System for deleting regions of DNA

Abstract

The present invention relates to a nucleic acid structure comprising a pair of guide RNAs operable to target and delete a region of DNA, the guide RNAs being separated by a spacer, and under the control of two different and convergent promoters, a vector comprising said nucleic acid, a library of such vectors, and methods of making such vectors. The present invention also relates to a system comprising the nucleic acid, vector or library and a nucleic acid encoding a Cas9 protein. The present invention further relates to methods of using the vector comprising the pair of guide RNAs to delete a region of DNA in a cell, and to large-scale methods of screening a genome using the library of vectors each comprising a different pair of guide RNAs to delete different regions of DNA in a plurality of cells.

Description

SYSTEM FOR DELETING REGIONS OF DNAFIELD OF THE INVENTION

[0001] The present invention relates to a nucleic acid structure comprising a pair of guide RNAs operable to target and delete a region of DNA, the guide RNAs being separated by a spacer, and under the control of two different and convergent promoters, a vector comprising said nucleic acid, a library of such vectors, and methods of making such vectors. The present invention also relates to a system comprising the nucleic acid, vector or library and a nucleic acid encoding a Cas9 protein. The present invention further relates to methods of using the vector comprising the pair of guide RNAs to delete a region of DNA in a cell, and to large- scale methods of screening a genome using the library of vectors each comprising a different pair of guide RNAs to delete different regions of DNA in a plurality of cells.INTRODUCTION

[0002] Recent advances in CRISPR genome editing have created new ways to study both the coding and non-coding parts of the genome19'22. CRISPR-Cas9 recognizes 20-bp genomic regions followed by PAM (5’-NGG-3’) and typically introduces genetic changes of a few nucleotide deletions or insertions around the targeting sites in the genome23. This is especially useful in studying gene functions as mutation-induced frameshifts in the coding regions render the encoded proteins non-functional. However for non-coding Regulatory elements (NCREs), the use of CRISPR gene editing is more complex.

[0003] Protein-coding genes only represent less than 2% of the human genome, and the rest is non-coding, many of which contain Regulatory elements that guide the transcription of genes at the right time and within the right tissue1,2. Based on the known functions, the non-coding Regulatory elements (NCREs) are categorized into various segments, such as non-coding RNAs, promoters, enhancers, silencers, insulators, etc2'7. Assigning and understanding the function of the non-coding regulatory genome have been one main focus in genetic research during the past decades2. With the efforts from individual research groups and large consortia such as ENCODE and Roadmap Epigenomics, putative biological roles have been assigned to many NCREs8'11. In general, different types of NCREs have unique combinations of epigenetic modifications. Therefore, using next-generation sequencing methods like ChlP-seq against various histone modifications or transcription factors, or methods to profile accessible"SUBSTITUTE SHEET RULE 26"chromatins such as DNase I hypersensitivity sites combined with sequencing, FAIRE-seq, and ATAC-seq, many NCREs have been mapped in the human genome12 13. For instance, insulator regions are often enriched for CTCF binding sites14 15, while enhancer regions are usually decorated with a combination of H3K27ac and H3K4mel16. Simultaneously, the recent development of massively parallel reporter systems also facilitates the direct biological activity measurement and identifications of enhancers and silencers4 17 18. However, none of these methods measure the biological functions of NCREs in their endogenous environment.

[0004] The application of CRISPR genome editing to NCREs is desirable. However, NCREs often range from 50-200 bp in length, with multiple transcription factor (TF) binding sites24. Therefore, a single CRISPR-Cas9-mediated genome editing must completely destroy the TF binding sites25,26to see any effect, which is also limited by the possible CRISPR-Cas9 targeting sites throughout the human genome. Due to such limitations, single guide RNAs tiling an entire testing region have often been used to study NCREs regulating a few important genes. The modified CRISPR system that uses catalytic inactive Cas9 proteins (dCas9), either linked to a transcription activation or repression system, can also be used to study the functions of enhancers and insulators26,27. However, pre-knowledge of NCRE functions is needed to select the proper CRISPR-dCas9-mediated activation or repression systems27,28. Such selection is further complicated by the recent evidence showing that certain NCREs could be bifunctional, e.g., functioning as enhancers or silencers depending on the cellular context7, 18. Similarly, the dCas9 system is also limited by the availability of single guide RNAs at the targeting regions.

[0005] More recently, several dual-guide CRISPR systems have been used to study noncoding RNAs or enhancers, however, their throughputs are still limited due to either the barcoding system or the design of the screening system; therefore only limited regions have been targeted29,30. Thus far, no systematic study of the NCREs in a genome-wide fashion, especially focusing on enhancers and silencers, has been performed.

[0006] There is a need in the art for an efficient CRISPR-mediated screening system that can delete thousands of regions of DNA in a systematic and genome-wide fashion, especially for the analysis of non-coding regions of DNA, irrespective of their biological functions which often may not be known."SUBSTITUTE SHEET RULE 26"STATEMENTS OF INVENTION

[0007] According to a first aspect of the present invention, there is provided a nucleic acid encoding a pair of guide RNAs operable to delete a region of DNA, wherein the nucleic acid encoding the pair of guide RNAs comprises: (i) a first gRNA comprising a first crRNA sequence and a first tracrRNA sequence, said sequences being operably linked to a first promoter, and (ii) a second gRNA comprising a second crRNA sequence and a second tracrRNA sequence said sequences being operably linked to a second promoter, wherein the first and second guide RNAs are separated by a spacer, and wherein the promoters are different and are convergent.

[0008] According to a second aspect of the present invention, there is provided a vector comprising the nucleic acid of the first aspect.

[0009] According to a third aspect of the present invention, there is provided a method of making a vector according to the second aspect, the method comprising: a. Providing a vector backbone comprising the first and second convergent promoters, and a first cleavage site downstream of the first promoter, and a second cleavage site downstream of the second promoter; b. Cleaving the backbone at the first and second cleavage sites; c. Inserting a nucleic acid comprising: the first crRNA sequence, the second crRNA sequence, a third cleavage site downstream of the first crRNA sequence, and a fourth cleavage site downstream of the second crRNA sequence, into the backbone between the first and second cleavage sites, such that the first crRNA sequence is downstream of the first promoter and the second crRNA sequence is downstream of the second promoter; d. Cleaving the backbone at the third and fourth cleavage sites; and e. Inserting a nucleic acid comprising: the first tracrRNA sequence and the second tracrRNA sequence separated by a spacer, into the backbone between the third and fourth cleavage sites, such that the first tracrRNA sequence is downstream of the first crRNA sequence and the second tracrRNA sequence is downstream of the second crRNA sequence.

[0010] According to a fourth aspect of the present invention, there is provided a library comprising a plurality of vectors according to the second aspect, wherein each vector in the"SUBSTITUTE SHEET RULE 26"library comprises a nucleic acid encoding a different pair of guide RNAs operable to delete a different region of DNA.

[0011] According to a fifth aspect of the present invention, there is provided a system for deleting a region of DNA, the system comprising (i) a nucleic acid according to the first aspect, a vector according to the second aspect, or a library according to the fourth aspect, for expressing one or more pairs of guide RNAs, and (ii) a nucleic acid encoding a Cas protein or a vector comprising said nucleic acid for expressing a Cas protein, or a pair of Cas proteins.

[0012] According to a sixth aspect of the present invention, there is provided a method of deleting one or more regions of DNA, the method comprising: a. Providing a cell with the system of the fifth aspect; b. Culturing the cell under suitable conditions to express the nucleic acids of the system and thereby produce one or more pairs of guide RNAs and a pair of Cas proteins; c. Culturing the cell under suitable conditions to allow the or each pair of guide RNAs and the pair of Cas proteins to associate to form a pair of CRISPR-Cas complexes and for the CRISPR-Cas complexes to delete one or more regions of DNA in the cell.

[0013] According to a seventh aspect of the present invention, there is provided a method of screening a genome, the method comprising: a. Providing a plurality of cells comprising the library of the fourth aspect, wherein each cell comprises a (i) vector of the library, and (ii) a nucleic acid encoding a Cas protein or a vector comprising said nucleic acid for expressing a Cas protein, or a pair of Cas proteins; b. Culturing the cells under suitable conditions to express, within each cell, the nucleic acids of the vector and optionally the nucleic acid encoding the Cas protein and thereby produce, within each cell, a pair of guide RNAs and a pair of Cas proteins; c. Culturing the cells under suitable conditions to allow the pair of guide RNAs and the pair of Cas proteins to associate to form a pair of CRISPR-Cas complexes and for the pair of CRISPR-Cas complexes to delete a region of DNA within each cell; and d. Performing (bulk or single cell) screening of the cells to determine the function of the one or more deleted regions of DNA."SUBSTITUTE SHEET RULE 26"Optionally the method of the seventh aspect may be a method of determining the function of one or more regions of DNA.

[0014] According to a further aspect of the invention there is provided the use of the nucleic acid, vector, library or system of the invention in a method of deleting a region of DNA, suitably a region of genomic DNA.

[0015] According to a further aspect of the invention there is provided the use of the nucleic acid, vector, library or system of the invention in a method of screening a genome. Suitably in a method of CRISPR screening, suitably in a method of genome-wide CRISPR screening.

[0016] In an embodiment of any of the above aspects, suitably a pair of Cas proteins are provided, suitably a first and a second Cas protein are provided. Suitably the pair of Cas proteins care capable of forming a pair of CRISPR-Cas complexes with the pair of guide RNAs, suitably of forming a first and a second CRISPR-Cas complex.

[0017] The present invention provides an efficient way of encoding pairs of guide RNAs and constructing vectors for their expression in cells, which may then be used to carry out deletions of genomic DNA, either in a targeted ‘knock-out’ fashion or in a genome- wide screen. The nucleic acid for expressing each pair of guide RNAs has been optimized by the inventors such that the risk of recombination is reduced, and that there are several advantages to the downstream sequencing processes used in screening methods. The inventors have found that a suitable spacer improves NGS sequencing accuracy, and that the use of specific convergent promoters allows for direct amplification of genomic DNA containing the guide RNAs and then pair-end sequencing which does not require barcodes.

[0018] The nucleic acid, vector and system of the invention are capable of being used to interrogate both previously annotated and unannotated genomic regions in an unbiased fashion. The present invention therefore provides a useful tool to study both the coding and the noncoding part (irrespective of the prior knowledge of function) of the genome at the multiple cell or down to the single-cell level. In particular, the present invention allows non-coding elements of the genome to be studied in their endogenous loci, and is capable of capturing clusters of such NCREs with redundant biological functions in a genome-wide and systematic study.

[0019] Further features and embodiments of one or more of the above aspects will now be described under the following headed sections. Any feature in any section may be combined with the above aspects in any workable combination."SUBSTITUTE SHEET RULE 26"DETAILED DESCRIPTION

[0020] Pair of Guide RNAs

[0021] The nucleic acid of the invention comprises and encodes a pair of guide RNAs comprising a first guide RNA and a second guide RNA which act together to target and cleave DNA, thereby deleting a region of DNA.

[0022] "Guide RNA" refers to a DNA-targ eting RNA forming part of a CRISPR-Cas complex, comprising broadly two sections: a DNA-targ eting section comprising a nucleotide sequence that is complementary to a target sequence in DNA and (ii) a protein-binding section that interacts with a Cas polypeptide, suitably comprising a dsRNA duplex.

[0023] " CRISPR-Cas complex" refers to the combination of a Cas polypeptide and a guide RNA. Suitably the Cas polypeptide is from a type II CRISPR system, suitable Cas polypeptides are described hereinbelow.

[0024] A guide RNA as used herein may refer to both a two-molecule / double-molecule guide RNA or a single molecule guide RNA (sgRNA).

[0025] Suitably the first guide RNA comprises a first crRNA and a first tracrRNA. Suitably the second guide RNA comprises a second crRNA and a second tracrRNA.

[0026] ‘ crRNA’ is a section of a guide RNA comprising the DNA-targeting section, and therefore comprises a nucleotide sequence that is complementary to a target sequence in DNA, the crRNA further comprises a nucleotide sequence operable to form a dsRNA duplex of the protein binding section with the tracrRNA. The crRNA may also be known as "CRISPR RNA" or "targeter-RNA" or "crRNA repeat".

[0027] ‘ tracrRNA’ is a section of a guide RNA comprising a nucleotide sequence operable to form a dsRNA duplex of the protein binding section with the crRNA. In a single guide RNA, the tracrRNA further comprises a linker sequence which links the nucleotide sequence operable to form a dsRNA duplex with the crRNA. The tracRNA typically further comprises one or more stem loops. The tracrRNA may also be known as "trans-acting CRISPR RNA" or “activator-RNA” or “scaffold”.

[0028] Suitably in a single molecule guide RNA, the crRNA and the tracrRNA are produced as one molecule, in which the nucleotide sequences operable to form a dsRNA duplex of the protein binding segment are linked via a linker sequence. Thus resulting in a loop structure. Suitably the linker sequence has a length of from about 3 nucleotides to about 100 nucleotides."SUBSTITUTE SHEET RULE 26"For example, the linker sequence can have a length of from about 3 nucleotides (nt) to about 90 nt, from about 3 nucleotides (nt) to about 80 nt, from about 3 nucleotides (nt) to about 70 nt, from about 3 nucleotides (nt) to about 60 nt, from about 3 nucleotides (nt) to about 50 nt, from about 3 nucleotides (nt) to about 40 nt, from about 3 nucleotides (nt) to about 30 nt, from about 3 nucleotides (nt) to about 20 nt or from about 3 nucleotides (nt) to about 10 nt. For example, the linker can have a length of from about 3 nt to about 5 nt, from about 5 nt to about 10 nt, from about 10 nt to about 15 nt, from about 15 nt to about 20 nt, from about 20 nt to about 25 nt, from about 25 nt to about 30 nt, from about 30 nt to about 35 nt, from about 35 nt to about 40 nt, from about 40 nt to about 50 nt, from about 50 nt to about 60 nt, from about 60 nt to about 70 nt, from about 70 nt to about 80 nt, from about 80 nt to about 90 nt, or from about 90 nt to about 100 nt.

[0029] Suitably the term ‘about’ as used herein refers to a range which may be + / -10% of the stated value.

[0030] Suitably in a two-molecule guide RNA, a stretch of nucleotides of the crRNA are complementary to and hybridize with a stretch of nucleotides of the tracrRNA molecule to form a complete guide RNA, suitably by forming the dsRNA duplex of the protein-binding segment of the guide RNA. As such, each crRNA molecule can be said to have a corresponding tracrRNA molecule which forms the guide RNA. The crRNAadditionally provides the DNA- targeting section nucleotide having a sequence that is complementary to a target sequence in DNA. Thus, in such a two-molecule format, a crRNA and a tracrRNA hybridize to form a guide RNA.

[0031] In one embodiment, the first and second guide RNAs of the invention are single molecule guide RNAs (sgRNA).

[0032] Suitably the first and second guide RNAs of the invention are operable to delete a region of DNA because the first and second crRNAs each comprise DNA-targeting sections having nucleotide sequences that are complementary to target sequences in the DNA. Suitably the first crRNA comprises a first DNA-targeting section having a nucleotide sequence that is complementary to a first target sequence in the DNA. Suitably the second crRNA comprises a second DNA-targeting section having a nucleotide sequence that is complementary to a second target sequence in the DNA. Suitably the target sequences in the DNA are upstream and downstream of the region of DNA to be deleted."SUBSTITUTE SHEET RULE 26"

[0033] Suitably the first target sequence in the DNA is upstream of the region of DNA to be deleted, and suitably the second target sequence in the DNA is downstream of the region of DNA to be deleted. Suitably "Upstream" refers to the nucleic acid sequence 5' of the relevant sequence when read in the direction of transcription. Suitably "Downstream" refers to the nucleic acid sequence 3' of the relevant sequence when read in the direction of transcription. Suitably the first crRNA comprises a first DNA-targeting section having a nucleotide sequence that is complementary to a first target sequence upstream or 5' of the region of DNA to be deleted, suitably proximal to the 5' end of the region to be deleted, suitably within lOOObp, suitably within 900bp, suitably within 800bp, suitably within 700bp, suitably within 600bp, suitably within 500bp, suitably within 400bp, suitably within 300bp, suitably within 200bp, suitably within lOObp, suitably within 50bp, suitably within 25bp, suitably immediately 5' of the region of DNA to be deleted. In one embodiment, the first crRNA comprises a first DNA- targeting section having a nucleotide sequence that is complementary to a first target sequence within 200bp of the 5' end of the region of DNA to be deleted. Suitably the second crRNA comprises a second DNA-targeting section having a nucleotide sequence that is complementary to a second target sequence downstream or 3' of the region of DNA to be deleted, suitably proximal to the 3' end of the region to be deleted, suitably within lOOObp, suitably within 900bp, suitably within 800bp, suitably within 700bp, suitably within 600bp, suitably within 500bp, suitably within 400bp, suitably within 300bp, suitably within 200bp, suitably within lOObp, suitably within 50bp, suitably within 25bp, suitably immediately 3' of the region of DNA to be deleted. In one embodiment, the second crRNA comprises a second DNA-targeting section having a nucleotide sequence that is complementary to a second target sequence within 200bp of the 5' end of the region of DNA to be deleted.

[0034] Suitably the first and second target sequences are adjacent to a protospacer adjacent motif (PAM) in the DNA. In order for a CRISPR-Cas complex comprising a guide RNA to target and cleave a target sequence, the target sequence should be associated with a PAM which is a short sequence recognized by the CRISPR-Cas complex. The precise sequence and length requirements for the PAM differ depending on the CRISPR enzyme used, but PAMs are typically 2-5 base pair sequences adjacent the protospacer (that is, the target sequence). Suitably the PAM sequence of the non-complementary strand of the DNA is 5'-XGG-3', where X is any DNA nucleotide and X is immediately 3' of the first and second target sequences of the non-complementary strand of the DNA. As such, the PAM sequence of the complementary"SUBSTITUTE SHEET RULE 26"strand is 5'-CCY-3', where Y is any DNA nucleotide and Y is immediately 5' of the first and second target sequences of the complementary strand of the DNA.

[0035] Suitably the nucleotide sequences of the DNA-targeting sections that are complementary to a first target sequence and a second target sequence in the DNA comprise a length of at least 10 nucleotides, between about 10-30 nucleotides, between about 10-25 nucleotides, between about 10-20 nucleotides. Suitably the nucleotide sequences of the DNA- targeting sections are at least 15, 16, 17, 18, 19, 20, 25 nucleotides, or between 10-30, or between 15-25, or between 15-20 nucleotides in length, preferably 20 nucleotides in length.

[0036] Suitably the nucleotide sequences of the DNA-targeting sections that are complementary to a first target sequence and a second target sequence in the DNA have a complementarity of at least 70% to the target sequence, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, up to 100% to the relevant target sequence.

[0037] Suitably the protein binding segment of each guide RNA comprises a dsRNA duplex (i.e., a hairpin) operable to bind to a Cas protein, wherein the dsRNA duplex is formed from a stretch of nucleotides of the crRNA which are complementary to and hybridize with a stretch of nucleotides of the tracrRNA. Suitably such stretches of nucleotides may be termed the duplexforming sections of the crRNA and the tracrRNA. Suitably the duplex forming sections have at least 70%, at least 80%, at least 90%, at least 95%, up to 100% complementarity. Suitably the dsRNA duplex comprises a length of 6 base pairs (bp) to about 50bp. For example, the dsRNA duplex can have a length from about 6 bp to about 40 bp, from about 6 bp to about 30bp, from about 6 bp to about 25 bp, from about 6 bp to about 20 bp, from about 6 bp to about 15 bp, from about 8 bp to about 40 bp, from about 8 bp to about 30bp, from about 8 bp to about 25 bp, from about 8 bp to about 20 bp or from about 8 bp to about 15 bp. For example, the dsRNA duplex can have a length from about from about 8 bp to about 10 bp, from about 10 bp to about 15 bp, from about 15 bp to about 18 bp, from about 18 bp to about 20 bp, from about 20 bp to about 25 bp, from about 25 bp to about 30 bp, from about 30 bp to about 35 bp, from about 35 bp to about 40 bp, or from about 40 bp to about 50 bp.

[0038] In some embodiments, a guide RNA of the invention may further comprise a capture sequence. Suitably one or both of the pair of guide RNAs may comprise a capture sequence. Sutiably both guide RNAs comprise a capture sequence. Suitably the capture sequence is comprised within the tracrRNA. Suitably the capture sequence is comprised within one of the stem loops of the tracrRNA, suitably either the second or third stem loop of the tracrRNA."SUBSTITUTE SHEET RULE 26"Alternatively, the capture sequence may be comprised at the 3' end of the tracrRNA. In one embodiment, the capture sequence is comprised within the second stem loop of the tracrRNA.

[0039] Suitable capture sequences are known in the art, and may be provided by 10X genomics for example GCTTTAAGGCCGGTCCTAGCAA (SEQ ID NO: 1) or GCTCACCTATTAGCGGCTAAGG (SEQ ID NO: 2). Suitably the first guide RNA comprises a first capture sequence and the second guide RNA comprises a second capture sequence. Suitably the first and second capture sequences are different.

[0040] Suitably the capture sequence facilitates binding of the guide RNAs for subsequent sequencing steps, suitably for single-cell sequencing steps. Suitably therefore, the pairs of guide RNAs used during single cell screening methods described herein each comprise a capture sequence as above.

[0041] Promoters

[0042] The nucleic acid of the invention further comprises a first promoter and a second promoter that are used to drive expression of the first and second guide RNAs.

[0043] As used herein, a “promoter” refers to a DNA regulatory region capable of binding RNA polymerase and initiating transcription of a downstream (3' direction) coding or noncoding sequence. For purposes of defining the present invention, the promoter is bounded at its 3' terminus by the transcription initiation site and extends upstream (5' direction) to include the minimum number of bases or elements necessary to initiate transcription at levels detectable above background. Suitably within the promoter is comprised transcription initiation site, as well as protein binding domains responsible for the binding of RNA polymerase. Eukaryotic promoters will often, but not always, contain "TATA" boxes and "CAT" boxes.

[0044] Suitably the promoters are chosen such that they are suitable to drive expression of the guide RNAs in the relevant cell in which it is desired to use the pair of guide RNAs to delete one or more regions of DNA. Suitably therefore the first and second promoters are eukaryotic promoters, suitably mammalian promoters.

[0045] Suitably the promoters may be constitutively active promoters (i.e., a promoter that is constitutively in an active / "ON" state), inducible promoters (i.e., a promoter whose state, active / "ON" or inactive / " OFF", is controlled by an external stimulus, e.g., the presence of a particular temperature, compound, or protein.), spatially restricted promoters (i.e., transcriptional control element, enhancer, etc.)(e.g., tissue specific promoter, cell type specific promoter, etc.), or temporally restricted promoters (i.e., the promoter is in the "ON" state or"SUBSTITUTE SHEET RULE 26""OFF" state during specific stages of embryonic development or during specific stages of a biological process, e.g., hair follicle cycle in mice).

[0046] Examples of inducible promoters include, but are not limited toT7 RNA polymerase promoter, T3 RNA polymerase promoter, Isopropyl-beta-D-thiogalactopyranoside (IPTG)- regulated promoter, lactose induced promoter, heat shock promoter, Tetracycline-regulated promoter, Steroid-regulated promoter, Metal-regulated promoter, estrogen receptor-regulated promoter, etc. Inducible promoters can therefore be regulated by molecules including, but not limited to, doxycycline; RNA polymerase, e.g., T7 RNA polymerase; an estrogen receptor; an estrogen receptor fusion; etc.

[0047] Examples of spatially restricted promoters include, but are not limited to, neuronspecific promoters, adipocyte-specific promoters, cardiomyocyte-specific promoters, smooth muscle-specific promoters, photoreceptor-specific promoters, etc. Neuron-specific spatially restricted promoters include, but are not limited to, a neuron-specific enolase (NSE) promoter (see, e.g., EMBL HSENO2, X51956); an aromatic amino acid decarboxylase (AADC) promoter; a neurofilament promoter (see, e.g., GenBank HUMNFL, L04147); a synapsin promoter (see, e.g., GenBank HUMSYNIB, M55301); a thy-1 promoter (see, e.g., Chen et al. (1987) Cell 51 :7-19; and Llewellyn, et al. (2010) Nat. Med. 16(10):l 161-1166); a serotonin receptor promoter (see, e.g., GenBank S62283); a tyrosine hydroxylase promoter (TH) (see, e.g., Oh et al. (2009) Gene Ther 16:437; Sasaoka et al. (1992) Mol. Brain Res. 16:274; Boundy et al. (1998) J. Neurosci. 18:9989; and Kaneda et al. (1991) Neuron 6:583-594); a GnRH promoter (see, e.g., Radovick et al. (1991) Proc. Natl. Acad. Sci. USA 88:3402-3406); an L7 promoter (see, e.g., Oberdick et al. (1990) Science 248:223-226); a DNMT promoter (see, e.g., Bartge et al. (1988) Proc. Natl. Acad. Sci. USA 85:3648-3652); an enkephalin promoter (see, e.g., Comb et al. (1988) EMBO J. 17:3793-3805); a myelin basic protein (MBP) promoter; a Ca2+-calmodulin-dependent protein kinase II-alpha (CamKIIa) promoter (see, e.g., Mayford et al. (1996) Proc. Natl. Acad. Sci. USA 93: 13250; and Casanova et al. (2001) Genesis 31:37); a CMV enhancer / platelet-derived growth factor-0 promoter (see, e.g., Liu et al. (2004) Gene Therapy 11:52-60); and the like.

[0048] Suitably the first and second promoters are RNA POL promoters, such as POL I, POLII, or POL III promoters. Examples of pol III promoters include, but are not limited to, U6 and Hl promoters. Examples of pol II promoters include, but are not limited to, the retroviral Rous sarcoma virus (RSV) LTR promoter (optionally with the RSV enhancer), the"SUBSTITUTE SHEET RULE 26"cytomegalovirus (CMV) promoter (optionally with the CMV enhancer) [see, e.g., Boshart et al, Cell, 41:521-530 (1985)], the SV40 promoter, the dihydrofolate reductase promoter, the 0-actin promoter, the phosphoglycerol kinase (PGK) promoter, and the EFla promoter. Suitably the first and second promoters are both POL III promoters.

[0049] Suitably the promoters are different, which advantageously avoids recombination events. In one embodiment the first promoter is U6. In one embodiment, the second promoter is Hl. Suitably it will be appreciated that the first and second promoters may be swapped, i.e. the first promoter may be Hl and the second promoter may be U6.

[0050] Suitably the first and second promoters are convergent, i.e. They are arranged in a face to face configuration such that the polymerase transcribes from the first promoter towards the second promoter and vice versa. Advantageously, this allows for direct amplification by PCR of genomic DNA containing the pair of guide RNAs after integration into the genome of a cell, and then pair-end sequencing of the amplified fragments without the need for barcodes.

[0051] Suitably therefore the first promoter is upstream of the nucleic acid encoding the first guide RNA. Suitably the first promoter is oriented 5' to 3', and suitably therefore the nucleic acid encoding the first guide RNA is oriented 5' to 3'.

[0052] Suitably the therefore the second promoter is upstream of the nucleic acid encoding the second guide RNA. Suitably the second promoter is oriented 3' to 5', and suitably therefore the nucleic acid encoding the first guide RNA is oriented 3' to 5'.

