Methods of identifying and correlating crispr-induced EXON skipping to phenotypic outcomes

Abstract

Disclosed herein are methods of identifying a guide RNA (gRNA) that induces exon skipping in a target gene. Also disclosed are methods of inducing exon skipping in a target gene methods of controlling heterogeneity of a phenotypic outcome.

Description

METHODS OF IDENTIFYING AND CORRELATING CRISPR-INDUCED EXON SKIPPING TO PHENOTYPIC OUTCOMESCROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Application No. 63 / 637,657, filed on April 23, 2024, which is incorporated herein, in its entirety, by reference.SUBMISSION OF SEQUENCE LISTING

[0002] The Sequence Listing associated with this application is filed in electronic format via Patent Center and hereby incorporated by reference into the specification in its entirety. The name of the text file containing the Sequence Listing is13094901920sequencelisting.xmL The size of the xml file is 17 KB, and the xml file was created on April 23, 2025.FIELD

[0003] The present disclosure relates to methods for inducing exon skipping to produce desired phenotypic outcomes using Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) / endonuclease gene editing.BACKGROUND

[0004] In the case of cancer cells and genetic disease, the phenotypes observed are typically as a result of gain or loss of function. With gene editing, the goal is to reverse these phenotypes, which include proliferation capacity, viability after chemotherapy exposure, invasion and migration capacities, immune escape, morphological changes, protein levels in cells or blood plasma, and transcriptional changes, among others.

[0005] Processing of eukaryotic pre-m RNAs is a complex process that requires a multitude of signals and protein factors to achieve appropriate mRNA splicing. Exon splicing by the spliceosome requires more than the canonical splicing signals whichdefine intron-exon boundaries. For example, one such additional signal is provided by cis-acting regulatory enhancer and silencer sequences. Exon splicing enhancers (ESE), exon splicing silencers (ESS), intron splicing enhancers (ISE), and intron splicing silencers (ISS) have been identified which either repress or enhance usage of splice donor sites or splice acceptor sites, depending on their site and mode of action.13Binding of specific proteins (trans-acting factors) to these regulatory sequences directs the splicing process, either promoting or inhibiting usage of particular splice sites and thus modulating the ratio of splicing products.14 15

[0006] Currently there are no effective means for controlling phenotypic heterogeneity by taking advantage of cis-acting regulatory enhancers of exon splicing.SUMMARY

[0007] One aspect is for a method of identifying a guide RNA (gRNA) that induces exon skipping in a target gene comprising: (a) identifying one or more gRNAs that bind near or in an exon splicing enhancer (ESE) or an exon splicing silencer (ESS) in the target gene; (b) contacting the target gene with one of the gRNAs identified in step (a) and a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, whereby the gRNA hybridizes to the target gene and the CRISPR- associated endonuclease cleaves the gene; (c) repeating step (b) with another gRNA of step (a) until all gRNAs of step (a) have been contacted with the target gene; (d) determining a percent rate of exon skipping generated by each of the one or more gRNAs; and (e) correlating a phenotypic change in a protein expressed by the target gene with the percent rate of exon skipping generated by each of the one or more gRNAs. In some embodiments, the method comprises after step (e) the step of administering to a subject a gRNA of step (d) determined to have at least a 10% rate of inducing exon skipping that correlates with the phenotypic change of step (e). In some embodiments, the one or more gRNAs each bind within 30 bases of the ESE or ESS.

[0008] Another aspect is for a method of inducing exon skipping in a target gene comprising: (a) identifying one or more gRNAs that bind near or in an exon splicing enhancer (ESE) or an exon splicing silencer (ESS) in the target gene; (b)contacting thetarget gene with one of the gRNAs identified in step (a) and a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, whereby the gRNA hybridizes to the target gene and the CRISPR-associated endonuclease cleaves the gene; (c) repeating step (b) with another gRNA of step (a) until all gRNAs of step (a) have been contacted with the target gene; (d) determining a percent rate of exon skipping generated by each of the one or more gRNAs; (e) correlating a phenotypic change in a protein expressed by an exon-skipped gene with the percent rate of exon skipping generated by each of the one or more gRNAs; and (f) administering to a subject the gRNA of step (d) determined to have at least a 10% rate of inducing exon skipping and at least a 10% rate of phenotypic change. In some embodiments, the one or more gRNAs each bind within 30 bases of the ESE or ESS.

[0009] A further aspect is for a method of inducing desirable splice variants of a target gene to control heterogeneity of a phenotypic outcome comprising: (a) identifying one or more gRNAs that bind near or in an exon splicing enhancer (ESE) or an exon splicing silencer (ESS) in the target gene; (b) contacting the target gene with one of the gRNAs identified in step (a) and a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, whereby the gRNA hybridizes to the target gene and the CRISPR-associated endonuclease cleaves the gene; (c) repeating step (b) with another gRNA of step (a) until all gRNAs of step (a) have been contacted with the target gene; (d) determining a percent rate of exon skipping generated by each of the one or more gRNAs; (e) correlating a desirable phenotypic outcome generated by a protein expressed by an exon-skipped gene with the percent rate of exon skipping generated by each of the one or more gRNAs; and (f) administering to a subject the gRNA of step (d) determined to have at least a 10% rate of inducing exon skipping and correlates with an at least 10% change in phenotypic heterogeneity. In some embodiments, the one or more gRNAs each bind within 30 bases of the ESE or ESS.

[0010] Other objects and advantages will become apparent to those skilled in the art upon reference to the detailed description that hereinafter follows.BRIEF DESCRIPTION OF THE DRAWINGS

[0011] Figure 1 : CRISPR design and NRF2 sequence target. The top panel displays the structural domains of the NRF2 protein aligned to the exons of the NRF2 gene. Three guide RNAs were designed to cleave within exon 2 and exon 4 of the gene. The top panel provides a schematic diagram of the cleavage sites of each guide RNA in relation to the gene and protein. The bottom panel presents sequence alignment and actual cleavage sites of each guide RNA. Guide RNA (1 ) was designed to target the beginning of exon 2 whereas guide RNA (2) and (3) were designed to target the beginning and end of exon 4, respectively.

[0012] Figure 2: Genomic, transcript and protein analyses of A549 clonal cell lines created by CRISPR targeting of exon 2 (Fig. 2A) or exon 4 (Fig. 2B). The first column lists the cell line and clonal identification number with the associated guide RNA used to target NRF2. The second column lists the genomic NRF2 sequence of each clone and the allele-specific indel patterns with the normal genomic sequence is listed before each set. The guide RNA sequence and the Pam are depicted with the cleavage site represented by the vertical line. Exonic splicing enhancers are highlighted within the second column. The third column lists the population of mRNA transcripts of each clonal cell population. Clonal cells were harvested for western blot analysis using an antibody directed against NRF2 and GAPDH was used as a loading control. The image displays the detection of NRF2 protein from A549 clonal cell lines targeted with a single or dual CRISPR guide RNA in both exon 2 (Fig. 2C) and exon 4 (Fig. 2D), as marked.

[0013] Figure 3: (Fig. 3A) Relative location of Exonic Splicing Enhancer regions in relation to various CRISPR target sites. The coding region of the NRF2 gene was analyzed for ESEs (highlighted). A second cell line, H1703, was also analyzed using gRNA (4) targeting exon 2 which resulted in similar exon skipping patterns seen in the A549 clonal cell lines. (Fig. 3B) Representative western blot analysis of CRISPR- engineered H1703 clonal cell lines. Clonal cells were harvested for western blot analysis using an antibody directed against NRF2 and GAPDH was used as a loading control.

[0014] Figure 4: Proliferation capacity of wild type and NRF2 modified A549 clonal cell lines in response to cisplatin treatment. (Fig. 4A) Schematic diagram of the splice variants present in each clonal cell line. (Fig. 4B) Proliferation was measured via bioreduction of MTS to a formazan product. Cells were treated with increasing concentrations of cisplatin for 72 hours then evaluated for cell proliferation. The average proliferation and viability of cells in response to cisplatin is graphed as relative absorbance values.

[0015] Figure 5: Transcript analysis for splice variants after CRISPR targeting. Lanes 1 - 4 show results from the KYSE cell line; lanes 5-8 show results from the FADU cell line. Lanes 1 and 5 show NRF2 cDNA amplicons from unedited cells with edited samples in lanes 2-4, 6-8 from targeting exon 2 or exon 4.

[0016] Figure 6: Comparison and characterization of the disruption of splicing motifs by CRISPR. (Fig. 6A) Schematic diagram of the gRNAs used to target exon 2 of NRF2 targeting two proximal ESE motifs (orange box, orange text). (Fig. 6B) Two clonal cell lines from targeting with gRNA (4). The indels that occur in these clones remove 1 base from an ESE motif, as shown in the first orange box. (Fig. 6C) Six clonal cell lines from targeting with R34G gRNA.

[0017] Figure 7: CRISPR / Cas targeting of EGFR exon 19 design. Alignment of the Exon 19 targeting CRISPR is depicted by the box and the line indicates the Cas9 cleavage site. The box indicates the ESE (Fig. 7A). Indel spectrum induced by targeting EGFR exon 19 with CRISPR in PC9 cells. Deletions are indicated by the dashes. The box highlights the ESE and deletions that span across it (Fig. 7B). Transcript analysis for splice variants after CRISPR targeting (Fig. 7C). Lanes 1 -4 show results from the mock CRISPR targeting in WT PC9 cells (WT-1 , WT-2) and two PC9 clonal derivatives (6-1 , 6-2); Lanes 5-8 show results from the EGFR Exon 19 gRNA1 CRISPR targeting in WT PC9 cells (WT-1 , WT-2) and two PC9 clonal derivatives (6-1 , 6-2).

[0018] Figure 8: (Fig. 8A) Schematic diagram of gRNAs (arrows indicate cleavage site) designed to target along exon 4 of the NRF2 gene. Along the DNA sequence, nucleotides in light grey depict predicted ESEs. (Fig. 8B) Exon 4 skipping of NRF2 gene after CRISPR / Cas9 disruption. cDNA from cells targeted with each respectivegRNA in exon 4 was collected at 72hr post-transfection and was used as the template for PCR amplification with primers that spanned from the 5’IITR region through Exon 5 (798bp) of the NRF2 gene. The image depicts an agarose gel of the cDNA amplicons from each gRNA (listed above the lane) targeting condition with editing efficiency (EE%) listed above each gRNA.DETAILED DESCRIPTION

[0019] Applicant has solved the stated problem.

[0020] Applicant herein discloses use of CRISPR / Cas systems to induce desirable splice variants to control the heterogeneity of phenotypic outcomes. Applicant discovered that exon skipping is not only active in response to CRISPR / Cas DNA cleavage, but in some cases altered transcripts dominate the population. The generation of transcripts missing individual exons could clearly influence the effectiveness of frontline therapy. Based on Applicant’s data, Applicant suggests that detailed analysis of DNA sequence alterations caused by CRISPR / Cas could have dramatic effects on phenotypic outcomes, which is dictated by the protein domains that are eliminated by exon skipping, as the absence of certain protein domains can lead to a diversity of responses to chemotherapy, radiation therapy or even immunotherapy.

[0021] NRF2 has five canonical exons, which have various functional domains associated with it. Neh4 / Neh5 domains have been previously validated in Applicant’s hands to hold high functional importance for disabling the function of the gene by reducing the transcriptional activity of NRF2 which is core to its function as a transcription factor. Applicant has screened several guide RNAs along the gene and have noticed a difference in exon skipping rates even within the same exon. Not all indels after CRISPR editing are made the same, therefore it is very important to characterize what the cell may do after a CRISPR edit with a particular indel. The indel occurs at the genomic level which is then transcribed into a mRNA that contains the indel which is then translated into a protein that contains the indel which ends up being a new form of the protein1. From Applicant’s observations, Applicant can identify gRNAs that disrupt splicing motifs or DNA elements which induce exon skipping ofcritical domains at a high rate regardless of the indel sizes generated, therefore Applicant can reduce the heterogeneity of outcomes a patient’s tumor may have after CRISPR in-vivo therapy.

[0022] Definitions

[0023] Applicant specifically incorporates the entire contents of all cited references in this disclosure. Further, when an amount, concentration, or other value or parameter is given as either a range or a list of upper values and lower values, this is to be understood as specifically disclosing all ranges formed from any pair of any upper range limit or value and any lower range limit or value, regardless of whether ranges are separately disclosed. Where a range of numerical values is recited herein, unless otherwise stated, the range is intended to include the endpoints thereof, and all integers and fractions within the range. It is not intended that the scope of the present disclosure be limited to the specific values recited when defining a range.

[0024] The indefinite articles “a” and “an”, as used herein in the specification and in the claims, unless clearly indicated to the contrary, should be understood to mean “at least one”.

