcription factor genes is within 550 base pairs upstream of the TSS of the gene.

[0013] In some of any embodiments, wherein the target site for each of the one or more transcription factor genes is selected from: (a) a target site for GATA3 having the sequence set forth in SEQ ID NO:28 or SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) a target site for GATA2 having the sequence set forth in SEQ ID NO:29, SEQ ID NO: 85 or SEQ ID NO:87, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (c) a target site for LM02 having the sequence set forth in any one of SEQ ID NOs:30, 80-82, and 94-113, 118, and 119, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (d) a target site for ZEB2 having the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (e) a target site for HEY2 having the sequence set forth in SEQ ID NO:32, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (f) a target site for CEBPD having the224742003140 sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (g) a target site for BMII having the sequence set forth in SEQ ID NO:34, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (h) a target site for LYL1 having the sequence set forth in SEQ ID NO:35 or SEQ ID NO: 83, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (i) a target site for TALI having the sequence set forth in SEQ ID NO:84 or SEQ ID NO: 86, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; or (j) a target site for CFOS having the sequence set forth in SEQ ID NO:88, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for each of the one or more transcription factor genes is selected from: (a) a target site for GATA3 having the sequence set forth in SEQ ID NO:28 or SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) a target site for GATA2 having the sequence set forth in SEQ ID NO:29, SEQ ID NO: 85 or SEQ ID NO:87, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (c) a target site for LM02 having the sequence set forth in SEQ ID NO:30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (d) a target site for ZEB2 having the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (e) a target site for HEY2 having the sequence set forth in SEQ ID NO:32, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (f) a target site for CEBPD having the sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (g) a target site for BMII having the sequence set forth in SEQ ID NO:34, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (h) a target site for LYL1 having the sequence set forth in SEQ ID NO:35 or SEQ ID NO: 83, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (i) a target site for TALI having the sequence set forth in SEQ ID NO:84 or SEQ ID NO: 86, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; or (j) a target site for224742003140CFOS having the sequence set forth in SEQ ID NO:88, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0014] In some of any embodiments, at least one of the one or more transcription factor genes is GATA3. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for GATA3 having the sequence set forth in SEQ ID NO:28 or SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0015] In some of any embodiments, at least one of the one or more transcription factor genes is GATA2. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for GATA2 having the sequence set forth in SEQ ID NO:29, SEQ ID NO: 85 or SEQ ID NO:87, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0016] In some of any embodiments, at least one of the one or more transcription factor genes is LM02. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for LM02, and wherein the target site for LM02 is within one of the regions defined by the following genomic coordinates: (a) chrl 1:33,867,158 to chrl 1:33,873,567; (b) chrl l: 33,876,536 to chrl 1:33,878,097; (c) chrl 1:33,890,358 to chrl 1:33,896,210; (d) chrl l: 33,906,104 to chrl 1:33,908,030; (e) chrl l: 33,911,674 to chrl 1:33,913,494; (f) chrl 1:33,920,461 to chrl 1:33,922,260; or (g) chrl 1:33,929,929 to chrl 1:33,932,651. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for LM02, and wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,867, 158 to chrl 1:33,873,567. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for LM02, and wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,867,215 to chrl 1:33,872,057. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for LM02, and wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,869,125. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for LM02, and wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,868,742.224742003140

[0017] In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for LM02 comprising the sequence set forth in any one of SEQ ID NOs: 30, 80-82, and 94-113, 118, and 119, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for LM02 comprising the sequence set forth in any one of SEQ ID NOs: 94-113, 118, and 119, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for LM02 comprises the sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 101, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for LM02 comprises the sequence set forth in SEQ ID NO: 101, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for LM02 comprises the sequence set forth in SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for LM02 having the sequence set forth in SEQ ID NO:30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0018] In some of any embodiments, one or more transcription factor genes is at least three genes GATA3, GATA2 and LM02.

[0019] In some of any embodiments, at least one of the one or more transcription factor genes is BMI1. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for BMI1 having the sequence set forth in SEQ ID NO: 34, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0020] In some of any embodiments, at least one of the one or more transcription factor genes is CEBPD. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for CEBPD having the sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.224742003140

[0021] In some of any embodiments, at least one of the one or more transcription factor genes is LYL1. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for LYL1 having the sequence set forth in SEQ ID NO:35 or SEQ ID NO: 83, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0022] In some of any embodiments, the one or more transcription factor genes is at least six genes GATA3, GATA2, LM02, BMI, CEBPD, and LYL1.

[0023] In some of any embodiments, at least one of the one or more transcription factor genes is HEY2. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for HEY2 having the sequence set forth in SEQ ID NO: 32, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0024] In some of any embodiments, at least one of the one or more transcription factor genes is ZEB2. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for ZEB2 having the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0025] In some of any embodiments, at least one of the one or more transcription factor genes is TALI. In some of any embodiments the target site for at least one of the one or more transcription factor genes is a target site for TALI having the sequence set forth in SEQ ID NO:84 or SEQ ID NO:86, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0026] In some of any embodiments, at least one of the one or more transcription factor genes is CFOS. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for CFOS having the sequence set forth in SEQ ID NO:88, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0027] In some of any embodiments, the DNA-targeting system does not introduce a genetic disruption or a DNA break.

[0028] In some of any embodiments, the fusion protein of each DNA-targeting module comprises a DNA-binding domain selected from: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or a variant thereof; a zinc finger protein (ZFP); a224742003140 transcription activator-like effector (TALE); a meganuclease; a homing endonuclease; or an I- Scel enzyme or a variant thereof, optionally wherein the DNA-binding domain comprises a catalytically inactive variant of any of the foregoing, wherein, when the DNA-binding domain of the fusion protein comprises a Cas protein, the DNA-targeting system further comprises one or more gRNAs, each capable of targeting the Cas protein to the target site for one of the one or more transcription factor genes.

[0029] In some of any embodiments, the DNA-binding domain of each of the one or more DNA-targeting modules is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof, and each of the one or more DNA-targeting modules further comprises one or more gRNAs for targeting the DNA-binding domain to the target site of one of the one or more transcription factor genes. In some of any embodiments, the Cas protein or variant thereof is a deactivated (dCas) protein. Provided herein is a DNA targeting system comprising: (a) a fusion protein comprising a DNA-binding domain comprising a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and (b) one or more gRNAs that target a target site for one or more transcription factor genes, wherein the DNA-targeting system increases transcription of the one or more transcription factor genes to promote differentiation of stem cells to HPCs. In some embodiments, the one or more transcription factor genes are selected from the group consisting of CEBPA, CEBPD, CFOS, BMI1, FOXQ1, GATA2, GATA3, GFI1, GFI1B, HEY2, HOXB9, LM02, LYL1, RUNX3, NFE2, SIRT1, SPI1, TALI and ZEB2.

[0030] Also provided herein is a DNA targeting system comprising: (a) a fusion protein comprising a DNA-binding domain comprising a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and (b) one or more gRNAs that target a target site for one or more transcription factor genes, wherein the one or more transcription factor genes are selected from the group consisting of CEBPA, CEBPD, CFOS, BMI1, FOXQ1, GATA2, GATA3, GFI1, GFI1B, HEY2, HOXB9, LM02, LYL1, RUNX3, NFE2, SIRT1, SPI1, TALI and ZEB2. Also provided herein is a DNA targeting system comprising: (a) a fusion protein comprising a DNA- binding domain comprising a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and (b) one or more gRNAs that target a target site for one or more transcription factor genes, wherein the DNA-targeting system increases transcription of the one or more transcription factor genes to promote differentiation of stem cells to HPCs, wherein the one or more transcription factors is at least three genes GATA3, GATA2 and LM02. Also224742003140 provided herein is a DNA targeting system comprising: (a) a fusion protein comprising a DNA- binding domain comprising a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and (b) one or more gRNAs that target a target site for one or more transcription factor genes, wherein the target site for at least one of the one or more transcription factor genes is a target site for LM02, and wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,869,125, wherein the DNA-targeting system increases transcription of the one or more transcription factor genes to promote differentiation of stem cells to HPCs.

[0031] In some of any embodiments, the dCas protein lacks nuclease activity. In some of any embodiments, the dCas protein is a dCas9 protein. In some of any embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein. In some of any embodiments, the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 41. In some of any embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 280, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any embodiments, the dSaCas9 protein is set forth in SEQ ID NO: 280. In some of any embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. In some of any embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 43. In some of any embodiments, the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 281, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any embodiments, the dSpCas9 protein is set forth in SEQ ID NO: 281.

[0032] In some of any embodiments, the one or more gRNAs comprise a gRNA spacer that is complementary to the target site of the gene. In some of any embodiments, the gRNA is selected from: (a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79, or a contiguous portion thereof of at least 14 nt; (b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77, or a contiguous portion thereof of at least 14 nt; (c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 21, 70-72, 120-139, 144, and 145, or a contiguous portion224742003140 thereof of at least 14 nt; (d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22, or a contiguous portion thereof of at least 14 nt; (e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23, or a contiguous portion thereof of at least 14 nt; (f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24, or a contiguous portion thereof of at least 14 nt; (g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; (h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73, or a contiguous portion thereof of at least 14 nt; (i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76, or a contiguous portion thereof of at least 14 nt; (j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; or (k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78, or a contiguous portion thereof of at least 14 nt. In some of any embodiments, the gRNA is selected from: (a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79, or a contiguous portion thereof of at least 14 nt; (b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77, or a contiguous portion thereof of at least 14 nt; (c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 126 or 127, or a contiguous portion thereof of at least 14 nt; (d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22, or a contiguous portion thereof of at least 14 nt; (e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23, or a contiguous portion thereof of at least 14 nt; (f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24, or a contiguous portion thereof of at least 14 nt; (g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; (h) a224742003140 gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73, or a contiguous portion thereof of at least 14 nt; (i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76, or a contiguous portion thereof of at least 14 nt; (j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; or (k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78, or a contiguous portion thereof of at least 14 nt. In some of any embodiments, the gRNA is selected from: (a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79, or a contiguous portion thereof of at least 14 nt; (b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77, or a contiguous portion thereof of at least 14 nt; (c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 21, SEQ ID NO: 70, SEQ ID NO:71 or SEQ ID NO:72, or a contiguous portion thereof of at least 14 nt; (d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22, or a contiguous portion thereof of at least 14 nt; (e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23, or a contiguous portion thereof of at least 14 nt; (f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24, or a contiguous portion thereof of at least 14 nt; (g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; (h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73, or a contiguous portion thereof of at least 14 nt; (i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76, or a contiguous portion thereof of at least 14 nt; (j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; or (k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78, or a contiguous portion thereof of at least22474200314014 nt. In some of any embodiments, the gRNA is selected from: (a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79; (b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77; (c) a gRNA targeting a target site for LMO2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 21, SEQ ID NO: 70, SEQ ID NO:71 or SEQ ID NO:72; (d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22; (e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23; (f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24; (g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25; (h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73; (i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76; (j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25; or (k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78.

[0033] In some of any embodiments, the gRNA comprises a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length. In some of any embodiments, the gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length. In some of any embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 91. In some of any embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 93. In some of any embodiments, the gRNA further comprises 2’ MeO modified bases and / or phosphorothiate backbone modifications.

[0034] In some of any embodiments, the one or more transcription factor genes include GATA2, GATA3, LM02 and BMI1. In some of any embodiments, the one or more transcription factor genes include GATA2, GATA3, LM02 and LYLE In some of any embodiments, the one or more transcription factor genes include GATA2, GATA3, LM02 and CEBPD. In some of any embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, LM02, BMI1, CEBPD, and LYLE224742003140In some of any embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, LM02, BMI1, CEBPD, and TALI. In some of any embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, LM02, BMI1, and TALI. In some of any embodiments, wherein the one or more transcription factor genes are BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, CEBPD, LYL1, TALI, HEY2, and CFOS. In some of any embodiments, the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, CEBPD, ZEB2, TALI, HEY2, and CFOS. In some of any embodiments, the one or more transcription factor genes include GATA2, GATA3, LM02, LYL1, CEBPD, ZEB2, TALI, HEY2, and CFOS.

[0035] In some of any embodiments, the at least one transcriptional activator effector domain is selected from the group consisting of: a VP64 domain, a p65 activation domain, a p300 domain, an Rta domain, a CBP domain, a VPR domain, a VPH domain, an HSF1 domain, a TET protein domain, optionally wherein the TET protein is TET1, a SunTag domain, or a domain, portion, variant, or truncation of any of the foregoing. In some of any embodiments, the at least one transcriptional activator effector domain comprises at least one VP 16 domain, and / or a VP16 tetramer (“VP64”) or a variant thereof. In some of any embodiments, the at least one transcriptional activator effector domain comprises a VP64 domain or a variant or portion thereof that exhibits transcriptional activation activity. In some of any embodiments, the at least one transcriptional activator effector domain is VP64. In some embodiments, the VP64 is positioned N-terminal and / or C-terminal to the DNA-binding domain. In some of any embodiments, the at least one transcriptional activator effector domain comprises two copies of VP64. In some of any embodiments, the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 47, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 47. In some of any embodiments, the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 48, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 48.224742003140

[0036] In some of any embodiments, the fusion protein comprises the sequence set forth in SEQ ID NO: 49, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

[0037] Also provided herein is a combination of gRNAs comprising two or more gRNAs, each selected from a gRNA that targets a target site for one or more transcription factor genes, wherein the one or more transcription factor genes are selected from the group consisting of CEBPA, CEBPD, CFOS, BMI1, FOXQ1, GATA2, GATA3, GFI1, GFI1B, HEY2, HOXB9, LM02, LYL1, RUNX3, NFE2, SIRT1, SPI1, TALI and ZEB2. Also provided herein is a combination of gRNAs comprising two or more gRNAs, each selected from a gRNA that targets a target site for one or more transcription factor genes, wherein the one or more transcription factor genes is at least GATA3, GATA2 and LMO2. Also provided herein is a combination of gRNAs comprising two or more gRNAs, each selected from a gRNA that targets a target site for one or more transcription factor genes, wherein the target site for at least one of the one or more transcription factor genes is a target site for LMO2, and wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,869,125. In some embodiments, the target site for each of the one or more transcription factor genes is in the gene or a regulatory DNA element thereof. In some embodiments, the regulatory DNA element is an enhancer or a promoter. In some of any embodiments, the regulatory DNA element is a promoter of the gene. In some of any embodiments, the target site for each of the one or more transcription factor genes is within 1000 base pairs of the transcription start site (TSS) of the gene. In some of any embodiments, the target site for each of the one or more transcription factor genes is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene. In some of any embodiments, the target site for each of the one or more transcription factor genes is with 550 base pairs upstream of the TSS of the gene.

[0038] In some of any embodiments, the combination of gRNAs comprises 2, 3, 4, 5, 6, 7, 8, or 9 different gRNAs, each that targets a target site of one of the one or more transcription factor genes. In some of any embodiments, the combination of gRNAs comprise 3, 4, 5, 6, 7, 8, or 9 In some of any embodiments, the one or more transcription factor genes is two genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the two genes are selected from the group consisting of BMI1,224742003140CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the one or more transcription factor genes is three genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the three genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the one or more transcription factor genes is four genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the four genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the one or more transcription factor genes is five genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the five genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the one or more transcription factor genes is six genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the six genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the one or more transcription factor genes is seven genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the seven genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the one or more transcription factor genes is eight genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the genes are BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the one or more transcription factor genes is nine genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2.

[0039] In some of any embodiments, the target site for each of the one or more transcription factor genes is selected from: (a) a target site for GATA3 having the sequence set forth in SEQ ID NO:28 or SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) a target site for GATA2 having the sequence set forth in SEQ ID NO:29, SEQ ID NO: 85 or SEQ ID NO:87, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of224742003140 the foregoing; (c) a target site for LM02 having the sequence set forth in any one of SEQ ID NOs:30, 80-82, 94-113, 118, and 119, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (d) a target site for ZEB2 having the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (e) a target site for HEY2 having the sequence set forth in SEQ ID NO:32, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (f) a target site for CEBPD having the sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (g) a target site for BMII having the sequence set forth in SEQ ID NO:34, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (h) a target site for LYL1 having the sequence set forth in SEQ ID NO:35 or SEQ ID NO: 83, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (i) a target site for TALI having the sequence set forth in SEQ ID NO:84 or SEQ ID NO: 86, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; or (j) a target site for CFOS having the sequence set forth in SEQ ID NO:88, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for each of the one or more transcription factor genes is selected from: (a) a target site for GATA3 having the sequence set forth in SEQ ID NO:28 or SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) a target site for GATA2 having the sequence set forth in SEQ ID NO:29, SEQ ID NO: 85 or SEQ ID NO:87, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (c) a target site for LM02 having the sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 101, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (d) a target site for ZEB2 having the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (e) a target site for HEY2 having the sequence set forth in SEQ ID NO:32, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (f) a target site for CEBPD having the sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (g) a target site for BMII having the sequence set forth in SEQ ID NO:34, a224742003140 contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (h) a target site for LYL1 having the sequence set forth in SEQ ID NO:35 or SEQ ID NO: 83, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (i) a target site for TALI having the sequence set forth in SEQ ID NO:84 or SEQ ID NO: 86, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; or (j) a target site for CFOS having the sequence set forth in SEQ ID NO:88, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for each of the one or more transcription factor genes is selected from: (a) a target site for GATA3 having the sequence set forth in SEQ ID NO:28 or SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (b) a target site for GATA2 having the sequence set forth in SEQ ID NO:29, SEQ ID NO: 85 or SEQ ID NO:87, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (c) a target site for LM02 having the sequence set forth in SEQ ID NO:30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (d) a target site for ZEB2 having the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (e) a target site for HEY2 having the sequence set forth in SEQ ID NO:32, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (f) a target site for CEBPD having the sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (g) a target site for BMII having the sequence set forth in SEQ ID NO:34, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (h) a target site for LYL1 having the sequence set forth in SEQ ID NO:35 or SEQ ID NO: 83, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; (i) a target site for TALI having the sequence set forth in SEQ ID NO:84 or SEQ ID NO: 86, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; or (j) a target site for CFOS having the sequence set forth in SEQ ID NO:88, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.224742003140

[0040] In some of any embodiments, at least one of the one or more transcription factor genes is GATA3. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for GATA3 having the sequence set forth in SEQ ID NO:28 or SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0041] In some of any embodiments, at least one of the one or more transcription factor genes is GATA2. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for GATA2 having the sequence set forth in SEQ ID NO:29, SEQ ID NO: 85 or SEQ ID NO:87, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0042] In some of any embodiments, at least one of the one or more transcription factor genes is LM02. In some of any embodiments, the target site for LM02 is within one of the regions defined by the following genomic coordinates: (a) chrl 1:33,867, 158 to chrl 1:33,873,567; (b) chrl l: 33,876,536 to chrl 1:33,878,097; (c) chrl 1:33,890,358 to chrl 1:33,896,210; (d) chrl l: 33,906,104 to chrl 1:33,908,030; (e) chrl l: 33,911,674 to chrl 1:33,913,494; (f) chrl 1:33,920,461 to chrl 1:33,922,260; or (g) chrl 1:33,929,929 to chrl 1:33,932,651. In some of any embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,867,158 to chrl 1:33,873,567. In some of any embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,867,215 to chrl 1:33,872,057. In some of any embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,869,125. In some of any embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,868,742. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for LM02 having the sequence set forth in any one of SEQ ID NOS: 30, 80-82, 94-113, 118, and 119, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for LM02 having the sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 101, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for LM02 having the sequence set forth in SEQ ID NO:224742003140101, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for LM02 having the sequence set forth in SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for LM02 having the sequence set forth in SEQ ID NO:30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0043] In some of any embodiments, one or more transcription factor genes is at least three genes GATA3, GATA2 and LM02.

[0044] In some of any embodiments, at least one of the one or more transcription factor genes is BMI1. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for BMI1 having the sequence set forth in SEQ ID NO: 34, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0045] In some of any embodiments, at least one of the one or more transcription factor genes is CEBPD. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for CEBPD having the sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0046] In some of any embodiments, at least one of the one or more transcription factor genes is LYL1. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for LYL1 having the sequence set forth in SEQ ID NO:35 or SEQ ID NO: 83, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0047] In some of any embodiments, one or more transcription factor genes is at least six genes GATA3, GATA2, LM02, BMI1, CEBPD, and LYL1.

[0048] In some of any embodiments, at least one of the one or more transcription factor genes is HEY2. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for HEY2 having the sequence set forth in SEQ ID NO: 32, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.224742003140

[0049] In some of any embodiments, at least one of the one or more transcription factor genes is ZEB2. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for ZEB2 having the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0050] In some of any embodiments, at least one of the one or more transcription factor genes is TALI. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for TALI having the sequence set forth in SEQ ID NO:84 or SEQ ID NO:86, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0051] In some of any embodiments, at least one of the one or more transcription factor genes is CFOS. In some of any embodiments, the target site for at least one of the one or more transcription factor genes is a target site for CFOS having the sequence set forth in SEQ ID NO:88, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

[0052] In some of any embodiments, the two or more gRNAs comprise a gRNA spacer that is complementary to the target site of the gene. In some of any embodiments, the gRNA is selected from: (a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79, or a contiguous portion thereof of at least 14 nt; (b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77, or a contiguous portion thereof of at least 14 nt; (c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 21, 70-72, 120-139, 144, and 145, or a contiguous portion thereof of at least 14 nt; (d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22, or a contiguous portion thereof of at least 14 nt; (e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23, or a contiguous portion thereof of at least 14 nt; (f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24, or a contiguous portion thereof of at least 14 nt; (g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof224742003140 of at least 14 nt; (h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73, or a contiguous portion thereof of at least 14 nt; (i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76, or a contiguous portion thereof of at least 14 nt; (j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; or (k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78, or a contiguous portion thereof of at least 14 nt. In some of any embodiments, the gRNA is selected from: (a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79, or a contiguous portion thereof of at least 14 nt; (b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77, or a contiguous portion thereof of at least 14 nt; (c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 126 or SEQ ID NO: 127, or a contiguous portion thereof of at least 14 nt; (d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22, or a contiguous portion thereof of at least 14 nt; (e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23, or a contiguous portion thereof of at least 14 nt; (f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24, or a contiguous portion thereof of at least 14 nt; (g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; (h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73, or a contiguous portion thereof of at least 14 nt; (i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76, or a contiguous portion thereof of at least 14 nt; (j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; or (k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78, or a contiguous portion thereof224742003140 of at least 14 nt. In some of any embodiments, the gRNA is selected from:(a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79, or a contiguous portion thereof of at least 14 nt; (b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77, or a contiguous portion thereof of at least 14 nt; (c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 21, SEQ ID NO: 70, SEQ ID NO:71 or SEQ ID NO:72, or a contiguous portion thereof of at least 14 nt; (d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22, or a contiguous portion thereof of at least 14 nt; (e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23, or a contiguous portion thereof of at least 14 nt; (f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24, or a contiguous portion thereof of at least 14 nt; (g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; (h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73, or a contiguous portion thereof of at least 14 nt;(i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76, or a contiguous portion thereof of at least 14 nt; (j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; or (k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78, or a contiguous portion thereof of at least 14 nt. In some of any embodiments, the gRNA is selected from: (a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79; (b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77; (c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 21, 70-72, 120-139, 144, and 145; (d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22; (e) a gRNA targeting a target site for224742003140HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23; (f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24; (g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25; (h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73; (i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76; (j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25; or (k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78. In some of any embodiments, the gRNA is selected from: (a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79; (b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77; (c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 126 or SEQ ID NO: 127; (d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22; (e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23; (f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24; (g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25; (h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73; (i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76; (j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25; or (k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78. In some of any embodiments, the gRNA is selected from: (a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79; (b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO:22474200314077; (c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 21, SEQ ID NO: 70, SEQ ID NO:71 or SEQ ID NO:72; (d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22; (e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23; (f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24; (g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25; (h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73; (i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76; (j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25; or (k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78.

[0053] In some of any embodiments, the gRNA comprises a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length. In some of any embodiments, the gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length. In some of any embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 91. In some of any embodiments, the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 93. In some of any embodiments, the combination of gRNAs comprise 2, 3, 4, 5, 6, 7, 8, or 9 different gRNAs, optionally each comprising the same scaffold sequence. In some of any embodiments, the combination of gRNAs comprise 3, 4, 5, 6, 7, 8, or 9 different gRNAs, optionally each comprising the same scaffold sequence. In some of any embodiments, the gRNA further comprises 2’ MeO modified bases and / or phosphorothiate backbone modifications.