[0053] Spacer

[0054] The nucleic acid of the invention further comprises a spacer, suitably located between the nucleic acid sequence encoding the first guide RNA and the nucleic acid sequence encoding the second guide RNA.

[0055] Suitably the spacer is any length of nucleic acid sequence located between the nucleic acid sequence encoding the first guide RNA and the nucleic acid sequence encoding the second guide RNA. Suitably between the 3' end of the first guide RNA and the 3' end of the second guide RNA. Suitably the spacer may comprise a nucleic acid sequence of between 50-500 nucleotides or base-pairs in length, suitably between 25-400, 50-350, 75-300, 100-250, 50-300, 50-250, 50-200, or about 50, about 200, about 300 or about 500 nucleotides or base-pairs in length. In one embodiment the spacer is about 200 nucleotides or base-pairs in length.Advantageously, this distance between the nucleic acid sequences encoding the guide RNAs has been found to be optimal for NGS sequencing, with good amplification efficiency."SUBSTITUTE SHEET RULE 26"

[0056] Suitably the spacer is a non-coding nucleic acid sequence. Suitably the spacer may comprise a nonsense nucleic acid sequence. Suitably the spacer comprises the following sequence: cgtctccACGTGGTGGCGCCCGgatcCCGACTGCTCCCTGAGTGTGGCTCTTTGTTCTGTCA ACGCCCGACCTTCATCGCGGCCGATTCCTTCTGCGGACCATGTCGTCCTGATACTTTG GCCATGTTTCCGTTGTAGGAGTGAAGCCACTTGGCTTTGCGCCGTAGTTCCAATGAA AAACCTATGGACTTTGTTTAGgatccGGTAGCATCAGGAcgagacg (SEQ ID NO: 3)

[0057] Nucleic Acid

[0058] Suitably the first aspect of the invention relates to a nucleic acid encoding and operable to express the pair of guide RNAs described above. "Nucleic acid" refers to a polymeric form of nucleotides of any length, either ribonucleotides or deoxyribonucleotides. Thus, this term includes, but is not limited to, single-, double-, or multi- stranded DNA or RNA, genomic DNA, cDNA, DNA-RNA hybrids, or a polymer comprising purine and pyrimidine bases or other natural, chemically or biochemically modified, non-natural, or derivatized nucleotide bases. The terms "polynucleotide" and "nucleic acid" are interchangeable and should be understood to include, as applicable to the embodiments being described, single-stranded (such as sense or antisense) and double-stranded polynucleotides.

[0059] Suitably the nucleic acid of the invention encodes the pair of guide RNAs, the first and second promoters, and the spacer.

[0060] Suitably the nucleic acid encoding the pair of guide RNAs comprises in the 5' to 3' direction: a first promoter, a first gRNA, a spacer, a second gRNA, a second promoter. Suitably wherein the first promoter, spacer and second promoter are as defined hereinabove.

[0061] Suitably the first promoter is operably linked to the first guide RNA, and the second promoter is operably linked to the second guide RNA. "Operably linked" refers to an arrangement of elements wherein the elements are in a relationship permitting them to function in their intended manner. For instance, a promoter is operably linked to a coding sequence if the promoter affects its transcription or expression.

[0062] Suitably the nucleic acid of the invention may be regarded as an expression construct. In a further aspect of the invention there is provided an expression construct comprising the nucleic acid of the first aspect.

[0063] Optionally the nucleic acid may further comprise one or more regulatory elements. "Regulatory element" refers to transcriptional and translational control sequences, such as"SUBSTITUTE SHEET RULE 26"promoters, enhancers, polyadenylation signals, terminators, UTRs, IRES, protein degradation signals, and the like, that provide for and / or regulate transcription of a non-coding sequence (e.g., a guide RNA) or a coding sequence (e.g., a Cas9 polypeptide) and / or regulate translation of an encoded polypeptide. Optionally the nucleic acid may further comprise one or more regulatory elements operably linked to the nucleic acid encoding the first guide RNA and / or the second guide RNA.

[0064] Vector and .Libra^

[0065] The present invention further provides a vector comprising the nucleic acid of the first aspect, or the expression construct thereof, “vector” refers to a nucleic acid molecule capable of transporting or carrying another nucleic acid to which it has been linked, typically into a cell.

[0066] Suitable vectors may be any known vector. Vectors include, but are not limited to, nucleic acid molecules that are single-stranded, double- stranded, or partially double-stranded; nucleic acid molecules that comprise one or more free ends, no free ends (e.g. circular); nucleic acid molecules that comprise DNA, RNA, or both; and other varieties of polynucleotides known in the art. One type of vector is a plasmid, which refers to a circular double stranded DNA loop into which additional DNA segments can be inserted, such as by standard molecular cloning techniques. Another type of vector is a viral vector, wherein virally-derived DNA or RNA sequences are present in the vector for packaging into a virus (e.g. retroviruses, replication defective retroviruses, adenoviruses, replication defective adenoviruses, and adeno- associated viruses). Viral vectors also include polynucleotides carried by a virus for transfection into a host cell. Certain vectors are capable of autonomous replication in a host cell into which they are introduced (e.g. bacterial vectors having a bacterial origin of replication and episomal mammalian vectors). Other vectors (e.g., non-episomal mammalian vectors) are integrated into the genome of a host cell upon introduction into the host cell, and thereby are replicated along with the host genome. Moreover, certain vectors are capable of directing the expression of genes to which they are operatively- linked. Such vectors are referred to herein as "expression vectors." Common expression vectors of utility in recombinant DNA techniques are often in the form of plasmids. Advantageous vectors include lentiviruses and adeno- associated viruses, and types of such vectors can also be selected for targeting particular types of cells.

[0067] In one embodiment, the vector is a mammalian vector, suitable for expression in mammalian cells. Suitable mammalian vectors include pCDM8 (Seed, 1987. Nature 329: 840)"SUBSTITUTE SHEET RULE 26"and pMT2PC (Kaufman, et al., 1987. EMBO J. 6: 187-195), pSpCas9(BB)-2A-Puro (PX459) 581 V2.0 (Addgene #62988), pUC19. Suitably mammalian vectors may be used in the methods of the invention for deleting a region of DNA, suitably a single region of DNA, for example in the sixth aspect.

[0068] In one embodiment, the vector is a viral vector, for example: Herpes Simplex virus, Epstein-Barr virus, Simian virus 40, Adenovirus, Adeno-associated virus, Vaccinia virus, Baculovirus, Sindbis virus, Semliki Forest virus, Retrovirus, Lentivirus, optionally which may be a replication defective virus. In one embodiment, the vector is a lentiviral vector, suitably for expression in mammalian cells. Suitable lentiviral vectors include the lentiGuide-Puro (Addgene #52963), LentiCas9-Blast (Addgene #52962). Suitably lentiviral vectors may be used to generate lentiviral particles, and may be used in the methods of the invention for screening a genome, for example in the seventh aspect.

[0069] Suitably the or each vector of the present invention comprises a selection marker. Suitably when more than one vector is used, each vector has a different selection marker. Suitable selection markers may be: drug resistance genes, fluorescent proteins, enzymes, antibiotic resistance genes. Suitably, selection markers are used to determine when a vector has successfully been transformed / transduced into a cell. Upon exposure of cells to a selective agent, only those cells which comprise the vector encoding the selective marker can be selected, typically because they are the only cells which survive. In one embodiment, the vector comprises an antibiotic resistance marker selected from: Blasticidin, Hygromycin, Zeocin, and Puromycin resistance.

[0070] In some cases when the vector of the invention is used in methods described herein, it only contains the nucleic acid of the invention or the expression construct thereof which encodes the pair of guide RNAs. Suitably in such an embodiment, when providing said vector to a cell to effect deletion of a region of DNA, a second vector is also provided comprising a nucleic acid or expression construct thereof which encodes a Cas polypeptide. This may be termed a double vector arrangement. Optionally, however, the vector comprising the nucleic acid of the invention or the expression construct thereof which encodes the pair of guide RNAs may further comprise a nucleic acid or an expression construct thereof encoding a Cas polypeptide. This may be termed a single vector arrangement.

[0071] The invention further provides a library of vectors in the fourth aspect. Suitably the library is formed of a plurality of vectors."SUBSTITUTE SHEET RULE 26"

[0072] Suitably each each vector in the library comprises a nucleic acid encoding a different pair of guide RNAs in accordance with the first aspect of the invention which is operable to target and delete a different region of DNA.

[0073] Suitably the library may be contained within a plurality of viral particles. Suitably the viral particles may be lentiviral or retroviral particles. In one embodiment the viral particles are lentiviral particles.

[0074] Suitably each viral particle comprises one vector of the library. Therefore a plurality of lentiviral particles comprising the library of the invention is provided as the fifth aspect herein.

[0075] Suitably lentiviral particles comprising the library of the invention may be made using a packaging cell line, according to known lentiviral production methods. Suitably the packaging cell line is a mammalian cell line comprising one or more packaging plasmids and one envelope plasmid, in addition to the lentiviral vector. Suitably the packaging plasmid may be psPAX2. Suitably the envelope plasmid may be pCMV-VSV-G.

[0076] One example of such a lentiviral production method as used in the examples is: HEK293T cells were grown in five T175 flasks at 50% confluency before transfection. For each flask of 293 T cells grown in 25 ml of fresh medium, 15 pg of library plasmids, 7 pg of psPAX2, 3.5 pg of pCMV-VSV-G, and 76.5 pl of 702 X-tremeGENE 9 DNA Transfection Reagent were mixed in 1 ml of serum-free medium and used for transfection. Fresh medium was added the day following transfection. Media supernatant containing virus particles was collected on the second and third days after transfection, pooled and further concentrated using Lenti-X according to the manufacturer’s protocol.

[0077] Suitably the lentiviral particles may be used to transduce a plurality of cells using known viral transduction methods, suitably such that each cell is transduced with a lentiviral particle, and each cell therefore comprises a vector of the library.

[0078] Suitably therefore a population of cells comprising the library of the invention is provided. Suitably each cell comprises one vector of the library. Suitably such a population of cells are used in the screening methods of the invention.

[0079] Method of Making a Vector

[0080] The present invention further provides a method of making the vector described above, via only two steps of integration. Advantageously the method provided is more efficient and"SUBSTITUTE SHEET RULE 26"simpler than prior methods to generate vectors encoding pairs of guide RNAs for use in CRISPR based deletions and screening methods.

[0081] Suitably, it will be understood that multiple vectors of the present invention may be made by the method described, in order to generate a library of such vectors.

[0082] Suitably the first step of the method inserts both the first and second crRNA sequences into a vector backbone.

[0083] Suitably the first step comprises: (a) Providing a vector backbone comprising the first and second convergent promoters, and a first cleavage site downstream of the first promoter, and a second cleavage site downstream of the second promoter; (b) Cleaving the backbone at the first and second cleavage sites; (c) Inserting a nucleic acid comprising: the first crRNA sequence, the second crRNA sequence, a third cleavage site downstream of the first crRNA sequence, and a fourth cleavage site downstream of the second crRNA sequence, into the backbone between the first and second cleavage sites, such that the first crRNA sequence is downstream of the first promoter and the second crRNA sequence is downstream of the second promoter.

[0084] Suitably the second step inserts both the first and second tracrRNA sequences into the vector backbone.

[0085] Suitably the second step comprises (a) Cleaving the backbone at the third and fourth cleavage sites; and (b) Inserting a nucleic acid comprising: the first tracrRNA sequence and the second tracrRNA sequence separated by a spacer, into the backbone between the third and fourth cleavage sites, such as that first tracrRNA sequence is downstream of the first crRNA sequence and the second tracrRNA sequence is downstream of the second crRNA sequence.

[0086] Suitably the vector backbone is an empty vector, suitably therefore it does not contain any coding nucleic acids, and may only contain regulatory elements. Suitably the vector backbone may be the backbone of any vector as defined elsewhere herein.

[0087] Suitably the first and second promoters are as defined elsewhere herein. Suitably "Downstream" refers to the nucleic acid sequence 3' of the relevant sequence when read in the direction of transcription. Suitably in the context of a promoter, downstream is the nucleic acid acid sequence for which transcription is controlled by the promoter sequence.

[0088] Suitably, the vector backbone in step (a) comprises from 5' to 3': the first promoter, a first cleavage site, a second cleavage site, and the second promoter."SUBSTITUTE SHEET RULE 26"

[0089] Suitably cleavage sites are sites in the vector backbone where cleavage of the vector backbone may take place, suitably where double stranded cleavage of the vector backbone may take place to open the vector backbone.

[0090] Suitably cleavage sites may be cleaved by an enzyme, suitably a restriction enzyme, suitably a restriction endonuclease, a REase, an ERNase or a restrictase. Suitably the enzyme is operable to cleave DNA. Suitably therefore the or each cleavage site may be a restriction site, suitably which is recognised by a restriction enzyme.

[0091] Suitable restriction enzymes may be selected from Type I, Type II, Type III, or Type IV enzymes. Suitably the restriction enzyme is a Type II restriction endonuclease. Suitably a Type II restriction endonuclease may be a Type IIB restriction endonuclease such as Bcgl and BpII, a Type IIE restriction endonuclease such as Nael, a Type IIF restriction endonuclease such as NgoMIV, a Type IIG enzyme such as RM.Eco57I, a Type IIM restriction endonuclease such as Dpnl, a Type IIS restriction endonuclease such as FokI, BbsI and BsmBI, or a Type IIT restriction endonuclease such as BpulOI and BsII. Suitably the restriction endonuclease is a Type IIS restriction endonuclease.

[0092] In one embodiment, the restriction endonuclease is BsmBI, or an isoschizomer thereof. Suitably the restriction endonuclease BsmBI, or isoschizomers thereof, recognise and cleave at a cleavage site having the sequence: CGTCTCN. Suitably therefore, the or each cleavage site in the vector backbone may comprise the sequence CGTCTCN.

[0093] In one embodiment, the restriction endonuclease is BbsI, or an isoschizomer thereof selected from BpuAI, and BstV2I. Suitably the restriction endonuclease BbsI, or isoschizomers thereof, recognise and cleave at a cleavage site having the sequence: GAAGACN2. Suitably therefore, the or each cleavage site in the vector backbone may comprise the sequence GAAGACN2

[0094] Suitably the first, second, third and fourth cleavage sites may be cleaved by the same restriction endonuclease enzyme, or by different restriction endonuclease enzymes. In one embodiment, the first, second, third and fourth cleavage sites are cleaved by a BsmBI restriction endonuclease, suitably therefore, these cleavage sites comprise the sequence CGTCTCN. Suitably the step of cleaving (b) and the step of cleaving (d) comprise exposing the vector backbone to a BsmBI restriction endonuclease under suitable conditions for the enzyme to cleave at the first and second cleavage site, and the third and fourth cleavage site respectively. Suitable conditions are known in the art, and may be: a mixture of 2 pg vector, 2"SUBSTITUTE SHEET RULE 26"|il Fastdigest BsmBI (THERMO SCI MOLECULAR BIO (FERM)Bpil), 2 pl FastAP (THERMO), 4 pl 10X FastDigest buffer, X pl water up to 40 pl total. Suitably the mixture is incubated at 37°C for 30 minutes.

[0095] Alternatively, the first and second cleavage sites may be cleaved by a BsmBI restriction endonuclease, suitably therefore, these cleavage sites comprise the sequence CGTCTCN. Suitably the step of cleaving (b) comprises exposing the vector backbone to a BsmBI restriction endonuclease under suitable conditions for the enzyme to cleave at the first and second cleavage site. Suitable conditions are known in the art, as noted above.

[0096] In such cases, the third and fourth cleavage sites may be cleaved by a BbsI restriction endonuclease, suitably therefore, these cleavage sites comprise the sequence GAAGACN2. Suitably the step of cleaving (d) comprises exposing the vector backbone to a BbsI restriction endonuclease under suitable conditions for the enzyme to cleave at the third and fourth cleavage site. Suitable conditions are known in the art, and may be: a mixture of 2 pg vector, 2 pl Fastdigest BbsI (THERMO SCI MOLECULAR BIO (FERM)Bpil, 2 pl FastAP (THERMO), 4 pl 10X FastDigest buffer, X pl water up to 40 pl total. Suitably the mixture is incubated at 37°C for 30 minutes.

[0097] It will be appreciated that the restriction endonucleases and their associated cleavage sequences may be swapped, such that the first and second cleavage sites may be interchangeable with the third and fourth cleavage sites.

[0098] Suitably the first and second crRNA and first and second tracrRNA are as defined elsewhere herein.

[0099] Suitably the vector backbone in step (c) comprises from 5' to 3': the first promoter, the first crRNA sequence, a third cleavage site, a fourth cleavage site, the second crRNA, the second promoter.

[0100] Suitably the vector backbone in step (e) comprises from 5' to 3': the first promoter, the first crRNA sequence, the first tracrRNA sequence, spacer, the second tracrRNA sequence, the second crRNA, the second promoter.

[0101] Suitably the steps of ‘inserting’ comprise ligating the vector backbone to the inserted nucleic acid. Suitably ligating the cleaved ends of the vector backbone to the nucleic acid to be inserted. Suitably such ligation is achieved by a ligase enzyme. Suitable ligase enzymes may be selected from any DNA ligase enzymes that catalyse the formation of phosphodiester bonds between two DNA strands. Suitable ligase enzymes may be a Gibson Assembly mix, T4 or T7"SUBSTITUTE SHEET RULE 26"DNA ligase. In one embodiment, the ligase used in the inserting steps is a T7 DNA ligase (NEB M0318). Alternatively, one or both steps of inserting a nucleic acid into the vector backbone may be achieved by Gibson assembly. Suitably in such embodiments, once the backbone of the vector is cleaved in steps (b) and / or (d), Gibson assembly may take place in steps (c) and / or (e). Suitably therefore step (c) and / or (e) may comprise inserting a nucleic acid by performing Gibson assembly, which may suitably be achieved by using a Gibson assembly mix. The Gibson assembly mix is well known in the art (for example from New England Biolabs) to comprise a ligase, as well as an exonuclease and a DNA polymerase, and can be used according to manufacturer's instructions. In one embodiemnt, step (e) is achieved by Gibson assembly.

[0102] Suitably therefore the inserting steps may each comprise the presence of Gibson Assembly mix or a ligase enzyme, suitably a T7 DNA ligase. Suitably the inserting steps may comprise contacting the open vector with the nucleic acid to be inserted and the Gibson Assembly mix or a ligase under suitable conditions for the nucleic acid to be ligated into the vector. Suitably therefore the or each inserting step may comprise ligating. Suitable conditions for ligation are known in the art, such as a mixture comprising: 20 pg of digested vector, 1.2 pg of the nucleic acid to be inserted, 200 pl of 2 T7 buffer and 40 pl T7 DNA ligase to produce a final volume of 400 pl. Suitably the ligation mixture is incubated at 16°C overnight.

[0103] Suitably in step (d) when a nucleic acid comprising: the first tracrRNA sequence and the second tracrRNA sequence separated by a spacer is inserted into the vector backbone, suitably the nucleic acid comprising the first tracrRNA sequence and the second tracrRNA sequence is provided into the reaction mixture. Suitably it is provided by cleaving a separate carrier vector comprising the nucleic acid comprising the first tracrRNA sequence and the second tracrRNA sequence separated by a spacer. Suitably the carrier vector comprises further cleavage sites located 5' and 3' of the nucleic acid comprising the first tracrRNA sequence and the second tracrRNA sequence separated by a spacer. Suitably said further cleavage sites are cleaved by a restriction endonculease enzyme, suitably to release the nucleic acid comprising the first tracrRNA sequence and the second tracrRNA sequence separated by a spacer for use in the ligation step (d). Suitably therefore step (d) may comprise an initial step prior to ligation of providing or releasing the nucleic acid comprising the first tracrRNA sequence and the second tracrRNA sequence separated by a spacer, suitably from a carrier vector. Suitably the further cleavage sites may be cleaved by an enzyme, suitably any restriction enzyme, suitably any"SUBSTITUTE SHEET RULE 26"restriction endonuclease. Suitably the further cleavage sites may be cleaved by a BbsI restriction endonuclease, suitably therefore, these cleavage sites comprise the sequence GAAGACN2.

[0104] Suitably the method may further comprise one or more steps of selecting vectors which successfully comprise the desired nucleic acid insert. Suitably this is accomplished by propagation of a cell comprising the vector, exposing said cells to a selective agent, and harvesting of vectors from surviving cells. Suitably such a selection step may take place between first and second steps, and after the second step of the method to ensure production of vectors with successful insertions. Suitably therefore after the first step of the method, the vector may be transformed into a host cell, and the host cell may be propagated for a suitable period of time in the presence of a selective agent. Suitably the vectors may then be isolated from the surviving host cells before proceeding with the second step of the method. Suitably after the second step of the method, the vector may be transformed into a host cell, and the host cell may be propagated for a suitable period of time in the presence of a selective agent. Suitably the final vectors may then be isolated from the host cells.

[0105] Cas Protein

[0106] As noted above, a "CRISPR-Cas complex" refers to the combination of a Cas polypeptide and a guide RNA, therefore a Cas polypeptide is required to form CRISPR-Cas complexes with the first and second guide RNAs.

[0107] Suitably each guide RNA associates with a Cas polypeptide, suitably via the protein binding segment thereof, suitably via the dsRNA duplex of the protein binding segment thereof. Suitably the first guide RNA associates with a first Cas polypeptide to form a first CRISPR-Cas complex and the second guide RNA associates with a second Cas polypeptide to form a second CRISPR-Cas complex. Suitably therefore the invention provides a pair of Cas proteins, suitably a first and a second Cas protein are provided. Suitably the pair of Cas proteins are capable of forming a pair of CRISPR-Cas complexes with the pair of guide RNAs, suitably of forming a first and a second CRISPR-Cas complex. Suitably a pair of Cas proteins may be provided to cells, and used in the methods herein, either in the form of a protein, or in the form of a nucleic acid encoding a Cas protein which may then suitably be transcribed multiple times to produce at least a pair of Cas proteins. Suitably any reference herein to ‘the Cas protein’ equally applies to the or each Cas protein, specifically to the first and second Cas proteins."SUBSTITUTE SHEET RULE 26"

[0108] Suitably therefore, a pair of CRISPR-Cas complexes are formed, which are operable together to delete a region of DNA in a further aspect of the invention.

[0109] Suitably the or each Cas polypeptide may be any Cas polypeptide operable to effect cleavage of a target sequence of DNA. Suitably both the first and second Cas polypeptides are the same Cas polypeptide.

[0110] Non-limiting examples of Cas proteins include Cascade, Casl, CaslB, Cas2, Cas3, Cas4, Cas5, Cas6, Cas7, Cas8, Cas9 (also known as Csnl and Csxl2), Cas 12a (Cpfl), Cas 12b (C2cl), Casl2c (C2c3), Casl3a (C2c2), Casl3b (C2c4), Casl3c (C2c7), Casl3d, CaslO, Csyl, Csy2, Csy3, Csel, Cse2, Cscl, Csc2, Csa5, Csn2, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, Csxl5, Csfl, Csf2, CsO, Csf4, homologues thereof, or modified versions thereof. In one embodiment the Cas polypeptide is a Cas9 polypeptide or a Cas 12a polypeptide. In one embodiment the Cas polypeptide is a Cas9 polypeptide. Suitably the skilled person will be aware that when using different Cas proteins, different guide RNA sequences and structures are required to form a functioning CRISPR-Cas complex.

[0111] In some cases, different Cas9 proteins (i.e., Cas9 proteins from various species) may be advantageous to use in the various provided methods in order to capitalize on various enzymatic characteristics of the different Cas9 proteins (e.g., for different PAM sequence preferences; for increased or decreased enzymatic activity; for an increased or decreased level of cellular toxicity). Suitable Cas9 polypeptides may be derived from different bacterial species, for example: S. pyogenes, S. aureus, S. thermophilus, L. innocua, and N. meningitidis. In one embodiment, the Cas9 polypeptide is the S. pyogenes Cas9 polypeptide, suitably which may be available from addgene #52962. S pyogenes Cas9 or spCas9 is included in SwissProt under accession number Q99ZW2.

[0112] Suitably the or each Cas polypeptide may be a natural or a modified Cas polypeptide. Suitably Cas polypeptides comprise two nuclease domains; a RuvC domain and an HNH domain. Suitably the RuvC domain acts to cleave the complementary strand of DNA. Suitably the HNH domain acts to cleave the non-complementary strand of DNA.

[0113] Suitably a nuclease domain of the Cas polypeptide may be modified. Suitably one or both of the nuclease domains may be modified. Suitably the nuclease domain may be modified to reduce or remove their nuclease activity. Suitably one of the RuvC or HNH domains may be modified to reduce the nuclease activity by 50% compared to a corresponding wild type Cas"SUBSTITUTE SHEET RULE 26"polypeptide. Suitably such that the Cas polypeptide is operable to carry out single-stranded cleavage of DNA, suitably of a target sequence of DNA. Suitably such a Cas polypeptide may be known as a nickase. Suitably both of the RuvC or HNH domains may be modified to remove the nuclease activity compared to a corresponding wild type Cas polypeptide. Suitably such that the Cas polypeptide is not operable to carry out cleavage of DNA, but can still bind to a target sequence of DNA. Suitably such a Cas polypeptide may be known as a dead Cas ‘dCas’.

[0114] Suitably the nuclease domains may be modified by one or more amino acid substitutions. Suitably the one or more amino acid substitutions are at positions selected from any of: DIO, G12, G17, E762, H840, N854, N863, H982, H983, A984, D986 and / or A987 (based on the amino acid position numbering of a S. pyogenes Cas9), or corresponding positions thereto. Suitably the one or more amino acid substitutions may be selected from any of: D10A, G12A, G17A, E762A, H840A, N854A, N863A, H982A, H983A, A984A, D986A and / or A987A (based on the amino acid position numbering of a S. pyogenes Cas9), or corresponding substitutions thereto. Optionally substitutions other than Alanine may be used. In some embodiments, the modified form of the Cas9 polypeptide comprises a D10A substitution (aspartate to alanine at amino acid position 10 of S. pyogenes Cas9) or the corresponding mutation thereto that can cleave the complementary strand of the target sequence of DNA but has reduced ability to cleave the non-complementary strand of the target sequence of DNA (thus resulting in a single strand break). In some embodiments, the modified form of the Cas9 polypeptide is a H840A (histidine to alanine at amino acid position 840 of S. pyogenes Cas9) or the corresponding mutation thereto that can cleave the non-complementary strand of the target sequence of DNA but has reduced ability to cleave the complementary strand of the target sequence of DNA (thus resulting in a single strand break). In one embodiment, the modified form of the Cas9 polypeptide comprises both D10A substitution (aspartate to alanine at amino acid position 10 of S. pyogenes Cas9) and a H840A (histidine to alanine at amino acid position 840 of S. pyogenes Cas9) or corresponding mutations thereto. Suitably such the Cas9 polypeptide is not able to cleave DNA but is able to target DNA at the target sequence.