[0025] The phrase “and / or”, as used herein in the specification and in the claims, should be understood to mean “either or both” of the elements so conjoined, i.e., elements that are conjunctively present in some cases and disjunctively present in other cases. Other elements may optionally be present other than the elements specifically identified by the “and / or” clause, whether related or unrelated to those elements specifically identified unless clearly indicated to the contrary. Thus, as a nonlimiting example, a reference to “A and / or B”, when used in conjunction with open- ended language such as “comprising” can refer, in one embodiment, to A without B (optionally including elements other than B); in another embodiment, to B without A (optionally including elements other than A); in yet another embodiment, to both A and B (optionally including other elements); etc.

[0026] As used herein in the specification and in the claims, “or” should be understood to have the same meaning as “and / or” as defined above. For example, whenseparating items in a list, “or” or “and / or” shall be interpreted as being inclusive, i.e., the inclusion of at least one, but also including more than one, of a number or list of elements, and, optionally, additional unlisted items. Only terms clearly indicated to the contrary, such as “only one of” or “exactly one of”, or, when used in the claims, “consisting of”, will refer to the inclusion of exactly one element of a number or list of elements. In general, the term “or” as used herein shall only be interpreted as indicating exclusive alternatives ( / .e., “one or the other but not both”) when preceded by terms of exclusivity, “either”, “one of”, “only one of”, “exactly one of”. “Consisting essentially of”, when used in the claims, shall have its ordinary meaning as used in the field of patent law.

[0027] The term “about” as used herein when referring to a measurable value such as an amount, a temporal duration, and the like, is meant to encompass variations of ±20%, ±10%, ±5%, ±1%, or ±0.1 % from the specified value, as such variations are appropriate to perform the disclosed methods.

[0028] A “Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)- associated endonuclease protein-binding domain” or “Cas binding domain” refers to a nucleic acid element or domain within a nucleic acid sequence or polynucleotide sequence that, in an effective amount, will bind or have an affinity for one or a plurality of CRISPR-associated endonuclease (or functional fragments thereof). In some embodiments, in the presence of the one or a plurality of proteins (or functional fragments thereof) and a target sequence, the one or plurality of proteins and the nucleic acid element forms a biologically active CRISPR complex and / or can be enzymatically active on a target sequence. In some embodiments, the CRISPR- associated endonuclease is a class 1 or class 2 CRISPR-associated endonuclease, and in some embodiments, a Cas9 or Cas12a endonuclease. The Cas9 endonuclease can have a nucleotide sequence identical to the wild type Streptococcus pyogenes sequence. In some embodiments, the CRISPR-associated endonuclease can be a sequence from other species, for example other Streptococcus species, such as thermophilus; Pseudomona aeruginosa, Escherichia coli, or other sequenced bacteria genomes and archaea, or other prokaryotic microorganisms. Such species include:Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., Cycliphilusdenitrificans, Aminomonas paucivorans, Bacillus cereus, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidates puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Dinoroseobacter shibae, Eubacterium dolichum, Gammaproteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, llyobacter polytropus, Kingella kingae, Lactobacillus crispatus, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Neisseria bacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactam ica, Neisseria meningitidis, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus aureus, Staphylococcus lugdunensis, Streptococcus sp., Subdoligranulum sp., Tistrella mobilis, Treponema sp., and Verminephrobacter eiseniae (or functional fragments or variants of any of the aforementioned sequences that have at least 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the aforementioned Cas9 endonucleases). In some embodiments, the CRISPR-associated endonuclease can be a Cas12a nuclease. The Cas12a nuclease can have a nucleotide sequence identical to a wild type Prevotella or Francisella sequence (or functional fragments or variants of any of the aforementioned sequences that have at least 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity to any of the aforementioned Cas12 endonucleases).

[0029] In some embodiments, the terms “(CRISPR)-associated endonuclease protein-binding domain” or “Gas binding domain” refer to a nucleic acid element or domain (e.g. and RNA element or domain) within a nucleic acid sequence that, in an effective amount, will bind to or have an affinity for one or a plurality of CRISPR-associated endonucleases (or functional fragments or variants thereof that are at least about 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% homologous to a CRISPR-associated endonuclease). In some embodiments, the Cas binding domain consists of at least or no more than about 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39,40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62,63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73, 74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85,86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96, 97, 98, 99, 100, 105, 1 10, 1 15, 120, 125, 130,135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, or 250 nucleotides and comprises at least one sequence that is capable of forming a hairpin or duplex that partially associates or binds to a biologically active CRISPR-associated endonuclease at a concentration and within a microenvironment suitable for CRISPR system formation.

[0030] The “Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) — CRISPR associated (Cas) (CRISPR-Cas) system guide RNA” or “CRISPR-Cas system guide RNA” may comprise a transcription terminator domain. The term “transcription terminator domain” refers to a nucleic acid element or domain within a nucleic acid sequence (or polynucleotide sequence) that, in an effective amount, prevents bacterial transcription when the CRISPR complex is in a bacterial species and / or creates a secondary structure that stabilizes the association of the nucleic acid sequence to one or a plurality of Cas proteins (or functional fragments thereof) such that, in the presence of the one or a plurality of proteins (or functional fragments thereof), the one or plurality of Cas proteins and the nucleic acid element forms a biologically active CRISPR complex and / or can be enzymatically active on a target sequence in the presence of such a target sequence and a DNA-binding domain. In some embodiments, the transcription terminator domain consists of at least or no more than about 25, 26, 27,28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50,51 , 52, 53, 54, 55, 56, 57, 58, 59, 60, 61 , 62, 63, 64, 65, 66, 67, 68, 69, 70, 71 , 72, 73,74, 75, 76, 77, 78, 79, 80, 81 , 82, 83, 84, 85, 86, 87, 88, 89, 90, 91 , 92, 93, 94, 95, 96,97, 98, 99, 100, 105, 110, 1 15, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170,175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, or 250 nucleotides and comprises at least one sequence that is capable of forming a hairpin or duplex that partially drives association of the nucleic acid sequence (sgRNA, crRNA with tracrRNA, or other nucleic acid sequence) to a biologically active CRISPR complex at a concentration and microenvironment suitable for CRISPR complex formation.

[0031] The term “DNA-binding domain” refers to a nucleic acid element or domain within a nucleic acid sequence {e.g. a guide RNA) that is complementary to a target gene. In some embodiments, the DNA-binding domain will bind or have an affinity for a target gene such that, in the presence of a biologically active CRISPR complex, one or plurality of Cas proteins can be enzymatically active on the target sequence. In some embodiments, the DNA binding domain comprises at least one sequence that is capable of forming Watson Crick base pairs with a target sequence as part of a biologically active CRISPR system at a concentration and microenvironment suitable for CRISPR system formation.

[0032] “CRISPR system” refers collectively to transcripts or synthetically produced transcripts and other elements involved in the expression of or directing the activity of CRISPR-associated (“Cas”) genes, including sequences encoding a Cas gene, a tracr (trans-activating CRISPR) sequence e.g. tracrRNA or an active partial tracrRNA), a tracr-mate sequence (encompassing a “direct repeat” and a tracrRNA-processed partial direct repeat in the context of an endogenous CRISPR system), a guide sequence (also referred to as a “spacer” in the context of an endogenous CRISPR system), or other sequences and transcripts from a CRISPR locus. In some embodiments, one or more elements of a CRISPR system is derived from a type I, type II, or type III CRISPR system. In some embodiments, one or more elements of a CRISPR system is derived from a particular organism comprising an endogenous CRISPR system, such as Streptococcus pyogenes. In general, a CRISPR system is characterized by elementsthat promote the formation of a CRISPR complex at the site of a target sequence (also referred to as a protospacer in the context of an endogenous CRISPR system). In the context of formation of a CRISPR complex, “target sequence” refers to a nucleic acid sequence to which a guide sequence is designed to have complementarity, where hybridization between a target sequence and a guide sequence promotes the formation of a CRISPR complex. Full complementarity is not necessarily required, provided there is sufficient complementarity to cause hybridization and promote formation of a CRISPR complex. A target sequence may comprise any polynucleotide, such as DNA or RNA polynucleotides. In some embodiments, the target sequence is a DNA polynucleotide and is referred to a DNA target sequence. In some embodiments, a target sequence comprises at least three nucleic acid sequences that are recognized by a Cas-protein when the Cas protein is associated with a CRISPR complex or system which comprises at least one sgRNA or one tracrRNA / crRNA duplex at a concentration and within an microenvironment suitable for association of such a system. In some embodiments, the target DNA comprises at least one or more proto-spacer adjacent motifs which sequences are known in the art and are dependent upon the Cas protein system being used in conjunction with the sgRNA or crRNA / tracrRNAs employed by this work. In some embodiments, the target DNA comprises NNG, where G is an guanine and N is any naturally occurring nucleic acid. In some embodiments the target DNA comprises any one or combination of NNG, NNA, GAA, NNAGAAW, NGGNG, and TTTV, where G is an guanine, A is adenine, T is thymine, N is any naturally occurring nucleic acid, and V is guanine, cytosine, or adenine.

[0033] In some embodiments, a target sequence is located in the nucleus or cytoplasm of a cell.

[0034] Typically, in the context of an endogenous CRISPR system, formation of a CRISPR complex (comprising a guide sequence hybridized to a target sequence and complexed with one or more Cas proteins) results in cleavage of one or both strands in or near (e.g. within 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 11 , 12, 13, 14, 15, 16, 17, 18, 19, 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, or more base pairs from) the target sequence. Without wishingto be bound by theory, the tracr sequence, which may comprise or consist of all or a portion of a wild-type tracr sequence (e.g. about or more than about 20, 26, 32, 45, 48, 54, 63, 67, 85, or more nucleotides of a wild-type tracr sequence), may also form part of a CRISPR complex, such as by hybridization along at least a portion of the tracr sequence to all or a portion of a tracr mate sequence that is operably linked to the guide sequence. In some embodiments, the tracr sequence has sufficient complementarity to a tracr mate sequence to hybridize and participate in formation of a CRISPR complex. As with the target sequence, it is believed that complete complementarity is not needed, provided there is sufficient to be functional (bind the Cas protein or functional fragment thereof). In some embodiments, the tracr sequence has at least 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of sequence complementarity along the length of the tracr mate sequence when optimally aligned. In some embodiments, one or more vectors driving expression of one or more elements of a CRISPR system are introduced into a host cell such that the presence and / or expression of the elements of the CRISPR system direct formation of a CRISPR complex at one or more target sites. For example, a Cas enzyme, a guide sequence linked to a tracr-mate sequence, and a tracr sequence could each be operably linked to separate regulatory elements on separate vectors. Alternatively, two or more of the elements expressed from the same or different regulatory elements, may be combined in a single vector, with one or more additional vectors providing any components of the CRISPR system not included in the first vector. With at least some of the modification contemplated by this disclosure, in some embodiments, the guide sequence or RNA or DNA sequences that form a CRISPR complex are at least partially synthetic. The CRISPR system elements that are combined in a single vector may be arranged in any suitable orientation, such as one element located 5' with respect to (“upstream” of) or 3' with respect to (“downstream” of) a second element. In some embodiments, the disclosure relates to a composition comprising a chemically synthesized guide sequence. In someembodiments, the chemically synthesized guide sequence is used in conjunction with a vector comprising a coding sequence that encodes a CRISPR enzyme, such as a class 2 Cas9 or Cas12a protein. In some embodiments, the chemically synthesized guide sequence is used in conjunction with one or more vectors, wherein each vector comprises a coding sequence that encodes a CRISPR enzyme, such as a class 2 Cas9 or Cas12a protein. The coding sequence of one element may be located on the same or opposite strand of the coding sequence of a second element, and oriented in the same or opposite direction. In some embodiments, a single promoter drives expression of a transcript encoding a CRISPR enzyme and one or more additional (second, third, fourth, etc.) guide sequences, tracr mate sequence (optionally operably linked to the guide sequence), and a tracr sequence embedded within one or more intron sequences (e.g. each in a different intron, two or more in at least one intron, or all in a single intron). In some embodiments, the CRISPR enzyme, one or more additional guide sequence, tracr mate sequence, and tracr sequence are each a component of different nucleic acid sequences. For instance, in the case of a tracr and tracr mate sequences and in some embodiments, the disclosure relates to a composition comprising at least a first and second nucleic acid sequence, wherein the first nucleic acid sequence comprises a tracr sequence and the second nucleic acid sequence comprises a tracr mate sequence, wherein the first nucleic acid sequence is at least partially complementary to the second nucleic acid sequence such that the first and second nucleic acid for a duplex and wherein the first nucleic acid and the second nucleic acid either individually or collectively comprise a DNA-targeting domain, a Cas protein binding domain, and a transcription terminator domain. In some embodiments, the CRISPR enzyme, one or more additional guide sequence, tracr mate sequence, and tracr sequence are operably linked to and expressed from the same promoter. In some embodiments, the disclosure relates to compositions comprising any one or combination of the disclosed domains on one guide sequence or two separate tracrRNA / crRNA sequences with or without any of the disclosed modifications. Any methods disclosed herein also relate to the use of tracrRNA / crRNA sequence interchangeably with the use of a guide sequence, such that a composition maycomprise a single synthetic guide sequence and / or a synthetic tracrRNA / crRNA with any one or combination of modified domains disclosed herein.