[0054] In some of any embodiments, the one or more transcription factor genes include GATA2, GATA3, LM02 and BMI1. In some of any embodiments, the one or more transcription factor genes include GATA2, GATA3, LM02 and LYLE In some of any embodiments, the one or more transcription factor genes include GATA2, GATA3, LM02 and CEBPD. In some of any embodiments, the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, CEBPD, and LYLE In some of any embodiments, the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, CEBPD, and TALI. In224742003140 some of any embodiments, the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, and LYL1. In some of any embodiments, the one or more transcription factor genes include BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2. In some of any embodiments, the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, CEBPD, LYL1, TALI, HEY2, and CFOS. In some of any embodiments, the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, CEBPD, ZEB2, TALI, HEY2, and CFOS. In some of any embodiments, the one or more transcription factor genes include GATA2, GATA3, LM02, LYL1, CEBPD, ZEB2, TALI, HEY2, and CFOS.

[0055] Also provided herein is a Cas-guide RNA (gRNA) combination comprising: (a) a fusion protein comprising a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof fused to a transcriptional activation domain; and (b) a combination of gRNAs of some of any embodiments. In some embodiments, the Cas protein or variant thereof is a deactivated (dCas) protein. In some embodiments, the dCas protein lacks nuclease activity. In some of any embodiments, the dCas protein is a dCas9 protein. In some embodiments, the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein. In some embodiments, the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 41. In some of any embodiments, the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 42, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any embodiments, the dSaCas9 protein is set forth in SEQ ID NO: 42. In some embodiments, the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein. In some embodiments, the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 43. In some of any embodiments, the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 44, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some of any embodiments, the dSpCas9 protein is set forth in SEQ ID NO: 44.

[0056] Also provided herein is a polynucleotide encoding the DNA-targeting system of some of any embodiments. Also provided herein is a polynucleotide encoding at least one fusion protein of the DNA-targeting system of some of any embodiments. Also provided herein is a polynucleotide encoding at least one DNA-targeting module of the DNA-targeting system of some of any embodiments. Also provided herein is a polynucleotide encoding at least one gRNA224742003140 of the combination of gRNAs of some of any embodiments. Also provided herein is a polynucleotide encoding the combination of gRNAs of some of any embodiments. Also provided herein is a polynucleotide encoding at least one fusion protein of the DNA-targeting system of some of any embodiments and at least one gRNA of the combination of gRNAs of some of any embodiments. Also provided herein is a plurality of polynucleotides, wherein a polynucleotide encodes at least one fusion protein of the DNA-targeting system of some of any embodiments or Cas-gRNA combination of some of any embodiments and a further polynucleotide encodes at least one gRNA of the combination of gRNAs of some of any embodiments. Also provided herein is a polynucleotide encoding the fusion protein and at least one gRNA of the Cas-gRNA combination of some of any embodiments.

[0057] Also provided herein is a vector comprising the polynucleotide of some of any embodiments. Also provided herein is a vector comprising the plurality of polynucleotides of some of any embodiments. In some of any embodiments, the vector is a viral vector. In some of any embodiments, the viral vector is an adeno-associated virus (AAV) vector. In some of any embodiments, the vector is a non-viral vector. In some of any embodiments, the non-viral vector is selected from: a lipid, a nanoparticle, a liposome, an exosome, or a cell penetrating peptide.

[0058] Also provided herein is a pharmaceutical composition comprising the DNA-targeting system of some of any embodiments, the Cas-gRNA combination of some of any embodiments, the polynucleotide of some of any embodiments, the plurality of polynucleotides of some of any embodiments, or the vector of some of any embodiments. In some embodiments, the pharmaceutical composition comprises a pharmaceutically acceptable excipient.

[0059] Also provided herein is a method of differentiating a population of stem cells comprising introducing the DNA-targeting system of some of any embodiments, the Cas-gRNA combination of some of any embodiments, the polynucleotide of some of any embodiments, the plurality of polynucleotides of some of any embodiments, the vector of some embodiments, or a combination thereof, into a population of stem cells, and culturing the stem cells under conditions for their differentiation. Also provided herein is a method of differentiating a population of stem cells comprising introducing the pharmaceutical composition of some of any embodiments into a population of stem cells and culturing the stem cells under conditions for their differentiation. In some embodiments, the population of stem cells are induced pluripotent stem cells (iPSCs). In some of any embodiments, cells of the population of stem cells differentiate into CD34+ cells. In some of any embodiments, cells of the population of stem cells224742003140 differentiate into CD43+ cells. In some of any embodiments, cells of the population of stem cells differentiate into hematopoietic progenitor cells (HPCs).

[0060] In some of any embodiments, cells of the population of stem cells differentiate into CD45+ cells. In some of any embodiments, cells of the population of stem cells differentiate into CD5+CD7+ cells. In some of any embodiments, cells of the population of stem cells differentiate into lymphoid progenitor cells.

[0061] In some of any embodiments, cells of the population of stem cells differentiate into CD34+ / CD43+ cells. In some of any embodiments, cells of the population of stem cells differentiate into CD34+ / CD43+ / CD235a- cells. In some of any embodiments, cells of the population of stem cells differentiate into CD34+, CD43+, and CD45+ cells. In some of any embodiments, cells of the population of stem cells differentiate into lymphoid cells. In some of any embodiments, cells of the population of stem cells differentiate into induced Natural Killer (iNK) cells. In some of any embodiments, cells of the population of stem cells differentiate into CD56+CD3- cells. In some of any embodiments, cells of the population of stem cells differentiate into cells that are characterized by one or more of the following: DNAM1+, NKG2D+, NKP30+ and / or CD16+. In some of any embodiments, cells of the population of stem cells differentiate into induced T (iT) cells. In some of any embodiments, cells of the population of stem cells differentiate into cells that are characterized by one or more of the following: CD45+, CD8A+, CD8B+, CD4+, and / or CD3+.

[0062] In some of any embodiments, the stem cells comprise cells engineered with a recombinant receptor, optionally a chimeric antigen receptor. In some of any embodiments, the differentiated cells comprise cells that express a recombinant receptor, optionally a chimeric antigen receptor.

[0063] Also provided herein is a method for generating hematopoietic progenitor cells (HPCs), the method comprising: (a) introducing any of the provided DNA-targeting system, Cas-gRNA combination, polynucleotide, plurality of polynucleotides, vector, or a combination thereof, into a population of induced pluripotent stem cells (iPSCs); and (b) culturing the iPSCs to differentiate cells of the population of iPSCs into hematopoietic progenitor cells (HPCs) to produce a population of cells comprising HPCs. Also provided herein is a method for generating hematopoietic progenitor cells (HPCs), the method comprising: (a) introducing a pharmaceutical composition as provided herein into a population of induced pluripotent stem cells (iPSCs); and (b) culturing the iPSCs to differentiate cells of the population of iPSCs into hematopoietic224742003140 progenitor cells (HPCs) to produce a population of cells comprising HPCs. In some of any embodiments, the iPSCs comprise cells engineered with a recombinant receptor. In some embodiments, the recombinant receptor is a chimeric antigen receptor. In some of any embodiments, the HPCs are CD34+ cells. In some of any embodiments, the HPCs are CD43+ cells.

[0064] In some of any such embodiments, the population of iPSCs are cultured for between 3 days and 12 days, such as between 4 days and 10 days or 5 days and 8 days. In some embodiments, the population of iPSCs are cultured for 5 days, 6 days, 7 days, or 8 days, or any value between any of the foregoing. In some embodiments, the population of iPSCs are cultured for 5 days. In some embodiments, the population of iPSCs are cultured for 6 days. In some embodiments, the population of iPSCs are cultured for 7 days. In some embodiment, the culture is carried out on a population of iPSCs that have been introduced with the DNA-binding systems (e.g., by transient delivery) and cultured in a media that supports stem cell culture. An exemplary media for supporting stem cell culture includes mTESR™ Plus media (STEMCELL Technologies). In some embodiments, other than the DNA-binding system, the media does not include, and is not supplemented with, any other exogenous extrinsic factor for supporting or promoting differentiation of the iPSCs to HPCs. In some embodiments, the culturing is carried out at a temperature to support growth and differentiation of the cells. In some embodiment, the temperature is or is about 32 °C to 40 °C, such as 35 °C to 39 °C. In some embodiments, the temperature is about 37 °C ± 2 °C.

[0065] Also provided herein is a population of hematopoietic progenitor cells produced by the method of some of any embodiments.

[0066] Also provided herein is a method for generating lymphoid progenitor cells (LPCs), the method comprising culturing the population of HPCs produced by the method of some of any embodiments, or the population of some embodiments under conditions to differentiate cells of the population of HPCs to lymphoid progenitor cells (LPCs) to produce a population comprising LPCs. In some embodiments, the lymphoid progenitor cells are CD45+ cells. In some of any embodiments, the lymphoid progenitor cells are CD5+CD7+ cells. In some of any embodiments, the LPCs express a recombinant receptor, optionally a chimeric antigen receptor.

[0067] Also provided herein is a population of lymphoid progenitor cells produced by the method of some of any embodiments. In some of any such embodiments, the population of HPCs are cultured for between 3 days and 12 days, such as between 4 days and 10 days or 6224742003140 days and 8 days. In some embodiments, the population of HPCs are cultured for 5 days, 6 days, 7 days, or 8 days, or any value between any of the foregoing. In some embodiments, the population of HPCs are cultured for 6 days. In some embodiments, the population of HPCs are cultured for 7 days. In some embodiments, the population of HPCs are cultured for 8 days. In some embodiment, the culturing is carried out in the further presence of one or more exogenous extrinsic factors to support the differentiation of cells of the population of HPCs into lymphoid progenitor cells. Various methods for supporting differentiation of HPCs into lymphoid progenitor cells are known to a skilled artisan, and include any of a variety of methods described herein. In some embodiments, the culturing is carried out at a temperature to support growth and differentiation of the cells. In some embodiment, the temperature is or is about 32 °C to 40 °C, such as 35 °C to 39 °C. In some embodiments, the temperature is about 37 °C ± 2 °C.

[0068] Also provided herein is a method of generating lymphoid cells (LCs), the method comprising culturing the population of LPCs produced by the method of some of any embodiments or the population of LPCs of some embodiments under conditions to differentiate cells of the population of LPCs to lymphoid cells (LCs) to produce a population comprising LCs. Also provided herein is a method of generating lymphoid cells (LCs), the method comprising culturing the population of HPCs produced by the method of some of any embodiments or the population of HPCs of some embodiments under conditions to differentiate cells of the population of HPCs to lymphoid cells (LCs) to produce a population comprising LCs. In some embodiments, the lymphoid cells (LCs) are induced Natural Killer (iNK) cells. In some of any embodiments, the lymphoid cells (LCs) are CD56+CD3- cells. In some of any embodiments, the lymphoid cells (LCs) are characterized by one or more of the following: DNAM1+, NKG2D+, NKP30+ and / or CD16+. In some embodiments, the lymphoid cells (LCs) are induced T (iT) cells. In some of any embodiments, the the LCs are characterized by one or more of the following: CD45+, CD8A+, CD8B+, CD4+, and / or CD3+. In some of any embodiments, the LCs express a recombinant receptor, optionally a chimeric antigen receptor.

[0069] In some of any such embodiments, the population of lymphoid progenitor cells are cultured for between 3 days and 12 days, such as between 4 days and 10 days or 6 days and 8 days. In some embodiments, the population of lymphoid progenitor cells are cultured for 5 days, 6 days, 7 days, or 8 days, or any value between any of the foregoing. In some embodiments, the population of lymphoid progenitor cells are cultured for 6 days. In some embodiments, the population of lymphoid progenitor cells are cultured for 7 days. In some embodiments, the224742003140 population of lymphoid progenitor cells are cultured for 8 days. In some embodiment, the culturing is carried out in the further presence of one or more exogenous extrinsic factors to support the differentiation of cells of the population of lymphoid progenitor cells into lymphoid cells. Various methods for supporting differentiation of lymphoid progenitor cells into lymphoid cells are known to a skilled artisan, and include any of a variety of methods described herein. In some embodiments, the culturing is carried out at a temperature to support growth and differentiation of the cells. In some embodiment, the temperature is or is about 32 °C to 40 °C, such as 35 °C to 39 °C. In some embodiments, the temperature is about 37 °C ± 2 °C. In some embodiments, certain available methods or reagents including commercial kits can be used to differentiate a population of HPCs to lymphoid cells, which then typically includes as part of an intermediate pathway of the process the differentiation to lymphoid progenitor cells. In such embodiments, the culturing is carried out for a duration of about 10 days to 18 days, such as 12 days to 16 days. In some embodiments, the culturing is carried out for 10 days, 11 days, 12 days, 13 days, 14 days, 15 days, 16 days or any value between any of the foregoing.

[0070] In some embodiments, any of the methods of culturing the cells can include steps of replacing the media in the cells. In some embodiments, the media can be supplemented with any necessary exogenous extrinsic factors, as necessary, to support the differentiation of cells as described.

[0071] Also provided herein is a population of lymphoid cells produced by the method of some of any embodiments. In some of any embodiments, the method is carried out in vitro or ex vivo. In some of any embodiments, the stem cells are human stem cells. In some of any embodiments, the introducing is by transient delivery into the population of stem cells. In some embodiments, the transient delivery comprises electroporation, transfection, or transduction. In some of any embodiments, the introducing increases transcription of the one or more transcription factors selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2.

[0072] Also provided herein is a population of differentiated cells produced by the method of some of any embodiments.

[0073] Also provided herein is a method of treating a disease or condition in a subject, the method comprising administering to the subject the population of the population of lymphoid cells of some embodiments or the population of differentiated cells of some embodiments.224742003140Brief Description of the Drawings

[0074] FIG. 1 shows a schematic of differentiation of induced pluripotent stem cells (iPSCs) into effector cells with some of the progenitor cells to be differentiated into along the way.

[0075] FIG. 2A shows the results of an activation screen of gRNAs with a DNA-targeting system for epigenetic transcriptional activation to identify transcription factors that regulate CD34 expression. Statistically significant hits are boxed and / or labeled.

[0076] FIG. 2B shows the results of a repression screen of gRNAs with a DNA-targeting system for epigenetic transcriptional activation to identify transcription factors that regulate CD34 expression. Statistically significant hits are boxed and / or labeled.

[0077] FIG. 3A-3B depict representative flow charts of CD34 expression for a multiplex screen of gRNAs with a DNA-targeting system for epigenetic transcriptional activation (FIG. 3A), along with some of the gRNAs that led to the largest shifts of CD34 expression (FIG. 3B).

[0078] FIG. 4A-4I depict representative flow charts for cells evaluated at 5 days (D5) and 7 days (D7) post electroporation of gRNAs and a DNA-targeting system for epigenetic transcriptional activation (FIG. 4A and 4B respectively), along with graphs showing the combinations of gRNAs tested and the percent positive of live cells for CD34 (FIG. 4C), percent positive of live cells for CD43 (FIG. 4D), percent positive of live cells for both CD34 and CD43 (FIG. 4E), the number of live cells (FIG. 4F), the total number of cells CD34 positive (FIG. 4G), and the number of cells CD43 positive (FIG. 4H). FIG. 41 shows a representative flow chart for CD34 and CD43 surface markers on undifferentiated iPSCs.

[0079] FIG. 5 shows HPC differentiation, as measured by CD34 expression, as compared to number of live HPCs per input iPSCs for HPCs 7 days (D7) post-delivery of a DNA-targeting system and one of six different combinations of multiplexed gRNAs targeting different combination of genes (A-F) by flow cytometry. These combinations were compared to how a DNA-targeting system targeting GATA2, GATA3, and LM02 (labeled “core”) has previously performed.

[0080] FIG. 6A shows the percentage of live cells that are CD34 positive cells for lymphoid progenitor cells derived from one of three engineered iPSC lines (Line 1, 2, or 3) 7 days (D7) post-delivery of a DNA-targeting system and one the following: (a) non-targeting gRNA (NT), (b) core set of gRNAs (Core), or (c) core set of gRNAs and BMI1, CEBPD, and LYL1 -targeting gRNAs (Core+). FIG. 6B shows the number of live cells per well for lymphoid progenitor cells derived from one of three engineered iPSC lines (Line 1, 2, or 3) 7 days (D7) post-delivery of a224742003140DNA-targeting system and one the following: (a) non-targeting gRNA (NT), (b) core set of gRNAs (Core), or (c) core set of gRNAs and BMI1, CEBPD, and LYL1 -targeting gRNAs (Core+). FIG. 6C shows the number of live cells per well for CD34 positive lymphoid progenitor cells derived from one of three engineered iPSC lines (Line 1, 2, or 3) 7 days (D7) post-delivery of a DNA-targeting system and one the following: (a) non-targeting gRNA (NT), (b) core set of gRNAs (Core), or (c) core set of gRNAs and BMI1, CEBPD, and LYL1 -targeting gRNAs (Core+).

[0081] FIG. 7A-7G depict the cell numbers of day 5 (D5) HPCs and day 7 (D7) HPCs incubated in lymphoid progenitor media (FIG. 7A and 7B, respectively), the percent of cells positive for CD45 for D5 HPCs and D7 HPCs incubated in lymphoid progenitor media (FIG. 7C and 7D, respectively) and the percent of CD45 positive cells that are positive for CD5 and CD7 for D5 HPCs and D7 HPCs incubated in lymphoid progenitor media (FIG. 7E and 7F, respectively). FIG. 7G shows a representative flow plot for CD5 and CD7 expression on epiedited HPCs that were differentiated into lymphoid progenitors. All cells were incubated for either 5 or 7 days following delivery of DNA-targeting systems using: gRNAs targeting GATA3, GATA2, and LM02 (core), gRNAs targeting core + LYL1, gRNAs targeting GATA3, GATA2, LM02, ZEB2, HEY2, CEBPD, BMI, LYL1, TALI, and CFOS (all), or non-targeting gRNAs (NT).

[0082] FIG. 8A shows lymphoid progenitor cell (LPC) differentiation, as measured by CD45 expression by flow cytometry after 7 days of hematopoietic progenitor cell (HPC) differentiation followed by 6 days of LPC differentiation (D7+6), as compared to percentage of live CD34 positive cells after 7 days of HPC differentiation (D7) following delivery of a DNA- targeting system and one of six different combinations of multiplexed gRNAs targeting different combination of genes (A-F). These combinations are compared to how a DNA-targeting system targeting GATA2, GATA3, and LM02 (labeled “core”) has previously performed.

[0083] FIG. 8B shows lymphoid progenitor cell (LPC) differentiation, as measured by CD45 expression by flow cytometry after 7 days of hematopoietic progenitor cell (HPC) differentiation followed by 6 days of LPC differentiation (D7+6) per input iPSC, as compared to number of live cells D7+6 following delivery of a DNA-targeting system and one of six different combinations of multiplexed gRNAs targeting different combination of genes (A-F). These combinations were compared to how a DNA-targeting system targeting GATA2, GATA3, and LM02 (labeled “core”) has previously performed.224742003140

[0084] FIG. 9A-9I show the total number of cells for lymphoid progenitor cells derived from day 5 (D5) HPCs and day 7 (D7) HPCs incubated in natural killer (NK) cell differentiation media (FIG. 9A), and shows the percent of live cells that are CD45 positive cells (FIG. 9B), percent of CD45 positive cells that are positive for CD56 and negative for CD3 (FIG. 9C), the number of CD45 and CD56 positive cells that are negative for CD3 (FIG. 9D), the percent of CD56 positive and CD3 negative cells that are DNAM1 positive (FIG. 9E), the percent of CD56 positive and CD3 negative cells that are NKG2D positive (FIG. 9F), the percent of CD56 positive and CD3 negative cells that are NKP30 positive (FIG. 9G), and the percent of CD56 positive and CD3 negative cells that are CD16 positive (FIG. 9H).FIG. 91 shows a representative flow plow of CD56 and CD3 surface expression on epi-edited HPCs that were differentiated into lymphoid progenitors before being differentiated into NK cells.

[0085] FIG. 10A-10K depict functional assays for NK cells differentiated from day 7 (D7) and day 5 (D5) HPCs, including the results of a cell killing assay where NK cells differentiated from D7 HPCs were incubated and stimulated with tumor cells as targets and the target cell number was monitored for a first killing assay (FIG. 10A) before the NK cells were transferred to fresh tumor cells for a second killing assay (FIG. 10B). In addition, concentrations of IFNy, IL-2, and TNFa were measured from the supernatant of the killing assay shown in FIG. 10A (depicted in FIG. 10C, FIG. 10D, and FIG. 10E respectively). Also, NK cells differentiated from epi-edited HPCs were also intracellularly stained for IL-2 and IFNy and the number of cells for each group of gRNAs that were positive for both was measured (FIG. 10F) with representative flow plots for cells that had been differentiated into HPCs using the core gRNAs (FIG. 10G), the core gRNAs plus CEBPD targeting gRNA (FIG. 10H), the core gRNAs plus LYL1 targeting gRNA (FIG. 101), the core gRNAs plus BMI1 targeting gRNA (FIG. 10J), and all gRNAs (FIG. 10K).

[0086] FIG. 10L depicts a functional assay for NK cells differentiated following delivery of dCas9-2xVP64 mRNA and gRNAs that targeted either the core targets (GATA2, LM02, GATA3) or a different set of three targets: GATA2, LM02, and LYL1, along with the following controls: conventional NK cells that were not delivered DNA-targeting systems, mock T cells, CAR T cells, and no treatment (target cells only).

[0087] FIG. 11 shows the LM02 gene region aligned with different informational tracks or annotations labeled (l)-(6). Information tracks or information include: (1) regulatory regions within the LM02 gene, where the transcriptional start site (TSS), enhancers, and DNA loops to224742003140 the TSS are further labeled; (2) peaks corresponding to human hematopoietic stem cell (HSC) RNA-seq signal; (3) peaks corresponding to human ATAC-seq data in HSCs, CD34, CD8, or CD4 cells, as labeled; (4) ChlP-seq data for H3K27me3, H3K4me3, or H3K27ac, as depicted, in human HSCs; (5) DNA loops between H3K27ac (putative enhancers) from H3K27ac HiChlP- seq; and (6) transcriptional factor ChlP-seq data in human HSCs corresponding to the depicted transcriptional factors. Regions of interests used to design a LM02 guide RNA (gRNA) library are also highlighted as shaded boxes on the LM02 region. These seven regions correspond to following genomic coordinates in chromosome 11 (chrl l): (1) chrl 1:33,867,158 to chrl 1:33,873,567; (2) chrl l: 33,876,536 to chrl 1:33,878,097; (3) chrl 1:33,890,358 to chrl 1:33,896,210; (4) chrl l: 33,906,104 to chrl 1:33,908,030; (5) chrl l: 33,911,674 to chrl 1:33,913,494; (6) chrl 1:33,920,461 to chrl 1:33,922,260; and (7) chrl 1:33,929,929 to chrl 1:33,932,651.

[0088] FIG. 12 shows the enrichment or depletion of LM02 guide RNAs (gRNAs) form a LM02 gRNA library screen following a CD34+ cell sort. Each dot corresponds to a tested LM02 gRNA from the library. Labeled gRNAs correspond to gRNAs that were significantly enriched (adjusted p-value, or padj, <0.01).

[0089] FIG. 13 shows the enriched gRNAs from the CD34+ cell sort from FIG. 13 aligned to the transcriptional start site (TSS) of the LM02 gene region and different informational tracks and annotations as depicted. Each labeled box represents a given gRNA.

[0090] FIG. 14 and FIG. 15 show the percentage of CD34+CD43+ cells (FIG. 14) or percentage of CD235a- cells within a CD34+CD43+ cell population (FIG. 15) for iPSCs following hematopoietic progenitor cell (HPC) differentiation for 7 days after delivery of a DNA-delivery system that included a dSpCas9-2xVP64 (SEQ ID NO: 40) and gRNAs targeting Core+ (GATA3, GATA2, BMI1, CEBPD, LYL1, and LM02) where the LMO2-targeting gRNA was either previously used reference gRNA or one of the depicted gRNAs upregulated in the LM02 gRNA library screen.

[0091] FIG. 16A and FIG. 16B show the percentage of CD5+CD7+ cells (FIG. 16A) or percentage of CD45+ cells (FIG. 16B) for iPSCs following hematopoietic progenitor cell (HPC) differentiation for 7 days then lymphoid progenitor cell (LPC) differentiation for an additional 6 days after delivery of a DNA-delivery system that included a dSpCas9-2xVP64 (SEQ ID NO: 40) and gRNAs targeting Core+ (GATA3, GATA2, BMI1, CEBPD, LYL1, and LM02) where224742003140 the LM02-targeting gRNA was either previously used reference gRNA or one of the depicted gRNAs upregulated in the LM02 gRNA library screen.