[0115] In one embodiment, the Cas polypeptide is not modified, and is suitably a wild type Cas polypeptide. Suitably therefore the Cas polypeptide is operable to carry out double stranded cleavage of DNA, suitably of a target sequence of DNA. Suitably the Cas polypeptide generates double stranded breaks at target sequences in the DNA which hybridize to the DNA-"SUBSTITUTE SHEET RULE 26"targeting section of the guide RNA and that have a protospacer-adjacent motif (PAM) sequence following the the target sequence. This embodiment is preferable such that the first and second CRISPR-Cas complexes used in the methods of the invention act to completely cleave a target DNA sequence either side of a region of DNA to be deleted, thereby excising the region of DNA. The break is then repaired by the cell using non-homologous end joining, such that the double-strand break is repaired by direct ligation of the cleaved ends to one another. More aspects of the CRISPR system are described in Karginov and Hannon, The CRISPR system: small RNA- guided defence in bacteria and archaea, Mole Cell 2010, January 15; 37(1): 7. In one embodiment, the or each Cas protein is a wild type Cas9 or Casl2a protein.

[0116] Suitably the Cas polypeptide, and therefore the CRISPR-Cas complexes used in the invention are operable to carry out cleavage of the target sequence of DNA within about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 100, 200, 500, or more base pairs from the first or last nucleotide of the target sequence.

[0117] Suitably however the Cas protein, whilst being a wild type protein, may still be codon optimised for expression in a Eukaryotic cell, suitably in a mammalian cell and suitably the mammalian cell is a human cell. This type of optimization is known in the art and entails the mutation of foreign-derived DNA to mimic the codon preferences of the intended host organism or cell while encoding the same protein. Thus, the codons are changed, but the encoded protein remains unchanged. Codon usage tables are readily available, for example, at the "Codon Usage Database" available at www.kazusa.orjp / codon / and these tables can be adapted in a number of ways. See Nakamura, Y., et al. "Codon usage tabulated from the international DNA sequence databases: status for the year 2000" Nucl. Acids Res. 28:292 (2000).

[0118] Suitably the Cas polypeptide may further comprise a heterologous sequence, and may therefore be considered a fusion protein. Suitably a heterologous sequence can provide for subcellular localization of the Cas polypeptide. Suitable heterologous sequences include: a nuclear localization signal (NLS) for targeting to the nucleus; a mitochondrial localization signal for targeting to the mitochondria; a chloroplast localization signal for targeting to a chloroplast; an ER retention signal. Suitably the heterologous sequence may be located at the N or the C terminus of the Cas polypeptide. In one embodiment, the Cas polypeptide comprises at least one NLS."SUBSTITUTE SHEET RULE 26"

[0119] Non-limiting examples of NLSs include an NLS sequence derived from: the NLS of the SV40 virus large T-antigen, having the amino acid sequence PKKKRKV (SEQ ID NO: 4); the NLS from nucleoplasms (e.g. the nucleoplasms bipartite NLS with the sequence KRP AATKKAGQ AKKKK (SEQ ID NO: 5) ; the c-myc NLS having the amino acid sequence PAAKRVKLD (SEQ ID NO: 6); the hRNPAl M9 NLS having the sequence NQSSNFGPMKGGNFGGRSSGPYGGGGQYFAKPRNQGGY (SEQ ID NO: 7); the sequence RMRIZFKNKGKDTAELRRRRVEVSVELRKAKKDEQILKRRNV (SEQ ID NO: 8) of the IBB domain from importin-alpha; the sequence RKLKKKJKKL (SEQ ID NO: 9) of the Hepatitis virus delta antigen; the sequence REKKKFLKRR (SEQ ID NO: 10) of the mouse Mxl protein; the sequence KRKGDEVDGVDEVAKKKSKK (SEQ ID NO: 11) of the human poly(ADP-ribose) polymerase; and the sequence RKCLQAGMNLEARKTKK SEQ ID NO: 12 of the steroid hormone receptors (human) glucocorticoid.

[0120] System

[0121] As explained elsewhere herein, the first and second guide RNAs must each associate with a Cas protein to form an active CRISPR-Cas complex complex. Therefore the nucleic acid encoding the first and second guide RNAs of the invention may be combined with a Cas protein, or with a further nucleic acid encoding a Cas protein, and provided as such.

[0122] Therefore, the invention further provides a system comprising both the nucleic acid encoding the pair of guide RNAs of the first aspect, and (i) a further nucleic acid encoding a Cas protein or (ii) one or more Cas proteins, suitably a first and a second Cas protein. Suitably, under option (i), the system is operable to express both the guide RNAs and the Cas protein, suitably the first and second Cas protein, so as to form a first and a second CRISPR-Cas complex. Suitably, under option (ii), the system is operable to express both the guide RNAs, and provides one or more Cas proteins, suitably a first and a second Cas protein, so as to form a first and a second CRISPR-Cas complex. In either case, the system is for assembling a first and a second CRISPR-Cas complex. Suitably therefore the system may be termed an expression system.

[0123] Suitably the nucleic acids of the system may be the same nucleic acid or different nucleic acids. In one embodiment, the nucleic acid encoding the or each Cas protein may be a separate nucleic acid to the nucleic acid encoding the guide RNAs. In another embodiment, the same nucleic acid may encode both the pair of guide RNAs of the first aspect, and the or each Cas protein."SUBSTITUTE SHEET RULE 26"

[0124] The system may be comprised on one or more vectors. In one embodiment, the system may be comprised on two separate vectors, suitably wherein the first vector comprises the nucleic acid encoding the pair of guide RNAs of the first aspect and the second vector comprises a nucleic acid encoding a Cas protein, or one or more Cas proteins. In another embodiment, the system may be comprised on one vector. Suitably the vector comprises both the nucleic acid encoding the pair of guide RNAs of the first aspect, and a further nucleic acid encoding a Cas protein, or one or more Cas proteins.

[0125] Suitably a cell may be provided with the system in any form such that it is able to express the components of the system, and form a first and a second CRISPR-Cas complex. Suitably a cell may be provided with a system comprising both the nucleic acid encoding the pair of guide RNAs of the first aspect, and (i) a further nucleic acid encoding a Cas protein or (ii) one or more Cas proteins, suitably a first and a second Cas protein. Suitably therefore, a cell comprising the system of the invention is a further aspect of the invention. Suitably means of providing the cell with the system, and suitable host cells are described elsewhere herein.

[0126] Region of DNA

[0127] The present invention relates to a nucleic acid structure comprising a pair of guide RNAs operable to target and delete a region of DNA, and to methods of using the pair of guide RNAs to delete a region of DNA in a cell, and further to large-scale methods of screening a genome using a library of vectors each comprising a different pair of guide RNAs to delete different regions of DNA in a plurality of cells. Suitably therefore, the present invention is for use in deleting one or more regions of DNA. Suitably on some occasions, it may be for use in deleting one region of DNA. Suitably on other occasions it may be for use in deleting multiple regions of DNA.

[0128] The function of the or each region of DNA may be known and may be targeted using a pair of guide RNAs of the invention, to achieve an intended effect in a cell. Alternatively, the function of the or each region of DNA may not be known, any may be targeted using a pair of guide RNAs of the invention to investigate the effect in a cell.

[0129] Suitably the or each region of DNA can be any region of DNA. Suitably the or each region of DNA may be a coding or a non-coding region of DNA. In some embodiments, the or each region of DNA is a non-coding Regulatory element (NCRE).

[0130] Non-coding Regulatory element (NCREs) may include any cis-regulatory element or module (CRE or CRM). Suitably the NCRE may be a positive transcriptional regulator, such as"SUBSTITUTE SHEET RULE 26"an enhancer; or may be a negative transcriptional regulator such as a silencer; or may be a genome structure regulator such as an insulator or operator. Suitable examples of non-coding Regulatory elements are described hereinabove, and may be selected from: promoters, enhancers, silencers, insulators, operators, AU-rich elements, upstream activation sequences, polyadenylation signals, terminators, UTRs, IRES, IREs, riboswitches, protein degradation signals, and the like. Suitable examples of non-coding Regulatory elements in DNA may be selected from: promoters, enhancers, silencers, insulators, and operators.

[0131] In one embodiment, the or each region of DNA is an enhancer. "Enhancer" refers to cis-acting DNA sequences that can increase the transcription of coding sequences such as genes. Suitably the enhancer may be an ultraconserved enhancer (UCEs) - "Ultraconserved enhancer" refers to non-coding genetic enhancers that are identical or near identical among different species. Due to their conservation, it is expected that many UCEs will have biological functions in different species that haven't yet been determined. Suitably therefore UCEs represent ideal regions of DNA to be targeted using the present invention.

[0132] Suitable NCREs that may be targeted for deletion in the present invention may be identified from UCNEbase31,VISTA Enhancer Browser32, or ENCODE16.

[0133] Suitably, the or each region of DNA may be a core sequence of a NCRE, suitably of an enhancer, suitably of a UCE. Suitably the core sequence is the central region of the sequence of the NCRE, suitably the central 70%, 75%, 80% 85%, or 90% of the full length sequence of the NCRE. Suitably the core sequence of a NCRE may be predicted or determined using the ENCODE16platform. In some embodiments, the or each target region of DNA may be the core sequence of a UCE.

[0134] Suitably a target region of DNA is deleted by using CRISPR-Cas complexes comprising the pair of guide RNAs described herein. Suitably each guide RNA comprises a crRNA which in turn comprises a DNA-targeting section, which comprises a nucleotide sequence complementary to a target sequence in the DNA. As described hereinabove, the target sequences of DNA to be cleaved by the first and second CRISPR-Cas complexes are selected to be upstream and downstream respectively of the region of DNA to be deleted, such that upon cleavage at the target sequences, the region of DNA is cleaved and removed.

[0135] Methods of Deleting Regions of DNA

[0136] The invention provides method of deleting one or more regions of DNA using the nucleic acid encoding the pair of guide RNAs. Essentially, the nucleic acid encoding the pair of"SUBSTITUTE SHEET RULE 26"guide RNAs and a vector comprising said nucleic acid can be used in methods to delete i.e. ‘knock-out’ one or more regions of DNA.

[0137] Suitably the method comprises step (a) of Providing a cell with the system described herein. Suitably said step comprises providing a cell with (i) the nucleic acid of the first aspect, encoding the pair of guide RNAs, optionally comprised upon a vector, and (ii) a nucleic acid encoding a Cas protein, or a Cas protein. Alternatively said step may comprise providing a cell which already comprises a nucleic acid encoding a Cas protein, or a Cas protein, with the nucleic acid of the first aspect encoding the pair of guide RNAs, optionally comprised upon a vector.

[0138] A suitable cell for the methods of the invention is a prokaryotic or eukaryotic cell. Suitable prokaryotic cells include bacterial cells. Suitably the cell is a eukaryotic cell. Suitable eukaryotic cells include: animal cells, plant cells, fungal cells, or algal cells. Suitable animal cells include vertebrate cells, invertebrate cells, mammalian cells, insect cells, fish cells, amphibian cells, nematode cells. In one embodiment the cell is a mammalian cell. Suitable mammalian cells include a somatic cell, a germ cell, a stem cell, suitably from any species of mammal such as a pig cell, a cow cell, a goat cell, a sheep cell, a rodent cell, a rat cell, a mouse cell, a non-human primate cell, and a human cell. In one embodiment, the cell is a human cell.

[0139] Suitably, the cell used in the methods of the invention maybe a K562 or HEK293T cell. Alternatively, the cell may be a stem cell, suitably a human stem cell, suitably a human embryonic stem cell or a human pluripotent stem cell or a human induced pluripotent stem cell. Suitably a mesenchymal stem cell or a hematopoietic stem cell. In some embodiments the cell is in vitro or ex vivo, suitably therefore the cell may be an isolated cell. In some embodiments the cell is not a human germ cell, a human zygote or a human embryo. In some embodiments the cell may be a non-human cell. In some embodiments the cell is not any part of the human body at the various stages of its formation and development.

[0140] Suitably the providing step of the method comprises introducing the nucleic acids of the system, which (as noted above) at least comprise a nucleic acid encoding the pair of guide RNAs of the first aspect or a vector thereof, into the cell. Sutiably introducing, may comprise transforming or transducing the cell with the system, suitably with the nucleic acids of the system, which (as noted above) at least comprise a nucleic acid encoding the pair of guide RNAs of the first aspect or a vector thereof. Suitably therefore the cell may be deemed a host cell."SUBSTITUTE SHEET RULE 26"

[0141] Suitable methods of transforming or transducing host cells are well known in the art, for example: microinjection, electroporation, sonoporation, biolistics, calcium phosphate- mediated transfection, cationic transfection, liposome transfection, dendrimer transfection, heat shock transfection, nucleofection transfection, magnetofection, lipofection, impalefection, optical transfection, proprietary agent-enhanced uptake of nucleic acids, and delivery via liposomes, irnmunoliposomes, virosomes, or artificial virions.

[0142] Suitably the providing step of the method comprises transforming a cell with the system. In some embodiments, transforming a cell with the system comprises transfecting a cell with the system. In some embodiments transfecting the cell is achieved by lentiviral transfection. Suitable production of lentiviral particles and their use in transfection of cells is described hereinbelow.

[0143] Suitably the cell is transiently or stably transformed with the system. Suitably the cell is stably transformed with the system, suitably therefore the system is stably integrated into the genome of the cell.

[0144] Optionally the method further comprises a step of selecting the cells that successfully contain the system, suitably such a selection step takes place after the providing step. Suitably the selection step comprises selecting the cells that successfully contain genomic integration of the system. Suitably this is achieved by exposing the cells to a selective agent and selecting those cells which survive. Suitably the exposure to a selective agent may take place during the culturing steps (b) and / or (c). In some embodiments, the selection step comprises exposing the cells to an antibiotic for a suitable period of time. Suitably in some cases the antibiotic may be puromycin, and the cells may be exposed to 2 mg ml-1for 6 days. Suitably selecting those cells that survive may take place after the culturing step (c). Suitably selecting those cells that survive may take place by centrifuging, for example using Histopaque-1077 (Sigma) and centrifuging cells at 400g for 30 minutes.

[0145] Suitably the method comprises step (b) of culturing the cell under suitable conditions to express the nucleic acids of the system to thereby produce one or more pairs of guide RNAs and a Cas protein.

[0146] Suitable culturing conditions will depend upon the cell in which the method is carried out. Suitable culturing conditions are known in the art for various cells. For example, for K562 cells, the culture conditions comprise: culturing the cells in RPMI 1640 + L-Glutamine (Gibco), 10% fetal bovine serum (Biowest), and 1% Penicillin-Streptomycin (Gibco). For"SUBSTITUTE SHEET RULE 26"HEK293T cells, the culture conditions comprise: culturing the cells in DMEM (Gibco), 10% FBS and 1% Penicillin- Streptomycin. For human embryonic stem cells (hESCs), the culture conditions comprise: culturing the cells in StemFlex™ Medium (Thermofisher A3349401) on Biolaminin (LN521-02) coated 6- well plate, and passaged using TrypLE Select (Thermofisher 12563011). Suitable cell density and culture conditions may be maintained according to the ENCODE Cell Culture Guidelines.

[0147] Suitably the cell is cultured under suitable conditions to produce one or more pairs of guide RNAs and a Cas protein within the cell.

[0148] Suitably the method comprises step (c) of culturing the cell under suitable conditions to allow the or each pair of guide RNAs and the Cas protein to associate and form CRISPR-Cas complexes, and for the CRISPR-Cas complexes to delete the one or more regions of DNA. Suitably each pair of guide RNAs associates with a pair of Cas proteins to form a first CRISPR-Cas complex and a second CRISPR-Cas complex within the cell. Suitably the first and the second CRISPR-Cas complexes act together to cleave DNA at the first and second target sequences, thereby deleting one or more regions of DNA.

[0149] Suitable culturing methods for step (c) are known in the art, and will depend on the cell being used in the methods, such as explained above. Suitably steps (b) and (c) may take place consecutively under the same culture conditions.

[0150] Suitably the cell is cultured for long enough such that the DNA has been cleaved at the target sequences, and the one or more regions of DNA have been deleted. Suitably the cell is cultured under suitable conditions in steps (b) and (c) for a total of between 5 to 15 days, between 6 to 13 days, between 7 to 11 days, between 8 to 9 days, suitably for about 8 days.

[0151] Suitably the method may further comprise one or more analysis steps to determine the function of the or each deleted region of DNA. Suitably therefore the method may comprise a step of determining the function of the one or more deleted regions of DNA. Suitably such analysis steps take place after step (c), suitably after expression of the CRISPR-Cas complexes in the cell. Suitably the analysis steps may comprise phenotypic and / or genotypic analysis of the cell.

[0152] Suitably phenotypic analysis may comprise: measuring growth of the cell, measuring cell surface markers, measuring excretions or secretions of the cell, measuring cell signaling, measuring transcriptional activities of the cell for example by using luciferase assays to test enhancer or silencer effects, measuring cell differentiation; measuring tumorigenic"SUBSTITUTE SHEET RULE 26"transformation; measuring resistance of the cell to drug treatment; measuring sensitivity of the cell to drug treatment; measuring disease phenotypes of the cell such as biomarkers.

[0153] Suitably genotypic analysis may comprise sequencing, suitably sequencing of the genetic information of the cell such as the genomic DNA or the mRNA of the cell. Suitable sequencing methods may comprise isolating the genomic DNA of the cell and sequencing thereof (whole genome sequencing), or isolating the mRNA of the cell and sequencing thereof (RNA-seq or sc-RNAseq). Genotypic analysis may further comprise determining epigenetic signatures of the DNA in the cell, measuring transcriptional changes in the cell by qPCR for example.

[0154] Suitably sequencing may be used to to determine the one or more deleted regions if unknown. Suitably therefore the method may also comprise a step of determining the location of the one or more deleted regions of DNA. Suitably this may be carried out alongside functional analysis steps.

[0155] Optionally the method may further comprise initial steps of (a) selecting one or more regions of DNA to be deleted and (b) designing a pair of guide RNAs to target the or each region of DNA to be deleted and (c) preparing a system comprising a nucleic acid encoding said pair of guide RNAs. Suitably such steps take place prior to providing the system to the cell.

[0156] Suitably designing a pair of guide RNAs may comprise designing a first gRNA having a crRNA with a DNA-targeting section complementary to a target sequence upstream of the region of DNA to be deleted, and designing a second guide RNA having a crRNA sequence with a DNA-targeting section complementary to a target sequence downstream of the region of DNA to be deleted. Suitable target sequences and their positions relative to the region of DNA to be deleted are described hereinabove, as are the requirements of the DNA-targeting section complementary thereto in the crRNA. Suitable means are known in the art to design guide RNAs which will target and bind to specific target sequences. For example, the software package CRISPRseek may be used to search for potential protospacer (target) sequences with the correct PAM and suggest guide RNAs which will bind thereto. Sutiably the method may comprise a step of selecting a pair of guide RNAs. Suitably this may take place after the step of designing the pairs of guide RNAs, suitably after designing a plurality of pairs of guide RNAs to target a region of DNA. Sutiably the selection step may comprise selecting the optimal pair of guide RNAs based on targeting efficiency and off-target effects."SUBSTITUTE SHEET RULE 26"

[0157] Suitably the method of deleting one or more regions of DNA in a cell functions as follows: the first and second guide RNAs associate with a Cas protein to form a first and second CRISPR-Cas complex, the first and second CRISPR-Cas complexes are targeted to the region of DNA to be deleted by the guide RNAs which bind to the target sequences in the region of DNA, once the first and second CRISPR-Cas complexes are bound to the target sequences the Cas protein cleaves the DNA at the target sequences, thereby generating a first cleavage point and a second cleavage point such that the region of DNA is cleaved, thereby deleting it.

[0158] Suitably the methods of the invention may be in vitro or ex vivo methods. Suitably the methods of the invention may be carried out in one or more cells in vitro or ex vivo. Suitably the methods of the invention are not methods for the treatment of the human or animal body by surgery or therapy practiced on the human or animal body. Suitably the methods of the invention are not methods of modifying the germ line genetic identity of human beings. Suitably the methods of the invention are not methods of modifying the genetic identity of animals which are likely to cause them suffering without any substantial medical benefit to man or animal.

[0159] Methods of Screening a Genome

[0160] The invention further provides a large-scale screening method for screening a genome by deleting many regions of DNA using a plurality of nucleic acids encoding different pairs of guide RNAs arranged in a library, and determining the function of said deleted regions.

[0161] Suitably such a large scale method is similar to the method of deleting one or more regions of DNA but is carried out as a screen across the genome of the cell, suitably where the function of the regions of DNA that are deleted are not necessarily known.

[0162] Suitably such a method may initially comprise a step of selecting a plurality of regions of DNA to be deleted. Suitable such regions of DNA are described hereinabove. Suitably the method may further comprise grouping the plurality of regions of DNA to be deleted. In one embodiment, the regions of DNA to be deleted are enhancers, optionally ultraconserved enhancers. Suitably such enhancers may be grouped into enhancer clusters to be deleted. Suitably such a grouping step may be carried out using known software programs, such as by distance metric computational models GSEA and MAGeCK RRA.

[0163] Suitably such a method may then comprise a step of designing one or more pairs of guide RNAs to target each region of DNA to be deleted, suitably a plurality of pairs of guide"SUBSTITUTE SHEET RULE 26"RNAs are designed to target each region of DNA to be deleted, suitably all possible pairs of guide RNAs are designed to target each region of DNA to be deleted. Suitably designing a pair of guide RNAs may comprise designing a first gRNA having a crRNA with a DNA-targeting section complementary to a target sequence upstream of the region of DNA to be deleted, and designing a second guide RNA having a crRNA sequence with a DNA-targeting section complementary to a target sequence downstream of the region of DNA to be deleted. Suitable target sequences and their positions relative to the region of DNA to be deleted are described hereinabove, as are the requirements of the DNA-targeting section complementary thereto in the crRNA. Suitable means are known in the art to design guide RNAs which will target and bind to specific target sequences. For example, the software package CRISPRseek may be used to search for potential protospacer (target) sequences with the relevant PAM and suggest guide RNAs which will bind thereto.

[0164] Suitably the method may then comprise a step of selecting a pair of guide RNAs. Suitably this may take place after the step of designing the one or more pairs of guide RNAs, suitably after designing a plurality of pairs of guide RNAs to target each region of DNA. Suitably the selection step may comprise selecting the optimal pair of guide RNAs to target each region of DNA to be deleted, suitably based on targeting efficiency and off-target effects.

[0165] Suitably the optimal guide RNAs are those that have high targeting efficiency and low off target effects. Suitable software programs are known in the art which may be used to determine guideRNA targeting efficiency and off-target effects. Suitably the following parameters may be used as a cutoff: gRNA efficacy > O.15,top50ff targetTotalScore < 47 and toplOOff targetTotalScore < 50. Suitably within the software program CRISPRseek.

[0166] Suitably, the method may then comprise a step of pairing the optimal guide RNA which targets upstream of each region of DNA to be deleted, and the optimal guide RNA which targets downstream of each region of DNA to be deleted. Suitably to form an optimal pair of guide RNAs for each region of DNA to be deleted.

[0167] Suitably the method then comprises inserting a nucleic acid encoding each of the optimal pairs of guide RNAs into a vector to form a library of vectors. Suitably this is carried out by the method of generating vectors described hereinabove. Suitably nucleic acids encoding each guide RNA may be ordered from known oligonucleotide synthesis companies such as CustomArray / GenScript. Suitably the nucleic acids encoding each guide RNA are compatible with the vector to be used. Suitably the nucleic acids encoding each guide RNA are compatible"SUBSTITUTE SHEET RULE 26"with a lentiviral vector. For example, the nucleic acid encoding the first guide RNA for use in a lentiviral vector may comprise the following sequence: ATCTTGTGGAAAGGACGAAACACCG-[gRNAl,20nt]- gttttagagctaGAAAtagcaagttGAGACG (SEQ ID NO: 13). For example, the nucleic acid encoding the second guide RNA for use in a lentiviral vector may comprise the following sequence: CGTCTCAACTTGCTATTTCTAGCTCTAAAAC-[reverse complement of gRNA2, 20nt]-CGGGAAAGAGTGGTCTCATACAGAACTTAT (SEQ ID NO: 14).

[0168] Suitably a library of vectors in which each vector comprises a nucleic acid encoding one pair of guide RNAs for targeting one region of DNA is formed. Suitably each vector in the library of vectors is generated using the method described hereinabove. Suitably the library of vectors is a library of lentiviral vectors.

[0169] Suitably the method comprises a step of: (a) Providing a plurality of cells comprising the library of the fourth aspect, wherein each cell comprises a vector of the library, and a nucleic acid encoding a Cas protein or a vector comprising said nucleic acid for expressing a Cas protein.

[0170] Suitably the providing step may comprise introducing the library into the plurality of cells. Suitably introducing may comprise transforming or transducing the plurality of cells with the library. Suitable methods of transforming or transducing are known in the art as described above. Suitably the plurality of cells are transfected, suitably by lentiviral transfection. Suitably therefore the plurality of cells are transfected with lentiviral particles comprising the library, according to the fifth aspect of the invention.

[0171] Suitably the technique of lentiviral particle production and transfection of cells therewith is known in the art. Suitably the lentiviral particles are produced in packaging cells. Suitably the packaging cells are incubated under suitable conditions with the library of vectors, one or more packaging plasmids and one or more envelope plasmids. Suitably the packaging plasmid may be psPAX2. Suitably the envelope plasmid may be pCMV-VSV-G. For example, the method may be carried out as follows: HEK293T cells were grown in five T175 flasks at 50% confluency before transfection. For each flask of 293T cells grown in 25 ml of fresh medium, 15 pg of library vectors, 7 pg of psPAX2, 3.5 pg of pCMV-VSV-G, and 76.5 pl of X- tremeGENE 9 DNA Transfection Reagent were mixed in 1 ml of serum-free medium and used for transfection. Fresh medium was added the day following transfection. Media supernatant"SUBSTITUTE SHEET RULE 26"containing virus particles was collected on the second and third days after transfection, pooled and further concentrated using Lenti-X according to the manufacturer’s protocol.

[0172] Suitably the plurality of cells are transfected with lentiviral particles comprising the library by incubating the plurality of cells with the lentiviral particles under suitable conditions. Suitably the cells are transfected with lentiviral particles by spin-infection. Suitably the cells are transfected with lentiviral particles at low Multiplicity of Infection (MOI) to ensure only one vector of the library is transfected per cell. Suitably the MOI is between 0.2 to 0.3. Suitably the method of spin-infection may comprise any suitable technique in the art, for example:3 x io6cells in each well of a 12- well plate are infected in 1 ml of medium containing 8 pg ml-1of polybrene. In total, four plates may be used for each infection to infect a total of 1.5 x 108cells, which would result in ~300x to ~500x coverage of the library.

[0173] Optionally, the method may then further comprise a step of selecting the cells that have successfully been transfected, and therefore contain a vector from the library. Suitably the step of selection comprises selecting those cells that successfully contain genomic integration of the system. Suitable selection techniques are described hereinabove. Suitably the selection step comprises exposing the cells to a selective agent under suitable conditions, suitably around 2 days after transfection. In some embodiments, the selection step comprises exposing the cells to an antibiotic for a suitable period of time. Suitably in some cases the antibiotic may be puromycin, and the cells may be exposed to 2 mg ml-1for 6 days.

[0174] Suitably only the surviving cells are retained. Suitably selecting those cells that survive may take place by centrifuging, for example using Histopaque-1077 (Sigma) and centrifuging cells at 400g for 30 minutes. Suitably the transfection step may be repeated one or more times. Suitably therefore the step of transfecting the plurality of cells with lentiviral particles comprising the library may be repeated.