[0035] In some embodiments, a guide RNA can be a short, synthetic, chimeric tracrRNA / crRNA (a “single-guide RNA” or “sgRNA”). A guide RNA may also comprise two short, synthetic tracrRNA / crRNAs (a “dual-guide RNA” or ‘dgRNA”).

[0036] The terms “cancer” or “tumor” are well known in the art and refer to the presence, e.g., in a subject, of cells possessing characteristics typical of cancercausing cells, such as uncontrolled proliferation, immortality, metastatic potential, rapid growth and proliferation rate, decreased cell death / apoptosis, and certain characteristic morphological features.

[0037] As used herein, “cancer” refers to all types of cancer or neoplasm or malignant tumors found in humans, including, but not limited to: leukemias, lymphomas, melanomas, carcinomas and sarcomas. As used herein, the terms or language “cancer,” “neoplasm,” and “tumor,” are used interchangeably and in either the singular or plural form, refer to cells that have undergone a malignant transformation that makes them pathological to the host organism. Primary cancer cells (that is, cells obtained from near the site of malignant transformation) can be readily distinguished from non- cancerous cells by well-established techniques, particularly histological examination. The definition of a cancer cell, as used herein, includes not only a primary cancer cell, but also cancer stem cells, as well as cancer progenitor cells or any cell derived from a cancer cell ancestor. This includes metastasized cancer cells, and in vitro cultures and cell lines derived from cancer cells. In certain embodiments, the cancer is a blood tumor ( / .e., a non-solid tumor). In some embodiments, the cancer is lymphoid neoplasm diffuse large B-cell lymphoma, cholangiocarcinoma, uterine carcinosarcoma, kidney chromophobe, uveal melanoma, mesothelioma, adrenocortical carcinoma, thymoma, acute myeloid leukemia, testicular germ cell tumor, rectum adenocarcinoma, pancreatic adenocarcinoma, phenochromocytoma and paraganglioma, esophageal carcinoma, sarcoma, kidney renal papillary cell carcinoma, cervical squamous cell carcinoma and endocervical adenocarcinoma, kidney renal clear cell carcinoma, liver hepatocellular carcinoma, glioblastoma multiforme, bladder urothelial carcinoma, colonadenocarcinoma, stomach adenocarcinoma, ovarian serous cystadenocarcinoma, skin cutaneous melanoma, prostate adenocarcinoma, thyroid carcinoma, lung squamous cell carcinoma, head and neck squamous cell carcinoma, brain lower grade glioma, uterine corpus endometrial carcinoma, lung adenocarcinoma, multiple myeloma, breast invasive carcinoma, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, Kaposi sarcoma, AIDS-related lymphoma, primary CNS lymphoma, anal cancer, astrocytoma, atypical teratoid / rhabdoid tumor, bile duct cancer, bladder cancer, bone cancer, brain tumor, breast cancer, bronchial tumors, carcinoid tumor, carcinoma of unknown primary, cardiac tumor, medulloblastoma, germ cell tumor, cervical cancer, chordoma, chronic lymphocytic leukemia, chronic myelogenous leukemia, chronic myeloproliferative neoplasm, colorectal cancer, craniopharyngioma, embryonal tumor, endometrial cancer, ependymoma, esophageal cancer, esthesioneuroblastoma, Ewing sarcoma, extracranial germ cell tumor, extragonadal germ cell tumor, intraocular melanoma, retinoblastoma, fallopian tube cancer, fibrous histiocytoma of bone, osteosarcoma, gallbladder cancer, gastric cancer, gastrointestinal carcinoid tumor, gastrointestinal stromal tumor, CNS germ cell tumor, ovarian germ cell tumor, testicular cancer, gestational trophoblastic disease, hairy cell leukemia, head and neck cancer, hepatocellular cancer, Langerhans cell histiocytosis, Hodgkin lymphoma, hypopharyngeal cancer, islet cell tumor, kidney cancer, laryngeal cancer, leukemia, lip and oral cavity cancer, lung cancer (non-small cell, small cell, pleuropulmonary blastoma, tracheobronchial tumor), lymphoma, male breast cancer, malignant fibrous histiocytoma of bone, melanoma, Merkel cell carcinoma, malignant mesothelioma, metastatic cancer, metastatic squamous cell neck cancer with occult primary, midline tract carcinoma, mouth cancer, multiple endocrine neoplasia, plasma cell neoplasm, mycosis fungoides, myelodysplastic syndrome, myelodysplastic neoplasm, nasal cavity and paranasal sinus cancer, nasopharyngeal cancer, neuroblastoma, non-Hodgkin lymphoma, oropharyngeal cancer, ovarian cancer, pancreatic cancer, pancreatic ductal adenocarcinoma, papillomatosis, paraganglioma, parathyroid cancer, pharyngeal cancer, pheochromocytoma, pituitary tumor, pleuropulmonary blastoma, primary peritoneal cancer, prostate cancer, rectal cancer,rhabdomyosarcoma, salivary gland cancer, Sezary syndrome, skin cancer, small intestine cancer, soft tissue sarcoma, testicular cancer, thymoma, thymic carcinoma, thyroid cancer, transitional cell cancer of the renal pelvis and ureter, urethral cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, vascular tumor, vulvar cancer, or Wilms tumor see, e.g., Kerins et al., Sci. Rep. 8:12846 (2018)).

[0038] In certain embodiments, the cancer is a solid tumor. A “solid tumor” is a tumor that is detectable on the basis of tumor mass; e.g., by procedures such as CAT scan, MR imaging, X-ray, ultrasound or palpation, and / or which is detectable because of the expression of one or more cancer-specific antigens in a sample obtainable from a patient. The tumor does not need to have measurable dimensions.

[0039] Specific criteria for the staging of cancer are dependent on the specific cancer type based on tumor size, histological characteristics, tumor markers, and other criteria known by those of skill in the art. Generally, cancer stages can be described as follows:

[0040] Stage 0 - Carcinoma in situ

[0041] Stage I, Stage II, and Stage III - Higher numbers indicate more extensive disease: Larger tumor size and / or spread of the cancer beyond the organ in which it first developed to nearby lymph nodes and / or tissues or organs adjacent to the location of the primary tumor

[0042] Stage IV - The cancer has spread to distant tissues or organs

[0043] As used herein, a “variant”, “mutant”, or “mutated” polynucleotide contains at least one polynucleotide sequence alteration as compared to the polynucleotide sequence of the corresponding wild-type or parent polynucleotide. Mutations may be natural, deliberate, or accidental. Mutations include substitutions, deletions, and insertions.

[0044] As used herein, the terms “treat,” “treating” or “treatment” refer to an action to obtain a beneficial or desired clinical result including, but not limited to, alleviation or amelioration of one or more signs or symptoms of a disease or condition (e.g., regression, partial or complete), diminishing the extent of disease, stability (i.e., not worsening, achieving stable disease) of the state of disease, amelioration or palliationof the disease state, diminishing rate of or time to progression, and remission (whether partial or total). “Treatment” of a disorder can also mean prolonging survival as compared to expected survival in the absence of treatment. Treatment need not be curative. In certain embodiments, treatment includes one or more of a decrease in pain or an increase in the quality of life (QOL) as judged by a qualified individual, e.g., a treating physician, e.g., using accepted assessment tools of pain and QOL. In certain embodiments, a decrease in pain or an increase in the QOL as judged by a qualified individual, e.g., a treating physician, e.g., using accepted assessment tools of pain and QOL is not considered to be a “treatment” of the disorder.

[0045] “Chemotherapeutic agent” refers to a drug used for the treatment of cancer. Chemotherapeutic agents include, but are not limited to, small molecules, hormones and hormone analogs, and biologies (e.g., antibodies, peptide drugs, nucleic acid drugs). In certain embodiments, chemotherapy does not include hormones and hormone analogs.

[0046] A “cancer that is resistant to one or more chemotherapeutic agents” is a cancer that does not respond, or ceases to respond to treatment with a chemotherapeutic regimen, i.e., does not achieve at least stable disease (i.e., stable disease, partial response, or complete response) in the target lesion either during or after completion of the chemotherapeutic regimen. Resistance to one or more chemotherapeutic agents results in, e.g., tumor growth, increased tumor burden, and / or tumor metastasis.

[0047] A “therapeutically effective amount” is that amount sufficient , at dosages and for periods of time necessary, to achieve a desired therapeutic result, such as for treatment of a disease (e.g. cancer), condition, or disorder, and / or pharmacokinetic or pharmacodynamic effect of the treatment in a subject. A therapeutically effective amount can be administered in one or more administrations. The therapeutically effective amount may vary according to factors such as the disease state, age, sex, and weight of the subject.

[0048] As used herein, the terms “exon splicing enhancer” or “ESE” mean a nucleotide sequence, which when present in the exon and accessible for binding of nuclear splicing regulatory proteins and / or by forming a secondary structure or a part thereof ofthe pre-mRNA stimulates inclusion of this exon into the final spliced mRNA during pre- mRNA splicing.

[0049] As used herein, the terms “exon splicing silencer” or “ESS” means a nucleotide sequence, which when present in the exon and accessible for binding of nuclear splicing regulatory proteins and / or by forming a secondary structure or a part thereof of the pre-mRNA inhibits inclusion of this exon into the final spliced mRNA during pre- mRNA splicing.

[0050] As used herein, a “splice variant” of a gene is the product of alternative splicing, resulting in loss of exons or inclusion of introns and an altered protein-coding sequence.

[0051] CRISPR / Endonucleases

[0052] CRISPR / endonuclease (e.g., CRISPR / Cas9) systems are known in the art and are described, for example, in U.S. Pat. No. 9,925,248, which is incorporated by reference herein in its entirety. CRISPR-directed gene editing can identify and execute DNA cleavage at specific sites within the chromosome at a surprisingly high efficiency and precision. The natural activity of CRISPR / Cas9 is to disable a viral genome infecting a bacterial cell. Subsequent genetic reengineering of CRISPR / Cas function in human cells presents the possibility of disabling human genes at a significant frequency.

[0053] In bacteria, the CRISPR / Cas loci encode RNA-guided adaptive immune systems against mobile genetic elements (viruses, transposable elements and conjugative plasmids). Three types (l-lll) of CRISPR systems have been identified. CRISPR clusters contain spacers, the sequences complementary to antecedent mobile elements. CRISPR clusters are transcribed and processed into mature CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) RNA (crRNA) containing a DNA binding region (spacer) which is complementary to the target gene. The CRISPR-associated endonuclease, Cas9, belongs to the type II CRISPR / Cas system and has strong endonuclease activity to cut target DNA. Cas9 is guided by a mature crRNA that contains about 20 base pairs (bp) of unique target sequence (called aspacer) and a trans-activated small RNA (tracrRNA) that serves as a guide for ribonuclease Ill-aided processing of pre-crRNA. The crRNA:tracrRNA duplex directs Cas9 to target DNA via complementary base pairing between the spacer on the crRNA and the complementary sequence (called protospacer) on the target DNA. Cas9 recognizes a trinucleotide (NGG) protospacer adjacent motif (PAM) to specify the cut site (the 3rd nucleotide from PAM).

[0054] The compositions described herein can include a nucleic acid encoding a CRISPR-associated endonuclease. The CRISPR-associated endonuclease can be, e.g., a class 1 CRISPR-associated endonuclease or a class 2 CRISPR-associated endonuclease. Class 1 CRISPR-associated endonucleases include type I, type III, and type IV CRISPR-Cas systems, which have effector molecules that comprise multiple subunits. For class 1 CRISPR-associated endonucleases, effector molecules can include, in some embodiments, Cas7 and Cas5, along with, in some embodiments, SS (Cas1 1 ) and Cas8a1 ; Cas8b1 ; Cas8c; Cas8u2 and Cas6; Cas3" and Cas10d; Cas SS (Cas1 1 ), Cas8e, and Cas6; Cas8f and Cas6f; Cas6f; Cas8-like (Csf1 ); SS (Cas11 ) and Cas8-like (Csf1 ); or SS (Cas1 1 ) and Casi o. Class 1 CRISPR-associated endonucleases also be associated with, in some embodiments, target cleavage molecules, which can be Cas3 (type I) or Cas10 (type III) and spacer acquisition molecules such as, e.g., Cas1 , Cas2, and / or Cas4. See, e.g., Koonin et al., Curr. Opin. Microbiol. 37:67-78 (2017); Strich & Chertow, J. Clin. Microbiol. 57:1307-18 (2019).