[0092] FIG. 17A and FIG. 17B show the percentage of CD45+ cells (FIG. 17A) or percentage of CD56+CD3- cells (FIG. 17B) for iPSCs following hematopoietic progenitor cell (HPC) differentiation for 7 days, lymphoid progenitor cell (LPC) differentiation for 6 more days, and lymphocytic differentiation into induced Natural Killer (iNK) cells for 7 additional days after delivery of a DNA-delivery system that included a dSpCas9-2xVP64 (SEQ ID NO: 40) and gRNAs targeting Core+ (GATA3, GATA2, BMI1, CEBPD, LYL1, and LM02) where the LMO2-targeting gRNA was either previously used reference gRNA or one of the depicted gRNAs upregulated in the LM02 gRNA library screen.

[0093] FIG. 18A-18D show the cell viability, as measured by fold change in live cells, for hematopoietic progenitor cells (HPCs) (FIG. 18A), lymphoid progenitor cells (LPCs) (FIG. 18B), induced Natural Killer cells (iNK) (FIG. 18C), and all cells (overall) (FIG. 18D) following delivery of a DNA-delivery system into iPSCs that included a dSpCas9-2xVP64 (SEQ ID NO: 40) and gRNAs targeting Core+ (GATA3, GATA2, BMI1, CEBPD, LYL1, and LM02) where the LMO2-targeting gRNA was either previously used reference gRNA or one of the depicted gRNAs upregulated in the LM02 gRNA library screen. As controls, the use of a nontargeting gRNA (NT) or removing any LMO2-targeting gRNA (No LM02) was also used.

[0094] FIG. 19 shows the LM02 expression in iPSCs, as normalized to GAPDH, 4 days after delivery of a DNA-delivery system that included a dSpCas9-2xVP64 (SEQ ID NO: 40) and gRNAs targeting Core+ (GATA3, GATA2, BMI1, CEBPD, LYL1, and LM02) where the LMO2-targeting gRNA was either previously used reference gRNA (Reference) or one of the depicted gRNAs upregulated in the LM02 gRNA library screen. As controls, the use of a nontargeting gRNA (NT), removal of a LMO2-targeting gRNA (No LM02), or no delivery of DNA-delivery systems (Cells Only) was also used.

[0095] FIG. 20 shows the percent expression of CD45+ T cells that were further positive for the indicated markers on the X axis after either 20 days (left) or 27 days (right) of T cell differentiation according to the Kaneko protocol. “TCRab+” refers to cells that were positive for expressing a T-cell receptor.Detailed Description224742003140

[0096] Provided herein, in some embodiments, are epigenetic-modifying DNA-targeting systems. In some embodiments, the DNA-targeting systems comprise one or more DNA- targeting modules, each composed of a fusion portion comprising: (a) a DNA-binding domain that binds to a target site for one or more transcription factor genes, and (b) at least one transcriptional activation domain that increases transcription of the one or more transcription factor genes. In some embodiments, the one or more transcription factor genes are genes that lead to differentiation of stem cells into hematopoietic progenitor cells (HPCs). In some embodiments, the increased transcription of the one or more transcription factor genes lead to differentiation of stem cells into HPCs. In some embodiments, the one or more transcription factor genes are genes that lead to upregulation of CD34, CD43, and / or CD45. In some embodiments, the one or more transcription factor genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, ZEB2, RUNX3, HOXB9, SIRT1, FOXQ1, GFI1, GFI1B, SPI1, and CFOS. In some embodiments, the one or more HPC differentiation genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, and ZEB2. In some embodiments, the one or more HPC differentiation genes comprise GATA2, GATA3, and LM02. In some embodiments, the one or more HPC differentiation genes comprise GATA2, GATA3, LMO2, and one or more additional HPC differentiation genes. In some embodiments, the one or more HPC differentiation genes comprise GATA2, GATA3, LMO2, BMI1, CEBPD, and LYLE In some embodiments, the target site for a gene may be within a regulatory region for the gene, such as a promotor or enhancer of the gene.

[0097] In some embodiments, the DNA-targeting systems are synthetic transcription factors that can increase (or upregulate) transcription of a gene in a targeted manner. In some embodiments, the DNA-binding domain of the DNA-targeting system is a nuclease-inactive Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein (e.g., a dCas protein) or variant thereof complexed with a guide RNA (gRNA). Also provided are gRNAs for targeting to a target site in one or more transcription factor genes or a regulatory DNA elements for the one or more transcription factor genes. Also provided are CRISPR- Cas / gRNA combinations thereof composed of the gRNA and a nuclease inactivated Cas, such as a dCas9. Also provided herein are polynucleotides encoding the DNA-targeting system or the fusion protein of the DNA-targeting system, and vectors and cells containing the same. Also provided herein are methods of using the DNA-targeting system for activating transcription of224742003140 transcription factor genes in stem cells and promoting HPC differentiation.

[0098] In some embodiments, the DNA-targeting system includes a single DNA-targeting module for targeting activation or increased expression of a single gene. In some embodiments, the gene is selected from the group genes consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, ZEB2, RUNX3, HOXB9, SIRT1, FOXQ1, GFI1, GFI1B, SPI1, and CFOS. In some embodiments, the DNA-targeting system includes a plurality of DNA- targeting modules, in which each DNA-targeting module is for targeting activation or increased expression of a different gene. In some embodiments, the DNA-targeting systems are multiplexed DNA-targeting systems, i.e. targeted to target sites for more than one gene. Hence, the terms DNA-targeting system may include a multiplexed DNA targeting system that includes more than one DNA-targeting module. A multiplexed DNA targeting system comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 11, or at least 12 DNA-targeting modules. In some embodiments, the plurality of DNA-targeting modules target activation of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, or 17 genes, selected from BMI1, CEBPD, GATA2, GATA3, HEY2, LMO2, LYL1, TALI, ZEB2, RUNX3, HOXB9, SIRT1, FOXQ1, GFI1, GFI1B, SPI1, and CFOS.

[0099] In some embodiments, any two DNA-targeting modules of a DNA-targeting system comprise separate (i.e. non-overlapping) components. In some embodiments, different DNA- targeting modules of a DNA-targeting system comprise separate (i.e. non-overlapping) components. For example, a DNA-targeting system may comprise a first DNA-targeting module comprising a first fusion protein comprising a DNA-binding domain (e.g. a ZFN or TALE-based DNA-binding domain) that targets a first target site, and a second DNA-targeting module comprising a second fusion protein comprising a second DNA-binding domain (e.g. a ZFN or TALE-based DNA-binding domain) that targets a second target site.

[0100] In some embodiments, any two DNA-targeting modules of a DNA-targeting system may comprise shared (i.e. overlapping) components. In some embodiments, different DNA- targeting modules of a DNA-targeting system comprise shared (i.e. overlapping) components. For example, in one aspect, a DNA-targeting system may comprise a first DNA-targeting module comprising (a) a fusion protein comprising a Cas protein and a transcriptional effector (e.g. activation) domain, and (b) a first gRNA that complexes with the Cas protein and targets a first target site, and a second DNA-targeting module comprising (a) the fusion protein of the first DNA-targeting module, and (b) a second gRNA that complexes with the Cas protein and224742003140 targets a second target site. It will be understood that providing two or more different gRNAs for a given Cas protein allows the same Cas protein to be targeted to the target sites of the two or more gRNAs. Conversely, different Cas protein variants (e.g. SpCas9 and SaCas9) are compatible with different gRNA scaffold sequences and PAMs. Thus, it is possible to engineer a single DNA-targeting system comprising multiple non-overlapping CRISPR / Cas-based DNA- targeting modules.

[0101] The provided embodiments relate to compositions and methods for promoting lymphoid cell differentiation through transcriptional activation of one or more transcription factor genes. In some embodiments, the methods can be used in connection with adoptive cell therapies. In some embodiments, activating transcription of the one or more transcription factor genes modulates the expression of cell-surface makers. In some embodiments, expression of a cell-surface marker is increased, such as CD34, CD43, and / or CD45 which are characteristic of HPCs. In some embodiments, activation of the one or more transcription factor genes promotes stem cell differentiation into HPCs without the provision of extrinsic signaling ligands.

[0102] The ability to differentiate HPCs from stem cells has tremendous therapeutic potential, including in regenerative medicine and adoptive cell therapy. Current protocols for differentiating stem cells into effector cell types and lineages, such as lymphoid cells, include differentiating stem cells into HPCs as an intermediate step, thus for differentiating stem cells into effector cells to for therapeutic purposes, stem cell first need to be differentiated into HPCs. Differentiating the stem cells into HPCs require intricate culturing conditions, such as frequent media changes, the use of co-culture with feeder cells, specialized culture vessels, and additives in the media, such as cytokines. Additionally, the differentiation of stem cells to HPCs is highly sensitive to setup parameters (e.g. input cell number) and is therefore not reproducible or robust. Further, differentiation from HPCs into effector cells is highly dependent on set up parameters, such as the quality of the HPCs, meaning that extensive optimization is needed after producing HPCs for the differentiation of the HPCs into effector cells. The simplified process of some of the provided embodiments for differentiation stem cells into HPCs will decrease the need for optimization for further differentiating the HPCs into effector cells and streamline the production of HPCs.

[0103] In some aspects, the provided embodiments relate to identification of genomic locations or genes, that when activated, drive the differentiation of stem cells into HPCs. In some aspects, the differentiation of stem cells into HPCs are identified by cell surface markers,224742003140 which may consist of increased expression of CD34, CD43, and / or CD45. In some embodiments, the identification of genomic locations or genes, that when activated, drive the differentiation of stem cells into HPCs may be derived from screens that examine activation of individual genes or multiplex and examine activation of multiple genes at a time to determine which genes, when activated alone or in combinations, lead to increased HPC differentiation from stem cells.

[0104] The provided embodiments can be used to simplify the differentiation of HPCs from stem cells and to perform the differentiation quicker. The provided embodiments simplify the differentiation process in terms of reagents and labor. In addition, the provided embodiments can simplify differentiation process of stem cells into HPCs, improving scale-up to 3D cultures.

[0105] All publications, including patent documents, scientific articles and databases, referred to in this application are incorporated by reference in their entirety for all purposes to the same extent as if each individual publication were individually incorporated by reference. If a definition set forth herein is contrary to or otherwise inconsistent with a definition set forth in the patents, applications, published applications and other publications that are herein incorporated by reference, the definition set forth herein prevails over the definition that is incorporated herein by reference.

[0106] The section headings used herein are for organizational purposes only and are not to be construed as limiting the subject matter described.I. DNA-TARGETING SYSTEMS

[0107] In some embodiments, provided are DNA-targeting systems capable of specifically targeting a target site for one or more transcription factor genes, and activating transcription of the one or more transcription factor genes. In some embodiments, the target site for a transcription factor gene is a target site in the gene or a regulatory DNA element thereof. In some embodiments, targeting a transcription factor gene includes targeting a site within the gene or a site within a regulatory element for the gene. In provided embodiments, for each transcription factor gene that is targeted, the DNA-targeting system includes a fusion protein that comprises a DNA-binding domain that binds to the target site for the gene, and at least one effector domain for activating transcription of the gene. In some embodiments, the provided DNA-targeting systems increase transcription of the one or more transcription factor genes in a stem cell. In some embodiments, transcriptional activation of gene expression by the DNA- targeting systems provided herein can promote hematopoietic progenitor cell (HPC)224742003140 differentiation. In some embodiments, transcriptional activation of target genes by the DNA- targeting systems provided herein can promote expression of CD34, CD43, and / or CD45.

[0108] In some embodiments, the at least one effector domain is a transcriptional activation domain for increasing transcription of the one or more transcription factor genes (e.g. activates or increases transcription of the one or more genes as compared to transcription of the gene in the absence of the DNA-targeting system), such as any effector domain for transcriptional activation described in Section I.E. In some embodiments, the effector domain is a transcriptional activator effector domain, and the one or more genes transcription factor genes are genes that when activated lead to increased expression of CD34, CD43, and / or CD45 in the cell. In some embodiments, the effector domain is a transcriptional activator effector domain, and the one or more transcription factor genes are selected from the group consisting of: BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, ZEB2, RUNX3, HOXB9, SIRT1, FOXQ1, GFI1, GFI1B, SPI1, and CFOS.

[0109] In some embodiments, the effector domain directly or indirectly leads to increased transcription of the gene. In some embodiments, the effector domain induces, catalyzes or leads to transcription activation. In some embodiments, the effector domain induces transcription activation. In some aspects, the effector domain comprises: a VP64 domain, a p65 activation domain, a p300 domain, an Rta domain, a CBP domain, a VPR domain, a VPH domain, an HSF1 domain, a TET protein domain, optionally wherein the TET protein is TET1, a SunTag domain, or a domain, portion, variant, or truncation of any of the foregoing. In some embodiments, the effector domain is VP64.

[0110] In some embodiments, the DNA-targeting system includes a fusion protein comprising (a) at least one DNA-binding domain capable of being targeted to the target site; and (b) at least one effector domain capable of modulating transcription of the gene. In some embodiments, the at least one effector domain is a transcriptional activation domain. The fusion protein can be any suitable fusion protein, for example as described in Section I.F.

[0111] In some embodiments, the DNA-binding domain comprises or is derived from a CRISPR associated (Cas) protein, a zinc finger protein (ZFP), a transcription activator-like effector (TALE), meganuclease, homing endonuclease, I-Scel enzyme, or variants thereof. In some embodiments, the DNA-binding domain comprises a catalytically inactive (e.g. nucleaseinactive or nuclease-inactivated) variant of any of the foregoing. In some embodiments, the DNA-binding domain comprises a deactivated Cas9 (dCas9) protein or variant thereof that is a224742003140 catalytically inactivated so that it is inactive for nuclease activity and is not able to cleave the DNA. The DNA-binding domain can be any suitable DNA-binding domain, for example as described in Sections I.C and I.D.

[0112] In some embodiments, the DNA-binding domain comprises or is derived from a Cas protein or variant thereof, such as a nuclease-inactive Cas or dCas (e.g. dCas9, and the DNA- targeting system comprises one or more guide RNAs (gRNAs), such as a combination of gRNAs (e.g. two gRNAs or three gRNAs). For example, DNA-targeting systems provided herein comprise two, three, four, give, or six gRNAs. In some embodiments, the gRNA comprises a spacer sequence that is capable of targeting and / or hybridizing to the target site. In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof. In some aspects, the gRNA directs or recruits the Cas protein or variant thereof to the target site. The gRNA can be any suitable gRNA, for example any gRNA described in section I.C.2.

[0113] In some embodiments, the DNA-targeting system is for increasing transcription of one or more genes, such as any described in Section I.B. In some embodiments, the fusion protein is any as described herein, for example in Section I.F., In some embodiments, the fusion protein of a DNA-targeting module thereof is a dCas9-VP64 fusion protein, such as a dCas9- 2xVP64 fusion protein.

[0114] Exemplary components and features of the DNA-targeting systems are provided below in the following subsections.A. DNA-Targeting Modules and Multiplexed DNA-Targeting Systems

[0115] In some embodiments, the DNA-targeting system contains at one or more DNA- targeting modules, where each DNA-targeting module of the system is a component of the DNA-targeting system that is independently capable of targeting one target site for a target gene. In some embodiments, each DNA-targeting module includes (a) a DNA-binding domain capable of being targeted to the target site, and (b) an effector domain for modulating transcription of the gene. In some embodiments, the DNA-targeting system comprises a single DNA-targeting module for targeted transcriptional modulation of a single gene.

[0116] In some embodiments, a DNA-targeting module is a CRISPR / Cas-based DNA- targeting module. In some embodiments, in a CRISPR / Cas-based DNA-targeting module, the DNA-binding domain of the fusion protein is a Cas protein or variant thereof (e.g., a dCas protein, such as dCas9) and the DNA-targeting module further comprises a gRNA for targeting the DNA-binding domain to the target site.224742003140

[0117] In some embodiments, a DNA-targeting module is a zinc finger protein (ZFP)-based DNA-targeting module. In some embodiments, in a ZFP-based DNA-targeting module, the DNA-binding domain of the fusion protein is an engineered zinc finger protein (eZFP).

[0118] In some embodiments, a DNA-targeting module is a transcription activator-like effector (TALE)-based DNA-targeting module. In some embodiments, in a TALE-based DNA- targeting module, the DNA-binding domain of the fusion protein is an engineered TALE.

[0119] In some embodiments, the DNA-targeting system includes a plurality of DNA- targeting modules, in which each DNA-targeting module targets a different target site. In some embodiments, one or more target sites are for different genes. In some embodiments, one or more target sites are for the same gene. In some embodiments, the DNA-targeting system is a multiplexed DNA-targeting system, i.e. is targeted to target sites for more than one gene. Hence, the term DNA-targeting system may include a multiplexed epigenetic-modifying DNA targeting system that includes more than one DNA-targeting module. In some embodiments, a multiplexed epigenetic-modifying DNA targeting system comprises at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, at least 10, at least 20, at least 30, or more DNA-targeting modules. In some embodiments, a multiplexed epigenetic-modifying DNA- targeting system comprises 2 DNA-targeting modules. In some embodiments, a multiplexed epigenetic-modifying DNA-targeting system comprises 3 DNA-targeting modules. In some embodiments, a multiplexed epigenetic -modifying DNA-targeting system comprises 4 DNA- targeting modules. In some embodiments, a multiplexed epigenetic-modifying DNA-targeting system comprises 5 DNA-targeting modules. In some embodiments, a multiplexed epigenetic- modifying DNA-targeting system comprises 6 DNA-targeting modules. In some embodiments, a multiplexed epigenetic-modifying DNA-targeting system comprises 7 DNA-targeting modules. In some embodiments, a multiplexed epigenetic-modifying DNA-targeting system comprises 8 DNA-targeting modules. In some embodiments, a multiplexed epigenetic-modifying DNA- targeting system comprises 9 DNA-targeting modules. In some embodiments, a multiplexed epigenetic-modifying DNA-targeting system comprises 10 DNA-targeting modules.

[0120] In some embodiments, any two DNA-targeting modules of a DNA-targeting system can comprise separate (i.e. non-overlapping) components. For example, a DNA-targeting system may comprise a first DNA-targeting module comprising a first fusion protein with a DNA- binding domain (e.g. a ZFN or TALE-based DNA-binding domain) that targets a first target site, and a second DNA-targeting module comprising a second fusion protein with a second DNA-224742003140 binding domain (e.g. a ZFN or TALE-based DNA-binding domain) that targets a second target site.

[0121] In some embodiments, any two DNA-targeting modules of a DNA-targeting system can comprise shared (i.e. overlapping) components. For example, a DNA-targeting system may comprise: i) a first DNA-targeting module comprising (a) a fusion protein comprising a Cas protein and an effector domain, and (b) a first gRNA that complexes with the Cas protein and targets a first target site, and ii) a second DNA-targeting module comprising (a) the fusion protein of the first DNA-targeting module, and (b) a second gRNA that complexes with the Cas protein and targets a second target site. It will be understood that providing two or more different gRNAs for a given Cas protein allows the Cas protein to be targeted to the target sites of the two or more gRNAs. Conversely, different Cas protein variants (e.g. SpCas9 and SaCas9) are compatible with different gRNA scaffold sequences and PAMs, as described herein. Thus, it is possible to engineer a single DNA-targeting system comprising multiple non-overlapping CRISPR / Cas-based DNA-targeting modules.

[0122] In some aspects, provided herein is an epigenetic-modifying DNA-targeting system comprising a plurality of DNA-targeting modules for modulating transcription of one or more genes. In some embodiments, the plurality of DNA-targeting modules comprises a first DNA- targeting module for modulating transcription of a first gene of the one or more genes, and a second DNA-targeting module for modulating transcription of a second gene of the one or more genes. In some embodiments, each DNA-targeting module comprises a fusion protein comprising: (a) a DNA-binding domain for targeting a target site of the target gene for the DNA-targeting module, and (b) at least one effector domain. In some embodiments, each DNA- targeting module comprises a transcriptional activator effector domain for increasing transcription of the one or more genes.B. Target Genes and Target Sites for Promoting Hematopoietic Progenitor Cell (HPC) Differentiation

[0123] In some aspects, provided herein are one or more target sites for one or more transcription factor genes for promoting differentiation of stem cells to HPCs in which modulation of the one or more transcription factor genes promotes hematopoietic progenitor cell differentiation. In some embodiments, the target site is targeted using any of the provided DNA- targeting systems.

[0124] In some embodiments, the one or more target sites are for one or more transcription224742003140 factor genes in which increased expression of the transcription factor genes promotes differentiation into hematopoietic progenitor cell. In some embodiments, the one or more target sites is for one or more transcription factor genes in which increased expression of the genes promote CD34, CD43, and / or CD45 expression. In some embodiments, the one or more target sites is for one or more transcription factor genes selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, ZEB2, RUNX3, H0XB9, SIRT1, FOXQ1, GFI1, GFI1B, SPI1, and CFOS. In some embodiments, the one or more target sites is for one or more transcription factor genes selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, and ZEB2. In some embodiments, the one or more target sites is for one or more transcription factor genes selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, LM02, and LYLE In some embodiments, the one or more target sites is for one or more transcription factor genes including GATA2, GATA3, and LM02. In some embodiments, the one or more target sites is for one or more transcription factor genes including GATA2, GATA3, LM02, and one or more additional transcription factor genes.

[0125] In some embodiments, the target site is targeted by a DNA-targeting system, such as by a DNA-targeting module of the DNA-targeting system, such as any described herein. In some embodiments, the target site for a gene is in the gene or a regulatory DNA element thereof. In some embodiments, provided herein are multiplexed DNA-targeting systems that target a combination of at one or more transcription factor genes or regulatory DNA elements thereof described herein.

[0126] In some embodiments, the DNA-targeting system targets to or binds to a target site in a gene or a regulatory element thereof, such as any described herein. In some embodiments, the target site is located in a regulatory DNA element of a transcription factor gene such as any described. In some embodiments, a regulatory DNA element is a sequence to which a gene regulatory protein may bind and affect transcription of the gene. In some embodiments, the regulatory DNA element is a cis, trans, distal, proximal, upstream, or downstream regulatory DNA element of a gene. In some embodiments, the regulatory DNA element is a promoter or enhancer of the gene. In some embodiments, the target site is located within a promoter, enhancer, exon, intron, untranslated region (UTR), 5’ UTR, or 3’ UTR of the gene. In some embodiments, the regulatory DNA element is a promoter. In some embodiments, a promoter is a nucleotide sequence to which RNA polymerase binds to begin transcription of the gene. In some embodiments, a promoter is a nucleotide sequence located within about 25bp, 50bp, 75bp,224742003140 lOObp, about 5OObp, about lOOObp, or more, of a transcriptional start site of the gene. In some embodiments, a promoter is within 5OObp of a transcriptional start site of the gene. In some embodiments the target site is located within a sequence of unknown or known function that is suspected of being able to control expression of a gene.

[0127] In some embodiments, the DNA-targeting system targets to or binds within about 25bp, within about 50bp, within about 75bp, within about lOObp, within about 250bp, within about 500bp, within about, 750bps, or within about lOOObp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds within about 25bp, within about 50bp, within about 75bp, within about lOObp, within about 250bp, within about 500bp, within about 750bps, or within about lOOObp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA- targeting system targets to or binds about 25bp- lOOObp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds within about 25bp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds within about 50bp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA- targeting system targets to or binds within about 75bp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds within about lOObp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds within about 250bp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds within about 500bp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds within about 750bp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds within about lOOObp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the target site is within the promoter of the transcription factor gene.

[0128] In some embodiments, the DNA-targeting system targets to or binds within about 20bp, within about 50bp, within about lOObp, within about 200bp, within about 300bp, within about 400bp, within about 500bp, or within about 600bps upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds about 20bp-600bp upstream of the transcriptional start site of the transcription factor224742003140 gene. In some embodiments, the DNA-targeting system targets to or binds within about 20bp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds within about 50bp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds within about lOObp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds within about 200bp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds within about 300bp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA- targeting system targets to or binds within about 400bp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds within about 500bp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds within about 600bp upstream of the transcriptional start site of the transcription factor gene. In some embodiments, the DNA-targeting system targets to or binds within about lOOObp upstream of the transcriptional start site of the transcription factor gene.