[0175] Suitably step (b) comprises culturing the cells under suitable conditions to express, within each cell, the nucleic acids of the vector and the nucleic acid encoding the Cas protein and thereby produce, within each cell, a pair of guide RNAs and a Cas protein. Suitable conditions for said culturing are known in the art and described hereinabove in relation to the method of deletion.

[0176] Suitably step (c) comprises culturing the cells under suitable conditions to allow the pair of guide RNAs and the Cas protein to associate and from a CRISPR-Cas complex, and to delete a region of DNA within each cell. Suitable conditions for said culturing are known in the"SUBSTITUTE SHEET RULE 26"art and described hereinabove. Suitably steps (b) and (c) may effectively be combined, and may be carried out in a continuous culture. Suitably steps (b) and (c) may take around 8 days as described for the method of deletion hereinabove.

[0177] Suitably step (d) comprises performing bulk or single cell screening of the surviving cells to determine the function of the one or more deleted regions of DNA.

[0178] Suitably bulk CRISPR screening comprises the steps of:

[0179] (i) Optionally taking a control sample. Suitably taking a control sample of the cells that have successfully been transfected. Suitably such a control sample is taken prior to step (b) of culturing the successfully transfected cells. Suitably the control sample cells contain a vector from the library but have not yet expressed the CRISPR-Cas complexes therefrom, therefore deletion of a region of DNA has not yet occurred. Suitably the control sample may be referred to as a day 0 sample. Suitably the control sample may comprise about 108cells. Suitably the control sample may be set and stored, for example by freezing.

[0180] (ii) Optionally sorting the cells. Suitably sorting the cells into plates, for example into 96 or 384 well plates. Suitably the cells may be sorted prior to the culturing step (c), or after the culturing step (c), but prior to the challenging step. Suitably the sorted cells may be challenged with different selective pressures as defined in the step below. Suitably therefore the method may be an arrayed method of screening the genome.

[0181] (iii) Challenging the cells with a selective pressure of interest. Suitably this step takes place during step (c) or after step (c). Suitably therefore, during culturing of the transfected cells, they may be challenged with a selective pressure, however preferably the step of challenging the cells takes place after step (c). Suitably therefore the cells already comprise a deletion of one or more regions of DNA when challenged with the selective pressure of interest.

[0182] Suitable selective pressures may comprise: one or more biotic or abiotic pressures. Suitably the selective pressure comprises exposure to one or more drugs, exposure to infection, depriving nutrients, excessive nutrients, depriving growth factors or cytokines, excessive growth factors or cytokines, exposure to cold or heat, exposure to acid or alkali, stimulating proliferation, stimulating differentiation of the cells, stimulating transformation of the cells etc. In one embodiment, the cells are challenged with a drug, suitably by exposing the cells to the drug under suitable conditions. Suitably exposure to a drug may comprise exposure to any type of drug, for example a chemical drug, protein or peptide drug, oligonucleotide drug etc."SUBSTITUTE SHEET RULE 26"Suitably the drug may be a therapeutic drug, suitably a commercial therapeutic drug or a therapeutic drug candidate. Suitably the cells may be exposed to a suitable concentration of the drug, in some cases to more than one concentration of the drug. Suitably the cells are challenged with the selective pressure for a period of time, suitably between 10 to 20 days, suitably between 12 to 18 days, suitably between 14 to 16 days, suitably for around 15 days. Suitably the cells may be challenged with the selective pressure one or more times across the period. In one embodiment, the cells are challenged with a tyrosine kinase inhibitor at a concentration of around 0.1 pM, for a period of about 15 days, suitably during step (c).

[0183] (iv) Optionally sorting the cells based on phenotype. Suitably after the step of challenging the cells, there may be a step of sorting the cells, suitably to isolate those cells that display a phenotype of interest. Suitably the phenotype of interest may be linked to the selective pressure of interest. Suitably the phenotype of interest may be any change in the cell behaviour after exposure to the selective pressure. For example, the phenotype of interest may be: increased growth or proliferation relative to a control population of cells, differentiation, increased nutrient uptake, decreased nutrient uptake, increased secretion, decreased secretion, expression of one or more cell markers. In one embodiment, the cells are sorted based on the expression of one or more cell markers.

[0184] (v) Isolate the genomic DNA of each cell that survives the challenge (and optionally which displays the phenotype of interest). Suitably after challenging the cells with a selective pressure, some cells do not survive, suitably because they contain a disadvantageous deletion effected by the pair of guide RNAs and therefore the CRISPR-Cas complexes expressed therein. Equally after challenging the cells with selective pressure, some cells survive, suitably because they contain an advantageous deletion effected by the pair of guide RNAs and therefore the CRISPR-Cas complexes expressed therein. Suitably genomic DNA may be isolated from the surviving cells by known techniques. Suitably by lysing the cells, and collecting the genomic DNA. For example by using the QIAamp DNA Blood Maxi Kit (QIAGEN). Optionally the cells that do not survive, i.e. Dead cells, may be removed, suitably by refreshing the medium.

[0185] (vi) Amplify and sequence the guide RNAs contained in the genomic DNA. Suitably the guide RNAs are amplified from the genomic DNA by carrying out PCR. Suitably the PCR is carried out using primers which anneal to the nucleic acid sequence encoding the pair of guide RNA sequences in the genomic DNA. Suitably the primers anneal to the first and second"SUBSTITUTE SHEET RULE 26"promoter sequences of the nucleic acid sequence encoding the pair of guide RNAs. Suitably the first primer anneals to the first promoter and the second primer anneals to the second promoter. Suitably the first primer is ATCTTGTGGAAAGGACGAAA (SEQ ID NO: 15), which suitably anneals to the U6 promoter. Suitably the second primer is ATAAGTTCTGTATGAGACCACTCTTTC (SEQ ID NO: 16), which suitably anneals to the Hl promoter. Optionally, stagger sequences may be introduced into the primers to increase the diversity of the next-generation sequencing libraries. Suitably therefore one or more first primers may be used, which anneal to the first promoter. Suitably one or more staggered first primers. Suitably the one or more staggered first primers may be selected from: gATCTTGTGGAAAGGACGAAA (SEQ ID NO: 17), eg ATCTTGTGGAAAGGACGAAA (SEQ ID NO: 18), teg ATCTTGTGGAAAGGACGAAA (SEQ ID NO: 19), atcgATCTTGTGGAAAGGACGAAA (SEQ ID NO: 20), tateg ATCTTGTGGAAAGGACGAAA (SEQ ID NO: 21), and ctatcgATCTTGTGGAAAGGACGAAA (SEQ ID NO: 22). Suitably therefore one or more second primers may be used, which anneal to the second promoter. Suitably one or more staggered second primers. Suitably the one or more staggered second primers may be selected from: gATAAGTTCTGTATGAGACCACTCTTTC (SEQ ID NO: 23), cgATAAGTTCTGTATGAGACCACTCTTTC (SEQ ID NO: 24), tcgATAAGTTCTGTATGAGACCACTCTTTC (SEQ ID NO: 25), atcgATAAGTTCTGTATGAGACCACTCTTTC (SEQ ID NO: 26), tateg ATAAGTTCTGTATGAGACCACTCTTTC (SEQ ID NO: 27), and ctatcgATAAGTTCTGTATGAGACCACTCTTTC (SEQ ID NO: 28).

[0186] Suitably therefore the genomic DNA containing a nucleic acid encoding a pair of guide RNAs, according to the first aspect, is amplified. Suitably the use of different and convergent promoter sequences in the nucleic acid encoding the pair of guideRNAs means that the primers will amplify genomic DNA fragments containing a nucleic acid encoding a pair of guide RNAs, each fragment having a unique sequence, thereby avoiding the use of any sequencing barcodes. Suitably to produce an amplified fragment of genomic DNA. Suitably the PCR is carried out using known techniques, for example, for each 100 pl PCR reaction, 10 pg of genomic DNA, 50 pl of 2x LongAmp master mix, 2 pl of 25 pM U6_stagger primer, 2 pl of 25 pM Hl_stagger primer were used. A suitable PCR procedure may be as follows: 94 °C for 30 s, 25 cycles of 94 °C for 15 s, 60 °C for 15 s and 65 °C for 60 s, and 65 °C for 10 min. Suitably the amplified"SUBSTITUTE SHEET RULE 26"fragments, which comprise the genomic DNA containing a nucleic acid encoding a pair of guide RNAs are then sequenced. Optionally prior to sequencing, the amplified fragments are cleaned up and gel-purified, for example using MinElute PCR Purification Kit. Suitable sequencing methods are well known in the art. One such method is Illumina sequencing. Suitably, to prepare for sequencing, each amplified fragment is ligated to a sequencing adapter. Suitably the sequencing adapter is an Illimna Truseq adaptor. Suitably sequencing libraries are then prepared, suitably according to the ENCODE protocol, and sequenced, for example using the Illumina HiSeq4000 sequencer.

[0187] (vi) Determine the frequency of each pair of guide RNA amongst the cells. Suitably determining the frequency of each pair of guide RNAs comprises determining which pairs of guide RNAs are depleted or enriched. Suitably determining the frequency of each pair of guideRNAs is carried out by counting the sequenced reads, and mapping the sequenced reads to the pairs of guideRNAs selected in the initial design step. Suitable software known in the art may be used to carry out such a counting step and to determine those pairs of guide RNAs that are depleted or enriched. Suitably those which are signficantly depleted or enriched, suitably compared to the control sample of day 0 cells. Suitably a computational method called Modelbased Analysis of Genome-wide CRISPR / Cas9 Knockout is used (MAGeCK). Suitably with a cutoff of RRA score < O.O140. Suitably this step may further comprise determining which enhancer clusters are depleted or enriched. Suitably in such an embodiment, the enhancers are first clustered, and then depletion or enrichment of said clusters is determined, suitably by MAGeCK and GSEA.

[0188] Suitably determining which pairs of guide RNAs are depleted or enriched in the cells can be used to infer the function of the regions of DNA they target and delete. Suitably, the depletion of a pair of guide RNAs identifies a region of DNA whose deletion sensitises cells to the selective pressure applied during the method. Suitably, the enrichment of a pair of guide RNAs identifies a region of DNA whose deletion confers a selective advantage to the selective pressure applied during the method.

[0189] Alternatively the cells comprising the library of vectors created in the method of screening, may undergo single cell CRISPR screening. Suitable single cell CRISPR screening is a different process compared to bulk screening, but may share some of the same steps.

[0190] Suitably single cell screening comprises the steps of:"SUBSTITUTE SHEET RULE 26"

[0191] (i) Challenging the cells with a selective pressure of interest. Suitably this step takes place during step (c) or after step (c). Suitably therefore, during culturing of the transfected cells, they may be challenged with a selective pressure. However preferably the step of challenging the cells takes place after step (c). Suitably therefore the cells already comprise a deletion of one or more regions of DNA when challenged with the selective pressure of interest.

[0192] Suitable selective pressures may comprise: one or more biotic or abiotic pressures. Suitably the selective pressure comprises exposure to one or more drugs, exposure to infection, depriving nutrients, excessive nutrients, depriving growth factors or cytokines, excessive growth factors or cytokines, exposure to cold or heat, exposure to acid or alkali, stimulating proliferation, stimulating differentiation of the cells, stimulating transformation of the cells etc. In one embodiment, the cells are challenged with a drug, suitably by exposing the cells to the drug under suitable conditions. Suitably exposure to a drug may comprise exposure to any type of drug, for example a chemical drug, protein or peptide drug, oligonucleotide drug etc. Suitably the drug may be a therapeutic drug, suitably a commercial therapeutic drug or a therapeutic drug candidate. Suitably the cells may be exposed to a suitable concentration of the drug, in some cases to more than one concentration of the drug. Suitably the cells are challenged with the selective pressure for a period of time, suitably between 10 to 20 days, suitably between 12 to 18 days, suitably between 14 to 16 days, suitably around 15 days. Suitably the cells may be challenged with the selective pressure one or more times across the period. In one embodiment, the cells are challenged with a tyrosine kinase inhibitor at a concentration of around 0.1 pM, for a period of about 15 days, suitably during step (c).

[0193] (ii) Optionally sorting the cells based on phenotype. Suitably after the step of challenging the cells, there may be a step of sorting the cells, suitably to isolate those cells that display a phenotype of interest. Suitably the phenotype of interest may be linked to the selective pressure of interest. Suitably the phenotype of interest may be any change in the cell behaviour after exposure to the selective pressure. For example, the phenotype of interest may be: increased growth or proliferation relative to a control population of cells, differentiation, increased nutrient uptake, decreased nutrient uptake, increased secretion, decreased secretion, expression of one or more cell markers. In one embodiment, the cells are sorted based on the expression of one or more cell markers."SUBSTITUTE SHEET RULE 26"

[0194] (iii) Isolating the RNA of each cell that survives the challenge (and optionally which displays the phenotype of interest). Suitably isolating the RNA of each cell comprises isolating the guide RNAs present in each cell and isolating the mRNA present in each cell. Suitably the guide RNAs are isolated using capture sequences present in each guide RNA sequence. Suitable capture sequences and their location in guide RNAs are explained elsewhere herein. Suitably mRNA and guide RNA capture is carried out according to the lOx Genomics protocol, suitably the lOx Genomics Chromium platform according to the manufacturer’s protocol (ChromiumNextGEMSingleCell3' v3.1). Optionally, the RNA may be isolated from each cell by lysing each cell as explained above. Optionally after isolating the guide RNAs and the mRNA from each cell, the RNA may be barcoded.

[0195] Suitably this step may comprise mixing the cells that survive with barcoded gel beads operable to bind to the capture sequences, optionally in the presence of a partitioning oil. Suitably to form an emulsion, suitably comprising a plurality of droplets, each droplet comprising a cell and a barcoded gel bead. Suitably the cells are then lysed, and the barcodes bind to the capture sequences to form barcoded RNA. Suitably therefore each droplet comprises the barcoded RNA of one cell. Suitably the droplets are then dissolved, or deemulsified, to release the barcoded RNA of each cell.

[0196] (iv) Sequence the gRNAs expressed in the cell and sequence the mRNA expressed in the cell. Suitably single-cell sequencing libraries of the isolated RNA from each cell are then prepared for example using the lOx Genomics Chromium platform according to the manufacturer’s protocol (ChromiumNextGEMSingleCell3’ v.3.1). Suitably the isolated RNA is then sequenced, for example on the Illumina NovaSeq 6000 sequencer. Suitably the RNA from each cell may undergo reverse transcription to produce cDNA. Suitably the barcoded RNA from each cell or the cDNA from each cell may be pooled. Suitably to produce a cDNA sequencing library, which is then sequenced.

[0197] (v) Correlate mRNA expression with guide RNA expression in each cell. Suitably the sequencing reads from the mRNA and the sequencing reads from the guide RNAs are correlated by creating a matrix from each individual cell. Suitably a matrix which correlates gene expression (by virtue of mRNA expression) to guideRNA expression. Suitably such a correlating step may be carried out using known techniques the art, for example using the computational program Seurat 4.0.2. Suitably this correlation step indicates which guideRNAs were expressed and which changes in gene expression were thereby affected. Suitably"SUBSTITUTE SHEET RULE 26"compared to a control cell, suitably compared to a control cell from the control sample taken at day 0 of the method (see above). Suitably this correlation step indicates which guideRNAs were expressed that caused one or more genes to be upregulated, or increased in expression, and which guide RNAs were expressed that caused one or more genes to be downregulated, or decreased in expression. Suitably compared to a control cell as defined above. Suitably the mRNAs that are increased in a cell indicate an increase in the expression of the related gene. Suitably the mRNAs that are decreased in a cell indicate a decrease in the expression of the related gene. Suitably the gene expression changes are quantified as the fold change of mean of normalized gene expression from the cell population divided by the mean of normalized gene expression from the control cell population. Suitably the changes in gene expression in each cell are caused by the deletion of a region of DNA effected by the pair of guide RNAs expressed in the cell. Suitably therefore the function of a region of DNA can be inferred from the mRNA expression resulting from its deletion using CRISPR-Cas complexes comprising the pairs of guideRNAs described herein.

[0198] Suitably, as described above, the methods of screening may comprise a step of challenging the cells with a selective pressure of interest, and selecting those cells that survive. Suitably during step (c) of the method. In some embodiments, the selective pressure may comprise exposure to a drug, either a known drug or a candidate drug. Suitably such an embodiment may be used to determine one or more regions of DNA involved in drug resistance. Suitably such information may be used to determine regions of DNA that may be desirable therapeutic targets, suitably to reduce drug resistance.

[0199] Suitably therefore, the invention may further comprise a method of screening a genome to determine one or more therapeutic targets comprising: (a) Providing a plurality of cells comprising the library of the fourth aspect, wherein each cell comprises a vector of the library, and a nucleic acid encoding a Cas9 protein or a vector comprising said nucleic acid for expressing a Cas9 protein; (b) Culturing the cells under suitable conditions to express, within each cell, the nucleic acids of the vector and the nucleic acid encoding the Cas protein and thereby produce, within each cell, a pair of guide RNAs and a Cas9 protein; (c) Culturing the cells under suitable conditions to allow the pair of guide RNAs and the Cas protein to associate and delete a region of DNA within each cell; (d) exposing the cells to a drug and selecting those cells that survive; and (e) Performing bulk or single cell screening of the surviving cells to determine the one or more deleted regions of DNA. Optionally wherein the one or more"SUBSTITUTE SHEET RULE 26"regions of DNA provide resistance to said drug. Optionally wherein the one or more regions of DNA are therapeutic targets, suitably towards which novel therapies may be developed.

[0200] The practice of the present invention employs, unless otherwise indicated, conventional techniques of immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA, which are within the skill of the art. See Sambrook, Fritsch and Mamatis, MOLECULAR CLONING: A LABORATORY MANUAL, 2nd edition (1989); CURRENT PROTOCOLS IN MOLECULAR BIOLOGY (F. M. Ausubel, et al. eds., (1987)); the series METHODS IN ENZYMOLOGY (Academic Press, Inc ): PCR 2: A PRACTICAL APPROACH (M.J. MacPherson, B.D. Hames and G.R. Taylor eds. (1995)), Harlow and Lane, eds. (1988) ANTIBODIES, A LABORATORY MANUAL, and ANIMAL CELL CULTURE (R.I. Freshney, ed. (1987)).BRIEF DESCRIPTION OF THE FIGURES

[0201] The invention will now be described with reference to the following figures, in which:

[0202] FIG. 1 shows Identification of essential UCEs and validated enhancers: (1A) MAGeCK algorithm was used to identify significant hits depleted from the cells cultured for an additional 15 days compared to the initial population. The Manhattan plot shows the distribution of all the target regions. Significant hits were above the dash line, indicating the cutoff of MAGeCK RRA score 0.01. Different colors represent different chromosomes. The UCEs selected for downstream analyses were indicated. The NCRE de novo l is an intergenic fragment included in the library design; (FIG. IB, FIG. 1C, FIG. ID, FIG. IE) Knockout (KO) essential NCREs from K562 cells using the dual-CRISPR system. Upper panel: K562 cells were transfected with the respective guide RNA pairs that target the indicated NCREs. Singlecell clones with the respective NCRE deletion were selected. The blue arrow indicates the intact genomic regions. The red arrow indicates the NCRE deletions. Lower panel: cell proliferation assay was performed by mixing the KO cell lines with cells expressing GFP at a 1 :1 ratio. The changes in GFP percentage were monitored at indicated time points by FACS. Cells with dual-CRISPR guide RNAs targeting GFP sequences served as negative controls (Ctrl KO). The y axis represents the relative ratio of the GFP negative cells to the positive cells. The ratio of cells in the initial mixture was set as 100% (n=3 biological independent samples; values are shown as the mean ± s.d.; **P < 0.01, ***P < 0.001, ****p < 0.0001, calculated using two-way ANOVA); (FIG. IF) Enhancer activities were measured by luciferase assay. The respective NCREs were cloned by PCR into the enhancer reporter plasmid pGL4.23"SUBSTITUTE SHEET RULE 26"with a minimal promoter. The empty pGL4.23 plasmid was used as the control for the baseline luciferase activities. The y axis represents the relative unit of luciferase activity compared to that of pGL4.23 empty plasmids (n = 3 biological independent samples; bars show mean value ± s.d.; **P < 0.01, calculated using unpaired t-test); (FIG. 1G) Silencer activities were measured by luciferase assay. The respective NCREs were cloned by PCR into the silencer reporter plasmid pGL4.53 with a PGK promoter. The empty pGL4.53 plasmid was used as the control for the baseline luciferase activities. The y axis represents the relative unit of luciferase activity compared to that of pGL4.53 empty plasmids (n = 3 biological independent samples; bars show mean value ± s.d.; ***P < 0.001, calculated using unpaired t-test); (FIG. 1H) TADs identified by Hi-C surrounding the NCRE de novo l are shown, and the location of de novo l is indicated by the vertical blue bar. Horizontal yellow and blue bars indicate distinct TADs. Transcription of nearby genes was quantified by qPCR (n=3 biological independent samples; bars show mean value ± s.d.; **P < 0.01, calculated using unpaired t- test); (FIG. II) The expression level of gene PTPRD was measured by qPCR for K562 cells and K562 cells with CRISPR / dCas9-SAM activation systems targeting the PTPRD gene (PTPRD SAM) (n=3 biological independent samples; dots represent mean value ± s.d.; *P < 0.05, **P < 0.01, calculated using unpaired t-test); (FIG. 1J) Cell proliferation assay for K562 WT and K562 cells with CRISPR / dCas9-SAM activation systems targeting the PTPRD gene (PTPRD SAM). The y axis represents the relative ratio of the GFP negative cells to the positive cells. The ratio of cells in the initial mixture was set as 100% (n=3 biological independent samples; values are shown as the mean ± s.d.; ***P < 0.001, ****p < 0.0001, calculated using two-way ANOVA);

[0203] FIG. 2 shows Drug resistance regulated by UCEs: (2A) UCE-drug interactions study in K562 cells. MAGeCK MLE algorithm was used to identify UCEs involved in imatinib resistance based on dual-CRISPR screenings. Three different cell populations were used: the day 0 population, the 15-day imatinib treatment population and the 15-day non-treatment control population. Beta score indicates the degree of selection upon UCE removal relative to the day 0 initial population. The y axis represents beta scores of thel5-day imatinib treatment. The x axis shows beta scores of the 15-day non-treatment condition. The horizontal and vertical dashed lines indicate the mean plus or minus one standard deviation of the 15-day imatinib treatment and the 15-day non-treatment control beta score, respectively. The diagonal dashed line indicates the mean plus or minus one standard deviation of the differential beta scores,"SUBSTITUTE SHEET RULE 26"which can be calculated by subtracting the 15-day non-treatment control from the 15-day imatinib treatment beta score. The orange group shows UCEs conferring imatinib resistance upon removal; the purple group shows UCEs sensitizing cells to imatinib treatment upon removal. Selected UCEs for downstream analyses were marked; (FIG. 2B) Enhancer activities were measured by luciferase assay. The respective UCEs were cloned by PCR into the enhancer reporter plasmid pGL4.23 with a minimal promoter. The empty pGL4.23 plasmid was used as the control for the baseline luciferase activities. The y axis represents the relative unit of luciferase activity compared to that of pGL4.23 empty plasmids, QKI Jo is significant (n = 3 biological independent samples; bars show mean value ± s.d.; **P < 0.01, n.s., not significant); (FIG. 2C) Silencer activities were measured by luciferase assay. The respective UCEs were cloned by PCR into the silencer reporter plasmid pGL4.53 with a PGK promoter. The empty pGL4.53 plasmid was used as the control for the baseline luciferase activities. The y axis represents the relative unit of luciferase activity compared to that of pGL4.53 empty plasmids (n = 3 biological independent samples; bars show mean value ± s.d.; ***P < 0.001, ****p < 0.0001, calculated using unpaired t-test); (FIG. 2D, E) Knockout (KO) ZNF503_Op and QKI Jo from K562 cells using the dual-CRISPR system. K562 cells were transfected with the respective guide RNA pairs that target the indicated UCEs. Single-cell clones with the respective UCE deletion were selected. The blue arrow indicates the intact genomic regions. The red arrow indicates the NCRE deletions; (FIG. 2F) (I) Drug resistance conferred by knockouts of ZNF503_Op and QKI Jo. The ZNF503_Op and QKI Jo KO cells were treated with 0.4 pM imatinib for three days. Cell viability was measured by CellTiter-Blue (n=3 biological independent samples; Bars show mean value ± s.d.; *P < 0.05, calculated using unpaired t-test), (II) Cell proliferation assay was performed by mixing the enhancer E22:23590 KO cell lines with cells expressing GFP. The changes in GFP percentage were monitored at indicated time points by FACS, to the positive cells. Cells with dual CRISPR guide RNAs targeting RNAs served as negative controls (Ctrl KO). The ratio of cells in the initial mixture was set as 100% (n = 3 independent biological samples; values are shown as mean ± s.d.;****P < 0.0001, calculated using two-way ANOVA); (FIG. 2G, H) (I) Potential genes regulated by the UCEs. TADs identified by Hi-C surrounding ZNF503_Op and QKI Jo are shown, and the locations are indicated by vertical blue bars. Transcriptions of nearby genes were quantified by qPCR (n = 3 biological independent samples; bars show mean value ± s.d; *P < 0.05, **P < 0.01, ***P < 0.001, calculated using unpaired t-test), (II) two replicates are shown in the bottom panels."SUBSTITUTE SHEET RULE 26"

[0204] FIG. 3 shows Identification of essential enhancers in K562 cells: (3 A) Outline of the design of dual-CRISPR libraries targeting all potential enhancers in K562 cells. Enhancers were predicted by ENCODE based mainly on the combination of H3K4mel and H3K27ac and other markers. Two strategies were used to design paired guide RNAs to target all the potential enhancers in K562 cells; (FIG. 3B) The outline of the optimization of the dual CRISPR system. To have an optimal NGS sequencing efficiency, the distance between the two scaffolds was increased from 50bp to 200bp by nonsense sequences; (FIG. 3C) Essential enhancers in K562 cells were identified using dual-CRISPR screenings. MAGeCK algorithm was used to identify significant hits depleted from cells cultured for an additional 15 days compared to the initial population. The Manhattan plot shows the distribution of all the target regions. Significant hits were above the dash line, indicating the cutoff of MAGeCK RRA score 0.01. Different colors represent different chromosomes. The essential enhancers selected for downstream analyses were indicated; (FIG. 3D) Cell proliferation assay was performed by mixing the enhancer E22:23590 KO cell lines with cells expressing GFP. The changes in GFP percentage were monitored at indicated time points by FACS. Cells with dual-CRISPR guide RNAs targeting GFP sequences served as negative controls (Ctrl KO). The y axis represents the relative ratio of the GFP negative cells to the positive cells. The ratio of cells in the initial mixture was set as 100% (n=3 biological independent samples; values are shown as the mean ± s .d; ****P < 0.0001, calculated using two-way ANOVA); (FIG. 3E) Enhancer activities were measured by luciferase assay. The essential K562 putative enhancers were cloned by PCR into the enhancer reporter plasmid pGL4.23 with a minimal promoter. The empty pGL4.23 plasmid was used as the control for the baseline luciferase activities. The y axis represents the relative unit of luciferase activity compared to that of pGL4.23 empty plasmids (n = 3 biological independent samples; bars show mean value ± s.d.; **P < 0.01, ***P < 0.001, ****p < 0.0001, calculated using unpaired t-test); (FIG. 3F) Silencer activities were measured by luciferase assay. The essential K562 putative enhancers were cloned by PCR into the silencer reporter plasmid pGL4.53 with a PGK promoter. The empty pGL4.53 plasmid was used as the control for the baseline luciferase activities. The y axis represents the relative unit of luciferase activity compared to that of pGL4.53 empty plasmids (n = 3 biological independent samples; bars show mean value ± s.d.; ***P < 0.001, **** < 0.0001, calculated using unpaired t-test); (FIG. 3G) TADs identified by Hi-C surrounding the E22:23590 are shown and the location of E22:23590 is indicated by the vertical blue bar. Horizontal yellow and blue bars indicate distinct TADs. Transcription of nearby genes was quantified by qPCR (n=3 biological independent samples;"SUBSTITUTE SHEET RULE 26"bars show mean value ± s.d.; **P < 0.01, ***P < 0.001, ****p < 0.0001, calculated using unpaired t-test).