[0055] Class 2 CRISPR-associated endonucleases include type I, type V, and type VI CRISPR-Cas systems, which have a single effector molecule. For class 2 CRISPR- associated endonucleases, effector molecules can include, in some embodiments, Cas9, Cas12a (cpf1 ), Cas12b1 (c2c1 ), Cas12a2, Cas12b2, Cas12c (c2c3), Cas12d (CasY), Cas12e (CasX), Cas12f1 (Cas14a), Cas12f2 (Cas14b), Cas12f3 (Cas14c), Cas12g, Cas12h, Cas12i, Cas12j (CasO), Cas12k (c2c5), Cas13a (c2c2), Cas13b1 (c2c6), Cas13b2 (c2c6), Cas13bt, Cas13c (c2c7), Cas13ct, Cas13d, Cas13X, Cas13Y, c2c4, c2c8, c2c9, and / or c2c10. See, e.g., Koonin etal., Curr. Opin. Microbiol. 37:67- 78 (2017); Strich & Chertow, J. Clin. Microbiol. 57:1307-18 (2019); Makarova etal., Nat. Rev. Microbiol. 18:67-83 (2020); Pausch etal., Science 369:333-37 (2020); Tonget a!., Cell Dev. Biol. 8:622103 (2021 ); Xu et a!., Nat. Meth. 18:499-506 (2021 ); Kannan etal., Nat. Biotechnol. 40:194-97 (2022); Liu et al., Mol. Cell 82:333-47 (2022).

[0056] In some embodiments, the CRISPR-associated endonuclease can be a Cas9 nuclease. The Cas9 nuclease can have a nucleotide sequence identical to the wild type Streptococcus pyogenes sequence. In some embodiments, the CRISPR-associated endonuclease can be a sequence from other species, for example other Streptococcus species, such as agalactiae, anginosis, canis, castoreus, constella, constellates, denstasini, devriesei, dysgalactiae, equi, equinus, gallolyticus, infantarius, iniae, lutetiensis, macacae, massiliensis, mitis, mutans, ovis, parasanguinis, parauberis, phocae, pseudoporcinus, plurextorum, ratti, sanguinis, sobrinus, suis, thermophilus, or tigurinus; Pseudomona aeruginosa, Escherichia coli, or other sequenced bacteria genomes and archaea; or other prokaryotic microorganisms. Such species include: Acidaminoccus sp., Acidovorax avenae, Actinobacillus pleuropneumoniae, Actinobacillus succinogenes, Actinobacillus suis, Actinomyces sp., Alicyclobacillus acidiphilus, Alicyclobacillus acidoterrestris, Aminomonas paucivorans, Bacillus cereus, Bacillus hisahsii, Bacillus smithii, Bacillus thuringiensis, Bacteroides sp., Blastopirellula marina, Bradyrhizobium sp., Brevibacillus laterosporus, Campylobacter coli, Campylobacter jejuni, Campylobacter lari, Candidates puniceispirillum, Clostridium cellulolyticum, Clostridium perfringens, Corynebacterium accolens, Corynebacterium diphtheria, Corynebacterium matruchotii, Cycliphilus denitrificans, Dinoroseobacter shibae, Dolosigranulum pigrum, Enterococcus durans, Enterococcus faecalis, Enterococcus faecium, Enterococcus hirae, Enterococcus italicus, Enterococcus mundtii, Enterococcus phoeniculicola, Enterococcus villorum, Eubacterium dolichum, Francisella novicida, Gammaproteobacterium, Gluconacetobacter diazotrophicus, Haemophilus parainfluenzae, Haemophilus sputorum, Helicobacter canadensis, Helicobacter cinaedi, Helicobacter mustelae, llyobacter polytropus, Kingella kingae, Lachnospiraceae bacterium, Lactobacillus apodemi, Lactobacillus animalis, Lactobacillus crispatus, Leptotrichia shahii, Listeria innocua, Listeria selligeri, Listeria ivanovii, Listeria monocytogenes, Listeriaceae bacterium, Methylocystis sp., Methylosinus trichosporium, Mobiluncus mulieris, Moraxella bovoculi, Neisseriabacilliformis, Neisseria cinerea, Neisseria flavescens, Neisseria lactamica, Neisseria meningitidis, Neisseria sp., Neisseria wadsworthii, Nitrosomonas sp., Parvibaculum lavamentivorans, Pasteurella multocida, Prevotella bryantii, Phascolarctobacterium succinatutens, Ralstonia syzygii, Rhodopseudomonas palustris, Rhodovulum sp., Simonsiella muelleri, Sphingomonas sp., Sporolactobacillus vineae, Staphylococcus aureus, Staphylococcus auricularis, Staphylococcus lugdunensis, Streptococcus canis, Streptococcus sp., Subdoligranulum sp., Sulfuricurvum sp., Tistrella mobilis, Treponema sp., and Verminephrobacter eiseniae.

[0057] Alternatively, the wild type Streptococcus pyogenes Cas9 sequence can be modified. The nucleic acid sequence can be codon optimized for efficient expression in mammalian cells, e.g., human cells. A Cas9 nuclease sequence codon optimized for expression in human cells sequence can be for example, the Cas9 nuclease sequence encoded by any of the expression vectors listed in Genbank accession numbers NZ_LS483338.1 GI:69900935, KM099231 .1 Gl:669193757; KM099232.1Gl:669193761 ; or KM099233.1 Gl:669193765. Alternatively, the Cas9 nuclease sequence can be, for example, the sequence contained within a commercially available vector such as pX458, pX330 or pX260 from Addgene (Cambridge, Mass.). In some embodiments, the Cas9 endonuclease can have an amino acid sequence that is a variant or a fragment of any of the Cas9 endonuclease sequences of Genbank accession numbers NZ_LS483338.1 GI:69900935, KM099231.1 Gl:669193757; KM099232.1 Gl:669193761 ; or KM099233.1 Gl:669193765 or Cas9 amino acid sequence of pX458, pX330 or pX260 (Addgene, Cambridge, Mass.). The Cas9 nucleotide sequence can be modified to encode biologically active variants of Cas9, and these variants can have or can include, for example, an amino acid sequence that differs from a wild type Cas9 by virtue of containing one or more mutations (e.g., an addition, deletion, or substitution mutation or a combination of such mutations). One or more of the substitution mutations can be a substitution (e.g., a conservative amino acid substitution). For example, a biologically active variant of a Cas9 polypeptide can have an amino acid sequence with at least or about 50% sequence identity (e.g., at least or about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %,62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to a wild type Cas9 polypeptide. See, e.g., US2019 / 0032036, US2023 / 0075913, US2023 / 0031899, US2023 / 0021641 , US2022 / 0307001 , US2022 / 0235340, US2022 / 0204954, US2022 / 0154158, US2022 / 0154157, US2021 / 0301269, US2021 / 0284978, US2021 / 0261932, US2021 / 0163907, US2021 / 0147861 , US2020 / 0332271 , US2020 / 0318086, US20200299657, US2020 / 0277586, US2020 / 0199552; each of which incorporated by reference herein in its entirety.

[0058] In some embodiments, the CRISPR-associated endonuclease can be a Cas12a nuclease. The Cas12a nuclease can have a nucleotide sequence identical to a wild type Prevotella or Francisella sequence. Alternatively, a wild type Prevotella or Francisella Cas12a sequence can be modified. The nucleic acid sequence can be codon optimized for efficient expression in mammalian cells, e.g., human cells. A Cas12a nuclease sequence codon optimized for expression in human cells sequence can be for example, the Cas12a nuclease sequence encoded by any of the expression vectors listed in Genbank accession numbers NZ_CP010070.1 Gl: 24818655, MF193599.1 Gl: 1214941796, KY985374.1 Gl: 1242863785, KY985375.1 Gl: 1242863787, or KY985376.1 Gl: 1242863789. Alternatively, the Cas12a nuclease sequence can be, for example, the sequence contained within a commercially available vector such as pAs-Cpf1 or pLb-Cpf1 from Addgene (Cambridge, Mass.). In some embodiments, the Cas12a endonuclease can have an amino acid sequence that is a variant or a fragment of any of the Cas12a endonuclease sequences of Genbank accession numbers NZ_CP010070.1 Gl: 24818655, MF193599.1 Gl: 1214941796, KY985374.1 Gl: 1242863785, KY985375.1 Gl: 1242863787, or KY985376.1 Gl: 1242863789 or Cas12a amino acid sequence of pAs-Cpf1 or pLb-Cpf1 (Addgene, Cambridge, Mass.). The Cas12a nucleotide sequence can be modified to encode biologically active variants of Cas12a, and these variants can have or can include, for example, an amino acid sequence that differs from a wild type Cas12a by virtue of containing one or more mutations (e.g., an addition, deletion, or substitution mutation ora combination of such mutations). One or more of the substitution mutations can be a substitution (e.g., a conservative amino acid substitution). For example, a biologically active variant of a 0as12a polypeptide can have an amino acid sequence with at least or about 50% sequence identity (e.g., at least or about 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or99% sequence identity) to a wild type Cas12a polypeptide. See, e.g., US2019 / 0233814, US2019 / 0264186, US2023 / 0040148, US2021 / 0348144, US2021 / 0309701 , US2021 / 0230567, US2021 / 015591 1 , US2020 / 0263190, US2020 / 0216825, US2021 / 0115421 , US2021 / 0079366, US2020 / 0255861 , LIS2019 / 0010481 ; each of which incorporated by reference herein in its entirety.

[0059] The compositions described herein may also include sequence encoding a gRNA comprising a DNA-binding domain that is complementary to a target domain in a gene, and a CRISPR-associated endonuclease protein-binding domain. In some embodiments. The guide RNA sequence can be a sense or anti-sense sequence. The guide RNA sequence may include a proto-spacer adjacent motif (PAM). The sequence of the PAM can vary depending upon the specificity requirements of the CRISPR endonuclease used. In the CRISPR-Cas system derived from S. pyogenes, the target DNA typically immediately precedes a 5'-NGG proto-spacer adjacent motif (PAM).Thus, for the S. pyogenes Cas9, the PAM sequence can be AGG, TGG, CGG or GGG. Other Cas9 orthologs may have different PAM specificities. The specific sequence of the guide RNA may vary, but, regardless of the sequence, useful guide RNA sequences will be those that minimize off-target effects while achieving high efficiency.

[0060] In some embodiments, the DNA-binding domain varies in length from about 20 to about 55 nucleotides, for example, about 20, about 21 , about 22, about 23, about 24, about 25, about 26, about 27, about 28, about 29, about 30, about 31 , about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41 , about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51 , about 52, about 53, about 54, or about 55 nucleotides. In someembodiments, the Cas protein-binding domain is from about 30 to about 55 nucleotides in length, for example, about 30, about 31 , about 32, about 33, about 34, about 35, about 36, about 37, about 38, about 39, about 40, about 41 , about 42, about 43, about 44, about 45, about 46, about 47, about 48, about 49, about 50, about 51 , about 52, about 53, about 54, or about 55 nucleotides.

[0061] In some embodiments, the compositions comprise one or more nucleic acid (i.e. DNA) sequences encoding the guide RNA and the CRISPR endonuclease. When the compositions are administered as a nucleic acid or are contained within an expression vector, the CRISPR endonuclease can be encoded by the same nucleic acid or vector as the guide RNA sequence. In some embodiments, the CRISPR endonuclease can be encoded in a physically separate nucleic acid from the guide RNA sequence or in a separate vector. The nucleic acid sequence encoding the guide RNA may comprise a DNA binding domain, a Cas protein binding domain, and a transcription terminator domain.

[0062] The nucleic acid encoding the guide RNA and / or the CRISPR endonuclease may be an isolated nucleic acid. An “isolated” nucleic acid can be, for example, a naturally-occurring DNA molecule or a fragment thereof, provided that at least one of the nucleic acid sequences normally found immediately flanking that DNA molecule in a naturally-occurring genome is removed or absent. Isolated nucleic acid molecules can be produced by standard techniques. For example, polymerase chain reaction (PCR) techniques can be used to obtain an isolated nucleic acid containing a nucleotide sequence described herein, including nucleotide sequences encoding a polypeptide described herein. PCR can be used to amplify specific sequences from DNA as well as RNA, including sequences from total genomic DNA or total cellular RNA. Various PCR methods are described in, for example, PCR Primer: A Laboratory Manual, Dieffenbach and Dveksler, eds., Cold Spring Harbor Laboratory Press, 1995. Generally, sequence information from the ends of the region of interest or beyond is employed to design oligonucleotide primers that are identical or similar in sequence to opposite strands of the template to be amplified. Various PCR strategies also are available by which sitespecific nucleotide sequence modifications can be introduced into a template nucleicacid.