[0129] In some embodiments, delivery of the DNA-targeting system increases transcription of one or more transcription factor genes leading to increased expression of CD34, CD43, and / or CD45 on the target cell. In some embodiments, delivery of the DNA-targeting system increases transcription of one or more transcription factor genes selected from the group consisting of: BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, ZEB2, RUNX3, HOXB9, SIRT1, FOXQ1, GFI1, GFI1B, SPI1, and CFOS. In some embodiments, delivery of the DNA- targeting system increases transcription of one or more transcription factor genes selected from the group consisting of: BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2. In some embodiments, delivery of the DNA-targeting system increases transcription of one or more transcription factor genes selected from the group consisting of: BMI1, CEBPD, GATA2, GATA3, LM02, or LYLE In some embodiments, delivery of the DNA-targeting system increases transcription of transcription factor genes GATA2, GATA3, and LM02. In some embodiments, delivery of the DNA-targeting system increases transcription of transcription factor genes GATA2, GATA3, LM02, and one or more transcription factor genes selected from the group consisting of: BMI1, CEBPD, HEY2, LYL1, TALI, ZEB2, RUNX3, HOXB9, SIRT1, FOXQ1, GFI1, GFI1B, SPI1, and CFOS. In some embodiments, provided224742003140 herein are target sites for one or more genes for which increased transcription promotes hematopoietic progenitor cell differentiation. In some embodiments, delivery of the DNA- targeting system increases transcription of transcription factor genes GATA2, GATA3, LM02, and one or more transcription factor genes selected from the group consisting of: BMI1, CEBPD, and LYL1. In some embodiments, delivery of the DNA-targeting system increases transcription of transcription factor genes GATA2, GATA3, LM02, and one or more transcription factor genes selected from the group consisting of: BMI1, CEBPD, HEY2, LYL1, TALI, and ZEB2. In some embodiments, delivery of the DNA-targeting system increases transcription of transcription factor genes GATA2, GATA3, LM02, BMI1, CEBPD, HEY2, LYL1, TALI, and ZEB2. In some embodiments, delivery of the DNA-targeting system increases transcription of transcription factor genes GATA2, GATA3, LM02, and one or more transcription factor genes selected from the group consisting of: BMI1, CEBPD, HEY2, LYL1, TALI, ZEB2, and CFOS. In some embodiments, delivery of the DNA-targeting system increases transcription of transcription factor genes GATA2, GATA3, LM02, BMI1, CEBPD, HEY2, LYL1, TALI, ZEB2, and CFOS. In some embodiments, delivery of the DNA-targeting system increases transcription of transcription factor genes in any combination listed in Table 1A and Table IB. In some embodiments, provided herein are target sites for one or more genes for which increased transcription promotes hematopoietic progenitor cell differentiation.

[0130] In some embodiments, the DNA-targeting system targets a target site. In some embodiments, the DNA-targeting system targets a target site for one or more transcription factor genes, wherein the one or more transcription factor genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, ZEB2, RUNX3, HOXB9, SIRT1, FOXQ1, GFI1, GFI1B, SPI1, and CFOS. In some embodiments, the DNA-targeting system targets a target site for one or more transcription factor genes, wherein the one or more transcription factor genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, and ZEB2. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, and ZEB2. In some embodiments, the DNA- targeting system targets a target site for 2 or more of the following transcription factor genes; BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, and ZEB2. In some embodiments, the DNA-targeting system targets a target site for 3 or more of the following transcription factor genes; BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, and224742003140ZEB2. In some embodiments, the DNA-targeting system targets a target site for 4 or more of the following transcription factor genes; BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, and ZEB2. In some embodiments, the DNA-targeting system targets a target site for 5 or more of the following transcription factor genes; BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, and ZEB2. In some embodiments, the DNA-targeting system targets a target site for 6 or more of the following transcription factor genes; BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, and ZEB2. In some embodiments, the DNA-targeting system targets a target site for 7 or more of the following transcription factor genes; BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, and ZEB2. In some embodiments, the DNA-targeting system targets a target site for 8 or more of the following transcription factor genes; BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, and ZEB2. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, and ZEB2.

[0131] In some embodiments, the DNA-targeting system targets a target site for 2 or more of the following transcription factor genes; BMI1, CEBPD, GATA2, GATA3, LM02, and LYL1. In some embodiments, the DNA-targeting system targets a target site for 3 or more of the following transcription factor genes; BMI1, CEBPD, GATA2, GATA3, LM02, and LYL1. In some embodiments, the DNA-targeting system targets a target site for 4 or more of the following transcription factor genes; BMI1, CEBPD, GATA2, GATA3, LM02, and LYL1. In some embodiments, the DNA-targeting system targets a target site for 5 or more of the following transcription factor genes; BMI1, CEBPD, GATA2, GATA3, LM02, and LYL1. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; BMI1, CEBPD, GATA2, GATA3, LM02, and LYL1.

[0132] In some embodiments, the DNA-targeting system targets a target site for one or more of the following transcription factor genes; GATA2, GATA3, and LM02, along with one or more transcription factor genes selected from the group consisting of BMI1, CEBPD, and LYL1. In some embodiments, the DNA-targeting system targets a target site for 2 or more of the following transcription factor genes; GATA2, GATA3, and LM02, along with one or more transcription factor genes selected from the group consisting of BMI1, CEBPD, and LYL1. In some embodiments, the DNA-targeting system targets a target site for 2 or more of the following transcription factor genes; GATA2, GATA3, and LM02, along with 2 or more224742003140 transcription factor genes selected from the group consisting of BMI1, CEBPD, and LYLl. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, and LM02, along with 2 or more transcription factor genes selected from the group consisting of BMI1, CEBPD, and LYLl. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, LM02, BMI1, CEBPD, and LYLl. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, LM02, and LYLl. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, LM02, and BMI1. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, LM02, and CEBPD.

[0133] In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, and LM02. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, and LM02, along with one or more transcription factor genes selected from the group consisting of BMI1, CEBPD, HEY2, LYLl, TALI, ZEB2, and CEOS. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, LM02, BMI1, CEBPD, LYLl, TALI, HEY2, and CEOS. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, LM02, BMI1, CEBPD, ZEB2, TALI, HEY2, and CEOS. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, LM02, LYLl, CEBPD, ZEB2, TALI, HEY2, and CEOS. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, and LM02, along with one or more transcription factor genes selected from the group consisting of BMI1, CEBPD, HEY2, LYLl, TALI, and ZEB2. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, and LM02, along with one or more transcription factor genes selected from the group consisting of BMI1, CEBPD, and TALI. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, LM02, BMI1, CEBPD, and TALI. In some embodiments, the DNA-targeting system targets a target site for224742003140 each of the following transcription factor genes; GATA2, GATA3, LM02, BMI1, and TALI. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, and LM02, along with one or more transcription factor genes selected from the group consisting of BMI1, CEBPD, and LYL1. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; GATA2, GATA3, BMI1, CEBPD, and LYL1. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; BMI1, CEBPD, and LYL1. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; BMI1 and LYL1. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; BMI1 and CEBPD. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; CEBPD and LYL1. In some embodiments, the DNA-targeting system targets a target site for CEBPD. In some embodiments, the DNA-targeting system targets a target site for LYL1. In some embodiments, the DNA-targeting system targets a target site for BMI1. In some embodiments, the DNA- targeting system targets a target site for a combination of target sites for any of the combination of genes listed in Table 1A or Table IB.

[0134] In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; LM02, GATA2, GATA3, BMI1, CEBPD, and LYL1. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; LM02, GATA3, BMI1, CEBPD, and LYL1. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; LM02, GATA2, BMI1, CEBPD, and LYL1. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; LM02, GATA2, GATA3, CEBPD, and LYL1. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; LM02, GATA2, GATA3, BMI1, and LYL1. In some embodiments, the DNA-targeting system targets a target site for each of the following transcription factor genes; LM02, GATA2, GATA3, BMI1, and CEBPD.Table 1A. Combinations of genes targeted by DNA-targeting system for increasing transcription of target genes224742003140224742003140224742003140Table 2A. Further combinations of genes targeted by DNA-targeting system for increasing transcription of target genes

[0135] In some embodiments, the DNA-targeting system targets a target site for one or more genes selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI, ZEB2, RUNX3, HOXB9, SIRT1, FOXQ1, GFI1, GFI1B, SPI1, and CFOS. In some embodiments, the target site comprises a sequence selected from any one of SEQ ID NOs: 28-35, 80-89, 94-113, 118, 119, and 252-256 or a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs: 28-35, 80-89, 94-113, 118, 119, and 252-256 that is 15, 16, 17, 18 or 19 nucleotides in length, or a complementary sequence of any of the foregoing. In some embodiments, the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence described herein above. In some embodiments, the target site is a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to all or a contiguous portion of a target site sequence as set forth in any one of SEQ ID NOs: 28-35, 80-89, 94-113, 118, 119, and 252-256. In some embodiments, the target site is the sequence set forth in any one of SEQ ID NOs:28-35 and 252-256. In some embodiments, the target site is the sequence set forth in any one of SEQ ID NOs:80-89. In some embodiments, the target site is the sequence set forth in any one of SEQ ID NOs:94-l 13, 118, and 119.

[0136] In some embodiments, the DNA-targeting system targets a target site for GATA3. In some embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOs:22474200314028, 89, and 252-256, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of the sequence set forth in any one of SEQ ID NOs: 28, 89, and 252-256 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in any one of SEQ ID NOs: 28, 89, and 252-256. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 28, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of SEQ ID NO: 28, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 28. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 28 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 28. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 89, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 89. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 89 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 89. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 252, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 252, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 252. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 252 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 252. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 253, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 253, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 253. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 253 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 253. In224742003140 some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 254, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 254, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 254. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 254 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 254. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 255, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 255, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 255. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 255 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 255. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 256, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 256, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 256. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 256 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 256.

[0137] In some embodiments, the DNA-targeting system targets a target site for GATA2. In some embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOs:29, 85, and 87, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of the sequence set forth in any one of SEQ ID NOs:29, 85, and 87 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in any one of SEQ ID NOs:29, 85, and 87. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 29, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 29, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID224742003140NO: 29. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 29 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in the SEQ ID NO: 29. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 85, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 85, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 85. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 85 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in the SEQ ID NO: 85. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 87, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of SEQ ID NO: 87, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 87. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 87 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in the SEQ ID NO: 87.

[0138] In some embodiments, the DNA-targeting system targets a target site for LM02. In some embodiments, the target site for LM02 is within one of the regions defined by the following genomic coordinates: (a) chrl 1:33,867, 158 to chrl 1:33,873,567; (b) chrl l: 33,876,536 to chrl 1:33,878,097; (c) chrl 1:33,890,358 to chrl 1:33,896,210; (d) chrl l: 33,906, 104 to chrl 1 :33,908,030; (e) chrl 1:33,911 ,674 to chrl 1 :33,913,494; (f) chrl 1:33,920,461 to chrl 1:33,922,260; or (g) chrl 1:33,929,929 to chrl 1:33,932,651. In some embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,867,158 to chrl 1:33,873,567. In some embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl l: 33,876,536 to chrl 1:33,878,097. In some embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,890,358 to chrl 1:33,896,210. In some embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl l: 33,906,104 to chrl 1:33,908,030. In some embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,911,674 to chrl 1:33,913,494. In some embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,920,461 to chrl 1:33,922,260. In some224742003140 embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,929,929 to chrl 1:33,932,651.

[0139] In some embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,867,158 to chrl 1:33,873,567. The region defined by the genomic coordinates chrl 1:33,867,158 to chrl 1:33,873,567 comprises the transcriptional start site (TSS) region, which is located between the following genomic coordinates: chrl 1:33,867,215 to chrl 1:33,872,057. In some embodiments, the target site for LM02 is within the TSS region. In some embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,867,215 to chrl 1:33,872,057. In some embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,869,125. In some embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,868,742. In some embodiments, the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,869,106 to chrl 1:33,869,125.

[0140] In some embodiments, the DNA-targeting system targets a target site for LM02. In some embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOs: 30, 80-82, and 94-113, 118, and 119, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in any one of SEQ ID NOs: 30, 80-82, and 94-113, 118, and 119 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOs: 30, 80-82, and 94-113, 118, and 119.

[0141] In some embodiments, the DNA-targeting system targets a target site for LM02. In some embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOs: 30 and 80-82, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in any one of SEQ ID NOs: 30 and 80-82 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in any one224742003140 of SEQ ID NOs: 30 and 80-82. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:30, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 30, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 30. In some embodiments, the target site comprises a contiguous portion of the sequence set forth SEQ ID NO: 30 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 30. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:80, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 80, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 80. In some embodiments, the target site comprises a contiguous portion of the sequence set forth SEQ ID NO: 80 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 80. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 81, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 81, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 81. In some embodiments, the target site comprises a contiguous portion of the sequence set forth SEQ ID NO: 81 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 81. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:82, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 82, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 82. In some embodiments, the target site comprises a contiguous portion of the sequence set forth SEQ ID NO: 82 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 82.

[0142] In some embodiments, the DNA-targeting system targets a target site for LM02. In some embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOs: 94-113, 118, and 119, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence224742003140 identity to any of the foregoing. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in any one of SEQ ID NOs: 94-113, 118, and 119 that is 14, 15, 16, 17, 18, or 19 nucleotides in length.. In some embodiments, the target site is the sequence set forth in any one of SEQ ID NOs: 94-113, 118, and 119. In some embodiments, the target site comprises any one of the sequences set forth in Table E2. In some embodiments, gRNAs targeting the target site are enriched following a CD34+ cell sort. In some embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOs: 94-113, 118, and 119. In some embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOs: 94-113.

[0143] In some embodiments, the DNA-targeting system targets a target site for LM02. In some embodiments, the target site comprises the sequence set forth in any one of SEQ ID NOs: 94-113, 118, and 119, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in any one of SEQ ID NOs: 94-113, 118, and 119 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in any one of SEQ ID NOs: 94-113, 118, and 119. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 94, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 94, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 94. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 94 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 94. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 95, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 95, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 95. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 95 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 95. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 96, a contiguous portion thereof of at least 14 nucleotides224742003140(nt), a complementary sequence of SEQ ID NO: 96, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 96. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 96 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 96. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 97, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 97, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 97. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 97 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 97. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 98, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 98, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 98. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 98 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 98.1n some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 99, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 99, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 99. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 99 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 99. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 100, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 100. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 100 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 100.. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 101,224742003140 a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 101, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 101. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 101 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 101. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 102, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 102, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 102. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 102 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 102.In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 103, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 103, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 103. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 103 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 103. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 104, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 104, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 104. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 104 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 104.In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 105, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 105, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 105. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 105 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 105. In some embodiments, the target site comprises the224742003140 sequence set forth in SEQ ID NO: 106, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 106, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 106. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 106 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 106.In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 107, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 107, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 107. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 107 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 107. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 108, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 108, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 108. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 108 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 108. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 109, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 109, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 109. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 109 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 109. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 110, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 110, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 110. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 110 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO:2247420031401 lO.In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 111, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 111, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 111. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 111 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 11 l.In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 112, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 112, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 112. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 112 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 112. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 113, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 113, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 113. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 113 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 113. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 118, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 118, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 118. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 118 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 118.1n some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 119, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 119, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 119. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 119 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site224742003140 is the sequence set forth in SEQ ID NO: 119. In some embodiments, the DNA-targeting system targets a target site for LM02. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 101, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 101 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 101. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 100, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 100. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 100 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 100. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 101, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 101, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 101. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 101 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 101.

[0144] In some embodiments, the DNA-targeting system targets a target site for ZEB2. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 31, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 31. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 31 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 31.

[0145] In some embodiments, the DNA-targeting system targets a target site for HEY2. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:32, a224742003140 contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 32, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 32. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 32 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 32.

[0146] In some embodiments, the DNA-targeting system targets a target site for CEBPD. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 33, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 33. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 33 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 33.

[0147] In some embodiments, the DNA-targeting system targets a target site for BMI1. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:34, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 34, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 34. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 34 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 34.

[0148] In some embodiments, the DNA-targeting system targets a target site for LYL1. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:35 or SEQ ID NO:83, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 83 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 35 or SEQ ID NO: 83. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:35, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID224742003140NO: 35, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 35. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 35 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 35. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:83, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 83, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 83. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 83 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 83.

[0149] In some embodiments, the DNA-targeting system targets a target site for TALI. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:84 or SEQ ID NO:86, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO:84 or SEQ ID NO:86 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO:84 or SEQ ID NO:86. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:84, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 84, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 84. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 84 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 84. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:86, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 86, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 86. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 86 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 86.224742003140

[0150] In some embodiments, the DNA-targeting system targets a target site for CFOS. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:88, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 88, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 88. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 88 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 88.C. CRISPR / Cas-Based DNA-Targeting Systems and DNA-Binding Domains

[0151] Provided herein are DNA-targeting systems based on CRISPR / Cas systems, i.e. CRISPR / Cas-based DNA-targeting systems, that are able to bind to a target site for a target gene, or to a combination of target sites, e.g. for a combination of target genes. In some embodiments, the CRISPR / Cas DNA-binding domain is nuclease inactive, such as includes a dCas (e.g. dCas9) so that the system binds to the target site for a target gene without mediating nucleic acid cleavage at the target site. The CRISPR / Cas-based DNA-targeting systems may be used to modulate expression of a target gene in a cell, such as a T cell. In some embodiments, the target gene may include any as described herein, including any described above in Section I.B. In some embodiments, the target site for the target gene may include any as described herein, including any described above in Section I.B. In some embodiments, the CRISPR / Cas- based DNA-targeting system can include any known Cas enzyme, and generally a nucleaseinactive or dCas. In some embodiments, the CRISPR / Cas-based DNA-targeting system includes a fusion protein of a nuclease-inactive Cas protein or a variant thereof and an effector domain, and at least one gRNA. In some embodiments, the effector domain increases transcription of the one or more genes (e.g. the effector domain is a transcriptional activator, such as any described in Section I.E).

[0152] The CRISPR system (also known as CRISPR / Cas system, or CRISPR-Cas system) refers to a conserved microbial nuclease system, found in the genomes of bacteria and archaea, that provides a form of acquired immunity against invading phages and plasmids. Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), refers to loci containing multiple repeating DNA elements that are separated by non-repeating DNA sequences called spacers. Spacers are short sequences of foreign DNA that are incorporated into the genome between CRISPR repeats, serving as a “memory” of past exposures. Spacers encode the DNA-targeting224742003140 portion of RNA molecules that confer specificity for nucleic acid cleavage by the CRISPR system. CRISPR loci contain or are adjacent to one or more CRISPR-associated (Cas) genes, which can act as RNA-guided nucleases for mediating the cleavage, as well as non-protein coding DNA elements that encode RNA molecules capable of programming the specificity of the CRISPR-mediated nucleic acid cleavage.

[0153] In Type II CRISPR / Cas systems with the Cas protein Cas9, two RNA molecules and the Cas9 protein form a ribonucleoprotein (RNP) complex to direct Cas9 nuclease activity. The CRISPR RNA (crRNA) contains a spacer sequence that is complementary to a target nucleic acid sequence (target site), and that encodes the sequence specificity of the complex. The transactivating crRNA (tracrRNA) base-pairs to a portion of the crRNA and forms a structure that complexes with the Cas9 protein, forming a Cas / RNA RNP complex.

[0154] Naturally occurring CRISPR / Cas systems, such as those with Cas9, have been engineered to allow efficient programming of Cas / RNA RNPs to target desired sequences in cells of interest, both for gene-editing and modulation of gene expression. The tracrRNA and crRNA have been engineered to form a single chimeric guide RNA molecule, commonly referred to as a guide RNA (gRNA), for example as described in WO 2013 / 176772, WO 2014 / 093661, WO 2014 / 093655, Jinek, M. et al. Science 337(6096):816-21 (2012), or Cong, L. et al. Science 339(6121): 819-23 (2013). The spacer sequence of the gRNA can be chosen by a user to target the Cas / gRNA RNP complex to a desired locus, e.g., a desired target site in the target gene.

[0155] Cas proteins have also been engineered to be catalytically inactivated or nuclease inactive to allow targeting of Cas / gRNA RNPs without inducing cleavage at the target site. Mutations in Cas proteins can reduce or abolish nuclease activity of the Cas protein, rendering the Cas protein catalytically inactive. Cas proteins with reduced or abolished nuclease activity are referred to as deactivated Cas (dCas), or nuclease-inactive Cas (iCas) proteins, as referred to interchangeably herein. An exemplary deactivated Cas9 (dCas9) derived from 5. pyogenes contains silencing mutations of the RuvC and HNH nuclease domains (D10A and H840A), for example as described in WO 2013 / 176772, WO 2014 / 093661, Jinek, M. et al. Science 337(6096):816-21 (2012), and Qi, L. et al. Cell 152(5): 1173-83 (2013). Exemplary dCas variants derived from the Casl2 system (i.e. Cpfl) are described, for example in WO 2017 / 189308 and Zetsche, B. et al. Cell 163(3):759-71 (2015). Conserved domains that mediate nucleic acid cleavage, such as RuvC and HNH endonuclease domains, are readily identifiable in224742003140Cas orthologues, and can be mutated to produce inactive variants, for example as described in Zetsche, B. et al. Cell 163(3):759-71 (2015).

[0156] dCas-fusion proteins with transcriptional and / or epigenetic regulators have been used as a versatile platform for ectopically regulating gene expression in target cells. These include fusion of a Cas with an effector domain, such as a transcriptional activator or transcriptional repressor. For example, fusing dCas9 with a transcriptional activator such as VP64 (a polypeptide composed of four tandem copies of VP 16, a 16 amino acid transactivation domain of the Herpes simplex virus) can result in robust induction of gene expression. Alternatively, fusing dCas9 with a transcriptional repressor such as KRAB (Kruppel associated box) can result in robust repression of gene expression. A variety of dCas-fusion proteins with effector domains can be engineered for regulation of gene expression, for example as described in WO 2014 / 197748, WO 2016 / 130600, WO 2017 / 180915, WO 2021 / 226555, WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2021 / 247570, Adli, M. Nat. Commun. 9, 1911 (2018), Perez-Pinera, P. et al. Nat. Methods 10, 973-976 (2013), Mali, P. et al. Nat. Biotechnol. 31, 833-838 (2013), Maeder, M. L. et al. Nat. Methods 10, 977-979 (2013), Gilbert, L. A. et al. Cell 154(2):442-451 (2013), and Nunez, J.K. et al. Cell 184(9):2503-2519 (2021).

[0157] In some aspects, provided is a DNA-targeting system comprising a fusion protein comprising a DNA-binding domain comprising a nuclease-inactive Cas protein or variant thereof, and an effector domain for increasing transcription or inducing transcriptional activation (i.e. a transcriptional activator) when targeted to a target gene in a cell (e.g., an induced pluripotent stem cell). In some embodiments, the dCas protein is any suitable dCas protein, such as any described in section I.C.l. In some embodiments, the dCas protein is a dCas9 protein, such dSpCas9 or dSaCas9. In some embodiments, the at least one effector domain is any suitable transcriptional activator effector domain, such as any described in Section I.E., such as VP64. In some embodiments, the at least one effector domain is VP64. In some embodiments, the fusion protein is a dCas9-VP64 fusion protein, for example as described in Section I.F. In such embodiments, the DNA-targeting system also includes one or more gRNAs (e.g., as described in Section I.C.2.), provided in combination or as a complex with the dCas protein or variant thereof, for targeting of the DNA-targeting system to the target site of the target gene. In some embodiments, the fusion protein is guided to a specific target site sequence of the target gene by the guide RNA, wherein the effector domain mediates targeted epigenetic modification to increase or activate transcription of the target gene. In some embodiments, a combination of224742003140 gRNAs guides the fusion protein to a combination of target site sequences in a combination of genes, wherein the effector domain mediates targeted epigenetic modification to increase or activate transcription of the combination of target genes. Any of a variety of effector domains that increase or activate transcription can be used as described further below. / . CP / SPP / Cas-Dased DNA-Dinding Domains

[0158] In some aspects, the DNA-binding domain comprises a CRISPR-associated (Cas) protein or variant thereof, or is derived from a Cas protein or variant thereof such as a Cas protein derived from or based on a naturally occurring Cas protein or portion thereof. In some embodiments, the variant Cas protein comprises one or more modifications, mutations, or amino acid substitutions in comparison to the naturally occurring Cas protein. In particular embodiments here, the Cas protein is nuclease-inactive (i.e. is a dCas protein).

[0159] In some embodiments, the Cas protein is derived from a Class 1 CRISPR system (i.e. multiple Cas protein system), such as a Type I, Type III, or Type IV CRISPR system. In some embodiments, the Cas protein is derived from a Class 2 CRISPR system (i.e. single Cas protein system), such as a Type II, Type V, or Type VI CRISPR system. In some embodiments, the Cas protein is from a Type V CRISPR system.