[0205] FIG. 4 shows Identifying essential cluster of enhancers with redundant functions: (4 A) Illustration of a cluster of enhancers regulating gene transcription; (FIG. 4B) Venn diagram indicates the overlapping clusters identified by GSEA and RRA. The plot shows the ranking of the individual enhancers from the top 3 clusters in the original screening analysis. The y axis represents the MAGeCK RRA score. The x axis represents the ranking of the individual enhancers based on the RRA score; (FIG. 4C) Enhancer activities were measured by luciferase assay. Individual putative enhancer elements from the top 3 enhancer clusters were cloned by PCR into the enhancer reporter plasmid pGL4.23 with a minimal promoter. The empty pGL4.23 plasmid was used as the control for the baseline luciferase activities. The y axis represents the relative unit of luciferase activity compared to that of pGL4.23 empty plasmids (n = 3 biological independent samples; bars show mean value ± s.d.; *P < 0.05, ** / ’ < 0.01, *** / > < 0.001, calculated using unpaired t-test); (FIG. 4D, E, F) Growth effect of one enhancer (d), two-enhancer (e), three- enhancer (f) removal from the cluster. Cell proliferation assay was performed by mixing the KO cell lines with cells expressing GFP at a 1 : 1 ratio. The changes in GFP percentage were monitored at indicated time points by FACS. Cells with dual-CRISPR guide RNAs targeting GFP sequences served as negative controls (Ctrl KO). The y axis represents the relative ratio of the GFP negative cells to the positive cells. The ratio of cells in the initial mixture was set as 100%. Rl, R2 and R3 represent the three individual potential enhancers targeted by the dual-CRISPR libraries (n=3 biological independent samples; values are shown as the mean ± s.d.; *P < 0.05, ****p < 0.0001, calculated using two-way ANOVA); (FIG. 4G) Upper panel, epigenetic signatures surrounding chr22 enhancer cluster. The RNA Pol2 ChlA-PET loops are shown. The red arrow indicates the location of gene SLC25A1. Lower panel, transcription of SLC25A1 in different enhancer KO clones were quantified by qPCR (n=3 biological independent samples; values are shown as the mean ± s.d. for each bar; * / ’ < 0.05, **P < 0.01, ***P < 0.001, calculated using unpaired t-test).

[0206] FIG. 5 shows Single-cell RNA-seq coupled with the dual-CRISPR system: (5A) Illustration of adapting the dual-CRISPR system with scRNA-seq. Two distinct capture sequences were inserted into the stem loops of the guide RNA scaffolds. The guide RNAs could then be captured together with the mRNA within individual single cells; (FIG. 5B) Transcriptional changes of genes surrounding PAX6_Ta measured by bulk RNA-seq. The y"SUBSTITUTE SHEET RULE 26"axis shows the logarithm fold change of genes between the PAX6_Ta KO clone and control K562 cells. The x axis indicates the genomic coordinate. The dashed line indicates the location of PAX6_Ta. Distinct colors represent different TADs; (FIG. 5C) Transcriptional changes of genes surrounding PAX6_Ta measured by scRNA-seq. The y axis shows the logarithm fold change of the captured genes between single cells with the guide RNA pair targeting PAX_Ta and control K562 single cells. The x axis indicates the genomic coordinate. Dashed line indicates the location of PAX6_Ta. Distinct colors represent different TADs; (FIG. 5D) The upper panel shows epigenetic signatures surrounding enhancer El 1 : 125334 as indicated by the red line. The lower panel shows the differential gene expression testing results. Violin plots show the normalized expression levels of candidate genes in perturbed (188 cells) and control (4282 cells) groups. The gene EI24 was significantly down-regulated (P value < 0.0097 calculated by MAST fitted model; n.s., not significant).

[0207] FIG. 6 shows UCE PAX6_Ta regulating the cardiomyocyte differentiation in hESCs: (6A) KEGG pathway enrichment analysis of genes differentially regulated in PAX6_Ta KO cells (+ / -5Mbp around PAX6_Ta). The Y-axis represents the significantly enriched KEGG pathways, and the x-axis represents their corresponding gene ratios, i.e., the ratio between the number of unique genes in a specific pathway and the number of unique genes mapped to all these pathways. The left panel presents all significantly up-regulated pathways, and the right panel presents all down-regulated ones. The dot size denotes the number of genes mapped in a pathway, and the color indicates the significance level of the enrichment (FDR < 0.25); (FIG. 6B) Silencer activities were measured by the repressive ability on the super-core (SC) promoter driving the expression of GFP. A previously published silencer served as the positive control (validated silencer). The empty vector (SC promoter) was used as the control to normalize the respective repressive activities (n=3 biological independent samples; values are shown as the mean ± s.d. for each bar; ***P < 0.001, ****p < 0.0001, calculated by using unpaired t-test); (FIG. 6C) Knockout (KO) PAX6_Ta in NKX2-5eGFP / whESC. PCR results show the removal of PAX6_Ta in NKX2-5eGFP / whESC bulk cells; (FIG. 6D) The removal of PAX6_Ta caused the defect of cardiomyocyte differentiation, as measured by NKX2-5 / GFP expression. Upper panel, NKX2-5eGFP / whESC cells (hESC_NKX) after differentiation; lower panel, NKX2-5eGFP / whESC PAX6_Ta KO bulk cells (PAX6_Ta_KO) after differentiation (Scale bar: 275 pm); (FIG. 6E) (I) Quantification of the cardiomyocyte differentiation as shown in (d) by FACS. The cardiomyocyte differentiation efficiency measured by GFP in NKX2-5eGFP / whESC was set to"SUBSTITUTE SHEET RULE 26"100%. (n=2 biological independent samples; values are shown as the mean ± s.d.; **P < 0.01, calculated using unpaired t-test), (II) the same quantification is shown in panel (II) based on n=3 biological independent samples; values are shown as the mean ± s.d.; **P < 0.001, calculated using unpaired t-test; (FIG. 6F) (I) The transcription of gene RCN1 was quantified by qPCR before and after differentiation in both NKX2-5eGFP / w hESC cells (hESC NKX) and NKX2-5eGFP / w hESC PAX6_Ta KO bulk cells (PAX6_Ta_KO) (n=2 biological independent samples; values are shown as the mean ± s.d.; *P < 0.05, calculated using unpaired t-test); (FIG. 6F) (II) the same quantification is shown in panel (II) based on n=3 biological independent samples; values are shown as the mean ± s.d.; **P < 0.001, calculated using unpaired t-test.

[0208] FIG 7 shows Design and testing of the dual CRISPR plasmid: (FIG. 7A) The expression of the two guide RNAs was driven by the human U6 and Hl promoters, respectively. The transcription driven by the U6 and Hl promoters is in a convergent direction, with transcription termination sequences at the end of each guide RNA scaffold sequence. (FIG.7B) K562 cells were transiently transfected with the designed plasmid containing two guide RNAs targeting a -250 bp sequence. A PCR amplicon of 600 bp in size (intact region) indicated the original DNA sequence in the genome, and the genomic sequence knockout (KO) by the two guide RNAs was shown as the PCR amplicon of 350 bp in size (KO region). (FIG.7C) Sanger sequencing of the PCR fragments in (B) showed a complete knockout by the designed two guide RNAs. (FIG.7D) Cloning strategy of the dual-CRISPR screening libraries. The U6 and Hl promoters drive the expression of two guide RNAs separately in a convergent orientation. To make the dual-CRISPR libraries, oligo pools that contain dual crRNAs and additional restriction sites were cloned between the two promoters. Then the plasmids containing the crRNAs were amplified and digested again to insert the dual-tracrRNA scaffolds to form the complete dual CRISPRs. (FIG.7E) Cells containing dual-CRISPR libraries could be used to study different biological pathways. Genomic DNA from different cell populations was then isolated, and the dual-CRISPR libraries could be amplified by direct PCR reactions, which would be ready for next-generation sequencing (NGS). The change in abundance of dual-guide-RNAs will then be calculated by different algorithms to identify potential hits.(FIG.7F) Similar to Fig.7B, K562 cells were transiently transfected with the designed plasmids containing two guide RNAs flanking the testing sequences. In the left panel, the PCR amplicon of 600 bp in size (intact region) indicates the original DNA sequence in the genome, and the"SUBSTITUTE SHEET RULE 26"genomic sequence knockout (KO) by the two guide RNAs is shown as the PCR amplicon of 350 bp in size (KO region). The differences in transcription and editing efficiency driven by U6 and Hl promoters were tested by swapping the respective gRNAs, indicated as U6_gRNAl / Hl_gRNA2 and U6_gRNA2 / Hl_gRNAl . In the right panel, the PCR amplicon of 1500bp in size (intact region) indicates the original sequence upstream of the Top2a gene, and the genomic sequence knockout by the two guide RNAs is shown as the PCR amplicon of 1000 bp in size (KO region).

[0209] FIG 8 shows UCE dual-CRISPR screening in K562 cells: (FIG.8A) Western blot to validate the Cas9-expressing K562 cells. The expression of Cas9-FLAG proteins in different clones was probed with the anti-FLAG antibody. Actin was used as the loading control. K562 _Cas9_FLAG clone #10 was used for subsequent screenings. (FIG.8B) Pearson correlation of the dual-CRISPR library biological replicates, (control, day 0 of the screening samples; 15 days, cells grew for another 15 days). (FIG.8C) Percentage of the recovered target UCEs and validated enhancers from different samples during the dual-CRISPR screenings in K562 cells. (FIG.8D) FACS gating examples of cell proliferation assays. (FIG.8E) Epigenetic signatures surrounding the UCEs PBX3 C1, FOXP1 F1, PAX6_Ta and the NCRE De_novo_l. (FIG.8F) Chromatin looping around NCRE de novo l, as shown by CTCF ChlA-PET loops and in situ Hi-C matrix. (FIG.8F) Similar to Fig. 8B, Spearman correlation between biological replicates of the dual-CIRPSR libraries (day-0: initial screening cell population; day-15: cells grew for another 15 days).

[0210] FIG 9 shows Essential UCEs shared between K562 and 293T cells: (FIG.9A) Essential UCEs in 293 T are identified using the dual-CRISPR screening. MAGeCK algorithm was used to identify significant hits depleted from the cells cultured for an additional 14 days compared to the initial population. The Manhattan plot was used to show the distribution of all the target regions. Significant hits were above the dashed line, indicating the cutoff MAGeCK RRA score of 0.01. Different colors represent different chromosomes. The top essential hits were shown. (FIG.9B) Venn diagram shows the shared essential UCEs between K562 (353 UCEs with RRA score < 0.01) and 293T (410 UCEs with RRA score < 0.01) cell lines, respectively. Only 26 (<10%) of the essential UCEs identified from the respective screenings were shared between K562 and 293T cells. (FIG.9C) Venn diagram shows the genes around the essential UCEs between K562 (4158 genes that are + / - IMbp around the essential UCEs) and 293T (4731 genes that are + / - IMbp around the essential UCEs) cell lines. 1629 (>30%) of"SUBSTITUTE SHEET RULE 26"the genes were shared between K562 and 293T cells. (FIG. 9D-9E) Over Representation Analysis of Gene Ontology ter— ms in Biological Process sub-ontology based on the potential genes that might be regulated by the essential UCEs in 293T cells (d) and K562 cells (e), respectively.

[0211] FIG 10 shows NCREs regulate their surrounding genes to exert their functions: (FIG.10A) TADs identified by Hi-C surrounding PAX6_Ta are shown, and the location of PAX6_Ta is indicated by the vertical blue bar. Horizontal yellow and blue bars indicate distinct TADs. Transcription of three nearby genes within the same TAD as PAX6_Ta was quantified by qPCR (n = 3 biological independent samples; bars show mean value ± s.d.; ****P < 0.0001, calculated using unpaired t-test). (FIG.10B) RNA-seq was performed to identify differentially expressed genes around NCRE PAX6_Ta from PAX6_Ta-KO cells versus K562 WT cells. The x axis represents the coordinates of genes surrounding PAX6_Ta ranging from - 5Mb to + 5 Mb. The y axis shows the -loglO(FDR) of nearby genes by DESeq2. The dashed horizontal line indicates the FDR cutoff of 0.05, and the dotted vertical line indicates the location of PAX6_Ta. Each dot represents one gene, the red dots represent significantly up-regulated genes, blue dots represent significantly down-regulated genes, and the gray ones are either nonsignificant genes or genes with |log2FC|<l.

[0212] FIG 11 shows Essential enhancers in K562 cells: (FIG. 11A-11E) The removal of essential enhancers E12: 123591 (a), E6:52372 (b), E14:71791 (c), E7: 135735 (d), E8: 124178 (e) caused cell growth disadvantage in K562 cells, while WT and control cells in every case show normal proliferation at about 100%. All clones were either 3 star or 4 star significance. Cell proliferation assay was performed by mixing the KO cell lines with cells expressing GFP at a 1 : 1 ratio. The changes in GFP percentage were monitored at indicated time points by FACS. Cells with dual-CRISPR guide RNAs targeting GFP sequences served as negative controls (Ctrl-KO). The y axis represents the relative ratio of the GFP negative cells to the positive cells. The ratio of cells in the initial mixture was set as 100% (n=3 biological independent samples; values are shown as the mean ± s.d.; ***P < 0.001, ****P < 0.0001, calculated using two-way ANOVA). (FIG. 11F-11K) Epigenetic signatures surrounding the identified essential enhancers in K562 cells. E12: 123591 (f), E6:52372 (g), E14:71791 (h), E7: 135735 (i), E8: 124178 (j) and E22:23590 (k).

[0213] FIG 12 shows Identifying essential enhancer clusters: (FIG. 12A-12C) Growth effect of one enhancer (a), two-enhancer (b), and three-enhancer (c) removal from the cluster in"SUBSTITUTE SHEET RULE 26"chromosome 6. Cell proliferation assay was performed by mixing the KO cell lines with cells expressing GFP at a 1 : 1 ratio. The changes in GFP percentage were monitored at indicated time points by FACS. Cells with dual-CRISPR guide RNAs targeting GFP sequences served as negative controls (Ctrl KO). The y axis represents the relative ratio of the GFP negative cells to the positive cells. The ratio of cells in the initial mixture was set as 100%. Rl, R2, and R3 represent the three individual potential enhancers targeted by the dual-CRISPR libraries (n=3 biological independent samples; values are shown as the mean ± s.d.; **P < 0.01, ***P < 0.001, ****p < 0.0001, calculated using two-way ANOVA). (FIG. 12D-F) Growth effect of one enhancer (d), two-enhancer (e), and three-enhancer (f) removal from the cluster in chromosome 10. Cell proliferation assay was performed by mixing the KO cell lines with cells expressing GFP at a 1 : 1 ratio. The changes in GFP percentage were monitored at indicated time points by FACS. Cells with dual-CRISPR guide RNAs targeting GFP sequences served as negative controls (Ctrl KO). The y axis represents the relative ratio of the GFP negative cells to the positive cells. The ratio of cells in the initial mixture was set as 100%. Rl, R2, and R3 represent the three individual potential enhancers targeted by the dual-CRISPR libraries (n=3 biological independent samples; values are shown as the mean ± s.d.; *P < 0.05, **P < 0.01, ***P < 0.001, calculated using two-way ANOVA).

[0214] FIG 13 shows Results of Single-cell dual-CRISPR screening: (FIG. 13A-F) Determining the lentiviral titer of the single-cell dual-CRISPR library (sc-library) in K562 cells, with a range of OpL (a), 0.1 pL (b), IpL (c), lOpL (d) and lOOpL (e) was tested. GFP expression from the sc-library was used as the marker to determine the virus infection rate. Individual plots show 0% GFP-expressing cells (a), 0,76% GFP-expressing cells (b), 8,63% GFP-expressing cells (c), 59,2% GFP-expressing cells (d), and 97,2% GFP-expressing cells (e), which was plotted in (f). A predicted MOI <0.3 using 5uL of sc-library lentivirus was then determined. (FIG. 13G) Cells containing the sc-library for the downstream analysis. FACS showed that 20% of total cells had the sc-library, as monitored by GFP expression, representing an MOI of 0.2 in the final experiment. This ensured that every single cell contained only one pair of guide RNAs from the sc-library. (FIG. 13H) Comparison of usable cells per paired guide RNA barcodes (pGBCs) between two batches of scRNA-seq data: A10 and A30 show 10,000 and 30,000 cells were loaded per 10X Genomics chip, respectively. 1,199 and 3,271 usable cells were retrieved from A10 and A30 batches, respectively. Wilcoxon signed-rank test was used to compare two datasets. (FIG. 131) Comparison of usable cells per target NCRE"SUBSTITUTE SHEET RULE 26"(multiple pairs of guide RNA were included in the sc-library) between two batches of scRNA- seq data. Wilcoxon signed-rank test was used to compare two batch data. (FIG. 13J) Basic quality control of 4,470 single cells containing the sc-library after cell filtering and multiplets filtering. Violin plots show the total number of genes, total UMIs of transcripts, the total number of unique sgRNA, total UMIs of sgRNA detected, and the percentage of transcripts encoding mitochondrial and ribosomal genes detected in every single cell. (FIG. 13K) The dual-CRISPRs targeting the promoters of ribosomal genes: RPL18A, RPL13, RPL21, and RPL8 served as the positive controls. The gene expression matrix was normalized using the LogNormalize method with a scale factor of 10,000. Identities of cell perturbation were assigned based on whether pGBCs were captured. Differential gene expression testing between perturbed cells (Perturb) and negative control cells (NCT) were done by the MAST algorithm with the number of gene UMIs observed per cell and the number of gRNA UMIs per cell as covariates (*****P<0.00001, *** / ’<0.001, ** / ’<0.01, *P<0.05). (FIG. 13L-N) The upper panel shows epigenetic signatures surrounding NCREs E16:30551 (L), E3: 126465 (m), and TSHZ1 Roman (n), indicated by the red line. The lower panel, or L(II), shows the differential gene expression testing results using MAST. Violin plots show the normalized expression levels of candidate genes in perturbed and control cells (P < 0.05, calculated by MAST fitted model; ns: not significant).

[0215] FIG 14 shows Removing UCE PAX6 affecting cardiac development: (FIG. 14A) FACS gating examples of the silencer reporter assay. (FIG. 14B) Sanger sequencing of the PAX6_Ta region showed that the two guide RNAs made the deletion in NKX2-5eGFP / w hESC. (FIG. 14C-D) The transcription of heart-related genes PAX6, RCN1, ELP, CACNG8, RYR2, MYH6, MYH7, ADRY2, and ADRB1 was quantified by qPCR before (b) and after (c) differentiation in both NKX2-5eGFP / w hESC cells (hESC_NKX) and NKX2-5eGFP / w hESC PAX6_Ta KO bulk cells (PAX6_Ta_KO) (n=2 biological independent samples; values are shown as the mean ± s.d. for each bar; *P < 0.05, **P < 0.01, calculated using unpaired t-test). (FIG. 14E-F) The same experiment as shown in Fig 14C-D was repeated with n=3 independent samples. The transcription of heart-related genes PAX6, RCN1, ELP, CACNG8, RYR2, MYH6, MYH7, ADRY2, and ADRB1 was quantified by qPCR before (f) and after (g) differentiation in both NKX2-5eGFP / w hESC cells (hESC_NKX) and NKX2-5eGFP / w hESC PAX6_Ta KO bulk cells (PAX6_Ta_KO) (n = 3 biological independent samples, values are shown as the mean ± s.d. for each bar; *P< 0.05, **P < 0.01, calculated using unpaired t-test)."SUBSTITUTE SHEET RULE 26"EXAMPLES

[0216] The invention will now be described by the following non-limiting examples which demonstrate various aspects and embodiments of the invention.

[0217] Methods

[0218] Cell culture

[0219] K562 cells were cultured in RPMI 1640 + L-Glutamine (Gibco), 10% fetal bovine serum (Biowest), and 1% Penicillin-Streptomycin (Gibco). 293T cells were cultured in DMEM (Gibco), 10% FBS and 1% Penicillin-Streptomycin. Cell density and culture conditions were maintained according to the ENCODE Cell Culture Guidelines. NKX2-5eGFP / w human embryonic stem cells (hESCs) were cultured in StemFlex™ Medium (Thermofisher A3349401) on Biolaminin (LN521-02) coated 6-well plate, and passaged using TrypLE Select (Thermofisher 12563011).

[0220] Dual-CRISPR .plasmid const^

[0221] The lentiviral dual-CRISPR plasmid used for the screening was made based on the lentiGuide-Puro (Addgene #52963). The human U6 and Hl promoters were cloned to replace the U6 promoter of the lentiGuide-Puro plasmid. The plasmid is referred to as Lenti-dual- CRISPR-U6-H1 (pBP43). Dual-CRISPR plasmid containing Cas9 used to generate knockout clones for screening validations was made based on the pSpCas9(BB)-2A-Puro (PX459) V2.0 (Addgene #62988). The human U6 and Hl promoters were cloned to replace the U6 promoter in the PX459V2. And the 3XFLAG tag on the Cas9 protein was replaced with the HA tag. The plasmid is referred to as Dual-CRISPR-Cas9-U6-Hl (pBP48).

[0222] The dual-scaffold was cloned to the pUC19 backbone. The plasmid is referred to as pUC19-dual-scaffold-1.0 (pBP44) or pUC19-dual-scafffold-2.0 (pBP49).

[0223] The gRNAs were designed and cloned as the following:

[0224] U6 side: GTGGAAAGGACGAAACACCGN20 (gRNA target sequence) GTTTTAGAGCTAGAAATAGC (SEQ ID NO: 29)

[0225] Hl side: TATGAGACCACTCTTTCCCG N20 (gRNA target sequence) GTTTTAGAGCTAGAAATAGC (SEQ ID NO: 30)

[0226] Dual-CRISPR-Cas9-U6-Hl was digested with BbsI, and the dual-scaffold was isolated from pUC19_dual_scaffold digested with BbsI. Then these four fragments were assembled using NEBuilder HiFi according to the manufacturer's protocol."SUBSTITUTE SHEET RULE 26"

[0227] DuabCRISPR .library design

[0228] The paired guide RNA sequences of the dual-CRISPR library targeting the ultraconserved elements (UCE) and some validated enhancers were from the published computation pipeline CRISPETa (referred as UCE library)39.

[0229] The predicted K562 enhancers based on ENCODE ChlP-seq data using machine learning model were selected as targets for dual CRISPR screening16. The software package CRISPRseek was used to search for potential protospacers sequences with PAM NGG pattern as the potential CRISPR targeting regions74. Guide RNAs with high predicted cleavage efficiency and specificity were chosen with the following parameters cutoff: gRNAefficacy > 0.15, top5OfftargetTotalScore < 47 and top 1 OOfftargetTotalScore < 50.

[0230] First, the immediate upstream and downstream flanking regions (200 bp in size) of the enhancers were selected for guide RNA design. Single gRNAs from the two flanking regions were paired, resulting in up to 25 pairs of gRNAs per targeting enhancer region (referred to as enhancer library 1 in FIG. 5A targeting 3,995 enhancers). Due to genomic repeats and other constraints, not all enhancers could be targeted by the previous design strategy. To target the rest of the predicted enhancers in K562 cells, pairs of guide RNAs were selected within the enhancer regions (5’ proximal and 3’ proximal of the enhancers), with around 14 pairs of guide RNAs per enhancer (referred as enhancer library 2 in FIG. 5B targeting 13,020 enhancers). In this way, all predicted enhancers in K562 cells by ENCODE were targeted.

[0231] The oligo pools were then designed and ordered from Custom Array / GenScript.

[0232] For UCE library and enhancer library 1, pairs of gRNAs were designed to be compatible with Lenti-dual-CRISPR-1.0 as following:

[0233] ATCTTGTGGAAAGGACGAAACACCG-[gRNAl, 20nt]- gttttgagacgggatccCGTCTCAAAAC (SEQ ID NO: 31)

[0234] [reverse complement of gRNA2, 20nt]-CGGGAAAGAGTGGTCTCATACAGAACTTAT (SEQ ID NO: 32)

[0235] For enhancer library 2, pairs of gRNAs were designed to be compatible with Lenti- dual-CRISPR-2.0 as following:

[0236] ATCTTGTGGAAAGGACGAAACACCG-[gRNAl ,20nt]- gttttagagctaGAAAtagcaagttGAGACG- [barcode, 1 Ont]-"SUBSTITUTE SHEET RULE 26"CGTCTCAACTTGCTATTTCTAGCTCTAAAAC-[reverse complement of gRNA2, 20nt]- CGGGAAAGAGTGGTCTCATACAGAACTTAT (SEQ ID NO: 33).

[0237] Each dual-CRISPR library also contains control paired guide RNAs from a previous study, which target promoters, exons, and introns of 17 ribosomal genes and 3 cancer-related genes (FOXA1, HOXB13 and EZH2), non-targeting guide RNAs, guide RNAs targeting the adeno-associated virus integration site 1 (AAVS1) loci29.

[0238] Single-cell .dual-CMSPR libra

[0239] For single-cell dual-CRISPR library design, 82 paired guide RNA sequences were selected from the pooled dual CRISPR screen experiments to target 42 significant NCREs affecting cell growth and imatinib resistance, with 1-3 unique pairs of guide RNAs chosen for each region. 10 extra pairs of guide RNAs were selected to target the AAVS1 region as negative controls. The oligo pool was then ordered and assembled into the single-cell dual- CRISPR library.