[0063] Isolated nucleic acids also can be chemically synthesized, either as a single nucleic acid molecule (e.g., using automated DNA synthesis in the 3' to 5' direction using phosphoramidite technology) or as a series of oligonucleotides. For example, one or more pairs of long oligonucleotides (e.g., >50-100 nucleotides) can be synthesized that contain the desired sequence, with each pair containing a short segment of complementarity (e.g., about 15 nucleotides) such that a duplex is formed when the oligonucleotide pair is annealed. DNA polymerase is used to extend the oligonucleotides, resulting in a single, double-stranded nucleic acid molecule per oligonucleotide pair, which then can be ligated into a vector. Isolated nucleic acids also can be obtained by mutagenesis of, e.g., a naturally occurring portion of a Cas9- encoding DNA (in accordance with, for example, the formula above).

[0064] Recombinant constructs are also provided herein and can be used to transform cells in order to express the CRISPR endonuclease and / or a guide RNA complementary to a gene. A recombinant nucleic acid construct may comprise a nucleic acid encoding a CRISPR endonuclease and / or a guide RNA complementary to a gene, operably linked to a promoter suitable for expressing the CRISPR endonuclease and / or a guide RNA complementary to the gene in the cell. In some embodiments the nucleic acid encoding a CRISPR endonuclease is operably linked to the same promoter as the nucleic acid encoding the guide RNA. In other embodiments, the nucleic acid encoding a CRISPR endonuclease and the nucleic acid encoding the guide RNA are operably linked to different promoters. In some embodiments, the nucleic acid encoding a CRISPR endonuclease and / or the nucleic acid encoding a guide RNA are operably linked to a lung specific promoter.

[0065] In some embodiments, one or more CRISPR endonucleases and one or more guide RNAs may be provided in combination in the form of ribonucleoprotein particles (RNPs). An RNP complex can be introduced into a subject by means of, e.g., injection, electroporation, nanoparticles, vesicles, and / or with the assistance of cell-penetrating peptides.

[0066] DNA vectors containing nucleic acids such as those described herein also arealso provided. A “DNA vector” is a replicon, such as a plasmid, phage, or cosmid, into which another DNA segment may be inserted so as to bring about the replication of the inserted segment. Generally, a DNA vector is capable of replication when associated with the proper control elements. Suitable vector backbones include, for example, those routinely used in the art such as plasmids, viruses, artificial chromosomes, BACs, YACs, or PACs. The term “DNA vector” includes cloning and expression vectors, as well as viral vectors and integrating vectors. An “expression vector” is a vector that includes a regulatory region. A wide variety of host / expression vector combinations may be used to express the nucleic acid sequences described herein. Suitable expression vectors include, without limitation, plasmids and viral vectors derived from, for example, bacteriophage, baculoviruses, and retroviruses. Numerous vectors and expression systems are commercially available from such corporations as Novagen (Madison, Wis.), Clontech (Palo Alto, Calif.), Stratagene (La Jolla, Calif.), and Invitrogen / Life Technologies (Carlsbad, Calif.).

[0067] The DNA vectors provided herein also can include, for example, origins of replication, scaffold attachment regions (SARs), and / or markers. A marker gene can confer a selectable phenotype on a host cell. For example, a marker can confer biocide resistance, such as resistance to an antibiotic (e.g., kanamycin, G418, bleomycin, or hygromycin). As noted above, an expression vector can include a tag sequence designed to facilitate manipulation or detection (e.g., purification or localization) of the expressed polypeptide. Tag sequences, such as green fluorescent protein (GFP), glutathione S-transferase (GST), polyhistidine, c-myc, hemagglutinin, or Flag™ tag (Kodak, New Haven, Conn.) sequences typically are expressed as a fusion with the encoded polypeptide. Such tags can be inserted anywhere within the polypeptide, including at either the carboxyl or amino terminus.

[0068] The DNA vector can also include a regulatory region. The term “regulatory region” refers to nucleotide sequences that influence transcription or translation initiation and rate, and stability and / or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, proteinbinding sequences, 5' and 3' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, nuclear localization signals, and introns.

[0069] As used herein, the term “operably linked” refers to positioning of a regulatory region (e.g. a promoter) and a sequence to be transcribed in a nucleic acid so as to influence transcription or translation of such a sequence. For example, to bring a coding sequence under the control of a promoter, the translation initiation site of the translational reading frame of the polypeptide is typically positioned between one and about fifty nucleotides downstream of the promoter. A promoter can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site or about 2,000 nucleotides upstream of the transcription start site. A promoter typically comprises at least a core (basal) promoter. A promoter also may include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). The choice of promoters to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and cell- or tissue-preferential expression. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning promoters and other regulatory regions relative to the coding sequence.

[0070] Vectors include, for example, viral vectors (such as adenoviruses (“Ad”), adeno- associated viruses (AAV), and vesicular stomatitis virus (VSV) and retroviruses), liposomes and other lipid-containing complexes, and other macromolecular complexes capable of mediating delivery of a polynucleotide to a host cell. Direct injection of adenoviral vectors into lung tumors has been a routine procedure in clinical trials evaluating gene therapy of lung cancer. Dong et al., J. Int. Med. Res. 36, 1273-1287 (2008); Li et al., Cancer Gene Then 20, 251-259 (2013); Zhou etal., Cancer Gene Ther. 23, 1 -6 (2016). Vectors can also comprise other components or functionalities that further modulate gene delivery and / or gene expression, or that otherwise provide beneficial properties to the targeted cells. As described and illustrated in more detail below, such other components include, for example, components that influence bindingor targeting to cells (including components that mediate cell-type or tissue-specific binding); components that influence uptake of the vector nucleic acid by the cell; components that influence localization of the polynucleotide within the cell after uptake (such as agents mediating nuclear localization); and components that influence expression of the polynucleotide. Such components also might include markers, such as detectable and / or selectable markers that can be used to detect or select for cells that have taken up and are expressing the nucleic acid delivered by the vector. Such components can be provided as a natural feature of the vector (such as the use of certain viral vectors which have components or functionalities mediating binding and uptake), or vectors can be modified to provide such functionalities. Other vectors include those described by Chen et al., BioTechniques, 34:167-71 (2003). A large variety of such vectors are known in the art and are generally available.

[0071] Suitable nucleic acid delivery systems include recombinant viral vector, typically sequence from at least one of an Ad, AAV, helper-dependent adenovirus, retrovirus, or hemagglutinating virus of Japan-liposome (HVJ) complex. In such cases, the viral vector comprises a strong eukaryotic promoter operably linked to the polynucleotide e.g., a cytomegalovirus (CMV) promoter. The recombinant viral vector can include one or more of the polynucleotides therein, in some embodiments about one polynucleotide. In embodiments in which the polynucleotide is to be administered with a non-viral vector, use of between from about 0.1 ng to about 4000 pg will often be useful e.g., about 0.1 ng to about 3900 pg, about 0.1 ng to about 3800 pg, about 0.1 ng to about 3700 pg, about 0.1 ng to about 3600 pg, about 0.1 ng to about 3500 pg, about 0.1 ng to about 3400 pg, about 0.1 ng to about 3300 pg, about 0.1 ng to about 3200 pg, about 0.1 ng to about 3100 pg, about 0.1 ng to about 3000 pg, about 0.1 ng to about 2900 pg, about 0.1 ng to about 2800 pg, about 0.1 ng to about 2700 pg, about 0.1 ng to about 2600 pg, about 0.1 ng to about 2500 pg, about 0.1 ng to about 2400 pg, about 0.1 ng to about 2300 pg, about 0.1 ng to about 2200 pg, about 0.1 ng to about 2100 pg, about 0.1 ng to about 2000 pg, about 0.1 ng to about 1900 pg, about 0.1 ng to about 1800 pg, about 0.1 ng to about 1700 pg, about 0.1 ng to about 1600 pg, about 0.1 ng to about 1500 pg, about 0.1 ng to about 1400 pg, about 0.1 ng toabout 1300 pg, about 0.1 ng to about 1200 pg, about 0.1 ng to about 1 100 pg, about 0.1 ng to about 1000 pg, about 0.1 ng to about 900 pg, about 0.1 ng to about 800 pg, about 0.1 ng to about 700 pg, about 0.1 ng to about 600 pg, about 0.1 ng to about 500 pg, about 0.1 ng to about 400 pg, about 0.1 ng to about 300 pg, about 0.1 ng to about 200 pg, about 0.1 ng to about 100 pg, about 0.1 ng to about 90 pg, about 0.1 ng to about 80 pg, about 0.1 ng to about 70 pg, about 0.1 ng to about 60 pg, about 0.1 ng to about 50 pg, about 0.1 ng to about 40 pg, about 0.1 ng to about 30 pg, about 0.1 ng to about 20 pg, about 0.1 ng to about 10 pg, about 0.1 ng to about 1 pg, about 0.1 ng to about 900 ng, about 0.1 ng to about 800 ng, about 0.1 ng to about 700 ng, about 0.1 ng to about 600 ng, about 0.1 ng to about 500 ng, about 0.1 ng to about 400 ng, about 0.1 ng to about 300 ng, about 0.1 ng to about 200 ng, about 0.1 ng to about 100 ng, about 0.1 ng to about 90 ng, about 0.1 ng to about 80 ng, about 0.1 ng to about 70 ng, about0.1 ng to about 60 ng, about 0.1 ng to about 50 ng, about 0.1 ng to about 40 ng, about0.1 ng to about 30 ng, about 0.1 ng to about 20 ng, about 0.1 ng to about 10 ng, about0.1 ng to about 1 ng, about 1 ng to about 4000 pg, about 1 ng to about 3900 pg, about1 ng to about 3800 pg, about 1 ng to about 3700 pg, about 1 ng to about 3600 pg, about 1 ng to about 3500 pg, about 1 ng to about 3400 pg, about 1 ng to about 3300 pg, about 1 ng to about 3200 pg, about 1 ng to about 3100 pg, about 1 ng to about 3000 pg, about 1 ng to about 2900 pg, about 1 ng to about 2800 pg, about 1 ng to about 2700 pg, about 1 ng to about 2600 pg, about 1 ng to about 2500 pg, about 1 ng to about 2400 pg, about 1 ng to about 2300 pg, about 1 ng to about 2200 pg, about 1 ng to about 2100 pg, about 1 ng to about 2000 pg, about 1 ng to about 1900 pg, about 1 ng to about 1800 pg, about 1 ng to about 1700 pg, about 1 ng to about 1600 pg, about 1 ng to about 1500 pg, about 1 ng to about 1400 pg, about 1 ng to about 1300 pg, about 1 ng to about 1200 pg, about 1 ng to about 1100 pg, about 1 ng to about 1000 pg, about 1 ng to about 900 pg, about 1 ng to about 800 pg, about 1 ng to about 700 pg, about 1 ng to about 600 pg, about 1 ng to about 500 pg, about 1 ng to about400 pg, about 1 ng to about 300 pg, about 1 ng to about 200 pg, about 1 ng to about100 pg, about 1 ng to about 90 pg, about 1 ng to about 80 pg, about 1 ng to about 70 pg, about 1 ng to about 60 pg, about 1 ng to about 50 pg, about 1 ng to about 40 pg,about 1 ng to about 30 pg, about 1 ng to about 20 pg, about 1 ng to about 10 pg, about 1 ng to about 1 pg, about 1 ng to about 900 ng, about 1 ng to about 800 ng, about 1 ng to about 700 ng, about 1 ng to about 600 ng, about 1 ng to about 500 ng, about 1 ng to about 400 ng, about 1 ng to about 300 ng, about 1 ng to about 200 ng, about 1 ng to about 100 ng, about 1 ng to about 90 ng, about 1 ng to about 80 ng, about 1 ng to about 70 ng, about 1 ng to about 60 ng, about 1 ng to about 50 ng, about 1 ng to about 40 ng, about 1 ng to about 30 ng, about 1 ng to about 20 ng, about 1 ng to about 10 ng, about 10 ng to about 4000 pg, about 20 ng to about 4000 pg, about 30 ng to about 4000 pg, about 40 ng to about 4000 pg, about 50 ng to about 4000 pg, about 60 ng to about 4000 pg, about 70 ng to about 4000 pg, about 80 ng to about 4000 pg, about 90 ng to about 4000 pg, about 100 ng to about 4000 pg, about 200 ng to about 4000 pg, about 300 ng to about 4000 pg, about 400 ng to about 4000 pg, about 500 ng to about 4000 pg, about 600 ng to about 4000 pg, about 700 ng to about 4000 pg, about 800 ng to about 4000 pg, about 900 ng to about 4000 pg, about 1 pg to about 4000 pg, 10 pg to about 4000 pg, 20 pg to about 4000 pg, 30 pg to about 4000 pg, 40 pg to about 4000 pg, 50 pg to about 4000 pg, 60 pg to about 4000 pg, 70 pg to about 4000 pg, 80 pg to about 4000 pg, 90 pg to about 4000 pg, 100 pg to about 4000 pg, 200 pg to about 4000 pg, 300 pg to about 4000 pg, 400 pg to about 4000 pg, 500 pg to about 4000 pg, 600 pg to about 4000 pg, 700 pg to about 4000 pg, 800 pg to about 4000 pg, 900 pg to about 4000 pg, 1000 pg to about 4000 pg, 1 100 pg to about 4000 pg, 1200 pg to about 4000 pg, 1300 pg to about 4000 pg, 1400 pg to about 4000 pg, 1500 pg to about 4000 pg, 1600 pg to about 4000 pg, 1700 pg to about 4000 pg, 1800 pg to about 4000 pg, 1900 pg to about 4000 pg, 2000 pg to about 4000 pg, 2100 pg to about 4000 pg, 2200 pg to about 4000 pg, 2300 pg to about 4000 pg, 2400 pg to about 4000 pg, 2500 pg to about 4000 pg, 2600 pg to about 4000 pg, 2700 pg to about 4000 pg, 2800 pg to about 4000 pg, 2900 pg to about 4000 pg, 3000 pg to about 4000 pg, 3100 pg to about 4000 pg, 3200 pg to about 4000 pg, 3300 pg to about 4000 pg, 3400 pg to about 4000 pg, 3500 pg to about 4000 pg, 3600 pg to about 4000 pg, 3700 pg to about 4000 pg, 3800 pg to about 4000 pg, or 3900 pg to about 4000 pg.