[0160] CRISPR / Cas systems may be multi-protein systems or single effector protein systems. Multi-protein, or Class 1, CRISPR systems include Type I, Type III, and Type IV systems. In some aspects, Class 2 systems include a single effector molecule and include Type II, Type V, and Type VI. In some embodiments, the DNA targeting system comprises components of CRISPR / Cas systems, such as a Type I, Type II, Type III, Type IV, Type V, or Type VI CRISPR system. In some embodiments, the Cas protein is from a Class 1 CRISPR system (i.e., multiple Cas protein system), such as a Type I, Type III, or Type IV CRISPR system. In some embodiments, the Cas protein is from a Class 2 CRISPR system (i.e., single Cas protein system), such as a Type II, Type V, or Type VI CRISPR system.

[0161] Various CRISPR / Cas systems and associated Cas proteins for use in gene editing and regulation have been described, for example in Moon, S.B. et al. Exp. Mol. Med. 51, 1-11 (2019), Zhang, F. Q. Rev. Biophys. 52, E6 (2019), and Makarova K.S. et al. Methods Mol. Biol. 1311:47-75 (2015).

[0162] Type I CRISPR / Cas systems employ a large multisubunit ribonucleoprotein (RNP) complex called Cascade that recognizes double-stranded DNA (dsDNA) targets. After target224742003140 recognition and verification, Cascade recruits the signature protein Cas3, a fused helicase- nuclease, to degrade DNA.

[0163] In some embodiments, the Cas protein is derived from a Type II CRISPR system. In some embodiments, the Cas protein is derived from a Cas9 protein or variant thereof, for example as described in WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2014 / 093655, Jinek, M. et al. Science 337(6096):816-21 (2012), Mali, P. et al. Science 339(6121):823-6 (2013), Cong, L. et al. Science 339(6121):819-23 (2013), Perez-Pinera, P. et al. Nat. Methods 10, 973-976 (2013), or Mali, P. et al. Nat. Biotechnol. 31, 833-838 (2013). In Type II CRISPR / Cas systems with the Cas protein Cas9, two RNA molecules and the Cas9 protein form a ribonucleoprotein (RNP) complex to direct Cas9 nuclease activity. The CRISPR RNA (crRNA) contains a spacer sequence that is complementary to a target nucleic acid sequence (target site), and that encodes the sequence specificity of the complex. The transactivating crRNA (tracrRNA) base-pairs to a portion of the crRNA and forms a structure that complexes with the Cas9 protein, forming a Cas / RNA RNP complex. Cas9 mediates cleavage of target DNA if a correct protospacer-adjacent motif (PAM) is also present at the 3' end of the protospacer. For protospacer targeting, the sequence must be immediately followed by the protospacer-adjacent motif (PAM), a short sequence recognized by the Cas9 nuclease that is required for DNA cleavage.

[0164] Different Type II systems have differing PAM requirements. The S. pyogenes CRISPR system may have the PAM sequence for this Cas9 (SpCas9) as 5'-NRG-3', where R is either A or G, and characterized the specificity of this system in human cells. A unique capability of the CRISPR / Cas9 system is the straightforward ability to simultaneously target multiple distinct genomic loci by co-expressing a single Cas9 protein with two or more sgRNAs. For example, the Streptococcus pyogenes Type II system typically prefers to use an “NGG” (SEQ ID NO: 45) sequence, where “N” can be any nucleotide, but also accepts other PAM sequences, such as “NAG” in engineered systems (Hsu et al., Nature Biotechnology (2013) doi:10.1038 / nbt.2647). Similarly, the Cas9 derived from Neisseria meningitidis (NmCas9) normally has a native PAM of NNNNGATT (SEQ ID NO: 184), but has activity across a variety of PAMs, including a highly degenerate NNNNGNNN (SEQ ID NO: 272) PAM (Esvelt et al. Nature Methods (2013) doi:10.1038 / nmeth.2681). In another example, the Cas9 derived from Campylobacter jejuni typically uses 5'-NNNNACAC-3' (SEQ ID NO: 273) or 5'-NNNNRYAC- 3' (SEQ ID NO: 185) PAM sequences, where “N” can be any nucleotide, “R” can be either224742003140 guanine (G) or adenine (A), and “Y” can be either cytosine (C) or thymine (T). In some aspects, the PAM sequences for spacer targeting depends on the type, ortholog, variant or species of the Cas protein.

[0165] In some embodiments, the Cas protein is derived from a Cas9 protein or variant thereof, for example as described in WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO 2014 / 093655, Jinek, M. et al. Science 337(6096):816-21 (2012), Mali, P. et al. Science 339(6121):823-6 (2013), Cong, L. et al. Science 339(6121):819-23 (2013), Perez-Pinera, P. et al. Nat. Methods 10, 973-976 (2013), or Mali, P. et al. Nat. Biotechnol. 31, 833-838 (2013). Various CRISPR / Cas systems and associated Cas proteins for use in gene editing and regulation have been described, for example in Moon, S.B. et al. Exp. Mol. Med. 51, 1-11 (2019), Zhang, F. Q. Rev. Biophys. 52, E6 (2019), and Makarova K.S. et al. Methods Mol. Biol. 1311:47-75 (2015).

[0166] In some embodiments, the Cas9 protein comprises a sequence from a Cas9 molecule of S. aureus. In some embodiments, the Cas9 protein comprises a sequence set forth in SEQ ID NO: 41 or SEQ ID NO: 274, or a variant thereof, such as an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 41 or SEQ ID NO: 274. In some embodiments, the Cas9 protein comprises a sequence from a Cas9 molecule of S. pyogenes. In some embodiments, the Cas9 protein comprises a sequence set forth in SEQ ID NO: 41 or SEQ ID NO: 274, or a variant thereof, such as an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 41 or SEQ ID NO: 274.

[0167] In Type III systems, the RNP complex is multimeric with a helicoid structure similar to Cascade. In contrast to Type I CRISPR / Cas systems, the Type III RNP complex recognizes complementary RNA sequences instead of dsDNA. RNA recognition stimulates a nonspecific DNA cleavage activity of the exemplary Type III Cas 10 nuclease that is part of the RNP complex, such that DNA cleavage is achieved cotranscriptionally.

[0168] In some embodiments, the Cas protein is from a Type V CRISPR system. Exemplary Cas proteins of a Type V CRISPR system include Casl2a (also known as Cpfl), Casl2b (also known as C2cl), Casl2e (also known as CasX), Casl2k (also known as C2c5), Casl4a, and Cas 14b. In some embodiments, the Cas protein is from a Cas 12 protein (i.e., Cpfl) or variant thereof, for example as described in WO 2017 / 189308, WO2019 / 232069 and Zetsche et al. Cell. 163(3):759-71 (2015).224742003140

[0169] Exemplary Type V systems include those based on a Casl2 effector, and the C- terminus with only one RuvC endonuclease domain is the defining characteristic of the Type V systems. The RuvC nuclease domain cleaves dsDNA adjacent to protospacer adjacent motif (PAM) sequences and single- stranded DNA (ssDNA) nonspecifically. The Type V systems can be further divided into subtypes, each characterized by different signature proteins, PAM sequences, and properties. Non-limiting exemplary Cas proteins derived from Type V CRISPR systems include Casl2a (Cpfl), UnlCasl2fl, Casl2j (CasPhi, such as CasPhi-2), Casl2k, and CasMini. For example, Type V-A includes, for example, Casl2a, which uses “TTTV” (SEQ ID NO: 183) PAM sequence, where “V” is adenine (A), cytosine (C), or guanine (G). Type V-F is includes, for example, Casl2f, which can use '"TTTR” (SEQ ID NO: 139), where “R” is G or A, or “TTTN” (SEQ ID NO: 276), where “N” is any nucleotide. Type V-K is includes, for example, Cas 12k, which uses “GGTT” (SEQ ID NO: 277) PAM sequence.

[0170] In some embodiments, the Cas 12a protein comprises a sequence from a Cas 12a molecule of Acidaminococcus sp, such as an AsCasl2a set forth in SEQ ID NO: 278 or SEQ ID NO: 279, or a variant thereof, such as an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 278 or SEQ ID NO: 279.

[0171] In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule, or variant thereof. In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule of .S'. pyogenes, S. thermophilus, S. aureus, C. jejuni, N. meningitidis, F. novicida, S. canis, S. auricularis, or variant thereof. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of .S', aureus. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of .S', pyogenes.

[0172] Non-limiting examples of Cas9 orthologs from other bacterial strains include but are not limited to: Cas proteins identified in Acaryochloris marina MBIC 11017; Acetohalobium arabaticum DSM 5501; Acidithiobacillus caldus; Acidithiobacillus ferrooxidans ATCC 23270; Alicyclobacillus acidocaldarius LAA1; Alicyclobacillus acidocaldarius subsp. Acidocaldarius DSM 446; Allochromatium vinosum DSM 180; Ammonifex degensii KC4; Anabaena variabilis ATCC 29413; Arthrospira maxima CS-328; Arthrospira platensis str. Paraca; Arthrospira sp. PCC 8005; Bacillus pseudomycoides DSM 12442; Bacillus selenitireducens MLS 10; Burkholderiales bacterium 1_1_47; Caldicelulosiruptor becscii DSM 6725; Candidatus224742003140Desulforudis audaxviator MP104C; Caldicellulosiruptor hydrothermalis 108; Clostridium phage c-st; Clostridium botulinum A3 str. Loch Maree; Clostridium botulinum Ba4 str. 657; Clostridium difficile QCD-63q42; Crocosphaera watsonii WH 8501; Cyanothece sp. ATCC 51142; Cyanothece sp. CCY0110; Cyanothece sp. PCC 7424; Cyanothece sp. PCC 7822; Exiguobacterium sibiricum 255-15; Finegoldia magna ATCC 29328; Ktedonobacter racemifer DSM 44963; Lactobacillus delbrueckii subsp. Bulgaricus PB2003 / 044-T3-4; Lactobacillus salivarius ATCC 11741; Listeria innocua; Lyngbya sp. PCC 8106; Marinobacter sp. ELB 17; Methanohalobium evestigatum Z-7303; Microcystis phage Ma-LMMOl; Microcystis aeruginosa NIES-843; Microscilla marina ATCC 23134; Microcoleus chthonoplastes PCC 7420; Neisseria meningitidis; Nitrosococcus halophilus Nc4; Nocardiopsis dassonvillei subsp. Dassonvillei DSM 43111; Nodularia spumigena CCY9414; Nostoc sp. PCC 7120; Oscillatoria sp. PCC 6506; Pelotomaculum_thermopropionicum SI; Petrotoga mobilis SJ95; Polaromonas naphthalenivorans CJ2; Polaromonas sp. JS666; Pseudoalteromonas haloplanktis TAC125;Streptomyces pristinaespiralis ATCC 25486; Streptomyces pristinaespiralis ATCC 25486; Streptococcus thermophilus; Streptomyces viridochromogenes DSM 40736; Streptosporangium roseum DSM 43021; Synechococcus sp. PCC 7335; and Thermosipho africanus TCF52B (Chylinski et al., RNA Biol., 2013; 10(5): 726-737).

[0173] In some embodiments, the DNA-targeting systems or fusion proteins comprise a Cas protein or variant thereof, such as a Cas protein or variant thereof set forth in any one of SEQ ID NOs: 41-44, 274, and 278-300, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any one of SEQ ID NOs: 41-44, 274, and 278-300. In some embodiments, the Cas protein or variant thereof of any of the DNA- targeting systems or fusion proteins provided herein comprise a sequence set forth in any one of SEQ ID NOs: 41-44, 274, and 278-300. In some aspects, the Cas protein or variant thereof lacks an initial methionine residue. In some aspects, the Cas protein or variant thereof comprises an initial methionine residue.

[0174] In some aspects, the Cas protein is a variant that lacks nuclease activity (i.e. is a dCas protein). In some embodiments, the Cas protein is mutated so that nuclease activity is reduced or eliminated. Such Cas proteins are referred to as deactivated Cas or dead Cas (dCas) or nucleaseinactive Cas (iCas) proteins, as referred to interchangeably herein. In some embodiments, the variant Cas protein is a variant Cas9 protein that lacks nuclease activity or that is a deactivated Cas9 (dCas9, or iCas9) protein.224742003140

[0175] In some embodiments, Cas proteins are engineered to be catalytically inactivated or nuclease inactive to allow targeting of Cas / gRNA RNPs without inducing cleavage at the target site. Mutations in Cas proteins can reduce or abolish nuclease activity of the Cas protein, rendering the Cas protein catalytically inactive. Cas proteins with reduced or abolished nuclease activity are referred to as deactivated Cas (dCas), or nuclease-inactive Cas (iCas) proteins, as referred to interchangeably herein. In some aspects, the dCas or iCas can still bind to target site in the DNA in a site- and / or sequence- specific manner, as long as it retains the ability to interact with the guide RNA (gRNA) which directs the Cas-gRNA combination to the target site.

[0176] In some aspects, the dCas or iCas exhibits reduced or no endodeoxyribonuclease activity. For example, an exemplary dCas or iCas, for example dCas9 or iCas9, exhibits less than about 20%, less than about 15%, less than about 10%, less than about 5%, less than about 1%, or less than about 0.1%, of the endodeoxyribonuclease activity of a wild-type Cas protein, e.g., a wild-type Cas9 protein. In some embodiments, the dCas or iCas, for example dCas9 or iCas9, exhibits substantially no detectable endodeoxyribonuclease activity. In some embodiments, an exemplary dCas or iCas, for example dCas9 or iCas9, comprises one or more amino acid mutations, substitutions, deletions or insertions at a position corresponding to a position selected from DIO, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986, and / or a A987, with reference to a wild-type Streptococcus pyogenes Cas9 (SpCas9), for example, with reference to numbering of positions of a SpCas9 sequence set forth in SEQ ID NO:43. In some aspects, the dCas9 or iCas9 comprises one or more amino acid mutations, substitutions, deletions or insertions corresponding to D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, and / or D986A, with reference to a wild-type Streptococcus pyogenes Cas9 (SpCas9), for example, with reference to numbering of positions of a SpCas9 sequence set forth in SEQ ID NO: 43. Corresponding positions for mutations can be determined based on sequence alignments and determination of sequence conservation, for example, as described in WO 2013 / 171772 for Cas9 proteins from various species. In some aspects, the dCas protein lacks an initial methionine residue. In some aspects, the dCas protein comprises an initial methionine residue.

[0177] In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule, or variant thereof. In some embodiments, the dCas9 protein can comprise a sequence derived from a naturally occurring Cas9 molecule of S. pyogenes, S. thermophilus, S. aureus, C. jejuni, N. meningitidis, F. novicida, S. canis, S. auricularis, or224742003140 variant thereof. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of S. aureus. In some embodiments, the dCas9 protein comprises a sequence derived from a naturally occurring Cas9 molecule of S. pyogenes. In some embodiments, the dCas9 protein comprises a sequence from a Cas9 molecule of C. jejuni.

[0178] Exemplary deactivated Cas9 (dCas9) derived from S. pyogenes contains silencing mutations of the RuvC and HNH nuclease domains (D10A and H840A), for example as described in WO 2013 / 176772, WO 2014 / 093661, Jinek et al. Science 337(6096):816-21 (2012), and Qi et al. Cell 152(5): 1173-83 (2013). Exemplary dCas variants derived from the Casl2 system (i.e. Cpfl) are described, for example in WO 2017 / 189308 and Zetsche et al. Cell 163(3):759-71 (2015). Conserved domains that mediate nucleic acid cleavage, such as RuvC and HNH endonuclease domains, are readily identifiable in Cas orthologues, and can be mutated to produce inactive variants, for example as described in Zetsche et al. Cell 163(3):759-71 (2015). Other exemplary Cas orthologs or variants include engineered variants based on a Casl2f (also known as Casl4), including those described in Xu et al., Mol. Cell 81(20):4333-4345 (2021).

[0179] In some embodiments, the DNA-binding domain comprises a Cas-gRNA combination that includes (a) a Cas protein or a variant thereof and (b) at least one gRNA. In some embodiments, the variant Cas protein lacks nuclease activity or is a deactivated Cas (dCas) protein. In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof. In some embodiments, the gRNA comprises a gRNA spacer sequence that is capable of hybridizing to the target site or is complementary to the target site at a target gene.

[0180] In some embodiments, the Cas protein or a variant thereof is a Cas9 protein or a variant thereof. In some embodiments, the variant Cas protein is a variant Cas9 protein that lacks nuclease activity or that is a deactivated Cas9 (dCas9) protein. In some embodiments, the Cas9 protein or a variant thereof is derived from a Staphylococcus aureus Cas9 (SaCas9) protein or a variant thereof. In some embodiments, the variant Cas9 is a Staphylococcus aureus dCas9 protein (dSaCas9) that comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO:41. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO:42 or SEQ ID NO: 280, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 280, which lacks an initial methionine residue. In some224742003140 embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 42, which includes an initial methionine residue.

[0181] In some embodiments, the Cas9 protein or variant thereof is derived from a Streptococcus pyogenes Cas9 (SpCas9) protein or a variant thereof. In some embodiments, the variant Cas9 is a Streptococcus pyogenes dCas9 (dSpCas9) protein that comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO:43. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 44 or SEQ ID NO: 281, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 281, which lacks an initial methionine residue. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 44, which includes an initial methionine residue.

[0182] In some embodiments, the Cas9 protein or variant thereof is a Campylobacter jejuni Cas9 (CjCas9) protein or a variant thereof. In some embodiments, the variant Cas9 comprises at least one amino acid mutation compared to the sequence set forth in SEQ ID NO: 282 or 283. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 294, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 295, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 295, which lacks an initial methionine residue. In some embodiments, the variant Cas9 protein comprises the sequence set forth in SEQ ID NO: 294, which includes an initial methionine residue.

[0183] In some embodiments, the Cas protein or a variant thereof is a Casl2a protein or a variant thereof. In some embodiments, the variant Cas protein is a variant Cas 12a protein that lacks nuclease activity or that is a deactivated Cas 12a (dCasl2a) protein. In some embodiments, the Cas 12a protein or variant thereof is a Acidaminococcus sp. Cas 12a (AsCasl2a) protein or a variant thereof. In some embodiments, the variant Cas 12a is a Acidaminococcus sp. dCasl2a (dAsCasl2a) protein that comprises at least one amino acid mutation compared to the sequence set forth in SEQ ID NO: 278 or 279. In some embodiments, the variant Cas 12a protein224742003140 comprises the sequence set forth in SEQ ID NO: 284, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant Casl2a protein comprises the sequence set forth in SEQ ID NO: 285, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant Casl2a protein comprises the sequence set forth in SEQ ID NO: 285, which lacks an initial methionine residue. In some embodiments, the variant Casl2a protein comprises the sequence set forth in SEQ ID NO: 284, which includes an initial methionine residue.

[0184] In some embodiments, the Cas protein or a variant thereof is a CasPhi-2 protein or a variant thereof. In some embodiments, the variant Cas protein is a variant CasPhi-2 protein that lacks nuclease activity or that is a deactivated CasPhi-2 (dCasPhi-2) protein. In some embodiments, the variant CasPhi-2 comprises at least one amino acid mutation compared to the sequence set forth in SEQ ID NO: 286 or 287. In some embodiments, the variant CasPhi-2 protein comprises the sequence set forth in SEQ ID NO: 288, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant CasPhi-2 protein comprises the sequence set forth in SEQ ID NO: 289, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant CasPhi-2 protein comprises the sequence set forth in SEQ ID NO: 289, which lacks an initial methionine residue. In some embodiments, the variant CasPhi-2 protein comprises the sequence set forth in SEQ ID NO: 288, which includes an initial methionine residue.

[0185] In some embodiments, the Cas protein or a variant thereof is a UnlCasl2fl protein or a variant thereof. In some embodiments, the variant Cas protein is a variant UnlCasl2fl protein that lacks nuclease activity or that is a deactivated UnlCasl2fl (dUnlCasl2fl) protein. In some embodiments, the variant UnlCasl2fl comprises at least one amino acid mutation compared to the sequence set forth in SEQ ID NO: 290 or 291. In some embodiments, the variant UnlCasl2fl protein comprises the sequence set forth in SEQ ID NO: 292, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant UnlCasl2fl protein comprises the sequence set forth in SEQ ID NO: 293, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the variant UnlCasl2fl protein comprises the sequence set forth in SEQ ID NO:224742003140293, which lacks an initial methionine residue. In some embodiments, the variant UnlCasl2fl protein comprises the sequence set forth in SEQ ID NO: 292, which includes an initial methionine residue.

[0186] In some embodiments, the Cas protein or a variant thereof is a Casl2k protein or a variant thereof. In some embodiments, the Cas 12k protein comprises the sequence set forth in SEQ ID NO: 296, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the Cas 12k protein comprises the sequence set forth in SEQ ID NO: 297, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the Cas 12k protein comprises the sequence set forth in SEQ ID NO: 297, which lacks an initial methionine residue. In some embodiments, the Cas 12k protein comprises the sequence set forth in SEQ ID NO: 296, which includes an initial methionine residue.

[0187] In some embodiments, the Cas protein or a variant thereof is a CasMini protein or a variant thereof, such as an engineered Cas protein or variant based on a Casl2f (also known as Cas 14), including those described in Xu et al., Mol. Cell 81(20):4333-4345 (2021) or set forth in SEQ ID NO: 300. In some embodiments, the variant Cas protein is a variant CasMini protein that lacks nuclease activity or that is a deactivated CasMini (dCasMini) protein. In some embodiments, the variant CasMini comprises at least one amino acid mutation compared to the sequence set forth in SEQ ID NO: 300. In some embodiments, the variant CasMini protein comprises the sequence set forth in SEQ ID NO: 300, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CasMini protein comprises the sequence set forth in SEQ ID NO: 300. In some embodiments, the variant CasMini protein comprises the sequence set forth in SEQ ID NO: 298 or 299, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto. In some embodiments, the CasMini protein comprises the sequence set forth in SEQ ID NO: 299, which lacks an initial methionine residue. In some embodiments, the CasMini protein comprises the sequence set forth in SEQ ID NO: 298, which includes an initial methionine residue.2. Guide RlVAs (g GVAs)

[0188] In some embodiments, the Cas protein (e.g. dCas9) is provided in combination or as a complex with one or more guide RNA (gRNA). In some aspects, the gRNA is a nucleic acid that promotes the specific targeting or homing of the gRNA / Cas RNP complex to the target site224742003140 of the target gene, such as any described above in Section LB. In some embodiments, a target site of a gRNA may be referred to as a protospacer.

[0189] Provided herein are gRNAs, such as gRNAs that target or bind to a target site for a gene, such as in a target gene or regulatory DNA element thereof, such as any described herein, for example in Section I.B. In some embodiments, the gRNA is capable of complexing with the Cas protein or variant thereof, such as any Cas protein or variant thereof described herein in Section I.C.l. In some embodiments, the gRNA is capable of complexing with a fusion protein comprising a Cas protein or variant thereof, such as any fusion protein described herein in Section I.F. In some embodiments, the gRNA comprises a gRNA spacer sequence (i.e. a spacer sequence or a guide sequence) that is capable of hybridizing to the target site, or that is complementary to the target site, such as any target site described herein. In some embodiments, the gRNA comprises a scaffold sequence that complexes with or binds to the Cas protein. In some embodiments, the scaffold is specific for a Cas protein. In some embodiments, the scaffold is specific for SpCas9 or a dSpCas9. In some embodiments the scaffold comprises the nucleic acid sequence set forth in SEQ ID NO: 37 and SEQ ID NO: 182. In some embodiments the scaffold comprises the nucleic acid sequence set forth in SEQ ID NO: 90 and SEQ ID NO: 91. In some embodiments, the scaffold is specific for a SaCas9 or a dSaCas9. In some embodiments, the scaffold comprises the nucleic acid sequence set forth in SEQ ID NO: 92 and SEQ ID NO: 93.

[0190] In some embodiments, the gRNAs provided herein are chimeric gRNAs. In general, gRNAs can be unimolecular (i.e. composed of a single RNA molecule), or modular (comprising more than one, and typically two, separate RNA molecules). Modular gRNAs can be engineered to be unimolecular, wherein sequences from the separate modular RNA molecules are comprised in a single gRNA molecule, sometimes referred to as a chimeric gRNA, synthetic gRNA, or single gRNA. In some embodiments, the chimeric gRNA is a fusion of two noncoding RNA sequences: a crRNA sequence and a tracrRNA sequence, for example as described in WO 2013 / 176772, or Jinek, M. et al. Science 337(6096):816-21 (2012). In some embodiments, the chimeric gRNA mimics the naturally occurring crRNA:tracrRNA duplex involved in the Type II Effector system, wherein the naturally occurring crRNA:tracrRNA duplex acts as a guide for the Cas9 protein.