[0240] Construction of the. ooled and. single ceJl.dual CMS.PR

[0241] The synthesized oligo pools were first amplified by PCR using the following primer:

[0242] Forward primer: ATCTTGTGGAAAGGACGAAA (SEQ ID NO: 15);

[0243] Reverse primer: ATAAGTTCTGTATGAGACCA (SEQ ID NO: 34);

[0244] For Lenti-dual-CRISPR-1.0 libraries, PCR procedures using NEBNext High-Fidelity 2X PCR Master Mix were: 98 °C for 30 s, 18 cycles of 98 °C for 10 s, 68 °C for 30 s and 72 °C for 30 s, and 72 °C for 5 min. For each reaction, 80 ng of the oligo pool was used for a 100 pl PCR reaction, and 20 reactions per library were pooled. The pooled PCR products were further size-selected and gel-purified using QIAGEN MinElute column. In the first step of library construction, the amplified oligo libraries were assembled into the digested Lenti-dual- CRISPR-U6-H1 plasmids using Gibson assembly. The assembly mix was made using 200 ng of digested dual-CRISPR-U6-Hl plasmids, 30 ng insert DNA (at molar ratio 1: 10) and 10 pl of 2x Gibson Assembly Master Mix to produce a final volume of 20 pl. The assembly mix was incubated at 50 °C for 60 min, and in total 10 reactions were pooled for each library. The pooled reaction products were purified by ethanol precipitation and resuspended in 10 pl water, from which 2 pl of the products was electroporated into 25 pl of Endura electrocompetent cells (Endura 60242-2). In total 5 electroporation reactions were pooled and grown in 5 ml SOC recovery medium for 2 h. Then 5 pl from the 5 ml SOC recovery medium was used to perform"SUBSTITUTE SHEET RULE 26"serial dilution plating to determine the transformation efficiency, and the rest was further cultured in 1000 ml LB medium with 100 pg ml-1carbenicillin overnight. The plasmids containing the oligos were extracted using the Qiagen Maxiprep kit, and were further digested with BsmBI to open the plasmids at the restriction enzyme sites placed on oligo inserts. To assemble the final Lenti-dual-CRISPR-1.0 libraries, the BsmBI-digested plasmids containing the oligos were ligated with the dual-scaffold fragments (digested and isolated from pUC19- dual-scaffold-1.0) using T7 DNA ligase (NEB M0318). The ligation mix was made using 20 pg of digested plasmids, 1.2 pg of the dual-scaffold fragment, 200 pl of 2 x T7 buffer and 40 pl T7 DNA ligase to produce a final volume of 400 pl. The ligation mix was incubated at 16°C overnight. The ligation reaction products were purified by ethanol precipitation, resuspended in 210 pl of water, and treated with Plasmid-Safe (Epicentre, E8101K). The products were then purified using QIAGEN MinElute column and eluted in 10 pl of water, which was electroporated into 50 pl of Endura electrocompetent cells and grown in 2 ml SOC recovery medium for 2 h. Then 2 pl from the 2 ml SOC recovery medium was used to perform serial dilution plating to determine the transformation efficiency, and the rest was further cultured in 500 ml LB medium with 100 pg ml-1carbenicillin. The final lenti-dual-CRISPR-1.0 libraries were extracted using the Qiagen Maxiprep Kit.

[0245] For Lenti-dual-CRISPR-2.0 libraries, the procedures to clone the oligo pools into the lenti-dual-CRISPR-U6-Hl plasmids were similar. Gibson assembly was used to clone the dual- scaffold-2.0 (with 200 bp random sequences inserted between the two scaffold sequences) to make the final lenti-dual-CRISPR-2.0 libraries.

[0246] Lentivirus production

[0247] For each library, the 293T cells were grown in five T175 flasks at 50% confluency before transfection. For each flask of 293 T cells grown in 25 ml of fresh medium, 15 pg of library plasmids, 7 pg of psPAX2, 3.5 pg of pCMV-VSV-G, and 76.5 pl of X-tremeGENE 9 DNA Transfection Reagent were mixed in 1 ml of serum-free medium and used for transfection. Fresh medium was added the day following transfection. Media supernatant containing virus particles was collected on the second and third days after transfection, pooled and further concentrated using Lenti-X according to the manufacturer’s protocol. Virus titer was then determined by making serial (10-3to 10-10) dilutions of 4 pl of frozen virus supernatant in media containing 8 pg ml-1of polybrene to infect 293T cells. Two days after"SUBSTITUTE SHEET RULE 26"infection, cells were selected with 2 pg ml1puromycin for an additional 7 d. Virus titer was then calculated based on the survival colonies and the related dilution.

[0248] Po Jed.dual-C

[0249] K562 / Cas9 cells were infected with the respective virus libraries at a multiplicity of infection = 0.2 by spin-infection. For spin-infection, 3 * 106cells in each well of a 12- well plate were infected in 1 ml of medium containing 8 pg ml-1of polybrene. In total, four plates were used for each infection to infect a total of 1.5 x 108cells, which would result in -300 to -500 coverage of the dual-CRISPR libraries. Two days after infection, cells were selected by 2 mg ml-1of puromycin for a further 6 d. Dead cells were then removed with Histopaque-1077 (Sigma) by centrifuging cells at 400g for 30 minutes at room temperature. For each biological replicate experiment, the lentivirus was produced again and infection was repeated.

[0250] After puromycin selection, an aliquot of 108K562 / Cas9 cells infected with the respective dual-CRISPR libraries were frozen as the control population (day 0). From the same cell population, 108K562 cells were further cultured for another 15 days to identify essential NCREs that affect cell growth; and another 108cells were cultured under 0.1 pM imatinib treatment for 15 days. Dead cells were removed, and drugs were refreshed during the subculture of imatinib-treated cells. On day 15, 108cells for each culture were collected and frozen for the next step. For 293T cells, procedures were similar, except that the dead cells were removed by refreshing the medium.

[0251] Single-cell RNA-seq combined with the dual-CRISPR assay

[0252] The single-cell dual-CRISPR library was introduced into K562-Cas9 expressing cells via spin infection at a final MOI of <0.3. After one week, cells that successfully incorporated the single-cell dual-CRISPR system were sorted based on GFP expression using FACS sort. To prepare for single-cell processing, 0.5 x 106cells at a concentration of 1,500 cells ml’1in 0.04% BSA-PBS were used for single-cell RNA and guide RNA capture according to the lOx Genomics protocol. The lOx Genomics Chromium platform was used to generate the single-cell libraries according to the manufacturer’s protocol, which were sequenced on the Illumina NovaSeq 6000 platform.

[0253] Library sequencing

[0254] Genomic DNA was isolated using the QIAamp DNA Blood Maxi Kit (QIAGEN). Dual-CRISPR regions integrated into the chromosomes were then PCR amplified using LongAmp Taq DNA Polymerase (NEB, #M0323) using primers annealing to U6 and Hl"SUBSTITUTE SHEET RULE 26"promoters (Table 1). Stagger sequences were introduced to the PCR primers to increase the diversity of the next-generation sequencing libraries, when the flowcell only contains the dual- CRISPR sequencing libraries75. The stagger primers are not necessary if libraries are pooled with other sequencing samples with diverse sequences, and a one-step PCR using primers containing Illumina sequencing adaptors will be sufficient. For each 100 pl PCR reaction, 10 pg of genomic DNA, 50 pl of 2 LongAmp master mix, 2 pl of 25 pM U6_stagger primer, 2 pl of 25 pM Hl stagger primer were used. In total, 7 PCR reactions with different pairs of stagger primer were used and pooled, assaying 70 pg of genomic DNA. PCR procedures were: 94 °C for 30 s, 25 cycles of 94 °C for 15 s, 60 °C for 15 s and 65 °C for 60 s, and 65 °C for 10 min. These fragments were then cleaned up and gel-purified using MinElute PCR Purification Kit. Then the Illumina TruSeq adapters were ligated, and sequencing libraries were prepared according to the ENCODE protocol and sequenced on the Illumina HiSeq4000 platform.

[0255] Table 1 : Primers used for dual-CRISPR system, PCR, luciferase assay and qPCR separated by Figure reference"SUBSTITUTE SHEET RULE 26"

[0256] Pooled .dual-CRISPR .screen analysis

[0257] Cutadapt 3.4 was used to extract the unique 20nt protospacer sequences from each pair of guide RNA sequences by locating the U6 / H1 promoter sequences from the 5’ end and scaffold sequence from the 3’ end of the 20nt protospacer sequence (U6 promoter, ATATATCTTGTGGAAAGGACGAAA, SEQ ID NO: 165; Hl promoter, ATAAGTTCTGTATGAGACCACTCTT, SEQ ID NO: 166)76. The read pairs that did not contain the correct promoter and scaffold sequences were not considered. To ensure functional CRISPR guide RNA sequences were counted, the protospacer sequence and the additional 20 bp (for both Lenti-dual-CRISRP-1.0 / Lenti-dual-CRISPR-2.0) into the CRISPR scaffold sequences were kept during the trimming of the sequence reads (tracrRNA sequence, AAGTTAAAAT, SEQ ID NO: 167). Trimmed reads were then mapped to the indexed paired protospacers references generated by Bowtie2 based on our initial library designs, and only aligned reads with mapping quality (MAPQ) score over 23 were used for downstream analyses77.

[0258] MAGeCK RRA was used to identify the significant hits depleted after 15-day culture compared with the day 0 initial cell population, with the cutoff of RRA score < 0.0140. MAGeCK MLE was performed to identify NCREs that confer imatinib resistance, by comparing the 15-day imatinib-treated cell population, 15-day culture cell population and day 0 initial cell population78. The NCREs whose loss confers Imatinib resistance were identified as regions that were positively selected (i.e., NCREs with beta scores > mean + 2 s.d.) in the 15- day imatinib-treated populations but are weakly selected in the 15-day culture populations.

[0259] Gene pathway over-representation analyses were based on GO term and KEGG pathway definitions with the cutoff of FDR < 0.25. Genes potentially affected by NCREs were identified as the ones located in the range of + / - 1Mb of the NCRE regions.

[0260] Single-cell dual-CRISPR screen analysis

[0261] Cell Ranger 6.0.1 pipeline was used to process Chromium single-cell data to align reads, generate feature-barcode matrices for the mRNAs, and capture guide RNAs. Seurat 4.0.2 was used to process the single-cell RNA-seq data79. Gene expression matrix was normalized using the “LogNormalize" method with scale factor of 10,000 and log-transformed. Cell-cycle- related scores were regressed during data scaling. Differential expression tests and p-value calculations were performed by the MAST-fitted model based on whole transcriptome data in all single cells except for genes with mean normalized expression < 0.0165."SUBSTITUTE SHEET RULE 26"

[0262] TADs around the candidate NCREs were used to narrow down the potential genes deregulated by CRISPR editing. The K562 TAD dataset was downloaded from 3D Genome Browser, and the median window size is around 1 Mbp. The neighboring gene coordinates were extracted using biomaRt80. The gene expression changes were quantified as the log2 fold change of mean of normalized gene expression from perturbed cell population divided by the mean of normalized gene expression from the negative control cell population.

[0264] RNA was isolated using ISOLATE II RNA Mini Kit (BIO-52073), and sequencing libraries were made with the Invitrogen Collibri Stranded RNA Library Preparation kit (A39003024), according to the manufacturer’s protocol. The whole-transcriptome sequencing was done on the Illumina NovaSeq 6000 platform.

[0265] Snakemake pipeline (https: / / github.com / snakemake-workflows / rna-seq-star-deseq2) was used to process the bulk RNA-seq sample. Briefly, Cutadapt v3.4 was used to trim adapters from reads, STAR was used to align the spliced transcripts to the reference genome (GROG 7 assembly release 75) and quantify the read counts per gene81. DESeq2 was used to perform differential expression analysis82. P-values were corrected by the BH procedure for FDR control.

[0266] The KEGG pathway enrichment analysis was based on the GSEA method, which integrates the expression level of individual genes and aggregates gene expression in the pathway analysis to manifest the phenotypic differences, i.e., to show if the pathway is activated or suppressed61.

[0267] Enhancer cluster analysis

[0268] Individual enhancers from the dual-CRISPR libraries (enhancer libraries 1 and 2) were first clustered based on the super-enhancer annotations in K562 cells60. Two computational methods GSEA and MAGeCK RRA were used to compute the depletion scores of enhancer clusters associated with cell growth40,61. P-values obtained by the two methods are corrected by the BH procedure for FDR control. Among the top 20 clusters identified from both methods, 10 clusters were shared, which were considered for further validation.

[0269] Luciferase assay

[0270] Candidate NCREs were amplified with primers containing a homologous arm from the genomic DNA of K562 cells. These fragments were then inserted in front of the promoters of"SUBSTITUTE SHEET RULE 26"the luciferase plasmids pGL4.23 (Pr omega, with some modification on the cloning sites, detecting the enhancer activity) and pGL4.53 (Promega, detecting the silencer activity) by using NEBuilder HiFi. Cells were then co-transfected with the pRL-CMV Renilla reporter vector and the pGL4.53 / modified pGL4.23 vectors with the NCRE sequence inserted. The luciferase assay was performed using the Dual-Luciferase Reporter Assay System from Promega according to the manufacturer’s protocol. Original luciferase plasmid without any insertion was used as the control. All luciferase assays were from three independent transfections performed on different days. All tested regions and associated primers are listed in Table 1.

[0271] Pual-CRISPR-Cas9-guided NCRE knockout

[0272] Paired guide RNAs targeting the 5' and 3' ends of the NCREs were selected from the screening libraries. The gRNA sequences were cloned into the dual-CRISPR-Cas9-U6-Hl (pBP48) plasmid containing the gRNA scaffold and Cas9 sequence. K562 cells were transfected with the plasmids containing the respective pairs of guide RNAs and then selected for successful transfection using puromycin. K562 cells transfected with the dual-CRISPR plasmid containing a pair of guide RNAs targeting the GFP sequences were used as the CRISPR-editing control (control KO). Single clones of cells were picked and verified using PCR and Sanger sequencing.

[0273] Quantitative PCR

[0274] RNA was extracted using ISOLATE II RNA Mini Kit (Bioline, BIO-52073), and the cDNA was synthesized using SuperScript IV VILO Master Mix (Invitrogen, 11756050). Realtime PCR was performed with SensiFAST SYBR No-ROX Kit (Bioline, BIO-98020) on Biorad CFX Opus 384 Real-Time PCR Systems system. The expression of the housekeeping gene GAPDH was used as the control.

[0275] Cell proliferation assay

[0276] Cell proliferation assay was performed by mixing the NCRE KO cell lines with cells expressing GFP at a 1 :1 ratio. The changes in GFP percentage were monitored at indicated time points by FACS. Ctrl KO cells were used as the negative control.

[0277] To test the imatinib response, NCRE KO clones and K562 cells were seeded into a 96- well plate and treated with 0,4 pM Imatinib (STI571, Selleckchem) for 3 days. The cell viability was then measured using the CellTiter-Blue viability assay (Promega, G8082)."SUBSTITUTE SHEET RULE 26"Relative survival was normalized to their respective untreated NCRE KO clones and K562 cells and corrected for background signal.

[0278] CMSPR-Cas9- uided .NCRE ..knockout i

[0279] The crRNAs targeting the 5' and 3' ends of UCE PAX6_Tarzan were ordered from Integrated DNA Technologies (IDT). To form the crRNA: tracrRNA duplex, 2.2 pl of crRNA and 2.2 pl of tracrRNA (IDT) were mixed in 0.6 pl Nuclease-free duplex buffer. The mix was heated at 95°C for 5 min, then cooled to room temperature for 10 min. Then 0.5 pl of crRNA: tracrRNA duplex, 0.24 pl of Cas9 Nuclease V3 (IDT), and 0.76 pl of buffer R (Neon™ Transfection System) were mixed and incubated for 20 min at room temperature to form the CRISPR-Cas9 complex. Electroporation was performed by mixing 0.25 million NKX2-5eGFP / whESCs in 22 pl buffer R with 2 pl of CRISPR-Cas9 complex, according to the protocol of Neon nucleofector. Cells were then transferred to a laminin-coated 12-well plate for culturing. Genomic DNA was isolated 4 days later, and the deletion of PAX6_Tarzan was verified using PCR and Sanger sequencing. Primer and guide RNA sequences are listed in Table 1. The maintenance of NKX2-5eGFP / w hESCs and cardiomyocyte differentiation were performed as described previously83.

[0280] Example 1: Development of the dual-CRISPR system

[0281] NCREs could be positive transcriptional regulators, i.e., enhancers; negative transcriptional regulators, i.e., silencers; or genome structure regulators, i.e. insulators. To systematically study NCREs irrespective of their different biological functions in their endogenous chromosome context, a versatile high-throughput dual-CRISPR screen system was developed. In this dual-CRISPR system, two different RNA polymerase III promoters, U6 and Hl, are positioned in a convergent orientation to drive the transcription of two CRISPR guide RNAs (FIG.7A). To test this plasmid system, two guide RNAs targeting the 5 and 3 prime ends of one DNA fragment in the genome were inserted. After transfecting the cells, two functional guide RNAs were expressed and able to successfully delete the targeted region from the human genome (FIG. 7B and 7C).

[0282] To target thousands of potential NCREs and test their functions in different contexts, a lentiviral system and cloning strategies analogous to the method described above were developed. In short, after NCREs were selected, all potential single guide RNAs that target both ends of each NCRE were designed. Guide RNAs were then selected based on their targeting efficiency and off-target potential. After that, guide RNAs were paired to be able to"SUBSTITUTE SHEET RULE 26"remove the targeting regions. Paired 20-nucleotide (nt) crRNA sequences are then properly orientated to follow the direction of the convergent promoters (FIG. 7D). A restriction enzyme site was placed between the paired protospacer sequences in order to open the plasmids to insert the guide RNA scaffolds in the subsequent steps, and oligo pools were ordered. A two- step cloning strategy was then used to assemble the full functional dual-CRISPR library (Methods). In brief, first, the oligo pool that contains only the paired 20-nt crRNA sequences were cloned into the lentiviral vector. After propagation, the plasmids containing the paired 20- nt crRNA sequences were opened up by restriction enzyme digestion, followed by the second round of cloning to insert the two tracrRNA scaffold sequences. The final plasmid libraries then contain paired functional guide RNAs that would remove the respective individual NCREs. There are several advantages of the new dual-CRISPR system. Two distinct promoter sequences should reduce the chance of potential recombination events in plasmids or infected cells. Furthermore, arranging guide RNAs in a convergent orientation allows direct PCR steps to amplify the fragments containing the paired guide RNA sequences from the infected cells, which are compatible with direct high-throughput pair-end sequencing (FIG. 7E).

[0283] Example 2: Identification of essential ultraconserved non-coding elements

[0284] UCEs are non-coding genetic sequences that are identical among different species33'35. Given such stringent conservation during evolution, it is expected that many UCEs should have biological functions and be pivotal in different species36,37. However, recent research showed that ultraconserved enhancers did not require perfect sequence matches to maintain their functions, when 23 of such enhancer UCEs were studied in vivo in mouse models38. To study the function of UCEs in a high-throughput fashion, a dual CRISPR library was assembled based on the published computation pipeline39, which targets 4,047 UCEs in the human genome from UCNEbase31, and 1,527 in vivo-validated conserved enhancers from VISTA Enhancer Browser32. In total 63,879 dual-CRISPRs were designed, including 1,070 control guides from a previous study29. The dual-CRISPR library was packaged into lentivirus, then used to infect K562 cells stably expressing Cas9 proteins (FIG. 8A). Infected cells were kept in culture for 15 days to identify NCREs that would affect cell growth. Genomic DNA was isolated, and PCR was performed to extract the dual-CRISPRs. The abundance of different dual-CRISPRs was compared to that from the initial population (day 0). Two biological replicate experiments were performed to extract reliable hits. The replicates correlated well with each other, indicating the screen system is stable and reliable (FIG. 8B). After filtering low coverage, more than 90"SUBSTITUTE SHEET RULE 26"percent of the target NCREs were matched with the paired guide RNAs across all replicates (FIG. 8C). The robust ranking algorithm MAGeCK was used to identify potential NCREs that affect cell growth based on the screen data40. There were 346 UCEs and other potential NCREs depleted in the cell population that further grew for 15 days, compared to the initial population, suggesting these UCEs are potentially essential NCREs in K562 cells (FIG. 1A). Unexpectedly, we also identified a previously unannotated intergenic region that affected the growth of K562 cells. Thus, the dual-CRISPR system was capable of interrogating both previously annotated and unannotated genomic regions in an unbiased fashion.

[0285] To validate the identified UCEs and the potential new NCRE, dual-CRISPR pairs targeting the potential essential NCREs were used to generate knockout clones in K562 cells (1 A, b, c, d and e; upper panel). We first determined cell growth rates in these clones. When UCEs PBX3_Claudia (referred as PBX3 C1), FOXPI Flora (referred as FOXP1 F1), PAX6_Tarzan (referred as PAX6_Ta) and one potential NCRE (referred as de novo l) were removed from K562 cells, respective clones grew significantly slower compared to the wildtype K562 cells, indicating these UCEs regulate important genes or pathways in K562 cells (FIG. IB, c, d and e; lower panel). We then surveyed the epigenetic modifications surrounding these UCEs. No clear combinations of epigenetic signatures were found to predict the function of these regions (Supplementary FIG. 2D), except that the de novo l region sits next to a CTCF binding site (within 1 kb to the center of the CTCF binding site) which does not directly involve in chromosome looping based on ChlA-PET data (FIG. 8E). As these UCEs are potential Regulatory elements, we then tested their transcriptional regulatory activities using luciferase assays. Genomic fragments containing the UCEs were cloned into two different luciferase reporter systems for the detection of enhancer and silencer activities respectively. Interestingly, no significant enhancer activity was observed from these regions using a commonly used minimal-promoter-driven luciferase reporter (FIG. IF). However, two UCEs showed silencer activity monitored by a PGK-promoter-driven luciferase reporter (FIG. 1G).

[0286] NCREs usually function in a tissue-specific manner. To test the functions of UCEs in a different cell type of origin, another NCRE essentiality screen was performed in 293T cells using the same dual-CRISPR library (FIG. 9A). As expected, only less than 10% of the essential UCEs were shared between 293T cells and K562 cells (FIG. 9B). However, when genes that fall within 1Mb surrounding the potential essential UCEs were compared, more than 30% of the genes were shared between the two cell lines (FIG. 9C), suggesting that although a"SUBSTITUTE SHEET RULE 26"different set of UCEs that may function in these two cell lines, there might still be some overlapping activities shared among these different UCEs that regulate a common set of genes that affect cell growth (FIG. 9D and E).

[0287] Example 3: UCEs regulate a cascade of gene pathways affecting cell growth

[0288] NCREs may regulate proximal and distal genes, especially genes that are within the same topologically associating domains (TAD)4,41. To find out which genes might be affected by the deletion of these identified NCREs and potentially lead to the growth disadvantage, we integrated Hi-C data to identify genes that share the same TAD with the respective NCREs42. Transcriptional changes of the genes in the same TAD with the NCRE and in a close-by TAD were monitored between WT and UCE-knockout K562 cells by qPCR. Transcription of PTPRD and RCN1 genes were significantly upregulated in the knockout clones of de novo l and PAX6_Ta respectively (FIG. 1H, and FIG. 10A). In addition, these results also indicate that some of these UCEs are potential silencers, as corroborated by the luciferase assay from de novo l (FIG. 1G). To survey the regulatory effects of NCRE on global transcription, RNA- seq was performed on K562_PAX6_Ta_KO clones. Transcriptional changes of genes that are 5 Mb surrounding the PAX6 regions were compared. RCN1 gene was significantly upregulated (FIG. 10B), confirming the qPCR results (FIG. 10A). Furthermore, CD59, EHF, ABTB2, CD44 and PRR5L genes were also significantly upregulated. Among these genes, EHF is a transcription factor, which may amplify the effects of PAX6_Ta regulation. PRR5L interacts with the mT0RC2 complex, and its upregulation would promote apoptosis43, which may also contribute to the growth delay phenotype. It should be noted that some of these de-regulated genes may be indirectly regulated by PAX6_Ta.

[0289] To link the cell growth phenotype to the genes regulated by the identified regions, we further studied the PTPRD gene that was upregulated by the de nono l NCRE removal. PTPRD gene codes a transmembrane receptor protein tyrosine phosphatase with tumor suppressor functions44. CRISPR activation system was used to upregulate the PTPRD gene directly in K562 cells (FIG. II), which mimics the effects of de nono l NCRE removal. A similar growth disadvantage was observed in cells with the PTPRD gene directly upregulated (FIG. 1 J), suggesting that the NCRE de novo l may impair cell growth by regulating the PTPRD gene.

[0290] Example 4: UCEs that function in drug-resistance"SUBSTITUTE SHEET RULE 26"

[0291] Mutations or genetic changes in the NCREs could contribute to different diseases45'48. However, it is unknown if NCREs are directly involved in drug responses. To test this, K562 cells infected with the dual-CRISPR library targeting UCEs were exposed to the tyrosine kinase inhibitor imatinib that targets the BCR-ABL fusion kinase in K562 cells and other related chronic myeloid leukemias. After 15 days, surviving cells were collected, and changes in the abundance of dual-CRISPRs were analyzed.

[0292] After comparing the initial screening cell population, cells growing for 15 days without any drug treatment and cells growing under imatinib for 15 days, 81 NCREs possibly involved in resistance to imatinib treatment were enriched (FIG. 2A)49. First, luciferase assays were performed to identify whether these regions serve as enhancers or silencers. Two UCEs ZNF503_Ophelia (ZNF503_Op) and QKI Jonathan (QKI Jo) were studied and showed significant silencer activities when using a PGK-promoter luciferase reporter (FIG. 2B and C). Individual K562 cell clones with UCEs ZNF503_Op and QKI Jo deleted were made respectively (FIG. 2D and e). These cells became more resistant to imatinib treatment than the control cells (FIG. 2F), indicating that the dual-CRISPR screenings identified potential NCREs that may play a role in imatinib resistance. To identify potential genes that these regions may regulate, transcription changes of the nearby genes were studied. In the ZNF503_Op KO clone, genes SAMD8, VDAC2 and ZNF503 were up-regulated (FIG. 2G), while in the QKI Jo KO clone, genes PACRG and QKI were upregulated (Fig. 2h). These data also corroborate that these two NCREs may function as silencers (FIG. 2C), which contribute survival advantages of these cells during imatinib treatment, directly or indirectly. For instance, gene ZNF503 is a transcriptional repressor that may regulate genes that drive tumor cell proliferation50,51.

[0293] Example 5: Identification of essential enhancers using optimized dual-CRISPR systems

[0294] Enhancer regions have been extensively studied and well-defined during the past few decades. However, most annotations are based on the combinations of key epigenetic modifications computationally or using ectopic enhancer reporter assays. Using the singleguide RNA CRISPR system, enhancers could be studied in their endogenous loci25,27. However, only limited regions could be studied due to technical issues or chosen biological readouts. It has been challenging to dissect the roles of enhancers in their endogenous loci in a comprehensive manner52, and therefore no related systematic study has been done yet."SUBSTITUTE SHEET RULE 26"

[0295] Using the dual CRISPR system, we designed 254,203 guide RNAs targeting all 13,539 potential enhancers in K562 cells predicted by the ENCODE project16. Because for many enhancers, it is not possible to identify paired guide RNAs to remove the enhancer regions completely, a complementary strategy was used to design guide RNA pairs targeting the inside of the enhancer regions to aim to remove the core sequences in the center (FIG. 3A). While constructing these libraries, we further optimized the cloning procedures and final structure of the dual CRISPR library system (FIG. 3B), which we named dual-CRISPR-2.0. The main improvement is that the distance between the two scaffolds was increased to 200 bp for optimal NGS sequencing efficiency (FIG. 3B). Using these genome-wide dual-CRISPR screening libraries, all potential enhancers in K562 cells were assayed and 1,005 enhancers were found to affect cell growth (FIG. 3C). We focused first on the top hits by deleting these potential essential enhancer regions, which caused decreased cell growth (FIG. 3D and FIG. 11A-11E), indicating these potential enhancer regions may regulate cell growth in K562 cells. Luciferase assays were then performed to test the enhancer activity of these regions, where 4 out 6 regions led to a strong and significant gene upregulation (FIG. 3E). These regions shared a typical enhancer signature in K562 cells (FIG. 11F-1 IK). We then tested the potential silencer activity of these regions. Interestingly 2 out of the 6 regions showed a significant silencer activity (FIG. 3F), but these 2 regions also exerted significant enhancer activity (FIG. 3E). These seemingly contradictory results in fact corroborate other recent studies where many NCREs were shown to have both enhancer and silencer activities in different biological contexts and when regulating different promoters7 18,53. In these studies, the promoters used for the enhancer and silencer reporter systems were different. Furthermore, another two NCRE regions did not show either enhancer or silencer activity, despite that clear growth disadvantage was observed when these two NCREs were removed (FIG. 3D and FIG. 1 ID). One possibility is that these NCREs do not regulate the specific promoters used in the luciferase assays. Therefore, studying the functions of NCREs in their endogenous loci also provides complementary data compared to other widely used experimental and computational methods to define NCREs.