[0072] Additional vectors include viral vectors, fusion proteins and chemical conjugates.Retroviral vectors include Moloney murine leukemia viruses and HIV-based viruses. One HIV-based viral vector comprises at least two vectors wherein the gag and pol genes are from an HIV genome and the env gene is from another virus. DNA viral vectors include pox vectors such as orthopox or avipox vectors, herpesvirus vectors such as a herpes simplex I virus (HSV) vector (Geller et al., J. Neurochem 64:487 (1995); Lim et al., in DNA Cloning: Mammalian Systems, D. Glover, Ed. (Oxford Univ. Press, Oxford England) (1995); Geller et al., Proc Natl. Acad. Sci. U.S.A.90:7603 (1993); Geller etal., Proc Natl. Acad. Sci USA 87:1 149 (1990)), Ad Vectors (LaSalle et al., Science 259:988 (1993); Davidson et al., Nat. Genet. 3:219 (1993); Yang etal., J. Virol. 69:2004 (1995)), and AAV Vectors (Kaplitt et al., Nat. Genet. 8:148 (1994)).

[0073] If desired, the polynucleotides described here may also be used with a microdelivery vehicle such as cationic liposomes, adenoviral vectors, and exosomes. For a review of the procedures for liposome preparation, targeting and delivery of contents, see Mannino and Gould-Fogerite, BioTechniques 6:682 (1988). See also, Feigner and Holm, Bethesda Res. Lab. Focus 1 1 :21 (1989) and Maurer, Bethesda Res. Lab. Focus 1 1 :25 (1989). In some embodiments, exosomes may be used for delivery of a nucleic acid encoding a CRISPR endonuclease and / or guide RNA to a target cell, e.g. a cancer cell. Exosomes are nanosized vesicles secreted by a variety of cells and are comprised of cellular membranes. Exosomes can attach to target cells by a range of surface adhesion proteins and vector ligands (tetraspanins, integrins, CD1 1 b and CD18 receptors), and deliver their payload to target cells. Several studies indicate that exosomes have a specific cell tropism, according to their characteristics and origin, which can be used to target them to disease tissues and / or organs. See Batrakova et al., J. Control. Release 219:396-405 (2015). For example, cancer-derived exosomes function as natural carriers that can efficiently deliver CRISPR / Cas9 plasmids to cancer cells. See Kim et al., J. Control. Release 266:8-16 (2017). In some embodiments, RNPs (discussed further below) are loaded into exosomes for delivery. See, e.g., Wan et al., Sci. Adv. 8:eabp9435 (2022).

[0074] Replication-defective recombinant adenoviral vectors, can be produced in accordance with known techniques. See Quantin et al., Proc. Natl. Acad. Sci. USA89:2581 -84 (1992); Stratford- Perricadet et a!., J. Clin. Invest. 90:626-30 (1992); and Rosenfeld et al., Cell 68143-55 (1992).

[0075] Another delivery method is to use single stranded DNA producing vectors which can produce the expressed products intracellularly. See, e.g., Chen et al., BioTechniques 34:167-71 (2003).

[0076] Introduction of CRISPR / Cas systems can be accomplished by lipid nanoparticle (LNP)-mediated delivery. For example, LNP-mediated delivery can be used to deliver a combination of Cas mRNA and guide RNA or a combination of Cas protein and guide RNA. Delivery through such methods results in transient Cas expression, and the biodegradable lipids improve clearance, improve tolerability, and decrease immunogenicity. Lipid formulations can protect biological molecules from degradation while improving their cellular uptake.

[0077] LNPs are particles comprising a plurality of lipid molecules physically associated with each other by intermolecular forces. These include microspheres (including unilamellar and multilamellar vesicles, e.g., liposomes), a dispersed phase in an emulsion, micelles, or an internal phase in a suspension. Such lipid nanoparticles can be used to encapsulate one or more nucleic acids or proteins for delivery.Formulations which contain cationic lipids are useful for delivering polyanions such as nucleic acids. Other lipids that can be included are neutral lipids (i.e., uncharged or zwitterionic lipids), anionic lipids, helper lipids that enhance transfection, and stealth lipids that increase the length of time for which nanoparticles can exist in vivo.Examples of suitable cationic lipids, neutral lipids, anionic lipids, helper lipids, and stealth lipids can be found in WO 2016 / 010840, herein incorporated by reference in its entirety for all purposes. An exemplary lipid nanoparticle can comprise a cationic lipid and one or more other components. In one embodiment, the other component can comprise a helper lipid such as cholesterol. In another embodiment, the other components can comprise a helper lipid such as cholesterol and a neutral lipid such as distearoylphosphatidylcholine (DSPC).

[0078] An LNP may contain one or more or all of the following: (i) a lipid for encapsulation and for endosomal escape; (ii) a neutral lipid for stabilization; (iii) ahelper lipid for stabilization; and (iv) a stealth lipid. See, e.g., Finn etal., Cell Rep. 22:1 - 9 (2018) and WO 2017 / 173054, each of which is herein incorporated by reference in its entirety for all purposes. In certain LNPs, the cargo can include a guide RNA or a nucleic acid encoding a guide RNA. In certain LNPs, the cargo can include an exogenous donor nucleic acid. In certain LNPs, the cargo can include a guide RNA or a nucleic acid encoding a guide RNA and a Gas protein or a nucleic acid encoding a Gas protein. In certain LNPs, the cargo can include a guide RNA or a nucleic acid encoding a guide RNA, a Gas protein or a nucleic acid encoding a Gas protein, and an exogenous donor nucleic acid.

[0079] The lipid for encapsulation and endosomal escape can be a cationic lipid. The lipid can also be a biodegradable lipid, such as a biodegradable ionizable lipid. One example of a suitable lipid is Lipid A or LP01 , which is (9Z,12Z)-3-((4,4- bis(octyloxy)butanoyl)oxy)-2-((((3-(diethylamino)propoxy-)carbonyl)oxy)methyl)propyl octadeca-9,12-dienoate, also called 3-((4,4-bis(octyloxy)butanoyl)oxy)-2-((((3- (diethylamino)propoxy)carbonyl- )oxy)methyl)propyl (9Z,12Z)-octadeca-9,12-dienoate. Another example of a suitable lipid is Lipid B, which is ((5-((dimethylamino)methyl)-1 ,3- phenylene)bis(oxy))bis(octane-8,1 -diyl)bis(decanoate), also called ((5- ((dimethylamino)methyl)-1 ,3-phenylene)bis(oxy))bis(octane-8,1 -diyl)bis(decanoate). Another example of a suitable lipid is Lipid C, which is 2-((4-(((3- (dimethylamino)propoxy)carbonyl)oxy)hexadecanoyl)oxy)propane-1 -,3- diyl(9Z,9Z',12Z,12Z')-bis(octadeca-9,12-dienoate). Another example of a suitable lipid is Lipid D, which is 3-(((3-(dimethylamino)propoxy)carbonyl)oxy)-13- (octanoyloxy)tridecyl 3-octylundecanoate. Other suitable lipids include heptatriaconta- 6,9,28,31 -tetraen-19-yl 4-(dimethylamino)butanoate (also known as Dlin-MC3-DMA (MC3))).

[0080] Cationic lipid can be present in embodiments of the composition and lipid particles can comprise an amount from about 30 to about 60 mole percent (“mol%”, or the percentage of the total moles that is of a particular component), from about 30 mol% to about 55 mol%, from about 30 mol% to about 50 mol%, from about 30 mol% to about 45 mol%, from about 30 mol% to about 40 mol%, from about 30 mol% toabout 35 mol%, from about 35 mol% to about 60 mol%, from about 40 mol% to about 60 mol%, from about 45 mol% to about 60 mol%, from about 50 mol% to about 60 mol%, from about 55 mol% to about 60 mol%, from about 35 mol% to about 55 mol%, from about 40 mol% to about 50 mol%. In in some embodiments, the cationic lipid is present in about 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, 40, 41 , 42, 43, 44, 45, 46, 47, 48, 49, 50, 51 , 52, 53, 54, 55, 56, 57, 58, 59, or 60 mol%.

[0081] Some such lipids suitable for use in the LNPs described herein are biodegradable in vivo. For example, LNPs comprising such a lipid include those where at least 75% of the lipid is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days. As another example, at least 50% of the LNP is cleared from the plasma within 8, 10, 12, 24, or 48 hours, or 3, 4, 5, 6, 7, or 10 days.

[0082] Such lipids may be ionizable depending upon the pH of the medium they are in. For example, in a slightly acidic medium, the lipids may be protonated and thus bear a positive charge. Conversely, in a slightly basic medium, such as, for example, blood where pH is approximately 7.35, the lipids may not be protonated and thus bear no charge. In some embodiments, the lipids may be protonated at a pH of at least about 9, 9.5, or 10. The ability of such a lipid to bear a charge is related to its intrinsic pKa. For example, the lipid may, independently, have a pKa in the range of from about 5.8 to about 6.2.

[0083] Neutral (also termed structural) lipids function to stabilize and improve processing of the LNPs. Examples of suitable neutral lipids include a variety of neutral, uncharged or zwitterionic lipids. Examples of neutral phospholipids suitable for use in the present disclosure include, but are not limited to, 5-heptadecylbenzene-1 ,3-diol (resorcinol), dipalmitoylphosphatidylcholine (DPPC), distearoylphosphatidylcholine (DSPC), phosphocholine (DOPC), dimyristoylphosphatidylcholine (DMPC), phosphatidylcholine (PLPC), 1 ,2-distearoyl-sn-glycero-3-phosphocholine (DAPC), phosphatidylethanolamine (PE), egg phosphatidylcholine (EPC), dilauryloylphosphatidylcholine (DLPC), dimyristoylphosphatidylcholine (DMPC), 1 - myristoyl-2-palmitoyl phosphatidylcholine (MPPC), 1 -palmitoyl-2-myristoyl phosphatidylcholine (PMPC), 1 -palmitoyl-2-stearoyl phosphatidylcholine (PSPC), 1 ,2-diarachidoyl-sn-glycero-3-phosphocholine (DBPC), 1 -stearoyl-2-palmitoyl phosphatidylcholine (SPPC), 1 ,2-dieicosenoyl-sn-glycero-3-phosphocholine (DEPC), palmitoyloleoyl phosphatidylcholine (POPC), lysophosphatidyl choline, dioleoyl phosphatidylethanolamine (DOPE), dilinoleoylphosphatidylcholine distearoylphosphatidylethanolamine (DSPE), dimyristoyl phosphatidylethanolamine (DMPE), dipalmitoyl phosphatidylethanolamine (DPPE), palmitoyloleoyl phosphatidylethanolamine (POPE), lysophosphatidylethanolamine, and combinations thereof. For example, the neutral phospholipid may be selected from the group consisting of distearoylphosphatidylcholine (DSPC) and dimyristoyl phosphatidyl ethanolamine (DMPE).