[0191] In some aspects, the spacer sequence of a gRNA is a polynucleotide sequence comprising at least a portion that has sufficient complementarity with the target site to hybridize224742003140 with the target site in the target gene and direct sequence-specific binding of a Cas / gRNA complex to the sequence of the target site. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization. In some embodiments, the gRNA comprises a spacer sequence that is complementary, e.g., at least 80%, 85%, 90%, 95%, 98%, 99%, or 100% (e.g., fully complementary), to the target site. The strand of the target nucleic acid comprising the target site sequence may be referred to as the “complementary strand” of the target nucleic acid.

[0192] In some aspects, a gRNA targets a target site in double- stranded DNA. Thus, in some aspects, the sequence of the target site may be defined by the sequence that the gRNA spacer hybridizes to, or by the sequence complementary to the sequence that the gRNA spacer hybridizes to. In some aspects, the sequence of the target site may be defined by the sequence that the gRNA spacer displaces in order to hybridize to the DNA. In some embodiments, the sequence of the target site is the sequence that the gRNA hybridizes to.

[0193] In some embodiments, the gRNA spacer sequence is between about 14 nucleotides (nt) and about 26 nt, or between 16 nt and 22 nt in length. In some embodiments, the gRNA spacer sequence is 14 nt, 15 nt, 16 nt, 17 nt, 18 nt, 19 nt, 20 nt, 21 nt or 22 nt, 23 nt, 24 nt, 25 nt, or 26 nt in length. In some embodiments, the gRNA spacer sequence is 18 nt, 19 nt, 20 nt, 21 nt or 22 nt in length. In some embodiments, the gRNA spacer sequence is 20 nt in length.

[0194] A target site of a gRNA may be referred to as a protospacer. In some aspects, the spacer is designed to target a protospacer with a specific protospacer-adjacent motif (PAM), i.e. a sequence immediately adjacent to the protospacer that contributes to and / or is required for Cas binding specificity. Different CRISPR / Cas systems have different PAM requirements for targeting. For example, in some embodiments, .S'. pyogenes Cas9 uses the PAM 5’-NGG-3’ (SEQ ID NO: 45), where N is any nucleotide. In some embodiments, .S'. aureus Cas9 uses the PAM 5’- NNGRRT-3’ (SEQ ID NO: 46), where N is any nucleotide, and R is G or A. In some embodiments, Cas proteins may use or be engineered to use different PAMs from those listed above. In some embodiments, the protospacer-adjacent motif (PAM) of a gRNA for complexing with .S'. pyogenes Cas9 or variant thereof is NGG, as set forth in SEQ ID NO: 45. In some embodiments, the PAM of a gRNA for complexing with .S', aureus Cas9 or variant thereof is NNGRRT, as set forth in SEQ ID NO: 46. In some embodiments, the PAM of a gRNA for complexing with a Type V CRISPR / Cas system, such as with Casl2a (also known as Cpfl) or variant thereof uses TTTV (SEQ ID NO: 183), where V is A, C, or G. In some embodiments, N.224742003140 meningitidis Cas9 uses the PAM 5'-NNNNGATT-3’ (SEQ ID NO: 184), where N is any nucleotide. In some embodiments, C. jejuni Cas9 uses the PAM 5'-NNNNRYAC-3' (SEQ ID NO: 185), where N is any nucleotide, R is G or A, and Y is C or T. In some embodiments, S. thermophilus Cas9 uses the PAM 5’-NNAGAAW-3’ (SEQ ID NO: 186), where N is any nucleotide and W is A or T. In some embodiments, F. Novicida Cas9 uses the PAM 5’-NGG-3’ (SEQ ID NO: 45), where N is any nucleotide. In some embodiments, T. denticola Cas9 uses the PAM 5’-NAAAAC-3’ (SEQ ID NO: 187), where N is any nucleotide. In some embodiments, Cas proteins may use or be engineered to use different PAMs from those listed above. For example, mutated SpCas9 proteins may use the PAMs 5’-NGG-3’ (SEQ ID NO:45), 5’-NGAN- 3’ (SEQ ID NO: 188), 5’-NGNG-3’ (SEQ ID NO: 189), 5’-NGAG-3’ (SEQ ID NO: 190), or 5’- NGCG-3’ (SEQ ID NO: 191), where N is any nucleotide. Methods for designing or identifying gRNA spacer sequences and / or protospacer sequences in a particular region, are known. gRNA spacer sequences and / or protospacer sequences can be determined based on the type of Cas protein used and the associated PAM sequence.

[0195] A spacer sequence may be selected to reduce the degree of secondary structure within the spacer sequence. Secondary structure may be determined by any suitable polynucleotide folding algorithm.

[0196] It is understood by a person of skill in the art that the gRNA (including the guide sequence) will comprise the base uracil (U), whereas DNA encoding the gRNA molecule will comprise the base thymine (T). While not wishing to be bound by theory, it is believed that the complementarity of the guide sequence with the target sequence contributes to specificity of the interaction of the gRNA molecule / Cas molecule complex with a target nucleic acid. It is understood that in a guide sequence and target sequence pair, the uracil bases in the guide sequence will pair with the adenine bases in the target sequence.

[0197] In some embodiments, one, more than one, or all of the nucleotides of a gRNA can have a modification, e.g., to render the gRNA less susceptible to degradation and / or improve bio-compatibility. By way of non-limiting example, the backbone of the gRNA can be modified with a phosphorothioate, or other modification(s). In some cases, a nucleotide of the gRNA can comprise a 2’ modification, e.g., a 2-acetylation, e.g., a 2’ methylation, or other modification(s).

[0198] Methods for designing gRNAs and exemplary targeting domains can include those described in, e.g., International PCT Pub. Nos. WO 2014 / 197748, WO 2016 / 130600, WO 2017 / 180915, WO 2021 / 226555, WO 2013 / 176772, WO 2014 / 152432, WO 2014 / 093661, WO2247420031402014 / 093655, WO 2015 / 089427, WO 2016 / 049258, WO 2016 / 123578, WO 2021 / 076744, WO 2014 / 191128, WO 2015 / 161276, WO 2017 / 193107, and WO 2017 / 093969.

[0199] In some aspects, the gRNA comprises scaffold sequences. In some aspects, the scaffold sequence (in some cases including a crRNA sequence and / or a tracrRNA sequence) will be different depending on the Cas protein. In some aspects, different CRISPR / Cas systems have different gRNA scaffold sequences for associating with Cas protein. In some embodiments, an exemplary scaffold sequence for S. aureus Cas9 comprises a sequence set forth in SEQ ID NO:93, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:93. In some embodiments, an exemplary scaffold sequence for S. aureus Cas9 comprises a sequence set forth in SEQ ID NO:93. In some embodiments, an exemplary scaffold sequence for S. pyogenes Cas9 comprises a sequence set forth in SEQ ID NO:91, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:91. In some embodiments, an exemplary scaffold sequence for S. pyogenes Cas9 comprises a sequence set forth in SEQ ID NO:91.

[0200] In some embodiments, an exemplary scaffold sequence for Acidaminococcus sp. Casl2a comprises a sequence set forth in SEQ ID NO: 192, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 192. In some embodiments, an exemplary scaffold sequence for CasPhi-2 comprises a sequence set forth in SEQ ID NO: 193, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 193. In some embodiments, an exemplary scaffold sequence for UnlCasl2fl comprises a sequence set forth in any one of SEQ ID NOs:194-196, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any one of SEQ ID NOs:194-196. In some embodiments, an exemplary scaffold sequence for UnlCasl2fl comprises a sequence set forth in SEQ ID NO: 194 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 194. In some embodiments, an exemplary scaffold sequence for UnlCasl2fl comprises a sequence set forth in SEQ ID NO: 195, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 195. In some embodiments, an exemplary scaffold sequence for UnlCasl2fl comprises224742003140 a sequence set forth in SEQ ID NO: 196, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 196. In some embodiments, an exemplary scaffold sequence for C. jejuni Cas9 comprises a sequence set forth in SEQ ID NO: 197, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 197. In some embodiments, an exemplary scaffold sequence for Casl2k comprises a sequence set forth in SEQ ID NO: 198, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 198. In some embodiments, an exemplary scaffold sequence for CasMini comprises a sequence set forth in SEQ ID NO: 199, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 199.

[0201] In some embodiments, a gRNA provided herein targets a target site for a gene for transcriptional activation, such as any target site or gene described in Section I.B. In some embodiments, a gRNA provided herein targets a target site for transcription factor genes, wherein the genes are selected from: BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2.

[0202] In some embodiments, the gRNA targets a target site that comprises a sequence selected from any one of SEQ ID NOs: 28-35, 80-89, 94-113, 118,119, and 252-256, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of any one of SEQ ID NOs: 28-35, SO- 89, 94-113, 118, 119, and 252-256 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is set forth in any one of SEQ ID NOs: 28-35, 80-89, 94-113, 118, 119, and 252-256.

[0203] In some embodiments, the gRNA comprises a spacer sequence selected from any one of SEQ ID NOs: 19-26, 70-79, 120-139, 144, 145, and 257-261, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOs: 19-26, 70-79, 120-139, 144, 145, and 257-261 that is 14, 15, 16, 17, 18 or 19224742003140 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOs: 19-26, 70-79, 120-139, 144, 145, and 257-261.

[0204] In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 1-8, 50-59, 146-165, 170, 171, and 262-266, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 1-8, 50-59, 146-165, 170, 171, and 262-266. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 1-8, 50-59, 146-165, 170, 171, and 262-266.

[0205] In some embodiments, the gRNA comprises modified nucleotides. In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 10-17, 60-69, 114-117, 140-143, 166-169, 172-181, and 267-271, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 10-17, 60-69, 114-117, 140-143, 166-169, 172-181, and 267-271. In some embodiments, the gRNA is set forth in any one of SEQ ID NOs: 10-17, 60-69, 114-117, 140-143, 166-169, 172-181, and 267-271.

[0206] In some embodiments, the gRNA targets a target site for GATA3. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in any one of SEQ ID NOs: 28, 89, and 252-256, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of the sequence set forth in any one of SEQ ID NOs: 28, 89, and 252-256 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in any one of SEQ ID NOs: 28, 89, and 252-256. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 28, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of SEQ ID NO: 28, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 28. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 28 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 28. In224742003140 some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 89, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 89. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 89 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 89. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 252, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 252, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 252. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 252 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 252. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 253, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 253, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 253. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 253 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 253. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 254, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 254, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 254. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 254 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 254. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 255, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 255, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 255. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 255 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 255. In some224742003140 embodiments, the target site comprises the sequence set forth in SEQ ID NO: 256, a contiguous portion thereof of at least 14 nucleotides, a complementary sequence of SEQ ID NO: 256, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 256. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 256 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 256.

[0207] In some embodiments, the gRNA targeting a target site for GATA3 comprises a spacer sequence set forth in any one of SEQ ID NOs: 19, 79, and 257-261, a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of the sequence set forth in any one of SEQ ID NOs: 19, 79, and 257-261 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOs: 19, 79, and 257-261. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO: 19, a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 19. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 19 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 19. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:79, a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:79. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:79 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:79. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:257, a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:257. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:257 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:257.224742003140In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:258, a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:258. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:258 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:258. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:259, a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:259. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:259 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:259. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:260, a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:260. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:260 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:260. In some embodiments, the spacer sequence comprises the sequence set forth in SEQ ID NO:261, a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:261. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO:261 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:261.

[0208] In some embodiments, the gRNA targeting a target site for GATA3 comprises the sequence set forth in any one of SEQ ID NOs: 1, 59, and 262-266, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in any one of SEQ ID NOs: 1, 59, and 262-266. In some embodiments, the gRNA is the sequence set forth in any one of SEQ ID NOs: 1, 59, and 262-266. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 1 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,22474200314099%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 1. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 1. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 1. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 59 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 59. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 59. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 59. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 262 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 262. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 262. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 262. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 263 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 263. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 263. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 263. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 264 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 264. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 264. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 264. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 265 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 265. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 265. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 265. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 266 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 266. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 266. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 266.

[0209] In some embodiments, the gRNA targeting a target site for GATA3 comprises224742003140 modified nucleotides. In some embodiments, the gRNA comprises a sequence set forth in any one of SEQ ID NOs: 10, 69, and 267-271 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 10, 69, and 267-271. In some embodiments, the gRNA is the sequence set forth in any one of SEQ ID NOs: 10, 69, and 267-271. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 10 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 10. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 10. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 10. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 69 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 69. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 69. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 69. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 267 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 267. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 267. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 267. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 268 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 268. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 268. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 268. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 269 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 269. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 269. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 269. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 270 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 270. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 270. In some embodiments, the gRNA comprises224742003140 a sequence set forth in SEQ ID NO: 270. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 271 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 271. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 271. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 271.

[0210] In some embodiments, the gRNA targets a target site for GATA2. In some embodiments, the gRNA targets the target site that comprises the sequence set forth in any one of SEQ ID NOs:29, 85, and 87, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site is a contiguous portion of the sequence set forth in any one of SEQ ID NOs:29, 85, and 87 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in any one of SEQ ID NOs:29, 85, and 87. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 29, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of SEQ ID NO: 29, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 29. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 29 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in the SEQ ID NO: 29. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 85, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of SEQ ID NO: 85, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 85. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 85 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the target site is the sequence set forth in the SEQ ID NO: 85. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 87, a contiguous portion thereof of at least 14 nucleotides, or a complementary sequence of SEQ ID NO: 87, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 87. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 87 that is 14, 15, 16, 17, 18 or 19 nucleotides in length. In some embodiments, the224742003140 target site is the sequence set forth in the SEQ ID NO: 87.

[0211] In some embodiments, the gRNA targeting a target site for GATA2 comprises a spacer sequence set forth in any one of SEQ ID NOs: 20, 75, and 77, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOs: 20, 75, and 77 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in any one of SEQ ID NOs: 20, 75, and 77. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 20 or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 20. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 20 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 20. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 75 or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 75. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 75 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 75. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 77 or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 77. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 77 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 77.

[0212] In some embodiments, the gRNA targeting a target site for GATA2 comprises a sequence selected from any one of SEQ ID NOs: 2, 55, and 57 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 2, 55, and 57. In some embodiments, the gRNA224742003140 is the sequence set forth in any one of SEQ ID NOs: 2, 55, and 57. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 2, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 2. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 2. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 2. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 55, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 55. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 55. In some embodiments, the gRNA is a sequence set forth in SEQ ID NO: 55. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 57, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 57. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 57. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 57.

[0213] In some embodiments, the gRNA targeting a target site for GATA2 comprises modified nucleotides. In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 11, 65, and 67 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 11, 65, and 67. In some embodiments, the gRNA is set forth in the sequence set forth in any one of SEQ ID NOs: 11, 65, and 67. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 11 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 11. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 11. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 11. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 65 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 65. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 65. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 65. In some embodiments, the gRNA comprises a sequence set forth in SEQ ID NO: 67 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,22474200314099%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 67. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 11. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 67.

[0214] In some embodiments, the gRNA targets a target site for LMO2, such as any target site for LMO2 described herein in Section I.B. In some specific examples, the target site comprises the sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 101, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of any of the foregoing, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the target site comprises a contiguous portion of the sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 101 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 101. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 100, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 100. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 100 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 100. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO: 101, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 101, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 101. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 101 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 101.

[0215] In some embodiments, the gRNA targeting a target site for LM02 comprises a spacer sequence selected from any one of SEQ ID NOs: 21, 70-72, 120-139, 144, and 145, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of any one of SEQ ID NOs: 21, 70-72, 120-139, 144, and 145 that is 14, 15, 16, 17, 18, or 19 nucleotides in length. In some embodiments, the spacer sequence of the gRNA is set forth224742003140 in any one of SEQ ID NOs: 21, 70-72, 120-139, 144, and 145. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 21, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 21. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 21. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 21. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 70, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 70. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 70. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 70. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 71, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 71. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 71. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 71. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 72, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 72. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 72. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 72. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 120, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO:120. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 120. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO:120. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO:121, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 121. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 121. In some embodiments, the spacer sequence of the224742003140 gRNA is set forth in SEQ ID NO: 121. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 122, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 122. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 122. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 122. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 123, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 123. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 123. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 123. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 124, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 124. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 124. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 124. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 125, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 125. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 125. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 125. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 126, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 126. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 126. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 126. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 127, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 127. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 127. In some embodiments, the spacer sequence224742003140 of the gRNA is set forth in SEQ ID NO: 127. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 128, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 128. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 128. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 128. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 129, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 129. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 129. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 129. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 130, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 130. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 130. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 130. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 131, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 131. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 131. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 131. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 132, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 132. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 132. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 132. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 133, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 133. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 133. In some embodiments, the spacer sequence224742003140 of the gRNA is set forth in SEQ ID NO: 133. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 134, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 134. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 134. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 134. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 135, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 135. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 135. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 135. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 136, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 136. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 136. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 136. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 137, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 137. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 137. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 137. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 138, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 138. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 138. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 138. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 139, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 139. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 139. In some embodiments, the spacer sequence224742003140 of the gRNA is set forth in SEQ ID NO: 139. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 144, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 144. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 144. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 144. In some embodiments, the gRNA comprises a spacer sequence set forth in SEQ ID NO: 145, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 145. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 145. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 145.

[0216] In some embodiments, the gRNA targeting a target site for LM02 comprises a sequence selected from any one of SEQ ID NOs: 3, 50-52, 146-165, 170, and 171 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of any one of SEQ ID NOs: 3, 50-52, 146-165, 170, and 171. In some embodiments, the gRNA is the sequence set forth in any one of SEQ ID NOs: 3, 50-52, 146-165, 170, and 171. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 3, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 3. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 3. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 3. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 50, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 50. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 50. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 50. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 51, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 51. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 51. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 51. In some embodiments, the gRNA comprises the sequence set forth in224742003140SEQ ID NO:52, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 52. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 52. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 52. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 146, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 146. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 146. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 146. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 147, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 147. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 147. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 147. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 148, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 148. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 148. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 148. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 149, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 149. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 149. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 149. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 150, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 150. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 150. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 150. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 151, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 151. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 151. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 151. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 152, or a sequence having at or at least 80%, 85%, 90%, 91%,22474200314092%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 152. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 152. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 152. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 153, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 153. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 153. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 153. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 154, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 154. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 154. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 154. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 155, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 155. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 155. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 155. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 156, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 156. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 156. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 156. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 157, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 157. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 157. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 157. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 158, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 158. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 158. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 158. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 159, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%,22474200314099.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 159. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 159. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 159. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 160, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 160. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 160. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 160. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 161, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 161. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 161. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 161. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 162, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 162. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 162. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 162. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 163, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 163. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 163. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 163. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 164, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 164. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 164. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 164. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 165, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 165. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 165. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 165. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 170, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ224742003140ID NO: 170. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 170. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 170. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 171, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 171. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 171. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 171.

[0217] In some embodiments, the gRNA targeting a target site for LMO2 comprises modified nucleotides. In some embodiments, the gRNA comprises a sequence selected from any one of SEQ ID NOs: 12, 60-62, 114-117, 140-143, 166-169, and 172-181 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to any of the foregoing. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in any one of SEQ ID NOs: 12, 60-62, 114-117, 140-143, 166-169, and 172-181. In some embodiments, the gRNA is the sequence set forth in any one of SEQ ID NOs: 12, 60-62, 114-117, 140-143, 166-169, and 172-181. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 12, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 12. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 12. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 12. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 60, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 60. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 60. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 60. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 61, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 61. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 61. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 61. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 62, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 62. In some embodiments, the gRNA is a contiguous portion224742003140 of the sequence set forth in SEQ ID NO: 62. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 62. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 114, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 114. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 114. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 114. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 115, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 115. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 115. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 115. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 116, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 116. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 116. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 116. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 117, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 117. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 117. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 117. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 140, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 140. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 140. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 140. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 141, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 141. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 141. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 141. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 142, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ224742003140ID NO: 142. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 142. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 142. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 143, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 143. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 143. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 143. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 166, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 166. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 166. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 166. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 167, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 167. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 167. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 167. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 168, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 168. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 168. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 168. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 169, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 169. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 169. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 169. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 172, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 172. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 172. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 172. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 173, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%,22474200314099%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 173. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 173. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 173. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 174, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 174. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 174. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 174. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 175, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 175. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 175. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 175. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 176, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 176. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 176. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 176. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 177, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 177. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 177. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 177. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 178, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 178. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 178. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 178. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 179, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 179. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 179. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 179. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 180, or a sequence having at or at224742003140 least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 180. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 180. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 180. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 181, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 181. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 181. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 181.

[0218] In some embodiments, the gRNA targets a target site for ZEB2. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 31, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 31. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 31 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 31.

[0219] In some embodiments, the gRNA targeting a target site for ZEB2 comprises a spacer sequence set forth in SEQ ID NO: 22, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 22. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 22. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 22.

[0220] In some embodiments, the gRNA targeting a target site for ZEB2 comprises a sequence set forth in SEQ ID NO: 4 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 4. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 4. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 4.

[0221] In some embodiments, the gRNA targeting a target site for ZEB2 comprises modified nucleotides. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 13 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 13. In some embodiments,224742003140 the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 13. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 13.

[0222] In some embodiments, the gRNA targets a target site for HEY2. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:32, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 32, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 32. In some embodiments, the target site is a contiguous portion of the sequence set forth in SEQ ID NO: 32 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 32.

[0223] In some embodiments, the gRNA targeting a target site for HEY2 comprises a spacer sequence set forth in SEQ ID NO: 23, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 23. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 23. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 23.

[0224] In some embodiments, the gRNA targeting a target site for HEY2 comprises a sequence set forth in SEQ ID NO: 5 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 5. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 5. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 5.

[0225] In some embodiments, the gRNA targeting a target site for HEY2 comprises modified nucleotides. In some embodiments, the gRNA comprises the sequence set forth in SEQ ID NO: 14 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 14. In some embodiments, the gRNA is a contiguous portion of the sequence set forth in SEQ ID NO: 14. In some embodiments, the gRNA is the sequence set forth in SEQ ID NO: 14.

[0226] In some embodiments, the gRNA targets a target site for CEBPD. In some embodiments, the target site comprises the sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), a complementary sequence of SEQ ID NO: 33, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 33. In some embodiments, the224742003140 target site is a contiguous portion of the sequence set forth in SEQ ID NO: 33 that is 14, 15, 16, 17, 18, or 19 nucleotides long. In some embodiments, the target site is the sequence set forth in SEQ ID NO: 33.

[0227] In some embodiments, the gRNA targeting a target site for CEBPD comprises a spacer sequence set forth in SEQ ID NO: 24, or a contiguous portion thereof of at least 14 nt, or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 24. In some embodiments, the spacer sequence of the gRNA is a contiguous portion of SEQ ID NO: 24. In some embodiments, the spacer sequence of the gRNA is set forth in SEQ ID NO: 24.

[0228] In some embodiments, the gRNA targeting a target site for CEBPD comprises a sequence set forth in SEQ ID NO: 6 or a sequence having at or at least 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.5%, 99.9%, or 100% sequence identity to SEQ ID NO: 6. In some embodiments, the gRNA is a contiguous portion of SEQ ID NO: 6. In ...

Claims

224742003140CLAIMSWHAT IS CLAIMED:

1. A DNA-targeting system comprising one or more DNA-targeting modules, wherein the one or more DNA-targeting modules target one or more transcription factor genes for promoting differentiation of stem cells to HPCs, and wherein each of the one or more DNA- targeting modules comprises a fusion protein comprising: (a) a DNA-binding domain that binds to a target site for one of the one or more transcription factor genes; and (b) at least one transcriptional activation domain that increases transcription of the one or more transcription factor genes, wherein the one or more transcription factor genes is at least three genes GATA3, GATA2 and LM02.

2. A DNA-targeting system comprising one or more DNA-targeting modules, wherein the one or more DNA-targeting modules target one or more transcription factor genes for promoting differentiation of stem cells to HPCs, and wherein each of the one or more DNA- targeting modules comprises a fusion protein comprising:(a) a DNA-binding domain that binds to a target site for one of the one or more transcription factor genes, wherein the target site for at least one of the one or more transcription factor genes is a target site for LM02, and wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,869,125; and(b) at least one transcriptional activation domain that increases transcription of the one or more transcription factor genes.

3. The DNA-targeting system of claim 1 or 2, wherein transient delivery of the DNA-targeting system to a stem cell promotes differentiation to a HPC.

4. The DNA-targeting system of any of claims 1-3, wherein transient delivery of the DNA-targeting system to a stem cell promotes differentiation to a CD34+ cell.