[0296] To gain more insights into the mechanisms of the growth defects, we further monitored the potential genes that may be regulated by some of these NCREs. For instance, enhancer E22:23590 is located within the BCR region, which forms chromosomal translocation with the Abl gene present in many patients with chronic myelogenous leukemia (CML)54,55and the K562 cell line. Although no clear regulatory functions were observed of this potential enhancer"SUBSTITUTE SHEET RULE 26"in K562, two nearby genes Rab36 and BCR-ABL were differentially deregulated (FIG. 3G). Gene Rab36 was downregulated, while BCR-ABL fusion gene was upregulated, suggesting a complex function of this NCRE. Downregulation of Rab36 was shown to inhibit cell growth56. Also, it has been shown that upregulation of BCR-ABL stimulated the TGF-0 pathway causing cell growth arrest57. Therefore, the deregulation of these genes may collectively result in the growth disadvantage induced by removing enhancer E22:23590.

[0297] Example 6: Inferring functional enhancer clusters containing redundant units

[0298] Multiple NCREs, especially enhancers, are often present in close vicinity surrounding their target genes. Often these enhancers play redundant roles in regulating the same gene or genes58,59, which complicates the assignment of the biological functions of such enhancers (FIG. 4A). So far, most other groups and we have focused on identifying single NCRE or enhancer with strong phenotypes, such as on cell growth. It has been challenging to study clusters of enhancers in a high-throughput and genome-wide fashion. We hypothesized that for a cluster of enhancers with redundant activity, individually each of them would show some but not significant screening enrichment scores. However, when these enhancers are considered as a cluster, their combined biological effects would stand out. Based on this, a new analysis was performed on the enhancer essentiality screening (FIG. 3A), where the targeted enhancers were further grouped according to previously defined clusters based on a distance metric60. To reliably capture the essential enhancer clusters, two computational models GSEA and MAGeCK RRA were applied40,61, and P-values obtained by the two methods were corrected by BH procedure for FDR control (Methods). The shared top clusters computed by both methods were then used for the downstream study (Venn diagram in FIG. 4B5). The individual enhancers from the top 3 enhancer clusters were further studied (FIG. 4B).Luciferase assays were performed to determine the enhancer activity of these individual regions, and only enhancers from chromosome 6 showed strong enhancer activity using the pGL.4.23 reporter (FIG. 4C).

[0299] To test the functions of these individual enhancers and the clusters they reside in their endogenous loci, first, only one enhancer was removed from the clusters containing three enhancers using dual-CRISPR targeting. Only moderate growth disadvantage was observed (FIG. 4D, and FIG. 12A and 12D). Then two enhancers were removed, which resulted in three possible combinations of two-enhancer deletion clones. In all cases, the two-enhancer deletion clones had stronger growth suppression compared to the deletion of any single enhancer (FIG."SUBSTITUTE SHEET RULE 26"4D and e, and FIG. 12A, 12B, 12D and 12E), suggesting that some redundant functions are shared between these enhancer archipelagos as described before49. When all three enhancers were removed, proliferation was strongly impaired (FIG. 4F, and FIG. 12C and 12F), possibly via regulating the SLC25A1 gene (FIG. 4G). These data indicate that the dual-CRISPR screening system is also capable of capturing clusters of NCREs with redundant biological functions in a genome- wide and systematic study.

[0300] Example 7: Studying the functions of NCREs using the dual-CRISPR system at the single-cell level

[0301] Coupling the NCREs with their respective regulated genes has been challenging. Assays such as ChlA-PET or Hi-C that probe the 3D chromatin interactions could provide indications for genome- wide gene-NCRE physical interactions, though no direct measurement of the transcriptional regulation could be made. Single-cell (sc) RNA-seq combined with CRISPR perturbation (Perturb-seq) has been used to couple genetic perturbation, either targeting genes or enhancers, with broader transcriptome changes in single cells62,63. However, these studies still relied on single-guide-RNA-mediated perturbation, and prior knowledge of the potential function of the NCREs is required to choose the right dCas9-repressor system. Removing a larger fragment of the genome while monitoring the transcriptome changes could provide a useful tool to study both the coding and non-coding part (irrespective of the prior knowledge of the NCRE) of the genome at the single-cell level. To explore this option, the dual-CRISPR screening system was modified to capture the transcriptome changes of single cells upon removal of NCREs. Two distinct capture sequences were added to the dual-scaffold of guide RNAs64, allowing direct capture of the two guide RNAs and mRNAs within a single cell (FIG. 5A). As a pilot test, 82 pairs of guide RNAs targeting 42 different NCREs from the top lists of our genome-wide screening were selected and cloned into the sc-dual-CRISPR system that contains GFP as a marker. K562 / Cas9 cells were infected with lentivirus containing the sc-dual-CRISPR library at a low MOI 0.3 to make sure each cell only contained one pair of guide RNAs. Then GFP positive cells were sorted and processed for scRNA-seq (FIG. 13A- 13F).

[0302] Two single-cell sequencing libraries, either aiming to target up to 10,000 cells per chip or 30,000 cells per chip were made. After perturbation index assignment and filtering multiplet cell data, 1,199 and 3,271 usable single cells were harvested from two batches respectively. Therefore, it is possible to retrieve more usable single cells per perturbation with a higher"SUBSTITUTE SHEET RULE 26"number of input cells (FIG. 131 and 13 J), when pairs of guide RNAs were used as additional cell barcodes. The two experiments were combined with a mean of 110 cells per NCRE target achieved for downstream analysis , and a median of 4,454 genes per cell and a median of 21,446 UMIs of mRNA molecules per cell were observed (FIG. 13 J). We observed a similar trend of deregulation of genes between bulk RNA-seq data (FIG. 5B) and scRNA-seq data where PAX6_Ta region was removed, around the TADs of PAX6_Ta (FIG. 5C; 127 cells). To identify significantly deregulated genes targeted by the tested NCREs around their vicinity, differential expression analyses were performed by MAST65, a method tailored to fit a two-part generalized linear model for zero-inflated and bimodal distributed single-cell gene expression data. Due to the detection limit on lowly expressed transcripts66, only genes with acceptable mean normalized expression levels and detected in a sufficient percentage of cells were used for DE test by MAST (Methods). Positive controls with guides targeting promoters of 4 ribosomal genes (RPL18A, RPL13, RPL21 and RPL8) confirmed the feasibility of the sc-dual- CRISPR system to capture target genes of NCREs (FIG. 13K). There were 22 significant NCRE-gene pairs identified using this method (FIG. 5D, and FIG. 13L, 13M and 13N). For example, a potential enhancer El 1 : 125334 that was identified to play a role in imatinib resistance was found to down-regulate gene E124 upon dual-CRISPR editing (FIG. 5D). Gene E124 is indeed associated with resistance to many chemotherapeutic drugs including imatinib67,68, suggesting the enhancer El 1 : 125334 may regulate E124 gene to exert imatinib resistance. In addition, a potential enhancer El 6: 30551 identified to affect cell growth was found to downregulate genes PPP4C and BOLA2B and simultaneously upregulate gene ZNF689 (FIG. 13L). For instance, gene PPP4C encodes protein phosphatase 4 catalytic subunit and PPP4C-deficient thymocytes showed decreased proliferation and enhanced apoptosis in vivo69. The gene BOLA2B representing BolA family member 2 was reported to associate with human hepatocellular carcinoma progression and the BOLA2B-knockout mice model shows a slow tumorigenicity70. Together, deregulation of these genes together upon El 6: 30551 deletion may lead to cell growth disadvantage. These data show that it is possible to combine dual- CRISPR-mediated NCRE deletion with scRNA-seq to identify potential genes regulated by NCREs.

[0303] Example 8: UCE in PAX6 region affects hESC cardiomyocyte differentiation

[0304] Assigning specific biological functions to NCREs is still challenging. UCEs are especially interesting as these regions are conserved among different species, and for a long"SUBSTITUTE SHEET RULE 26"time these regions have been speculated to play fundamental functions in evolution. However, recent research indicated that mice with a few UCEs removed showed no abnormalities, and sequence conservation did not play an important role in the enhancer function within UCEs in mice71,72. A caveat though is that only a small percentage of the UCEs have been studied, and only the ones with enhancer function were considered. It is possible that other molecular functions may be associated with these UCEs in different developmental stages. It is therefore important to first define the potential functions of UCEs, which could be facilitated by systematically studying the functions of UCEs in different biological contexts using the dual- CRISPR system.

[0305] Global transcriptome analyses showed that in K562_PAX6_Ta_KO clones, several heart-related pathways were enriched (FIG. 6A). It suggests that this UCE may play a role in cardiomyocyte function. Although no enhancer or silencer activity of PAX6_Ta was shown in canonical luciferase assays in K562 cells (FIG. 1G and h), both qPCR and RNA-seq data measuring the surrounding gene expression of this UCE suggest that this UCE might be a silencer (FIG. 10A|). As NCREs often function in a tissue- and promoter-specific manner, we then constructed a silencer reporter driven by a super-core promoter SCP1 for expressing GFP, which measures silencer activity on a different promoter from the PGK promoter used in luciferase assays. A reduction of GFP fluorescence was observed, indicating UCE PAX6 may be a potential silencer in this context in K562 cells (FIG. 6B).

[0306] To further investigate the potential function of PAX6_Ta in cardiac development, NKX2-5(eGFP / w) human embryonic stem cells (hESCs) were transfected with dual-CRISPR targeting the PAX6_Ta region73. Efficient genome editing and complete knockout were observed in the PAX6_Ta_KO_bulk hESCs (FIG. 6C and FIG. 14A). Then both the parental NKX2-5(eGFP / w) hESCs and the PAX6_Ta_KO_bulk hESCs were subjected to cardiomyocyte differentiation. The parental NKX2-5(eGFP / w) hESCs became GFP+upon expression of the cardiac marker gene NKX2-5, once they committed into cardiomyocytes successfully73. In contrast, a significant reduction of GFP+cells was observed in PAX6_Ta_KO_bulk hESCs that underwent cardiac differentiation (FIG. 6D and e). Furthermore, the small percentage of the GFP+cardiomyocytes formed from the PAX6_Ta_KO_bulk hESCs showed irregular beat patterns compared to WT NKX2-5(eGFP / w) hESCs (Movie 1, 2, 3 and 4). When the transcription changes of the genes surrounding UCE PAX6_Ta were measured, gene RCN1 was significantly upregulated in PAX6_Ta_KO_bulk hESCs compared to the parental cells after"SUBSTITUTE SHEET RULE 26"cardiomyocyte differentiation. In contrast, no transcriptional changes were observed before differentiation (FIG. 6F). Furthermore, several genes contributing to cardiomyocyte functionality such as CACNG8, RYR2 and MYH6 genes were also similarly de-regulated (FIG. 14B and 14C). These data suggest that PAX6_Ta may be a key NCRE in cardiomyocyte differentiation via regulation of surrounding genes and possibly other genes that are key to heart development and physiology.SEQUENCES

[0307] Lenti_U6_Hl_for_dualGuide-Puro-screening vector. cccgggtgcaaagatggataaagttttaaacagagaggaatctttgcagctaatggaccttctaggtcttgaaaggagtgggaattggctc cggtgcccgtcagtgggcagagcgcacatcgcccacagtccccgagaagttggggggaggggtcggcaattgatccggtgcctagag aaggtggcgcggggtaaactgggaaagtgatgtcgtgtactggctccgcctttttcccgagggtgggggagaaccgtatataagtgcagt agtcgccgtgaacgttctttttcgcaacgggtttgccgccagaacacaggtaagtgccgtgtgtggttcccgcgggcctggcctctttacg ggttatggcccttgcgtgccttgaattacttccacctggctgcagtacgtgattcttgatcccgagcttcgggttggaagtgggtgggagagt tcgaggccttgcgcttaaggagccccttcgcctcgtgcttgagttgaggcctggcctgggcgctggggccgccgcgtgcgaatctggtg gcaccttcgcgcctgtctcgctgctttcgataagtctctagccatttaaaatttttgatgacctgctgcgacgctttttttctggcaagatagtctt gtaaatgcgggccaagatctgcacactggtatttcggtttttggggccgcgggcggcgacggggcccgtgcgtcccagcgcacatgttc ggcgaggcggggcctgcgagcgcggccaccgagaatcggacgggggtagtctcaagctggccggcctgctctggtgcctggcctcg cgccgccgtgtatcgccccgccctgggcggcaaggctggcccggtcggcaccagttgcgtgagcggaaagatggccgcttcccggcc ctgctgcagggagctcaaaatggaggacgcggcgctcgggagagcgggcgggtgagtcacccacacaaaggaaaagggcctttccg tcctcagccgtcgcttcatgtgactccacggagtaccgggcgccgtccaggcacctcgattagttctcgagcttttggagtacgtcgtcttta ggttggggggaggggttttatgcgatggagtttccccacactgagtgggtggagactgaagttaggccagcttggcacttgatgtaattctc cttggaatttgccctttttgagtttggatcttggttcattctcaagcctcagacagtggttcaaagtttttttcttccatttcaggtgtcgtgacgta cggccaccatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgcg ttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacgc gcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgccgtggcggtctggaccacgccggagagcgtcgaagc gggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctcct ggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggagtctcgcccgaccaccagggcaagggtctgggcagcg ccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccgcaacctccccttcta cgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgcctg aacgcgttaagtcgacaatcaacctctggattacaaaatttgtgaaagattgactggtattcttaactatgttgctccttttacgctatgtggata cgctgctttaatgcctttgtatcatgctattgcttcccgtatggctttcattttctcctccttgtataaatcctggttgctgtctctttatgaggagttg tggcccgttgtcaggcaacgtggcgtggtgtgcactgtgtttgctgacgcaacccccactggttggggcattgccaccacctgtcagctcc"SUBSTITUTE SHEET RULE 26"tttccgggactttcgctttccccctccctattgccacggcggaactcatcgccgcctgccttgcccgctgctggacaggggctcggctgttg ggcactgacaattccgtggtgttgtcggggaaatcatcgtcctttccttggctgctcgcctgtgttgccacctggattctgcgcgggacgtcc ttctgctacgtcccttcggccctcaatccagcggaccttccttcccgcggcctgctgccggctctgcggcctcttccgcgtcttcgccttcgc cctcagacgagtcggatctccctttgggccgcctccccgcgtcgactttaagaccaatgacttacaaggcagctgtagatcttagccactttt taaaagaaaaggggggactggaagggctaattcactcccaacgaagacaagatctgctttttgcttgtactgggtctctctggttagaccag atctgagcctgggagctctctggctaactagggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccg tctgttgtgtgactctggtaactagagatccctcagacccttttagtcagtgtggaaaatctctagcagtacgtatagtagttcatgtcatcttat tattcagtatttataacttgcaaagaaatgaatatcagagagtgagaggaacttgtttattgcagcttataatggttacaaataaagcaatagca tcacaaatttcacaaataaagcatttttttcactgcattctagttgtggtttgtccaaactcatcaatgtatcttatcatgtctggctctagctatcc cgcccctaactccgcccatcccgcccctaactccgcccagttccgcccattctccgccccatggctgactaattttttttatttatgcagaggc cgaggccgcctcggcctctgagctattccagaagtagtgaggaggcttttttggaggcctagggacgtacccaattcgccctatagtgagt cgtattacgcgcgctcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgccttgcagcacatccc cctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcgaatgggacgcgc cctgtagcggcgcattaagcgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttc gctttcttcccttcctttctcgccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacgg cacctcgaccccaaaaaacttgattagggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtc cacgttctttaatagtggactcttgttccaaactggaacaacactcaaccctatctcggtctattcttttgatttataagggattttgccgatttcg gcctattggttaaaaaatgagctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgcttacaatttaggtggcacttttcgggga aatgtgcgcggaacccctatttgtttatttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatatt gaaaaaggaagagtatgagtattcaacatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgc tggtgaaagtaaaagatgctgaagatcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagtttt cgccccgaagaacgttttccaatgatgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaact cggtcgccgcatacactattctcagaatgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaa ttatgcagtgctgccataaccatgagtgataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgctttttt gcacaacatgggggatcatgtaactcgccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacg atgcctgtagcaatggcaacaacgttgcgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatgga ggcggataaagttgcaggaccacttctgcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtct cgcggtatcattgcagcactggggccagatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaa cgaaatagacagatcgctgagataggtgcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaa acttcatttttaatttaaaaggatctaggtgaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtca gaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcg gtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgt"SUBSTITUTE SHEET RULE 26"agccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggc gataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacaca gcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaa ggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatag tcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcg gcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttga gtgagctgataccgctcgccgcagccgaacgaccgagcgcagcgagtcagtgagcgaggaagcggaagagcgcccaatacgcaaa ccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggcagtgagcgcaacgcaat taatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgtgagcggataacaatttc acacaggaaacagctatgaccatgattacgccaagcgcgcaattaaccctcactaaagggaacaaaagctggagctgcaagcttaatgt agtcttatgcaatactcttgtagtcttgcaacatggtaacgatgagttagcaacatgccttacaaggagagaaaaagcaccgtgcatgccga ttggtggaagtaaggtggtacgatcgtgccttattaggaaggcaacagacgggtctgacatggattggacgaaccactgaattgccgcatt gcagagatattgtatttaagtgcctagctcgatacataaacgggtctctctggttagaccagatctgagcctgggagctctctggctaactag ggaacccactgcttaagcctcaataaagcttgccttgagtgcttcaagtagtgtgtgcccgtctgttgtgtgactctggtaactagagatccct cagacccttttagtcagtgtggaaaatctctagcagtggcgcccgaacagggacttgaaagcgaaagggaaaccagaggagctctctcg acgcaggactcggcttgctgaagcgcgcacggcaagaggcgaggggcggcgactggtgagtacgccaaaaattttgactagcggagg ctagaaggagagagatgggtgcgagagcgtcagtattaagcgggggagaattagatcgcgatgggaaaaaattcggttaaggccagg gggaaagaaaaaatataaattaaaacatatagtatgggcaagcagggagctagaacgattcgcagttaatcctggcctgttagaaacatca gaaggctgtagacaaatactgggacagctacaaccatcccttcagacaggatcagaagaacttagatcattatataatacagtagcaaccc tctattgtgtgcatcaaaggatagagataaaagacaccaaggaagctttagacaagatagaggaagagcaaaacaaaagtaagaccacc gcacagcaagcggccgctgatcttcagacctggaggaggagatatgagggacaattggagaagtgaattatataaatataaagtagtaaa aattgaaccattaggagtagcacccaccaaggcaaagagaagagtggtgcagagagaaaaaagagcagtgggaataggagctttgttc cttgggttcttgggagcagcaggaagcactatgggcgcagcgtcaatgacgctgacggtacaggccagacaattattgtctggtatagtg cagcagcagaacaatttgctgagggctattgaggcgcaacagcatctgttgcaactcacagtctggggcatcaagcagctccaggcaag aatcctggctgtggaaagatacctaaaggatcaacagctcctggggatttggggttgctctggaaaactcatttgcaccactgctgtgcctt ggaatgctagttggagtaataaatctctggaacagatttggaatcacacgacctggatggagtgggacagagaaattaacaattacacaag cttaatacactccttaattgaagaatcgcaaaaccagcaagaaaagaatgaacaagaattattggaattagataaatgggcaagtttgtgga attggtttaacataacaaattggctgtggtatataaaattattcataatgatagtaggaggcttggtaggtttaagaatagtttttgctgtactttct atagtgaatagagttaggcagggatattcaccattatcgtttcagacccacctcccaaccccgaggggacccagagagggcctatttccca tgattccttcatatttgcatatacgatacaaggctgttagagagataattagaattaatttgactgtaaacacaaagatattagtacaaaatacgt gacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgtaacttgaaagtatttcgat ttcttggctttatatatcttGTGGAAAGGACGAAACACCgGAGACGGTTGTAAATGAGCACACAA"SUBSTITUTE SHEET RULE 26"AATACACATGCTAAAATATTATATTCTATGACCTTTATAAAATCAACCAAAATCTTCTTTTTAATAACTTTAGTATCAATAATTAGAATTTTTATGTTCCTTTTTGCAAACTTTTAATAAAAATGAGCAAAATAAAAAAACGCTAGTTTTAGTAACTCGCGTTGTTTTCTTCACCTTTAATAATAGCTACTCCACCACTTGTTCCTAAGCGGTCAGCTCCTGCTTCAATCATTTTTTGAGCATCTTCAAATGTTCTAACTCCACCAGCTGCTTTAACTAAAGCATTGTCTTTAACAACTGACTTCATTAGTTTAACATCTTCAAATGTTGCACCTGATTTTGAAAATCCTGTTGATGTTTTAACAAATTCTAATCCAGCTTCAACAGCTATTTCACAAGCTTTCATGATTTCTTCTTTTGTTAATAAACAATTTTCCATAATACATTTAACAACATGTGATCCAGCTGCTTTTTTTACAGCTTTCATGTCTTCTAAAACTAATTCATAATTTTTGTCTTTTAATGCACCAATATTTAATACCATATCAATTTCTGTTGCACCATCTTTAATTGCTTCAGAAACTTCGAATGCTTTTGTAGCTGTTGTGCATGCACCTAGAGGAAAACCTACAACATTTGTTATTCCTACATTTGTGCCTTTTAATAATTCTTTACAATAGCTTGTTCAATATGAATTAACACAAACTGTTGCAAAATCAAATTCAATTGCTTCATCACATAATTGTTTAATTTCAGCTTTCGTAGCATCTTGTTTTAATAATGTGTGATCTATATATTTGTTTAGTTTCATTTTTTCTCCTATATATTCATTTTTAATTTTAATTCTTTAATAATTTCGTCTACTTTAACTTTAGCGTTTTGAACAGATTCACCAACACCTATAAAATAAATTTTTAGTTTAGGTTCAGTTCCACTTGGGCGAACAGCAAATCATGACTTATCTTCTAAATAAAATTTTAGTAAGTCTTGTCCTGGCATATTATACATTCCATCGATGTAGTCTTCAACATTAACAACTTTAAGTCCAGCAATTTGAGTTAAGGGTGTTGCTCTCAATGATTTCATTAATGGTTCAATTTTTAATTTCTTTTCTTCTGGTTTAAAATTCAAGTTTAAAGTGAAAGTGTAATATGCACCCATTTCTTTAAATAAATCTTCTAAATAGTCTACTAATGTTTTATTTTGTTTTTTATAAAATCAAGCAGCCTCTGCTATTAATATAGAAGCTTGTATTCCATCTTTATCTCTAGCTGAGTCATCAATTACATATCCATAACTTTCTTCATAAGCAAAAACAAAATTTAATCCGTTATCTTCTTCTTTAGCAATTTCTCTACCCATTCATTTAAATCCAGTTAAAGTTTTTACAATATTAACTCCATATTTTTCATGAGCGATTCTATCACCCAAATCACTTGTTACAAAACTTGAATATAGAGCCGGATTTTTTGGAATGCTATTTAAGCGTTTTAGATTTGATAATTTTCAATCAATTAAAATTGGTCCTGTTTGATTTCCATCTAATCTTACAAAATGACCATCATGTTTTATTGCCATTCCAAATCTGTCAGCATCTGGGTCATTCATAATAATAATATCTGCATCATGTTTAATACCATATTCAAGCGGTATTTTTCATGCAGGATCAAATTCTGGATTTGGATTTACAACATTTTTAAATGTTTCATCTTCAAATGCATGCTCTTCAACCTCAATAACGTTATATCCTGATTCACGTAATATTTTTGGGGTAAATTTAGTTCCTGTTCCATTAACTGCGCTAAAAATAATTTTTAAATCTTTTTTAGCTTCTTGCTCTTTTTTGTACGTCTCTcGGGAAA"SUBSTITUTE SHEET RULE 26"GAGTGGTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTAT GGTGATTTCCCAGAACACATAGCGACATGCAAATATTGCAGGGCGCCACTCCCCTGT CCCTCACAGCCATCTTCCTGCCAGGGCGCACGCGCGCTGGGTGTTCCCGCCTAGTGA CACTGGGCCCGCGATTCCTTGGAGCGGGTTGATGACGTCAGCGTTCaagcttggcgtaactaga tcttgagacaaatggcagtattcatccacaattttaaaagaaaaggggggattggggggtacagtgcaggggaaagaatagtagacataa tagcaacagacatacaaactaaagaattacaaaaacaaattacaaaaattcaaaattttcgggtttattacagggacagcagagatccacttt ggcgccggctcgaggggg (SEQ ID NO: 168)

[0308] pUCl 9_dual_scafffold_spacer_200bp gcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggca gtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgt gagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcttgcatgcctgcaggtcgactctagagGAAGAC gtgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcTTTTTTcgt ctccACGTGGTGGCGCCCGgatcCCGACTGCTCCCTGAGTGTGGCTCTTTGTTCTGTCAAC GCCCGACCTTCATCGCGGCCGATTCCTTCTGCGGACCATGTCGTCCTGATACTTTGGC CATGTTTCCGTTGTAGGAGTGAAGCCACTTGGCTTTGCGCCGTAGTTCCAATGAAAA ACCTATGGACTTTGTTTAGgatccGGTAGCATCAGGAcgagacgAAAAAAGCACCGACTCG GTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCT CTAAAACACGTCTTCgtaccgagctcgaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttaccca acttaatcgccttgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcag cctgaatggcgaatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgc tctgatgccgcatagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttac agacaagctgtgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgat acgcctatttttataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgttt atttttctaaatacattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattca acatttccgtgtcgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaaga tcagttgggtgcacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatga tgagcacttttaaagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcaga atgacttggttgagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagt gataacactgcggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactc gccttgatcgttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttg cgcaaactattaactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttct gcgctcggcccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggcca"SUBSTITUTE SHEET RULE 26"gatggtaagccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggt gcctcactgattaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggt gaagatcctttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatct tcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctac caactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaaga actctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggact caagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctaca ccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggca gggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttg agcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctgg ccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccg aacgaccgagcgcagcgagtcagtgagcgaggaagcggaaga (SEQ ID NO: 169)