[0084] In certain embodiments, a neutral lipid is present in the lipid particle in an amount from about 20 mol% to about 40 mol%, from about 20 mol% to about 35 mol%, from about 20 mol% to about 30 mol%, from about 20 mol% to about 25 mol%, from about 25 mol% to about 40 mol%, from about 30 mol% to about 40 mol%, from about 30 mol% to about 40 mol%, from about 35 mol% to about 40 mol%, from about 25 mol% to about 35 mol%. In in some embodiments, the cationic lipid is present in about 20, 21 , 22, 23, 24, 25, 26, 27, 28, 29, 30, 31 , 32, 33, 34, 35, 36, 37, 38, 39, or 40 mol%.

[0085] In some embodiments, the lipids can be any of the lipids disclosed in US20210251898, US20210220449, US20210128488, US20210122703, US20210122702, US20210113483, US20210107861 , US20210095309, US20210087135, US20190292566 each incorporated herein by reference in its entirety.

[0086] Commercially available LNPs include, e.g., Lipofectamine™ CRISPRMAX™ Cas9 Transfection Reagent (available from ThermoFisher Scientific, Waltham, MA), Pro-DeliverIN™ CRISPR Transfection Reagent (available from Oz Biosciences, San Diego, CA), and NanoAssemblr® LNPs (available from Precision NanoSystems, Vancouver, BC).

[0087] Helper lipids include lipids that enhance transfection. The mechanism by which the helper lipid enhances transfection can include enhancing particle stability. In certain cases, the helper lipid can enhance membrane fusogenicity. Helper lipidsinclude steroids, sterols, and alkyl resorcinols. Examples of suitable helper lipids suitable include cholesterol, 5-heptadecylresorcinol, and cholesterol hemisuccinate. In one example, the helper lipid may be cholesterol or cholesterol hemisuccinate.

[0088] Stealth lipids include lipids that alter the length of time the nanoparticles can exist in vivo. Stealth lipids may assist in the formulation process by, for example, reducing particle aggregation and controlling particle size. Stealth lipids may modulate pharmacokinetic properties of the LNP. Suitable stealth lipids include lipids having a hydrophilic head group linked to a lipid moiety. The hydrophilic head group of stealth lipid can comprise, for example, a polymer moiety selected from polymers based on polyethylene glycol), poly(oxazoline), poly(vinyl alcohol), poly(glycerol), poly(N- vinylpyrrolidone), polyaminoacids, and poly N-(2-hydroxypropyl)methacrylamide.

[0089] The lipid moiety of the stealth lipid may be derived, for example, from diacylglycerol or diacylglycamide, including those comprising a dialkylglycerol or dialkylglycamide group having alkyl chain length independently comprising from about C4 to about C40 saturated or unsaturated carbon atoms, wherein the chain may comprise one or more functional groups such as, for example, an amide or ester. The dialkylglycerol or dialkylglycamide group can further comprise one or more substituted alkyl groups.

[0090] In some embodiments, the stealth lipid may be PEG-dilauroylglycerol, PEG- dimyristoylglycerol (PEG-DMG), PEG-dipalmitoylglycerol, PEG-distearoylglycerol (PEG-DSPE), PEG-dilaurylglycamide, PEG-dimyristylglycamide, PEG- dipalmitoylglycamide, and PEG-distearoylglycamide, PEG-cholesterol (1 -[8'-(Cholest-5- en-3[beta]-oxy)carboxamido-3',6'-dioxaoctanyl]carbamoyl-( )-methyl-poly(ethylene glycol), PEG-DMB (3,4-ditetradecoxylbenzyl-w-methyl-poly(ethylene glycol)ether), 1 ,2- dimyristoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethylene glycol)-2000] (PEG2k-DMG), 1 ,2-distearoyl-sn-glycero-3-phosphoethanolamine-N- [methoxy(polyethylene glycol)-2000] (PEG2k-DSPE), 1 ,2-distearoyl-sn-glycerol, methoxypoly ethylene glycol (PEG2k-DSG), polyethylene glycol)-2000-dimethacrylate (PEG2k-DMA), or 1 ,2-distearyloxypropyl-3-amine-N-[methoxy(polyethylene glycol)- 2000] (PEG2k-DSA).

[0091] The LNPs can have different ratios between the positively charged amine groups of the biodegradable lipid (N) and the negatively charged phosphate groups (P) of the nucleic acid to be encapsulated. This may be mathematically represented by the equation N / P. For example, the N / P ratio may be from about 0.5 to about 100, from about 1 to about 50, from about 1 to about 25, from about 1 to about 10, from about 1 to about 7, from about 3 to about 5, from about 4 to about 5, about 4, about 4.5, or about 5.

[0092] In some LNPs, the cargo can comprise Gas mRNA and gRNA. The Gas mRNA and gRNAs can be in different ratios. For example, the LNP formulation can include a ratio of Cas mRNA to gRNA nucleic acid ranging from about 25:1 to about 1 :25, ranging from about 10:1 to about 1 :10, ranging from about 5:1 to about 1 :5, or about 1 :1 . Alternatively, the LNP formulation can include a ratio of Cas mRNA to gRNA nucleic acid from about 1 :1 to about 1 :5, or about 10:1 . Alternatively, the LNP formulation can include a ratio of Cas mRNA to gRNA nucleic acid of about 1 :10, 25:1 , 10:1 , 5:1 , 3:1 , 1 :1 , 1 :3, 1 :5, 1 :10, or 1 :25.

[0093] In some LNPs, the cargo can comprise exogenous donor nucleic acid and gRNA. The exogenous donor nucleic acid and gRNAs can be in different ratios. For example, the LNP formulation can include a ratio of exogenous donor nucleic acid to gRNA nucleic acid ranging from about 25:1 to about 1 :25, ranging from about 10:1 to about 1 :10, ranging from about 5:1 to about 1 :5, or about 1 :1 . Alternatively, the LNP formulation can include a ratio of exogenous donor nucleic acid to gRNA nucleic acid from about 1 :1 to about 1 :5, about 5:1 to about 1 :1 , about 10:1 , or about 1 :10. Alternatively, the LNP formulation can include a ratio of exogenous donor nucleic acid to gRNA nucleic acid of about 1 :10, 25:1 , 10:1 , 5:1 , 3:1 , 2.5:1 , 1 :1 , 1 :2.5, 1 :3, 1 :5, 1 :10, or 1 :25.

[0094] In some embodiments, one or more CRISPR endonucleases and one or more guide RNAs may be provided in combination in the form of ribonucleoprotein particles (RNPs). An RNP complex can be introduced into a subject by means of, e.g., injection, electroporation, nanoparticles (including, e.g., lipid nanoparticles), vesicles, and / or with the assistance of cell-penetrating peptides. See, e.g., Lin et al., ELife 3:e04766 (2014);Sansbury et a!., CRISPR J. 2:121 -32 (2019); US2019 / 0359973).

[0095] LNP particles can have a diameter of less than 1000 nm, 500 nm, 250 nm, 200 nm, 150 nm, 100 nm, 75 nm, 50 nm, or 25 nm. Alternatively, a nanoparticle may range in size from 1 -1000 nm, 1 -500 nm, 1 -250 nm, 25-200 nm, 25-100 nm, 35-75 nm, or 25- 60 nm. LNPs may be made from cationic, anionic, or neutral lipids. Neutral lipids, such as the fusogenic phospholipid DOPE or the membrane component cholesterol, may be included in LNPs as “helper lipids” to enhance transfection activity and nanoparticle stability. LNPs may also be comprised of hydrophobic lipids, hydrophilic lipids, or both hydrophobic and hydrophilic lipids.

[0096] In certain embodiments, the cationic lipid N-[1 -(2,3-dioleyloxy)propyl]-N,N,N- trimethylammonium chloride (DOTMA) can be used. DOTMA can be formulated alone or combined with the neutral lipid, dioleoylphosphatidyl-ethanolamine (DOPE) or other cationic or non-cationic lipids into a liposomal transfer vehicle or a lipid nanoparticle, and such liposomes can be used to enhance the delivery of nucleic acids into target cells. Other suitable cationic lipids include, but are not limited to, 5- carboxyspermylglycinedioctadecylamide, 2,3-dioleyloxy-N-[2(spermine- carboxamido)ethyl]-N,N-dimethyl-1 -propanaminium, 1 ,2-Dioleoyl-3- Dimethylammonium-Propane, 1 ,2-Dioleoyl-3-Trimethylammonium-Propane. Contemplated cationic lipids also include 1 ,2-distearyloxy-N,N-dimethyl-3- aminopropane, 1 ,2-dioleyloxy-N,N-dimethyl-3-aminopropane, 1 ,2-dilinoleyloxy-N,N- dimethyl-3-aminopropane, 1 ,2-dilinolenyloxy-N,N-dimethyl-3-aminopropane, N-dioleyl- N,N-dimethylammonium chloride, N,N-distearyl-N,N-dimethylammonium bromide, N- (1 ,2-dimyristyloxyprop-3-yl)-N,N-dimethyl-N-hydroxyethyl ammonium bromide, 3- dimethylamino-2-(cholest-5-en-3-beta-oxybutan-4-oxy)-1 -(cis, ci- s-9,12- octadecadienoxy)propane, 2-[5'-(cholest-5-en-3-beta-oxy)-3'-oxapentoxy)-3-dimethyl-1 - (cis,cis-9',12'-octadecadienoxy)propane, N,N-dimethyl-3,4-dioleyloxybenzylamine, 1 ,2- N,N'-dioleylcarbamyl-3-dimethylaminopropane, 2,3-Dilinoleoyloxy-N,N- dimethylpropylamine, 1 ,2-N,N'-Dilinoleylcarbamyl-3-dimethylaminopropane, 1 ,2- Dilinoleoylcarbamyl-3-dimethylaminopropane, 2,2-dilinoleyl-4-dimethylaminomethyl- [1 ,3]-dioxolane, 2,2-dilinoleyl-4-dimethylaminoethyl-[1 ,3]-dioxolane, and 2-(2,2-di ((9Z, 12Z)-octadeca-9,12-dien-1 -yl)-1 ,3-dioxolan-4-yl)-N,N-dimethylethanamine (DLin- KC2-DMA)), or mixtures thereof.

[0097] In some embodiments, non-cationic lipids can be used. As used herein, the phrase “non-cationic lipid” refers to any neutral, zwitterionic, or anionic lipid. As used herein, the phrase “anionic lipid” refers to any of a number of lipid species that carry a net negative charge at a selected pH, such as physiological pH. Non-cationic lipids include, but are not limited to, distearoylphosphatidylcholine (DSPC), dioleoylphosphatidylcholine (DOPC), dipalmitoylphosphatidylcholine (DPPC), dioleoylphosphatidylglycerol (DOPG), dipalmitoylphosphatidylglycerol (DPPG), DOPE, palmitoyloleoylphosphatidylcholine (POPC), palmitoyloleoyl-phosphatidylethanolamine (POPE), dioleoyl-phosphatidylethanolamine 4-(N-maleimidomethyl)-cyclohexane-1 - carboxylate (DOPE-mal), dipalmitoyl phosphatidyl ethanolamine (DPPE), dimyristoylphosphoethanolamine (DMPE), distearoyl-phosphatidylethanolamine (DSPE), 16-O-monomethyl PE, 16-O-dimethyl PE, 18-1 -trans PE, 1 -stearoyl-2-oleoyl- phosphatidyethanolamine (SOPE), cholesterol, or a mixture thereof. Such non-cationic lipids may be used alone or can be used in combination with other excipients, for example, cationic lipids.

[0098] Virus-like particles

[0099] A recombinant expression vector sequence can be packaged into a virus or virus-like particle (also referred to herein as a “particle” or “virion”) for subsequent infection and transformation of a cell, ex vivo, in vitro, or in vivo. Such particles or virions will typically include proteins that encapsidate or package the vector genome. Suitable expression vectors may include viral expression vectors based on vaccinia virus; poliovirus; adenovirus; a retroviral vector (e.g., Murine Leukemia Virus), spleen necrosis virus, and vectors derived from retroviruses such as Rous Sarcoma Virus, Harvey Sarcoma Virus, avian leukosis virus, a lentivirus, human immunodeficiency virus, myeloproliferative sarcoma virus, and mammary tumor virus; and the like. In some embodiments, a recombinant expression vector of the present disclosure is a recombinant adeno-associated virus (AAV) vector. In some embodiments, arecombinant expression vector of the present disclosure is a recombinant lentivirus vector. In some embodiments, a recombinant expression vector of the present disclosure is a recombinant retroviral vector.

[0100] In other embodiments, suitable vectors may include virus-like particles (VLP). Virus-like particles (VLPs) are particles that closely resemble viruses, but do not contain viral genetic material and are therefore non-infectious. In some embodiments, VLPs comprise a polynucleotide encoding a transgene of interest, for example any Cas protein and / or a gRNA embodiments, and, optionally, donor template polynucleotides described herein, packaged with one or more viral structural proteins.