5. The DNA-targeting system of any of claims 1-3, wherein transient delivery of the DNA-targeting system to a stem cell promotes differentiation to a CD43+ cell.2247420031406. The DNA-targeting system of any of claims 1-3, wherein transient delivery of the DNA-targeting system to a stem cell promotes differentiation to a CD34+ / CD43+ cell.

7. The DNA-targeting system of any of claims 3-6, wherein the stem cell is an induced pluripotent stem cell (iPSC).

8. The DNA-targeting system of any of claims 1-7, wherein the one or more DNA targeting modules is a plurality of DNA-targeting modules for increasing transcription of one or more transcription factor genes, wherein each DNA-targeting module targets a target site for one of the one or more genes.

9. The DNA-targeting system of claim 8, wherein the plurality of DNA-targeting modules is 2, 3, 4, 5, 6, 7, 8, or 9 DNA-targeting modules, each targeting one of the one or more transcription factor genes.

10. The DNA-targeting system of claim 8 or claim 9, wherein the plurality of DNA- targeting modules is 2 DNA-targeting modules, each targeting one of the one or more transcription factor genes.

11. The DNA-targeting system of any of claims 2-10, wherein the one or more transcription factor genes is two genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the two genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2.

12. The DNA-targeting system of claim 8 or claim 9, wherein the plurality of DNA- targeting modules is 3 DNA-targeting modules, each targeting one of the one or more transcription factor genes.

13. The DNA-targeting system of any of claims 2-9 and 12, wherein the one or more transcription factor genes is three genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the three224742003140 genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2.

14. The DNA-targeting system of claim 8 or claim 9, wherein the plurality of DNA- targeting modules is 4 DNA-targeting modules, each targeting one of the one or more transcription factor genes.

15. The DNA-targeting system of any of claims 1-9 and 14, wherein the one or more transcription factor genes is four genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the four genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2.

16. The DNA-targeting system of claim 8 or claim 9, wherein the plurality of DNA- targeting modules is 5 DNA-targeting modules, each targeting one of the one or more transcription factor genes.

17. The DNA-targeting system of any of claims 1-9 and 16, wherein the one or more transcription factor genes is five genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the five genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2.

18. The DNA-targeting system of claim 8 or claim 9, wherein the plurality of DNA- targeting modules is 6 DNA-targeting modules, each targeting one of the one or more transcription factor genes.

19. The DNA-targeting system of any of claims 1-9 and 18, wherein the one or more transcription factor genes is six genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the six genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2.22474200314020. The DNA-targeting system of claim 8 or claim 9, wherein the plurality of DNA- targeting modules is 7 DNA-targeting modules, each targeting one of the one or more transcription factor genes.

21. The DNA-targeting system of any of claims 1-9 and 20, wherein the one or more transcription factor genes is seven genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the seven genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2.

22. The DNA-targeting system of claim 8 or claim 9, wherein the plurality of DNA- targeting modules is 8 DNA-targeting modules, each targeting one of the one or more transcription factor genes.

23. The DNA-targeting system of any of claims 1-9 and 22, wherein the one or more transcription factor genes is eight genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the genes are BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2.

24. The DNA-targeting system of claim 8 or claim 9, wherein the plurality of DNA- targeting modules is 9 DNA-targeting modules, each targeting one of the one or more transcription factor genes.

25. The DNA-targeting system of any of claims 1-9 and 24, wherein the one or more transcription factor genes is nine genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2.

26. The DNA-targeting system of any of claims 1-25, wherein the target site for each of the one or more transcription factor genes is in the gene or a regulatory DNA element thereof.22474200314027. The DNA-targeting system of claim 26, wherein the regulatory DNA element is an enhancer or a promoter of the gene.

28. The DNA-targeting system of claim 27, wherein the regulatory DNA element is a promoter of the gene.

29. The DNA-targeting system of any of claims 1-28, wherein the target site for each of the one or more transcription factor genes is within 1000 base pairs of the transcription start site (TSS) of the gene.

30. The DNA-targeting system of any of claims 1-29, wherein the target site for each of the one or more transcription factor genes is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene.

31. The DNA-targeting system of any of claims 1-30, wherein the target site for each of the one or more transcription factor genes is within 550 base pairs upstream of the TSS of the gene.

32. The DNA-targeting system of any of claims 1-31, wherein the target site for each of the one or more transcription factor genes is selected from:(a) a target site for GATA3 having the sequence set forth in SEQ ID NO:28 or SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(b) a target site for GATA2 having the sequence set forth in SEQ ID NO:29, SEQ ID NO: 85 or SEQ ID NO:87, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(c) a target site for LM02 having the sequence set forth in any one of SEQ ID NOs:30, 80-82, and 94-113, 118, and 119, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;224742003140(d) a target site for ZEB2 having the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(e) a target site for HEY2 having the sequence set forth in SEQ ID NO:32, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(f) a target site for CEBPD having the sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(g) a target site for BMII having the sequence set forth in SEQ ID NO:34, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(h) a target site for LYL1 having the sequence set forth in SEQ ID NO:35 or SEQ ID NO: 83, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(i) a target site for TALI having the sequence set forth in SEQ ID NO:84 or SEQ ID NO: 86, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; or(j) a target site for CFOS having the sequence set forth in SEQ ID NO:88, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

33. The DNA-targeting system of any of claims 1 and 3-32, wherein the target site for each of the one or more transcription factor genes is selected from:(a) a target site for GATA3 having the sequence set forth in SEQ ID NO:28 or SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(b) a target site for GATA2 having the sequence set forth in SEQ ID NO:29, SEQ ID NO: 85 or SEQ ID NO:87, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;224742003140(c) a target site for LM02 having the sequence set forth in SEQ ID NO:30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(d) a target site for ZEB2 having the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(e) a target site for HEY2 having the sequence set forth in SEQ ID NO:32, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(f) a target site for CEBPD having the sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(g) a target site for BMII having the sequence set forth in SEQ ID NO:34, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(h) a target site for LYL1 having the sequence set forth in SEQ ID NO:35 or SEQ ID NO: 83, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(i) a target site for TALI having the sequence set forth in SEQ ID NO:84 or SEQ ID NO: 86, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; or(j) a target site for CFOS having the sequence set forth in SEQ ID NO:88, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

34. The DNA-targeting system of any of claims 2-33, wherein at least one of the one or more transcription factor genes is GATA3.

35. The DNA-targeting system of any of claims 1-34, wherein the target site for at least one of the one or more transcription factor genes is a target site for GATA3 having the sequence set forth in SEQ ID NO:28 or SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.22474200314036. The DNA-targeting system of any of claims 2-35, wherein at least one of the one or more transcription factor genes is GATA2.

37. The DNA-targeting system of any of claims 1-36, wherein the target site for at least one of the one or more transcription factor genes is a target site for GATA2 having the sequence set forth in SEQ ID NO:29, SEQ ID NO: 85 or SEQ ID NO:87, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

38. The DNA-targeting system of any of claims 1 and 3-37, wherein the target site for LM02 is within one of the regions defined by the following genomic coordinates:(a) chrl 1:33,867,158 to chrl 1:33,873,567;(b) chrl l: 33,876,536 to chrl 1:33,878,097;(c) chrl 1:33,890,358 to chrl 1:33,896,210;(d) chrl l: 33,906,104 to chrl 1:33,908,030;(e) chrl l: 33,911,674 to chrl 1:33,913,494;(f) chrl 1:33,920,461 to chrl 1:33,922,260; or(g) chrl 1:33,929,929 to chrl 1:33,932,651.

39. The DNA-targeting system of any of claims 1 and 3-38, wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,867,158 to chrl 1:33,873,567.

40. The DNA-targeting system of any of claims 1 and 3-39, wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,867,215 to chrl 1:33,872,057.22474200314041. The DNA-targeting system of any of claims 1 and 3-40, wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,869,125.

42. The DNA-targeting system of any of claims 1-41, wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,868,742.

43. The DNA-targeting system of any of claims 1 and 3-38, wherein the target site for at least one of the one or more transcription factor genes is a target site for LM02 comprising the sequence set forth in any one of SEQ ID NOs: 30, 80-82, 94-113, 118, and 119, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

44. The DNA-targeting system of any of claims 1, 3-39, and 43, wherein the target site for at least one of the one or more transcription factor genes is a target site for LM02 comprising the sequence set forth in any one of SEQ ID NOs: 94-113, 118, and 119, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

45. The DNA-targeting system of any of claims 1-41, 43, and 44, wherein the target site for LM02 comprises the sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 101, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

46. The DNA-targeting system of any of claims 1-45, wherein the target site for LM02 comprises the sequence set forth in SEQ ID NO: 101, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

47. The DNA-targeting system of any of claims 1-41 and 43-45, wherein the target site for LM02 comprises the sequence set forth in SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.22474200314048. The DNA-targeting system of any of claims 1 and 3-38, wherein the target site for at least one of the one or more transcription factor genes is a target site for LM02 having the sequence set forth in SEQ ID NO:30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

49. The DNA-targeting system of any of claims 2-9 and 12-48, wherein one or more transcription factor genes is at least three genes GATA3, GATA2 and LM02.

50. The DNA-targeting system of any of claim 1-49, wherein at least one of the one or more transcription factor genes is BMI1.

51. The DNA-targeting system of any of claims 1-50, wherein the target site for at least one of the one or more transcription factor genes is a target site for BMI1 having the sequence set forth in SEQ ID NO: 34, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

52. The DNA-targeting system of any of claim 1-51, wherein at least one of the one or more transcription factor genes is CEBPD.

53. The DNA-targeting system of any of claims 1-52, wherein the target site for at least one of the one or more transcription factor genes is a target site for CEBPD having the sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

54. The DNA-targeting system of any of claims 1-53, wherein at least one of the one or more transcription factor genes is LYLE55. The DNA-targeting system of any of claims 1-54 wherein the target site for at least one of the one or more transcription factor genes is a target site for LYL1 having the224742003140 sequence set forth in SEQ ID NO:35 or SEQ ID NO: 83, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

56. The DNA-targeting system of any of claims 1-9 and 18-55, wherein one or more transcription factor genes is at least six genes GATA3, GATA2, LM02, BMI, CEBPD, and LYLE57. The DNA-targeting system of any of claims 1-56, wherein at least one of the one or more transcription factor genes is HEY2.

58. The DNA-targeting system of any of claims 1-57, wherein the target site for at least one of the one or more transcription factor genes is a target site for HEY2 having the sequence set forth in SEQ ID NO: 32, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

59. The DNA-targeting system of any of claims 1-58, wherein at least one of the one or more transcription factor genes is ZEB2.

60. The DNA-targeting system of any of claims 1-59, wherein the target site for at least one of the one or more transcription factor genes is a target site for ZEB2 having the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

61. The DNA-targeting system of any of claims 1-60, wherein at least one of the one or more transcription factor genes is TALI.

62. The DNA-targeting system of any of claims 1-61, wherein the target site for at least one of the one or more transcription factor genes is a target site for TALI having the sequence set forth in SEQ ID NO:84 or SEQ ID NO:86, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.22474200314063. The DNA-targeting system of any of the claims 1-62, wherein at least one of the one or more transcription factor genes is CFOS.

64. The DNA-targeting system of any of claims 1-63, wherein the target site for at least one of the one or more transcription factor genes is a target site for CFOS having the sequence set forth in SEQ ID NO:88, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

65. The DNA-targeting system of any of claims 1-64, wherein the DNA-targeting system does not introduce a genetic disruption or a DNA break.

66. The DNA-targeting system of any of claims 1-65, wherein the fusion protein of each DNA-targeting module comprises a DNA-binding domain selected from: a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or a variant thereof; a zinc finger protein (ZFP); a transcription activator- like effector (TALE); a meganuclease; a homing endonuclease; or an I-Scel enzyme or a variant thereof, optionally wherein the DNA- binding domain comprises a catalytically inactive variant of any of the foregoing, wherein, when the DNA-binding domain of the fusion protein comprises a Cas protein, the DNA-targeting system further comprises one or more gRNAs, each capable of targeting the Cas protein to the target site for one of the one or more transcription factor genes.

67. The DNA-targeting system of any of claims 1-66, wherein the DNA-binding domain of each of the one or more DNA-targeting modules is a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof, and each of the one or more DNA-targeting modules further comprises one or more gRNAs for targeting the DNA- binding domain to the target site of one of the one or more transcription factor genes.

68. The DNA-targeting system of claim 66 or claim 67, wherein the Cas protein or variant thereof is a deactivated (dCas) protein.

69. A DNA-targeting system comprising:224742003140(a) a fusion protein comprising a DNA-binding domain comprising a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and(b) one or more gRNAs that target a target site for one or more transcription factor genes, wherein the DNA-targeting system increases transcription of the one or more transcription factor genes to promote differentiation of stem cells to HPCs, wherein the one or more transcription factors is at least three genes GATA3, GATA2 and LM02.

70. A DNA-targeting system comprising:(a) a fusion protein comprising a DNA-binding domain comprising a deactivated Cas (dCas) protein and at least one transcriptional activation domain; and(b) one or more gRNAs that target a target site for one or more transcription factor genes, wherein the target site for at least one of the one or more transcription factor genes is a target site for LM02, and wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,869, 125, wherein the DNA-targeting system increases transcription of the one or more transcription factor genes to promote differentiation of stem cells to HPCs.

71. The DNA-targeting system of any of claims 68-70, wherein the dCas protein lacks nuclease activity.

72. The DNA-targeting system of claim any of claims 68-71, wherein the dCas protein is a dCas9 protein.

73. The DNA-targeting system of any of claims 68-72, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

74. The DNA-targeting system of claim 73, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 41.22474200314075. The DNA-targeting system of claim 73 or claim 74, wherein the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 280, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

76. The DNA-targeting system of any of claims 73-75, wherein the dSaCas9 protein is set forth in SEQ ID NO: 280.

77. The DNA-targeting system of any of claims 68-72, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

78. The DNA-targeting system of claim 77, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 43.

79. The DNA-targeting system of claim 77 or claim 78, wherein the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 281, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

80. The DNA-targeting system of any of claims 77-79, wherein the dSpCas9 protein is set forth in SEQ ID NO: 281.

81. The DNA-targeting system of any of claims 66-80, wherein the one or more gRNAs comprise a gRNA spacer that is complementary to the target site of the gene.

82. The DNA-targeting system of any of claims 66-81, wherein the gRNA is selected from:(a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79, or a contiguous portion thereof of at least 14 nt;(b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77, or a contiguous portion thereof of at least 14 nt;224742003140(c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 21, 70-72, 120-139, 144, and 145, or a contiguous portion thereof of at least 14 nt;(d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22, or a contiguous portion thereof of at least 14 nt;(e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23, or a contiguous portion thereof of at least 14 nt;(f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24, or a contiguous portion thereof of at least 14 nt;(g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt;(h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73, or a contiguous portion thereof of at least 14 nt;(i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76, or a contiguous portion thereof of at least 14 nt;(j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; or(k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78, or a contiguous portion thereof of at least 14 nt.

83. The DNA-targeting system of any of claims 66-82, wherein the gRNA is selected from:224742003140(a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79, or a contiguous portion thereof of at least 14 nt;(b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77, or a contiguous portion thereof of at least 14 nt;(c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 126 or 127, or a contiguous portion thereof of at least 14 nt;(d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22, or a contiguous portion thereof of at least 14 nt;(e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23, or a contiguous portion thereof of at least 14 nt;(f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24, or a contiguous portion thereof of at least 14 nt;(g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt;(h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73, or a contiguous portion thereof of at least 14 nt;(i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76, or a contiguous portion thereof of at least 14 nt;(j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; or224742003140(k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78, or a contiguous portion thereof of at least 14 nt.

84. The DNA-targeting system of any of claims 66-69 and 71-82, wherein the gRNA is selected from:(a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79, or a contiguous portion thereof of at least 14 nt;(b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77, or a contiguous portion thereof of at least 14 nt;(c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 21, SEQ ID NO: 70, SEQ ID NO:71 or SEQ ID NO:72, or a contiguous portion thereof of at least 14 nt;(d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22, or a contiguous portion thereof of at least 14 nt;(e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23, or a contiguous portion thereof of at least 14 nt;(f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24, or a contiguous portion thereof of at least 14 nt;(g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt;(h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73, or a contiguous portion thereof of at least 14 nt;224742003140(i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76, or a contiguous portion thereof of at least 14 nt;(j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; or(k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78, or a contiguous portion thereof of at least 14 nt.

85. The DNA-targeting system of any of claims 66-82, wherein the gRNA is selected from:(a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79;(b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77;(c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 21, 70-72, 120-139, 144, and 145;(d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22;(e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23;(f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24;(g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25;(h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73;(i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76;(j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25; or224742003140(k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78.

86. The DNA-targeting system of any of claims 66-83 and 85, wherein the gRNA is selected from:(a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79;(b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77;(c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 126 or SEQ ID NO: 127;(d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22;(e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23;(f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24;(g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25;(h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73;(i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76;(j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25; or(k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78.

87. The DNA-targeting system of any of claims 66-69, 71-82, 84, and 85, wherein the gRNA is selected from:(a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79;224742003140(b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77;(c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 21, SEQ ID NO: 70, SEQ ID NO:71 or SEQ ID NO:72;(d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22;(e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23;(f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24;(g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25;(h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73;(i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76;(j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25; or(k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78.

88. The DNA-targeting system of any of claims 66-87, wherein the gRNA comprises a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length.

89. The DNA-targeting system of any of claims 66-88, wherein the gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

90. The DNA-targeting system of any of claims 69-72 and 77-89, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 91.22474200314091. The DNA-targeting system of any of claims 69-76 and 81-89, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 93.

92. The DNA-targeting system of any of claims 69-91, wherein the gRNA further comprises 2’ MeO modified bases and / or phosphorothiate backbone modifications.

93. The DNA-targeting system of any of claims 1-92, wherein the one or more transcription factor genes include GATA2, GATA3, LM02 and BMI1.

94. The DNA-targeting system of any of claims 1-92, wherein the one or more transcription factor genes include GATA2, GATA3, LM02 and LYLl.

95. The DNA-targeting system of any of claims 1-92, wherein the one or more transcription factor genes include GATA2, GATA3, LM02 and CEBPD.

96. The DNA-targeting system of any of claims 1-92, wherein the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, CEBPD, and LYLl.

97. The DNA-targeting system of any of claims 1-92, wherein the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, CEBPD, and TALI.

98. The DNA-targeting system of any of claims 1-92, wherein the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, and TALI.

99. The DNA-targeting system of any of claims 1-95, wherein the one or more transcription factor genes are BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYLl, and ZEB2.

100. The DNA-targeting system of any of claims 1-92, wherein the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, CEBPD, LYLl, TALI, HEY2, and CEOS.224742003140101. The DNA-targeting system of any of claims 1-92, wherein the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, CEBPD, ZEB2, TALI, HEY2, and CFOS.

102. The DNA-targeting system of any of claims 1-92, wherein the one or more transcription factor genes include GATA2, GATA3, LM02, LYL1, CEBPD, ZEB2, TALI, HEY2, and CFOS.

103. The DNA-targeting system of any of claims 1-102, wherein the at least one transcriptional activator effector domain is selected from the group consisting of: a VP64 domain, a p65 activation domain, a p300 domain, an Rta domain, a CBP domain, a VPR domain, a VPH domain, an HSF1 domain, a TET protein domain, optionally wherein the TET protein is TET1, a SunTag domain, or a domain, portion, variant, or truncation of any of the foregoing.

104. The DNA-targeting system of any of claims 1-103, wherein the at least one transcriptional activator effector domain comprises at least one VP 16 domain, and / or a VP 16 tetramer (“VP64”) or a variant thereof.

105. The DNA-targeting system of any of claims 1-104, wherein the at least one transcriptional activator effector domain comprises a VP64 domain or a variant or portion thereof that exhibits transcriptional activation activity.

106. The DNA-targeting system of any of claims 1-105, wherein the at least one transcriptional activator effector domain is VP64.

107. The DNA-targeting system of claim 106, wherein the VP64 is positioned N- terminal and / or C-terminal to the DNA-binding domain.

108. The DNA-targeting system of any of claims 1-107, wherein the at least one transcriptional activator effector domain comprises two copies of VP64.224742003140109. The DNA-targeting system of any of claims 1-108, wherein the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 47, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 47.

110. The DNA-targeting system of any of claims 1-109, wherein the at least one transcriptional activator effector domain comprises the amino acid sequence set forth in SEQ ID NO: 48, a portion thereof, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to SEQ ID NO: 48.

111. The DNA-targeting system of any of claims 1-110, wherein the fusion protein comprises the sequence set forth in SEQ ID NO: 40, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

112. A combination of gRNAs comprising two or more gRNAs, each selected from a gRNA that targets a target site for one or more transcription factor genes, wherein the one or more transcription factor genes is at least three genes GATA3, GATA2 and LM02.

113. A combination of gRNAs comprising two or more gRNAs, each selected from a gRNA that targets a target site for one or more transcription factor genes, wherein the target site for at least one of the one or more transcription factor genes is a target site for LM02, and wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,869, 125.

114. The combination of gRNAs of claim 112 or claim 113, wherein the target site for each of the one or more transcription factor genes is in the gene or a regulatory DNA element thereof.

115. The combination of gRNAs of claim 114, wherein the regulatory DNA element is an enhancer or a promoter.224742003140116. The combination of gRNAs of claim 114 or claim 115, wherein the regulatory DNA element is a promoter of the gene.

117. The combination of gRNAs of any of claims 112- 116, wherein the target site for each of the one or more transcription factor genes is within 1000 base pairs of the transcription start site (TSS) of the gene.

118. The combination of gRNAs of any of claims 112-117, wherein the target site for each of the one or more transcription factor genes is within 20 base pairs, 50 base pairs, 100 base pairs, 200 base pairs, 300 base pairs, 400 base pairs, 500 base pairs, 600 base pairs, or any value between any of the foregoing, of the TSS of the gene.

119. The combination of gRNAs of any of claims 112-118, wherein the target site for each of the one or more transcription factor genes is with 550 base pairs upstream of the TSS of the gene.

120. The combination of gRNAs of any of claims 113-119 comprising 2, 3, 4, 5, 6, 7, 8, or 9 different gRNAs, each that targets a target site of one of the one or more transcription factor genes.

121. The combination of gRNAs of any of claims 113- 120, wherein the one or more transcription factor genes is two genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the two genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2.

122. The combination of gRNAs of any of claims 112- 119 comprising 3, 4, 5, 6, 7, 8, or 9 different gRNAs, each that targets a target site of one of the one or more transcription factor genes.

123. The combination of gRNAs of any of claims 113- 120 and 122, wherein the one or more transcription factor genes is three genes selected from the group consisting of BMI1,224742003140CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the three genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2.

124. The combination of gRNAs of any of claims 112- 120 and 122, wherein the one or more transcription factor genes is four genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the four genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2.

125. The combination of gRNAs of any of claims 112-120 and 122, wherein the one or more transcription factor genes is five genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the five genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2.

126. The combination of gRNAs of any of claims 112-120 and 122, wherein the one or more transcription factor genes is six genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the six genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2.

127. The combination of gRNAs of any of claims 112-120 and 122, wherein the one or more transcription factor genes is seven genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the seven genes are selected from the group consisting of BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2.

128. The combination of gRNAs of any of claims 112-120 and 122, wherein the one or more transcription factor genes is eight genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2, optionally wherein the genes are BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2.224742003140129. The combination of gRNAs of any of claims 112-120 and 122, wherein the one or more transcription factor genes is nine genes selected from the group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2.