[0309] SingleCell pUCl 9_dual_scafffold_spacer_200bp gcgcccaatacgcaaaccgcctctccccgcgcgttggccgattcattaatgcagctggcacgacaggtttcccgactggaaagcgggca gtgagcgcaacgcaattaatgtgagttagctcactcattaggcaccccaggctttacactttatgcttccggctcgtatgttgtgtggaattgt gagcggataacaatttcacacaggaaacagctatgaccatgattacgccaagcttgcatgcctgcaggtcgactctagagGAAGAC gtgttttagagctaGAAAtagcaagttaaaataaggctagtccgttatcaacttggccGCTTTAAGGCCGGTCCTAGC AAggccaagtggcaccgagtcggtgcTTTTTTcgtctccACGTGGTGGCGCCCGgatcCCGACTGCTCCC TGAGTGTGGCTCTTTGTTCTGTCAACGCCCGACCTTCATCGCGGCCGATTCCTTCTGC GGACCATGTCGTCCTGATACTTTGGCCATGTTTCCGTTGTAGGAGTGAAGCCACTTG GCTTTGCGCCGTAGTTCCAATGAAAAACCTATGGACTTTGTTTAGgatccGGTAGCATC AGGAcgagacgAAAAAAGCACCGACTCGGTGCCACTTggccCCTTAGCCGCTAATAGGTG AGCggcCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC ACGTCTTCgtaccgagctcgaattcactggccgtcgttttacaacgtcgtgactgggaaaaccctggcgttacccaacttaatcgcct tgcagcacatccccctttcgccagctggcgtaatagcgaagaggcccgcaccgatcgcccttcccaacagttgcgcagcctgaatggcg aatggcgcctgatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatatggtgcactctcagtacaatctgctctgatgccgc atagttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctg tgaccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctattttt ataggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaata cattcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgt cgcccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtg cacgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcactttta"SUBSTITUTE SHEET RULE 26"aagttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggtt gagtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactg cggccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcg ttgggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactat taactggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggc ccttccggctggctggtttattgctgataaatctggagccggtgagcgtgggtctcgcggtatcattgcagcactggggccagatggtaag ccctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactg attaagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcct ttttgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagat cctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttt tccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtag caccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacga tagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactg agatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcgga acaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcga tttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgct cacatgttctttcctgcgttatcccctgattctgtggataaccgtattaccgcctttgagtgagctgataccgctcgccgcagccgaacgacc gagcgcagcgagtcagtgagcgaggaagcggaaga (SEQ ID NO: 170)

[0310] dual_U6_Hl_PX459V2HA (with Cas9; plasmid for transient expression) gagggcctatttcccatgattccttcatatttgcatatacgatacaaggctgttagagagataattggaattaatttgactgtaaacacaaagat attagtacaaaatacgtgacgtagaaagtaataatttcttgggtagtttgcagttttaaaattatgttttaaaatggactatcatatgcttaccgta acttgaaagtatttcgatttcttggctttatatatcttGTGGAAAGGACGAAACACCggGTCTTCgAAAAggatcc ATGAGCAAAATAAAAAAACGCTAGTTTTAGTAACTCGCGTTGTTTTCTTCACCTTTAA TAATAGCTACTCCACCACTTGTTCCTAAGCGGaTCcAGCTaGAAGACtcGGGAAAGAGT GGTCTCATACAGAACTTATAAGATTCCCAAATCCAAAGACATTTCACGTTTATGGTG ATTTCCCAGAACACATAGCGACATGCAAATATTGCAGGGCGCCACTCCCCTGTCCCT CACAGCCATCTTCCTGCCAGGGCGCACGCGCGCTGGGTGTTCCCGCCTAGTGACACT GGGCCCGCGATTCCTTGGAGCGGGTTGATGACGTCAGCGTTCgttttagagctagaaatagcaagtt aaaataaggctagtccgtTTTTagcgcgtgcgccaattctgcagacaaatggctctagaggtacccgttacataacttacggtaaatgg cccgcctggctgaccgcccaacgacccccgcccattgacgtcaatagtaacgccaatagggactttccattgacgtcaatgggtggagta tttacggtaaactgcccacttggcagtacatcaagtgtatcatatgccaagtacgccccctattgacgtcaatgacggtaaatggcccgcct ggcattGtgcccagtacatgaccttatgggactttcctacttggcagtacatctacgtattagtcatcgctattaccatggtcgaggtgagcc"SUBSTITUTE SHEET RULE 26"ccacgttctgcttcactctccccatctcccccccctccccacccccaattttgtatttatttattttttaattattttgtgcagcgatgggggcggg gggggggggggggcgcgcgccaggcggggcggggcggggcgaggggcggggcggggcgaggcggagaggtgcggcggcag ccaatcagagcggcgcgctccgaaagtttccttttatggcgaggcggcggcggcggcggccctataaaaagcgaagcgcgcggcggg cgggagtcgctgcgacgctgccttcgccccgtgccccgctccgccgccgcctcgcgccgcccgccccggctctgactgaccgcgttac tcccacaggtgagcgggcgggacggcccttctcctccgggctgtaattagctgagcaagaggtaagggtttaagggatggttggttggtg gggtattaatgtttaattacctggagcacctgcctgaaatcactttttttcaggttGGaccggtATGTACCCATACGATGTT CCTGACTATGCGGGCTATCCCTATGACGTCCCGGACTATGCAGGATCGTATCCTTATGACGTTCCAGATTACGCTATGGCCCCAAAGAAGAAGCGGAAGGTCGGTATCCACGGAGTCCCAGCAGCCGACAAGAAGTACAGCATCGGCCTGGACATCGGCACCAACTCTG TGGGCTGGGCCGTGATCACCGACGAGTACAAGGTGCCCAGCAAGAAATTCAAGGTGCTGGGCAACACCGACCGGCACAGCATCAAGAAGAACCTGATCGGAGCCCTGCTGTTCGACAGCGGCGAAACAGCCGAGGCCACCCGGCTGAAGAGAACCGCCAGAAGAAGATACACCAGACGGAAGAACCGGATCTGCTATCTGCAAGAGATCTTCAGCAACGAGAT GGCCAAGGTGGACGACAGCTTCTTCCACAGACTGGAAGAGTCCTTCCTGGTGGAAGAGGATAAGAAGCACGAGCGGCACCCCATCTTCGGCAACATCGTGGACGAGGTGGCCT ACCACGAGAAGTACCCCACCATCTACCACCTGAGAAAGAAACTGGTGGACAGCACCGACAAGGCCGACCTGCGGCTGATCTATCTGGCCCTGGCCCACATGATCAAGTTCCGGGGCCACTTCCTGATCGAGGGCGACCTGAACCCCGACAACAGCGACGTGGACAAGCTGTTCATCCAGCTGGTGCAGACCTACAACCAGCTGTTCGAGGAAAACCCCATCAACGCCAGCGGCGTGGACGCCAAGGCCATCCTGTCTGCCAGACTGAGCAAGAGCAGACGGCTGGAAAATCTGATCGCCCAGCTGCCCGGCGAGAAGAAGAATGGCCTGTTCGGAAACCTGATTGCCCTGAGCCTGGGCCTGACCCCCAACTTCAAGAGCAACTTCGACCTGGCC GAGGATGCCAAACTGCAGCTGAGCAAGGACACCTACGACGACGACCTGGACAACCTGCTGGCCCAGATCGGCGACCAGTACGCCGACCTGTTTCTGGCCGCCAAGAACCTGTCCGACGCCATCCTGCTGAGCGACATCCTGAGAGTGAACACCGAGATCACCAAGGCCCCCCTGAGCGCCTCTATGATCAAGAGATACGACGAGCACCACCAGGACCTGACCCTGCTGAAAGCTCTCGTGCGGCAGCAGCTGCCTGAGAAGTACAAAGAGATTTTCTTCGACCAGAGCAAGAACGGCTACGCCGGCTACATTGACGGCGGAGCCAGCCAGGAAGAGTTCTACAAGTTCATCAAGCCCATCCTGGAAAAGATGGACGGCACCGAGGAACTGCTCGTGAAGCTGAACAGAGAGGACCTGCTGCGGAAGCAGCGGACCTTCGACAACGGCAGCATCCCCCACCAGATCCACCTGGGAGAGCTGCACGCCATTCTGCGGCGGCAGGAAGATTTTTACCCATTCCTGAAGGACAACCGGGAAAAGATCGAGAAGATCCTGACCTTCCGCA"SUBSTITUTE SHEET RULE 26"TCCCCTACTACGTGGGCCCTCTGGCCAGGGGAAACAGCAGATTCGCCTGGATGACCAGAAAGAGCGAGGAAACCATCACCCCCTGGAACTTCGAGGAAGTGGTGGACAAGGGCGCTTCCGCCCAGAGCTTCATCGAGCGGATGACCAACTTCGATAAGAACCTGCCCAACGAGAAGGTGCTGCCCAAGCACAGCCTGCTGTACGAGTACTTCACCGTGTATAACGAGCTGACCAAAGTGAAATACGTGACCGAGGGAATGAGAAAGCCCGCCTTCCTGAGCGGCGAGCAGAAAAAGGCCATCGTGGACCTGCTGTTCAAGACCAACCGGAAAGTGACCGTGAAGCAGCTGAAAGAGGACTACTTCAAGAAAATCGAGTGCTTCGACTCCGTGGAAATCTCCGGCGTGGAAGATCGGTTCAACGCCTCCCTGGGCACATACCACGATCTGCTGAAAATTATCAAGGACAAGGACTTCCTGGACAATGAGGAAAACGAGGACATTCTGGAAGATATCGTGCTGACCCTGACACTGTTTGAGGACAGAGAGATGATCGAGGAACGGCTGAAAACCTATGCCCACCTGTTCGACGACAAAGTGATGAAGCAGCTGAAGCGGCGGAGATACACCGGCTGGGGCAGGCTGAGCCGGAAGCTGATCAACGGCATCCGGGACAAGCAGTCCGGCAAGACAATCCTGGATTTCCTGAAGTCCGACGGCTTCGCCAACAGAAACTTCATGCAGCTGATCCACGACGACAGCCTGACCTTTAAAGAGGACATCCAGAAAGCCCAGGTGTCCGGCCAGGGCGATAGCCTGCACGAGCACATTGCCAATCTGGCCGGCAGCCCCGCCATTAAGAAGGGCATCCTGCAGACAGTGAAGGTGGTGGACGAGCTCGTGAAAGTGATGGGCCGGCACAAGCCCGAGAACATCGTGATCGAAATGGCCAGAGAGAACCAGACCACCCAGAAGGGACAGAAGAACAGCCGCGAGAGAATGAAGCGGATCGAAGAGGGCATCAAAGAGCTGGGCAGCCAGATCCTGAAAGAACACCCCGTGGAAAACACCCAGCTGCAGAACGAGAAGCTGTACCTGTACTACCTGCAGAATGGGCGGGATATGTACGTGGACCAGGAACTGGACATCAACCGGCTGTCCGACTACGATGTGGACCATATCGTGCCTCAGAGCTTTCTGAAGGACGACTCCATCGACAACAAGGTGCTGACCAGAAGCGACAAGAACCGGGGCAAGAGCGACAACGTGCCCTCCGAAGAGGTCGTGAAGAAGATGAAGAACTACTGGCGGCAGCTGCTGAACGCCAAGCTGATTACCCAGAGAAAGTTCGACAATCTGACCAAGGCCGAGAGAGGCGGCCTGAGCGAACTGGATAAGGCCGGCTTCATCAAGAGACAGCTGGTGGAAACCCGGCAGATCACAAAGCACGTGGCACAGATCCTGGACTCCCGGATGAACACTAAGTACGACGAGAATGACAAGCTGATCCGGGAAGTGAAAGTGATCACCCTGAAGTCCAAGCTGGTGTCCGATTTCCGGAAGGATTTCCAGTTTTACAAAGTGCGCGAGATCAACAACTACCACCACGCCCACGACGCCTACCTGAACGCCGTCGTGGGAACCGCCCTGATCAAAAAGTACCCTAAGCTGGAAAGCGAGTTCGTGTACGGCGACTACAAGGTGTACGACGTGCGGAAGATGATCGCCAAGAGCGAGCAGGAAATCGGCAAGGCTACCGCCAAGTACTTCTTCTACAGCAACATCATGAACTTTTTCAAGACC"SUBSTITUTE SHEET RULE 26"GAGATTACCCTGGCCAACGGCGAGATCCGGAAGCGGCCTCTGATCGAGACAAACGG CGAAACCGGGGAGATCGTGTGGGATAAGGGCCGGGATTTTGCCACCGTGCGGAAAG TGCTGAGCATGCCCCAAGTGAATATCGTGAAAAAGACCGAGGTGCAGACAGGCGGC TTCAGCAAAGAGTCTATCCTGCCCAAGAGGAACAGCGATAAGCTGATCGCCAGAAA GAAGGACTGGGACCCTAAGAAGTACGGCGGCTTCGACAGCCCCACCGTGGCCTATTC TGTGCTGGTGGTGGCCAAAGTGGAAAAGGGCAAGTCCAAGAAACTGAAGAGTGTGA AAGAGCTGCTGGGGATCACCATCATGGAAAGAAGCAGCTTCGAGAAGAATCCCATC GACTTTCTGGAAGCCAAGGGCTACAAAGAAGTGAAAAAGGACCTGATCATCAAGCT GCCTAAGTACTCCCTGTTCGAGCTGGAAAACGGCCGGAAGAGAATGCTGGCCTCTGC CGGCGAACTGCAGAAGGGAAACGAACTGGCCCTGCCCTCCAAATATGTGAACTTCCT GTACCTGGCCAGCCACTATGAGAAGCTGAAGGGCTCCCCCGAGGATAATGAGCAGA AACAGCTGTTTGTGGAACAGCACAAGCACTACCTGGACGAGATCATCGAGCAGATC AGCGAGTTCTCCAAGAGAGTGATCCTGGCCGACGCTAATCTGGACAAAGTGCTGTCCGCCTACAACAAGCACCGGGATAAGCCCATCAGAGAGCAGGCCGAGAATATCATCCA CCTGTTTACCCTGACCAATCTGGGAGCCCCTGCCGCCTTCAAGTACTTTGACACCACC ATCGACCGGAAGAGGTACACCAGCACCAAAGAGGTGCTGGACGCCACCCTGATCCA CCAGAGCATCACCGGCCTGTACGAGACACGGATCGACCTGTCTCAGCTGGGAGGCG ACAAAAGGCCGGCGGCCACGAAAAAGGCCGGCCAGGCAAAAAAGAAAAAGgaattcG GCAGTGGAGAGGGCAGAGGAAGTCTGCTAACATGCGGTGACGTCGAGGAGAATCCT GGCCCAatgaccgagtacaagcccacggtgcgcctcgccacccgcgacgacgtccccagggccgtacgcaccctcgccgccgc gttcgccgactaccccgccacgcgccacaccgtcgatccggaccgccacatcgagcgggtcaccgagctgcaagaactcttcctcacg cgcgtcgggctcgacatcggcaaggtgtgggtcgcggacgacggcgccgcggtggcggtctggaccacgccggagagcgtcgaag cgggggcggtgttcgccgagatcggcccgcgcatggccgagttgagcggttcccggctggccgcgcagcaacagatggaaggcctc ctggcgccgcaccggcccaaggagcccgcgtggttcctggccaccgtcggAgtctcgcccgaccaccagggcaagggtctgggcag cgccgtcgtgctccccggagtggaggcggccgagcgcgccggggtgcccgccttcctggagacctccgcgccccGcaacctcccctt ctacgagcggctcggcttcaccgtcaccgccgacgtcgaggtgcccgaaggaccgcgcacctggtgcatgacccgcaagcccggtgc ctgagaattctaaCTAGAGCTCGCTGATCAGCCTCGACTGTGCCTTCTAGTTGCCAGCCATCTGTTGTTTGCCCCTCCCCCGTGCCTTCCTTGACCCTGGAAGGTGCCACTCCCACTGTCC TTTCCTAATAAAATGAGGAAATTGCATCGCATTGTCTGAGTAGGTGTCATTCTATTCT GGGGGGTGGGGTGGGGCAGGACAGCAAGGGGGAGGATTGGGAAGAgAATAGCAGG CATGCTGGGGAgcggccgcaggaacccctagtgatggagttggccactccctctctgcgcgctcgctcgctcactgaggccg ggcgaccaaaggtcgcccgacgcccgggctttgcccgggcggcctcagtgagcgagcgagcgcgcagctgcctgcaggggcgcct"SUBSTITUTE SHEET RULE 26"gatgcggtattttctccttacgcatctgtgcggtatttcacaccgcatacgtcaaagcaaccatagtacgcgccctgtagcggcgcattaag cgcggcgggtgtggtggttacgcgcagcgtgaccgctacacttgccagcgccctagcgcccgctcctttcgctttcttcccttcctttctcg ccacgttcgccggctttccccgtcaagctctaaatcgggggctccctttagggttccgatttagtgctttacggcacctcgaccccaaaaaa cttgatttgggtgatggttcacgtagtgggccatcgccctgatagacggtttttcgccctttgacgttggagtccacgttctttaatagtggact cttgttccaaactggaacaacactcaaccctatctcgggctattcttttgatttataagggattttgccgatttcggcctattggttaaaaaatga gctgatttaacaaaaatttaacgcgaattttaacaaaatattaacgtttacaattttatggtgcactctcagtacaatctgctctgatgccgcata gttaagccagccccgacacccgccaacacccgctgacgcgccctgacgggcttgtctgctcccggcatccgcttacagacaagctgtga ccgtctccgggagctgcatgtgtcagaggttttcaccgtcatcaccgaaacgcgcgagacgaaagggcctcgtgatacgcctatttttata ggttaatgtcatgataataatggtttcttagacgtcaggtggcacttttcggggaaatgtgcgcggaacccctatttgtttatttttctaaatacat tcaaatatgtatccgctcatgagacaataaccctgataaatgcttcaataatattgaaaaaggaagagtatgagtattcaacatttccgtgtcg cccttattcccttttttgcggcattttgccttcctgtttttgctcacccagaaacgctggtgaaagtaaaagatgctgaagatcagttgggtgca cgagtgggttacatcgaactggatctcaacagcggtaagatccttgagagttttcgccccgaagaacgttttccaatgatgagcacttttaaa gttctgctatgtggcgcggtattatcccgtattgacgccgggcaagagcaactcggtcgccgcatacactattctcagaatgacttggttga gtactcaccagtcacagaaaagcatcttacggatggcatgacagtaagagaattatgcagtgctgccataaccatgagtgataacactgcg gccaacttacttctgacaacgatcggaggaccgaaggagctaaccgcttttttgcacaacatgggggatcatgtaactcgccttgatcgttg ggaaccggagctgaatgaagccataccaaacgacgagcgtgacaccacgatgcctgtagcaatggcaacaacgttgcgcaaactatta actggcgaactacttactctagcttcccggcaacaattaatagactggatggaggcggataaagttgcaggaccacttctgcgctcggccc ttccggctggctggtttattgctgataaatctggagccggtgagcgtggaagccgcggtatcattgcagcactggggccagatggtaagc cctcccgtatcgtagttatctacacgacggggagtcaggcaactatggatgaacgaaatagacagatcgctgagataggtgcctcactgat taagcattggtaactgtcagaccaagtttactcatatatactttagattgatttaaaacttcatttttaatttaaaaggatctaggtgaagatcctttt tgataatctcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcct ttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttcc gaaggtaactggcttcagcagagcgcagataccaaatactgtccttctagtgtagccgtagttaggccaccacttcaagaactctgtagca ccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgata gttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgag atacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaac aggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgattt ttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctca catgt (SEQ ID NO: 171)REFERENCES"SUBSTITUTE SHEET RULE 26"

[0311] 1 Nurk, S. et al. The complete sequence of a human genome. Science 376, 44-53, doi: 10.1126 / science.abj6987 (2022).

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Claims

CLAIMSWhat is claimed is:

1. A nucleic acid encoding a pair of guide RNAs operable to delete a region of DNA, wherein the nucleic acid encoding the pair of guide RNAs comprises: (i) a first guide RNA comprising a first crRNA sequence and a first tracrRNA sequence, said sequences being operably linked to a first promoter, and (ii) a second guide RNA comprising a second crRNA sequence and a second tracrRNA sequence, said sequences being operably linked to a second promoter, wherein the first and second guide RNAs are separated by a spacer, and wherein the promoters are different and are convergent.

2. The nucleic acid of claim 1 wherein the first guide RNA and / or the second guide RNA further comprises a capture sequence.

3. The nucleic acid of claim 2, wherein the capture sequence is comprised in the first tracrRNA sequence and / or the second tracrRNA sequence, preferably within a stem loop or 3’UTR of the first tracrRNA sequence and / or the second tracrRNA sequence.

4. The nucleic acid of any preceding claim, wherein the spacer has a length of between 50-200 nucleotides, preferably the spacer has a length of about 200 nucleotides.

5. The nucleic acid of any preceding claim, wherein the first and second promoters are POL III promoters.

6. The nucleic acid of any preceding claim, wherein the first promoter is U6, and the second promoter is Hl, or vice versa.

7. The nucleic acid of any preceding claim wherein the first and second crRNA sequences each comprise a DNA-targeting section, wherein each DNA-targeting section comprises a nucleotide sequence complementary to a suitable target sequence in the DNA.

8. The nucleic acid of claim 7, wherein the first crRNA sequence comprises a first DNA- targeting section comprising a nucleotide sequence complementary to a target sequence upstream of the region of DNA to be deleted."SUBSTITUTE SHEET RULE 26"9. The nucleic acid of claim 7 or claim 8, wherein the second crRNA sequence comprises a second DNA-targeting section comprising a nucleotide sequence complementary to a target sequence downstream of the region of DNA to be deleted.

10. A vector comprising the nucleic acid of any preceding claim.

11. A method of making a vector according to claim 10, the method comprising:(a) Providing a vector backbone comprising the first and second promoters, and a first cleavage site downstream of the first promoter, and a second cleavage site downstream of the second promoter;(b) Cleaving the backbone at the first and second cleavage sites;(c) Inserting a nucleic acid comprising: the first crRNA sequence, the second crRNA sequence, a third cleavage site downstream of the first crRNA sequence, and a fourth cleavage site downstream of the second crRNA sequence, into the backbone between the first and second cleavage sites, such that the first crRNA sequence is downstream of the first promoter and the second crRNA sequence is downstream of the second promoter;(d) Cleaving the backbone at the third and fourth cleavage sites; and(e) Inserting a nucleic acid comprising: the first tracrRNA sequence and the second tracrRNA sequence separated by a spacer, into the backbone between the third and fourth cleavage sites, such that the first tracrRNA sequence is downstream of the first crRNA sequence and the second tracrRNA sequence is downstream of the second crRNA sequence.

12. The method of claim 11, wherein the or each cleavage site is a restriction site, preferably wherein the or each cleavage site is recognised by a restriction endonuclease enzyme, more preferably wherein the or each cleavage site is recognised by a Type IIS restriction endonuclease enzyme.

13. The method of claim 12, wherein the first and second cleavage sites are recognised by BsmBI endonuclease, and step (b) comprises cleaving the backbone at the first and second sites by exposing the vector backbone to a BsmBI restriction endonuclease under suitable conditions for the enzyme to cleave at the first and second cleavage sites.

14. The method of claim 12 or claim 13, wherein the third and fourth cleavage sites are recognised by BsmBI endonuclease, and step (d) comprises cleaving the backbone at the third"SUBSTITUTE SHEET RULE 26"and fourth sites by exposing the vector backbone to a BsmBI restriction endonuclease under suitable conditions for the enzyme to cleave at the third and fourth cleavage sites.

15. The method of any of claim 11 to claim 14, wherein inserting steps (c) and (e) comprise ligating the nucleic acid into the vector backbone, preferably by contacting the nucleic acid and the vector backbone with a ligase enzyme under suitable conditions for the ligase to ligate the nucleic acid into the vector.

16. The method according to any of claim 11 to claim 15, wherein the vector produced by the method comprises, from 5' to 3': the first promoter, the first crRNA sequence, the first tracrRNA sequence, the spacer, the second tracrRNA sequence, the second crRNA, the second promoter, preferably wherein the first and second promoters are convergent.

17. A library comprising a plurality of vectors according to claim 10, wherein each vector in the library comprises a nucleic acid according to any of claims 1 -9 encoding a different pair of guide RNAs operable to delete a different region of DNA.

18. A system for deleting a region of DNA, the system comprising (i) a nucleic acid according to any of claim 1 to claim 9, a vector according to claim 10, or a library according to claim 17, for expressing one or more pairs of guide RNAs, and (ii) a nucleic acid encoding a Cas protein, a vector comprising said nucleic acid for expressing a Cas protein, or a pair of Cas proteins.

19. A method of deleting one or more regions of DNA, the method comprising:(a) Providing a cell with the system of claim 18;(b) Culturing the cell under suitable conditions to express the nucleic acids of the system and thereby produce one or more pairs of guide RNAs and a pair of Cas proteins; and(c) Culturing the cell under suitable conditions to allow the or each pair of guide RNAs and the pair of Cas proteins to associate to form a pair of CRISPR-Cas complexes and for the CRISPR-Cas complexes to delete one or more regions of DNA in the cell.

20. A method of screening a genome, the method comprising:(a) Providing a plurality of cells comprising the library of claim 17, wherein each cell comprises (i) a vector of the library, and (ii) a nucleic acid encoding a Cas protein or a vector comprising said nucleic acid for expressing a Cas protein, or a pair of Cas proteins;"SUBSTITUTE SHEET RULE 26"(b) Culturing the cells under suitable conditions to express, within each cell, the nucleic acids of the vector and optionally the nucleic acid encoding the Cas protein and thereby produce, within each cell, a pair of guide RNAs and a pair of Cas proteins;(c) Culturing the cells under suitable conditions to allow the pair of guide RNAs and the pair of Cas proteins to associate to form a pair of CRISPR-Cas complexes and for the pair of CRISPR-Cas complexes to delete a region of DNA within each cell; and(d) Performing bulk or single cell screening of the cells to determine the function of the one or more deleted regions of DNA.

21. A method according to claim 18, claim 19, or claim 20, wherein the or each Cas protein is a Cas9 protein, preferably wherein the or each Cas9 protein is the S. pyogenes Cas9 protein.

22. A method according to claim 19 or claim 20, wherein the one or more regions of DNA are non-coding regions of DNA, preferably wherein the one or more regions of DNA are noncoding regulatory elements, more preferably wherein the one or more regions of DNA are enhancers.

23. A method according to any of claim 20 to claim 22, wherein the method further comprises challenging the cells with a selective pressure of interest, preferably during step (c) or after step (c), preferably wherein the selective pressure of interest is selected from: exposure to one or more drugs, exposure to infection, depriving nutrients, excessive nutrients, depriving growth factors or cytokines, excessive growth factors or cytokines, exposure to cold or heat, exposure to acid or alkali, stimulating proliferation, stimulating transformation of the cells, and / or stimulating differentiation of the cells.

24. A method according to any of claim 20 to claim 23, wherein step (d) comprises performing bulk screening of the cells, preferably comprising the steps of (i) Isolating the genomic DNA of each cell, (ii) amplifying and sequencing the guide RNAs present in the genomic DNA, (iii) determining which pairs of guide RNAs are depleted or enriched in the cells relative to a control population of cells from step (b).

25. A method according to any of claim 20 to claim 23, wherein step (d) comprises performing single-cell screening of the cells, preferably comprising the steps of (i) Isolating the mRNA and guide RNAs present in each cell, (ii) amplifying and sequencing the mRNA and guide RNAs isolated from each cell, (iii) correlating mRNA expression with guide RNA expression."SUBSTITUTE SHEET RULE 26"

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