130. The combination of gRNAs of any of claims 112- 129, wherein the target site for each of the one or more transcription factor genes is selected from:(a) a target site for GATA3 having the sequence set forth in SEQ ID NO:28 or SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(b) a target site for GATA2 having the sequence set forth in SEQ ID NO:29, SEQ ID NO: 85 or SEQ ID NO:87, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(c) a target site for LM02 having the sequence set forth in any one of SEQ ID NOs:30, 80-82, 94-113, 118, and 119, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(d) a target site for ZEB2 having the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(e) a target site for HEY2 having the sequence set forth in SEQ ID NO:32, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(f) a target site for CEBPD having the sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(g) a target site for BMII having the sequence set forth in SEQ ID NO:34, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(h) a target site for LYL1 having the sequence set forth in SEQ ID NO:35 or SEQ ID NO: 83, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;224742003140(i) a target site for TALI having the sequence set forth in SEQ ID NO:84 or SEQ ID NO: 86, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; or(j) a target site for CFOS having the sequence set forth in SEQ ID NO:88, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

131. The combination of gRNAs of any of claims 112-130, wherein the target site for each of the one or more transcription factor genes is selected from:(a) a target site for GATA3 having the sequence set forth in SEQ ID NO:28 or SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(b) a target site for GATA2 having the sequence set forth in SEQ ID NO:29, SEQ ID NO: 85 or SEQ ID NO:87, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(c) a target site for LM02 having the sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 101, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(d) a target site for ZEB2 having the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(e) a target site for HEY2 having the sequence set forth in SEQ ID NO:32, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(f) a target site for CEBPD having the sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(g) a target site for BMII having the sequence set forth in SEQ ID NO:34, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;224742003140(h) a target site for LYL1 having the sequence set forth in SEQ ID NO:35 or SEQ ID NO: 83, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(i) a target site for TALI having the sequence set forth in SEQ ID NO:84 or SEQ ID NO: 86, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; or(j) a target site for CFOS having the sequence set forth in SEQ ID NO:88, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

132. The combination of gRNAs of any of claims 112 and 114-130, wherein the target site for each of the one or more transcription factor genes is selected from:(a) a target site for GATA3 having the sequence set forth in SEQ ID NO:28 or SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(b) a target site for GATA2 having the sequence set forth in SEQ ID NO:29, SEQ ID NO: 85 or SEQ ID NO:87, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(c) a target site for LM02 having the sequence set forth in SEQ ID NO:30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(d) a target site for ZEB2 having the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(e) a target site for HEY2 having the sequence set forth in SEQ ID NO:32, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(f) a target site for CEBPD having the sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;224742003140(g) a target site for BMII having the sequence set forth in SEQ ID NO:34, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(h) a target site for LYL1 having the sequence set forth in SEQ ID NO:35 or SEQ ID NO: 83, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing;(i) a target site for TALI having the sequence set forth in SEQ ID NO:84 or SEQ ID NO: 86, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing; or(j) a target site for CFOS having the sequence set forth in SEQ ID NO:88, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

133. The combination of gRNAs of any of claims 113-132, wherein at least one of the one or more transcription factor genes is GATA3.

134. The combination of gRNAs of any of claims 112-133, wherein the target site for at least one of the one or more transcription factor genes is a target site for GATA3 having the sequence set forth in SEQ ID NO:28 or SEQ ID NO: 89, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

135. The combination of gRNAs of any of claims 113-134, wherein at least one of the one or more transcription factor genes is GATA2.

136. The combination of gRNAs of any of claims 112-135, wherein the target site for at least one of the one or more transcription factor genes is a target site for GATA2 having the sequence set forth in SEQ ID NO:29, SEQ ID NO: 85 or SEQ ID NO:87, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

137. The combination of gRNAs of any of claims 112 and 114-136, wherein the target site for LM02 is within one of the regions defined by the following genomic coordinates:(a) chrl 1:33,867,158 to chrl 1:33,873,567;224742003140(b) chrll: 33,876,536 to chrl 1:33,878,097;(c) chrl 1:33,890,358 to chrl 1:33,896,210;(d) chrl l: 33,906,104 to chrl 1:33,908,030;(e) chrl l: 33,911,674 to chrl 1:33,913,494;(f) chrl 1:33,920,461 to chrl 1:33,922,260; or(g) chrl 1:33,929,929 to chrl 1:33,932,651.

138. The combination of gRNAs of any of claims 112 and 114-137, wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,867,158 to chrl 1:33,873,567.

139. The combination of gRNAs of any of claims 112 and 114-138, wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,867,215 to chrl 1:33,872,057.

140. The combination of gRNAs of any of claims 112 and 114-139, wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,869,125.

141. The combination of gRNAs of any of claims 112-140, wherein the target site for LM02 is within a region defined by the following genomic coordinates: chrl 1:33,868,723 to chrl 1:33,868,742.

142. The combination of gRNAs of any of claims 112 and 114-137, wherein the target site for at least one of the one or more transcription factor genes is a target site for LM02 having the sequence set forth in any one of SEQ ID NOS: 30, 80-82, 94-113, 118, and 119, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.224742003140143. The combination of gRNAs of any of claims 112- 140, wherein the target site for at least one of the one or more transcription factor genes is a target site for LM02 having the sequence set forth in SEQ ID NO: 100 or SEQ ID NO: 101, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

144. The combination of gRNAs of any of claims 112- 143, wherein the target site for at least one of the one or more transcription factor genes is a target site for LM02 having the sequence set forth in SEQ ID NO: 101, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

145. The combination of gRNAs of any of claims 112-140 and 142-144, wherein the target site for at least one of the one or more transcription factor genes is a target site for LM02 having the sequence set forth in SEQ ID NO: 100, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

146. The combination of gRNAs of any of claims 112, 114-137, and 142, wherein the target site for at least one of the one or more transcription factor genes is a target site for LM02 having the sequence set forth in SEQ ID NO:30, SEQ ID NO: 80, SEQ ID NO: 81 or SEQ ID NO: 82, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

147. The combination of gRNAs of any of claims 113- 146, wherein one or more transcription factor genes is at least three genes GATA3, GATA2 and LM02.

148. The combination of gRNAs of any of claims 112-147, wherein at least one of the one or more transcription factor genes is BMI1.

149. The combination of gRNAs of any of claims 112-148, wherein the target site for at least one of the one or more transcription factor genes is a target site for BMI1 having the sequence set forth in SEQ ID NO: 34, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.224742003140150. The combination of gRNAs of any of claims 112- 149, wherein at least one of the one or more transcription factor genes is CEBPD.

151. The combination of gRNAs of any of claims 112-150, wherein the target site for at least one of the one or more transcription factor genes is a target site for CEBPD having the sequence set forth in SEQ ID NO:33, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

152. The combination of gRNAs of any of claims 112-151, wherein at least one of the one or more transcription factor genes is LYLE153. The combination of gRNAs of any of claims 112-152, wherein the target site for at least one of the one or more transcription factor genes is a target site for LYL1 having the sequence set forth in SEQ ID NO:35 or SEQ ID NO: 83, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

154. The combination of gRNAs of any of claims 112-153, wherein one or more transcription factor genes is at least six genes GATA3, GATA2, LM02, BMI1, CEBPD, and LYLE155. The combination of gRNAs of any of claims 112-154, wherein at least one of the one or more transcription factor genes is HEY2.

156. The combination of gRNAs of any of claims 112-155, wherein the target site for at least one of the one or more transcription factor genes is a target site for HEY2 having the sequence set forth in SEQ ID NO: 32, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

157. The combination of gRNAs of any of claims 112-156, wherein at least one of the one or more transcription factor genes is ZEB2.224742003140158. The combination of gRNAs of any of claims 112-157, wherein the target site for at least one of the one or more transcription factor genes is a target site for ZEB2 having the sequence set forth in SEQ ID NO:31, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

159. The combination of gRNAs of any of claims 112-158, wherein at least one of the one or more transcription factor genes is TALI.

160. The combination of gRNAs of any of claims 112-159, wherein the target site for at least one of the one or more transcription factor genes is a target site for TALI having the sequence set forth in SEQ ID NO:84 or SEQ ID NO:86, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

161. The combination of gRNAs of any of claims 112-160, wherein at least one of the one or more transcription factor genes is CFOS.

162. The combination of gRNAs of any of claims 112-161, wherein the target site for at least one of the one or more transcription factor genes is a target site for CFOS having the sequence set forth in SEQ ID NO:88, a contiguous portion thereof of at least 14 nucleotides (nt), or a complementary sequence of any of the foregoing.

163. The combination of gRNAs of any of claims 112-162, wherein the two or more gRNAs comprise a gRNA spacer that is complementary to the target site of the gene.

164. The combination of gRNAs of any of claims 112-163, wherein the gRNA is selected from:(a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79, or a contiguous portion thereof of at least 14 nt;(b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77, or a contiguous portion thereof of at least 14 nt;224742003140(c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 21, 70-72, 120-139, 144, and 145, or a contiguous portion thereof of at least 14 nt;(d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22, or a contiguous portion thereof of at least 14 nt;(e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23, or a contiguous portion thereof of at least 14 nt;(f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24, or a contiguous portion thereof of at least 14 nt;(g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt;(h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73, or a contiguous portion thereof of at least 14 nt;(i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76, or a contiguous portion thereof of at least 14 nt;(j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; or(k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78, or a contiguous portion thereof of at least 14 nt.

165. The combination of gRNAs of any of claims 112-164, wherein the gRNA is selected from:224742003140(a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79, or a contiguous portion thereof of at least 14 nt;(b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77, or a contiguous portion thereof of at least 14 nt;(c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 126 or SEQ ID NO: 127, or a contiguous portion thereof of at least 14 nt;(d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22, or a contiguous portion thereof of at least 14 nt;(e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23, or a contiguous portion thereof of at least 14 nt;(f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24, or a contiguous portion thereof of at least 14 nt;(g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt;(h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73, or a contiguous portion thereof of at least 14 nt;(i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76, or a contiguous portion thereof of at least 14 nt;(j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; or224742003140(k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78, or a contiguous portion thereof of at least 14 nt.

166. The combination of gRNAs of any of claims 112 and 114-164, wherein the gRNA is selected from:(a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79, or a contiguous portion thereof of at least 14 nt;(b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77, or a contiguous portion thereof of at least 14 nt;(c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 21, SEQ ID NO: 70, SEQ ID NO:71 or SEQ ID NO:72, or a contiguous portion thereof of at least 14 nt;(d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22, or a contiguous portion thereof of at least 14 nt;(e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23, or a contiguous portion thereof of at least 14 nt;(f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24, or a contiguous portion thereof of at least 14 nt;(g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt;(h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73, or a contiguous portion thereof of at least 14 nt;224742003140(i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76, or a contiguous portion thereof of at least 14 nt;(j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25, or a contiguous portion thereof of at least 14 nt; or(k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78, or a contiguous portion thereof of at least 14 nt.

167. The combination of gRNAs of any of claims 112-164, wherein the gRNA is selected from:(a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79;(b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77;(c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in any one of SEQ ID NOs: 21, 70-72, 120-139, 144, and 145;(d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22;(e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23;(f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24;(g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25;(h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73;(i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76;(j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25; or224742003140(k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78.

168. The combination of gRNAs of any of claims 112-165 and 167, wherein the gRNA is selected from:(a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79;(b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77;(c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 126 or SEQ ID NO: 127;(d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22;(e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23;(f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24;(g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25;(h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73;(i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76;(j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25; or(k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78.

169. The combination of gRNAs of any of claims 112 and 114-164, 166, and 167, wherein the gRNA is selected from:(a) a gRNA targeting a target site for GATA3 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 19 or SEQ ID NO: 79;224742003140(b) a gRNA targeting a target site for GATA2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 20, SEQ ID NO: 75 or SEQ ID NO: 77;(c) a gRNA targeting a target site for LM02 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 21, SEQ ID NO: 70, SEQ ID NO:71 or SEQ ID NO:72;(d) a gRNA targeting a target site for ZEB2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 22;(e) a gRNA targeting a target site for HEY2 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 23;(f) a gRNA targeting a target site for CEBPD comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 24;(g) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25;(h) a gRNA targeting a target site for LYL1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 26 or SEQ ID NO:73;(i) a gRNA targeting a target site for TALI comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 74 or SEQ ID NO: 76;(j) a gRNA targeting a target site for BMI1 comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 25; or(k) a gRNA targeting a target site for CFOS comprising a gRNA spacer sequence comprising the sequence set forth in SEQ ID NO: 78.

170. The combination of gRNAs of any of claims 112-169, wherein the gRNA comprises a spacer sequence between 14 nt and 24 nt, or between 16 nt and 22 nt in length.

171. The combination of gRNAs of any of claims 112-170, wherein the gRNA comprises a spacer sequence that is 18 nt, 19 nt, 20 nt, 21 nt, or 22 nt in length.

172. The combination of gRNAs of any of claims 112-171, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 91.224742003140173. The combination of gRNAs of any of claims 112-171, wherein the gRNA further comprises a scaffold sequence set forth in SEQ ID NO: 93.

174. The combination of gRNAs of any of claims 113-173, comprising 2, 3, 4, 5, 6, 7, 8, or 9 different gRNAs, optionally each comprising the same scaffold sequence.

175. The combination of gRNAs of any of claims 112-174, comprising 3, 4, 5, 6, 7, 8, or 9 different gRNAs, optionally each comprising the same scaffold sequence.

176. The combination of gRNAs of any of claims 112-175, wherein the gRNA further comprises 2’ MeO modified bases and / or phosphorothiate backbone modifications.

177. The combination of gRNAs of any of claims 112- 176, wherein the one or more transcription factor genes include GATA2, GATA3, LM02 and BMI1.

178. The combination of gRNAs of any of claims 112- 176, wherein the one or more transcription factor genes include GATA2, GATA3, LM02 and LYLl.

179. The combination of gRNAs of any of claims 112- 176, wherein the one or more transcription factor genes include GATA2, GATA3, LM02 and CEBPD.

180. The combination of gRNAs of any of claims 112- 176, wherein the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, CEBPD, and LYLl.

181. The combination of gRNAs of any of claims 112-176, wherein the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, CEBPD, and TALI.

182. The combination of gRNAs of any of claims 112- 176, wherein the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, and TALI.224742003140183. The combination of gRNAs of any of claims 112- 176, wherein the one or more transcription factor genes include BMI1, CEBPD, GATA2, GATA3, HEY2, LM02, LYL1, and ZEB2.

184. The combination of gRNAs of any of claims 112- 176, wherein the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, CEBPD, LYL1, TALI, HEY2, and CFOS.

185. The combination of gRNAs of any of claims 112- 176, wherein the one or more transcription factor genes include GATA2, GATA3, LM02, BMI1, CEBPD, ZEB2, TALI, HEY2, and CFOS.

186. The combination of gRNAs of any of claims 112- 176, wherein the one or more transcription factor genes include GATA2, GATA3, LM02, LYL1, CEBPD, ZEB2, TALI, HEY2, and CFOS.

187. A Cas-guide RNA (gRNA) combination comprising:(a) a fusion protein comprising a Clustered Regularly Interspaced Short Palindromic Repeats associated (Cas) protein or variant thereof fused to a transcriptional activation domain; and(b) a combination of gRNAs any of claims 112-186.

188. The Cas-gRNA combination of claim 187, wherein the Cas protein or variant thereof is a deactivated (dCas) protein.

189. The Cas-gRNA combination of claim 188, wherein the dCas protein lacks nuclease activity.

190. The Cas-gRNA combination of claim 188 or claim 189, wherein the dCas protein is a dCas9 protein.224742003140191. The Cas-gRNA combination of claim 190, wherein the dCas9 protein is a Staphylococcus aureus dCas9 (dSaCas9) protein.

192. The Cas-gRNA combination of claim 191, wherein the dSaCas9 comprises at least one amino acid mutation selected from D10A and N580A, with reference to numbering of positions of SEQ ID NO: 41.

193. The Cas-gRNA combination of claim 191 or claim 192, wherein the dSaCas9 protein comprises the sequence set forth in SEQ ID NO: 280, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

194. The Cas-gRNA combination of any of claims 191-193, wherein the dSaCas9 protein is set forth in SEQ ID NO: 280.

195. The Cas-gRNA combination of claim 190, wherein the dCas9 protein is a Streptococcus pyogenes dCas9 (dSpCas9) protein.

196. The Cas-gRNA combination of claim 195, wherein the dSpCas9 protein comprises at least one amino acid mutation selected from D10A and H840A, with reference to numbering of positions of SEQ ID NO: 43.

197. The Cas-gRNA combination any of claim 195 or claim 196, wherein the dSpCas9 protein comprises the sequence set forth in SEQ ID NO: 281, or an amino acid sequence that has at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity thereto.

198. The Cas-gRNA combination of any of claims 195-197, wherein the dSpCas9 protein is set forth in SEQ ID NO: 281.

199. A polynucleotide encoding the DNA-targeting system of any of claims 1-111.

200. A polynucleotide encoding at least one fusion protein of the DNA-targeting system of any of claims 1-111.224742003140201. A polynucleotide encoding at least one DNA-targeting module of the DNA- targeting system of any of claims 1-111.

202. A polynucleotide encoding at least one gRNA of the combination of gRNAs of any of claims 112-186.

203. A polynucleotide encoding the combination of gRNAs of any of claims 112-186.

204. A polynucleotide encoding at least one fusion protein of the DNA-targeting system of any of claims 1-111 and at least one gRNA of the combination of gRNAs of any of claims 112-186.

205. A polynucleotide encoding the fusion protein and at least one gRNA of the Cas- gRNA combination of any of claims 187-198.

206. A plurality of polynucleotides, wherein a polynucleotide encodes at least one fusion protein of the DNA-targeting system of any of claims 1-111 or Cas-gRNA combination of any of claims 187-198 and a further polynucleotide encodes at least one gRNA of the combination of gRNAs of any of claims 112-186.

207. A vector comprising the polynucleotide of any of claims 199-205.

208. A vector comprising the plurality of polynucleotides of claim 206.

209. The vector of claim 207 or claim 208, wherein the vector is a viral vector.

210. The vector of claim 209, wherein the viral vector is an adeno-associated virus (AAV) vector.

211. The vector of claim 207 or claim 208, wherein the vector is a non- viral vector.224742003140212. The vector of claim 211, wherein the non- viral vector is selected from: a lipid nanoparticle, a liposome, an exosome, or a cell penetrating peptide.

213. A pharmaceutical composition comprising the DNA-targeting system of any of claims 1-111, the Cas-gRNA combination of any of claims 187-198, the polynucleotide of any of claims 199-205, the plurality of polynucleotides of claim 206, or the vector of any of claims 207-212.

214. The pharmaceutical composition of claim 213 comprising a pharmaceutically acceptable excipient.

215. A method of differentiating a population of stem cells comprising introducing the DNA-targeting system of any one of claims 1-111, the Cas-gRNA combination of any of claims 187-198, the polynucleotide of any of claims 199-205, the plurality of polynucleotides of claim 206, the vector of any of claims 207-212, or a combination thereof, into a population of stem cells, and culturing the stem cells under conditions for their differentiation.

216. A method of differentiating a population of stem cells comprising introducing the pharmaceutical composition of claim 213 or claim 214 into a population of stem cells, and culturing the stem cells under conditions for their differentiation.

217. The method of claim 215 or claim 216, wherein the population of stem cells are induced pluripotent stem cells (iPSCs).

218. The method of any of claims 215-217, wherein cells of the population of stem cells differentiate into CD34+ cells.

219. The method of any of claims 215-218, wherein cells of the population of stem cells differentiate into CD43+ cells.

220. The method of any of claims 215-219, wherein cells of the population of stem cells differentiate into hematopoietic progenitor cells (HPCs).224742003140221. The method of any of claims 215-217, wherein cells of the population of stem cells differentiate into CD45+ cells.

222. The method of any of claims 215-217 and 221, wherein cells of the population of stem cells differentiate into CD5+CD7+ cells.

223. The method of any of claims 215-217, 221 and 222, wherein cells of the population of stem cells differentiate into lymphoid progenitor cells.

224. The method of any of claims 215-220, wherein cells of the population of stem cells differentiate into CD34+ / CD43+ cells.

225. The method of any of claims 215-220, wherein cells of the population of stem cells differentiate into CD34+ / CD43+ / CD235a- cells.

226. The method of any of claims 215-220, wherein cells of the population of stem cells differentiate into CD34+, CD43+, and CD45+ cells.

227. The method of any of claims 215-217 and 221, wherein cells of the population of stem cells differentiate into lymphoid cells.

228. The method of any of claims 215-217, 221, and 227, wherein cells of the population of stem cells differentiate into induced Natural Killer (iNK) cells.

229. The method of any of claims 215-217, 221, and 227, wherein cells of the population of stem cells differentiate into CD56+CD3- cells.

230. The method of any of claims 215-217, 221, 227, and 229, wherein cells of the population of stem cells differentiate into cells that are characterized by one or more of the following: DNAM1+, NKG2D+, NKP30+ and / or CD16+.224742003140231. The method of any of claims 215-217, 221, and 227, wherein cells of the population of stem cells differentiate into induced T (iT) cells.

232. The method of any of claims 215-217, 221, and 227, wherein cells of the population of stem cells differentiate into cells that are characterized by one or more of the following: CD45+, CD8A+, CD8B+, CD4+, and / or CD3+.

233. The method of any of claims 215-232, wherein the stem cells comprise cells engineered with a recombinant receptor, optionally a chimeric antigen receptor.

234. The method of any of claims 215-233, wherein the differentiated cells comprise cells that express a recombinant receptor, optionally a chimeric antigen receptor.

235. A method for generating hematopoietic progenitor cells (HPCs), the method comprising: (a) introducing the DNA-targeting system of any one of claims 1-111, the Cas- gRNA combination of any of claims 187-198, the polynucleotide of any of claims 199-205, the plurality of polynucleotides of claim 206, the vector of any of claims 207-212, or a combination thereof, into a population of induced pluripotent stem cells (iPSCs); and (b) culturing the iPSCs to differentiate cells of the population of iPSCs into hematopoietic progenitor cells (HPCs) to produce a population of cells comprising HPCs.

236. A method for generating hematopoietic progenitor cells (HPCs), the method comprising: (a) introducing the pharmaceutical composition of claim 213 or claim 214 into a population of induced pluripotent stem cells (iPSCs); and (b) culturing the iPSCs to differentiate cells of the population of iPSCs into hematopoietic progenitor cells (HPCs) to produce a population of cells comprising HPCs.

237. The method of claim 235 or claim 236, wherein the iPSCs comprise cells engineered with a recombinant receptor, optionally a chimeric antigen receptor.

238. The method of any of claims 235-237, wherein the HPCs are CD34+ cells.224742003140239. The method of any of claims 235-238, wherein the HPCs are CD43+ cells.

240. A population of hematopoietic progenitor cells produced by the method of any of claims 235-239.

241. A method for generating lymphoid progenitor cells (LPCs), the method comprising culturing the population of HPCs produced by the method of any of claims 235-239 or the population of HPCs of claim 240 under conditions to differentiate cells of the population of HPCs to lymphoid progenitor cells (LPCs) to produce a population comprising LPCs.

242. The method of claim 241, wherein the LPCs are CD45+ cells.

243. The method of claim 241 or claim 242, wherein the LPCs are CD5+CD7+ cells.

244. The method of any of claims 241-243, wherein the LPCs express a recombinant receptor, optionally a chimeric antigen receptor.

245. A population of lymphoid progenitor cells (LPCs) produced by the method of any of claims 241-244.

246. A method of generating lymphoid cells (LCs), the method comprising culturing the population of LPCs produced by the method of any of claims 241-244 or the population of LPCs of claim 245 under conditions to differentiate cells of the population of LPCs to lymphoid cells (LCs) to produce a population comprising LCs.

247. A method of generating lymphoid cells (LCs), the method comprising culturing the population of HPCs produced by the method of any of claims 235-239 or the population of HPCs of claim 240 under conditions to differentiate cells of the population of HPCs to lymphoid cells (LCs) to produce a population comprising LCs.

248. The method of claim 246 or claim 247, wherein the LCs are induced Natural Killer (iNK) cells.224742003140249. The method of any of claims 246-248, wherein the LCs are CD56+CD3- cells.

250. The method of any of claims 246-249, wherein the LCs are characterized by one or more of the following: DNAM1+, NKG2D+, NKP30+ and / or CD16+.

251. The method of claim 246 or claim 247, wherein the LCs are induced T (iT) cells.

252. The method of any of claims 246, 247, and 251, wherein the LCs are characterized by one or more of the following: CD45+, CD8A+, CD8B+, CD4+, and / or CD3+.

253. The method of any of claims 246-252, wherein the LCs express a recombinant receptor, optionally a chimeric antigen receptor.

254. A population of lymphoid cells produced by the method of any of claims 246- 253.

255. The method of any of claims 215-239, 241-244, and 246-253 that is carried out in vitro or ex vivo.

256. The method of any of claims 215-239, 241-244, 246-253, and 255, wherein the stem cells are human stem cells.

257. The method of any of claims 215-239, 241-244, 246-253, 255, and 256, wherein the introducing is by transient delivery into the population of stem cells.

258. The method of claim 257, wherein the transient delivery comprises electroporation, transfection, or transduction.

259. The method of any of claims 215-239, 241-244, 246-253, and 255-258, wherein the introducing increases transcription of the one or more transcription factors selected from the224742003140 group consisting of BMI1, CEBPD, CFOS, GATA2, GATA3, HEY2, LM02, LYL1, TALI and ZEB2.

260. A population of differentiated cells produced by the method of any of claims 215- 234.

261. A method of treating a disease or condition in a subject, the method comprising administering to the subject the population of the population of lymphoid cells of claim 253 or the population of differentiated cells of claim 260.

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