[0101] In general, VLPs are constructed by producing viral structural proteins and purifying resulting viral particles. Then, following purification, a cargo / payload e.g., any of the engineered nucleic acids described herein) is encapsulated within the purified particle ex vivo. Accordingly, production of VLPs maintains separation of the nucleic acids encoding viral structural proteins and the nucleic acids encoding the cargo / payload. The viral structural proteins used in VLP production can be produced in a variety of expression systems, including mammalian, yeast, insect, bacterial, or in vivo translation expression systems. The purified viral particles can be denatured and reformed in the presence of the desired cargo to produce VLPs using methods known to those skilled in the art. Production of VLPs are described in more detail in Seow et al. (Mol Ther. 17: 767-77 (2009)).

[0102] Pharmaceutical Compositions

[0103] Any of the pharmaceutical compositions disclosed herein can be formulated for use in the preparation of a medicament, and particular uses are indicated below in the context of treatment, e.g., the treatment of a subject having cancer. When employed as pharmaceuticals, any of the nucleic acids and vectors can be administered in the form of pharmaceutical compositions. Administration may be pulmonary e.g., by inhalation or insufflation of powders or aerosols, including by nebulizer; intratracheal, intranasal, epidermal and transdermal), topical (including ophthalmic and to mucous membranes including intranasal, vaginal and rectal delivery), ocular, oral or parenteral. Parenteraladministration includes intravenous, intra-arterial, subcutaneous, intraperitoneal or intramuscular injection or infusion; or intracranial, e.g., intrathecal or intraventricular administration. Parenteral administration can be in the form of a single bolus dose, or may be, for example, by a continuous perfusion pump. Pharmaceutical compositions and formulations for topical administration may include transdermal patches, ointments, lotions, creams, gels, drops, suppositories, sprays, liquids, powders, and the like. Conventional pharmaceutical carriers, aqueous, powder or oily bases, thickeners and the like may be necessary or desirable.

[0104] In some embodiments, pharmaceutical compositions can contain, as the active ingredient, nucleic acids, vectors, and / or RNPs described herein in combination with one or more pharmaceutically acceptable carriers. The term “pharmaceutically acceptable” refers to molecular entities and compositions that do not produce an adverse, allergic or other untoward reaction when administered to an animal or a human, as appropriate. The term “pharmaceutically acceptable carrier,” as used herein, includes any and all solvents, dispersion media, coatings, antibacterial, isotonic and absorption delaying agents, buffers, excipients, binders, lubricants, gels, surfactants and the like, that may be used as media for a pharmaceutically acceptable substance. In making the pharmaceutical compositions disclosed herein, the active ingredient is typically mixed with an excipient, diluted by an excipient or enclosed within such a carrier in the form of, for example, a capsule, tablet, sachet, paper, or other container. When the excipient serves as a diluent, it can be a solid, semisolid, or liquid material (e.g., normal saline), which acts as a vehicle, carrier or medium for the active ingredient. Thus, the compositions can be in the form of tablets, pills, powders, lozenges, sachets, cachets, elixirs, suspensions, emulsions, solutions, syrups, aerosols (as a solid or in a liquid medium), lotions, creams, ointments, gels, soft and hard gelatin capsules, suppositories, sterile injectable solutions, and sterile packaged powders. As is known in the art, the type of diluent can vary depending upon the intended route of administration. The resulting compositions can include additional agents, such as preservatives. In some embodiments, the carrier can be, or can include, a lipid-based or polymer-based colloid. In some embodiments, the carrier material can be a colloidformulated as a liposome, a hydrogel, a microparticle, a nanoparticle, or a block copolymer micelle. As noted, the carrier material can form a capsule, and that material may be a polymer-based colloid.

[0105] The nucleic acid sequences disclosed herein can be delivered to an appropriate cell of a subject, e.g. a cancer cell. This can be achieved by, for example, the use of a polymeric, biodegradable microparticle or microcapsule delivery vehicle, sized to optimize phagocytosis by phagocytic cells such as macrophages. Delivery of “naked DNA” ( / .e., without a delivery vehicle) to an intramuscular, intradermal, or subcutaneous site, is another means to achieve in vivo expression. In the relevant polynucleotides (e.g., expression vectors) the nucleic acid sequence encoding the isolated nucleic acid sequence comprising a sequence encoding a CRISPR-associated endonuclease and a guide RNA can be operatively linked to a promoter or enhancer-promoter combination. Promoters and enhancers are described above.

[0106] In some embodiments, the pharmaceutical compositions can be formulated as a nanoparticle, for example, nanoparticles comprised of a core of high molecular weight linear polyethylenimine (LPEI) complexed with DNA and surrounded by a shell of polyethyleneglycol-modified (PEGylated) low molecular weight LPEI.

[0107] The nucleic acids, vectors, and RNPs may also be applied to a surface of a device (e.g., a catheter) or contained within a pump, patch, or other drug delivery device. The nucleic acids and vectors disclosed herein can be administered alone, or in a mixture, in the presence of a pharmaceutically acceptable excipient or carrier (e.g., physiological saline). The excipient or carrier is selected on the basis of the mode and route of administration. Suitable pharmaceutical carriers, as well as pharmaceutical necessities for use in pharmaceutical formulations, are described in, e.g., Remington: The Science and Practice of Pharmacy (Adejare, ed., 23rdEdition, Nov. 13, 2020) and in the USP / NF (United States Pharmacopeia and the National Formulary).

[0108] In some embodiments, the compositions can be formulated as a nanoparticle encapsulating a nucleic acid encoding a CRISPR-associated endonuclease and a guide RNA sequence.

[0109] The concentration of anti-cancer therapies can vary widely, and will be selectedprimarily based on fluid volumes, viscosities, body weight and the like in accordance with the particular mode of administration selected and the biological system’s needs. Generally, the amount of the anti-cancer therapy or therapies present in a pharmaceutical composition will be that which will produce a therapeutic effect. For example, in some embodiments, the weight per volume (w / v) or weight percent (wt %) concentration of an anti-cancer therapy or therapies in a pharmaceutical composition may be between about 0.001 % to 100%, 0.001% to 90%, 0.001 % to 80%, 0.001 % to 70%, 0.001 % to 60%, 0.001 % to 50%, 0.001 % to 40%, 0.001 % to 30%, 0.001 % to 20%, 0.001 % to 10%, 0.001 % to 1 %, 0.01% to 100%, 0.01 % to 90%, 0.01 % to 80%, 0.01 % to 70%, 0.01 % to 60%, 0.01% to 50%, 0.01 % to 40%, 0.01 % to 30%, 0.01 % to 20%, 0.01 % to 10%, 0.01% to 1%, 0.1% to 100%, 0.1% to 90%, 0.1 % to 80%, 0.1 % to 70%, 0.1% to 60%, 0.1% to 50%, 0.1% to 40%, 0.1% to 30%, 0.1 % to 20%, 0.1 % to 10%, 0.1 % to 1 %, 1 % to 100%, 1 % to 90%, 1 % to 80%, 1 % to 70%, 1 % to 60%, 1 % to 50%, 1 % to 40%, 1 % to 30%, 1 % to 20%, 1 % to 10%, 1 % to 5%, 1 % to 4%, 1 % to 3%, 1 % to 2%, 0.1% to 0.9%, 0.1% to 0.8%, 0.1% to 0.7%, 0.1% to 0.6%, 0.1% to 0.5%, 0.1 % to 0.4%, 0.1 % to 0.3%, 0.1 % to 0.2%, 0.2% to 1%, 0.3% to 1%, 0.4% to 1 %, 0.5% to 1%, 0.6% to 1 %, 0.7% to 1%, 0.8% to 1 %, or 0.9% to 1%.

[0110] In other embodiments, the concentration of an anti-cancer therapy or therapies in a pharmaceutical composition may be about 1 nM, 2 nM, 3 nM, 4 nM, 5 nM, 6 nM, 7 nM, 8 nM, 9 nM, 10 nM, 20 nM, 30 nM, 40 nM, 50 nM, 60 nM, 70 nM, 80 nM, 90 nM, 100 nM, 200 nM, 300 nM, 400 nM, 500 nM, 600 nM, 700 nM, 800 nM, 900 nM, 1 pM, 2 pM, 3 pM, 4 pM, 5 pM, 6 pM, 7 pM, 8 pM, 9 pM, 10 pM, 20 pM, 30 pM, 40 pM, 50 pM, 60 pM, 70 pM, 80 pM, 90 pM, 100 pM, 200 pM, 300 pM, 400 pM, 500 pM, 600 pM, 700 pM, 800 pM, 900 pM, 1 mM, 2 mM, 3 mM, 4 mM, 5 mM, 6 mM, 7 mM, 8 mM, 9 mM, 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, 100 mM, 200 mM, 300 mM, 400 mM, 500 mM, 600 mM, 700 mM, 800 mM, 900 mM, or 1 M. In some aspects, the concentration (molarity or wt%) of an anti-cancer therapy or therapies that produces a therapeutic effect in a subject (e.g., a human or other mammal) can be extrapolated from in vitro or in v / vo data, from cell culture and / or animal experiments.

[0111] In some embodiments, diseases / disorders include non-cancerous geneticdisorders, including but not limited to, chondroplasia, achromatopsia, acid maltase deficiency, adrenoleukodystrophy, aicardi syndrome, alpha-1 antitrypsin deficiency, alpha-thalassemia, androgen insensitivity syndrome, apert syndrome, arrhythmogenic right ventricular, dysplasia, ataxia telangictasia, barth syndrome, beta-thalassemia, blue rubber bleb nevus syndrome, canavan disease, chronic granulomatous diseases (CGD), cri du chat syndrome, cystic fibrosis, dercum’s disease, ectodermal dysplasia, fanconi anemia, fibrodysplasia ossificans progressive, fragile X syndrome, galactosemis, Gaucher’s disease, generalized gangliosidoses (e.g., GM1 ), hemochromatosis, the hemoglobin C mutation in the 6th codon of beta-globin (HbC), hemophilia, Huntington’s disease, Hurler Syndrome, hypophosphatasia, Klinefleter syndrome, Krabbes Disease, Langer-Giedion Syndrome, leukodystrophy, long QT syndrome, Marfan syndrome, Moebius syndrome, mucopolysaccharidosis (MPS), nail patella syndrome, nephrogenic diabetes insipdius, neurofibromatosis, Neimann-Pick disease, osteogenesis imperfecta, porphyria, Prader-Willi syndrome, progeria, Proteus syndrome, retinoblastoma, Rett syndrome, Rubinstein-Taybi syndrome, Sanfilippo syndrome, severe combined immunodeficiency (SCID), Shwachman syndrome, sickle cell disease (sickle cell anemia), Smith-Magenis syndrome, Stickler syndrome, Tay- Sachs disease, Thrombocytopenia Absent Radius (TAR) syndrome, Treacher Collins syndrome, trisomy, tuberous sclerosis, Turner's syndrome, urea cycle disorder, von Hippel-Landau disease, Waardenburg syndrome, Williams syndrome, Wilson’s disease, and Wiskott-Aldrich syndrome.

[0112] Methods of inducing exon skipping

[0113] In certain aspects, the disclosure relates to methods of identifying a guide RNA (gRNA) that induces exon skipping in a target gene comprising: (a) identifying one or more gRNAs that bind near or in an exon splicing enhancer (ESE) or an exon splicing silencer (ESS) in the target gene; (b) contacting the target gene with one of the gRNAs identified in step (a) and a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, whereby the gRNA hybridizes to the target gene and the CRISPR-associated endonuclease cleaves the gene; (c) repeating step (b)with another gRNA of step (a) until all gRNAs of step (a) have been contacted with the target gene; (d) determining a percent rate of exon skipping generated by each of the one or more gRNAs; and (e) correlating a phenotypic change in a protein expressed by the target gene with the percent rate of exon skipping generated by each of the one or more gRNAs.

[0114] Potential gRNAs can be identified in a target sequencing by examining such target sequence for ESEs and / or ESSs and locating a PAM site in such ESEs and / or ESSs or by locating a PAM site within 30, 29, 28, 27, 26, 25, 24, 23, 22, 21 , 20, 19, 18, 17, 16, 15, 14, 13, 12, 11 , 10, 9, 8, 7, 6, 5, 4, 3, 2, or 1 bases 3' or 5' of such ESEs and / or ESSs. 1 , 2, 3, 4, 5, 6, 7, 8, 9, 10, 1 1 , 12, 13, 14, 15, 16, 17, 18, 19, 20 or more gRNAs can be identified in a target sequence for determination of exon skipping capability.

[0115] In some embodiments, the identified gRNAs are one or more of SEQ ID NOs: 1 - 4, or 10 in Table 1 .

[0116] Table 1. Description of Sequences

[0117] Identified gRNAs are contacted the target gene in the presence of a Cas endonuclease, whereby the gRNA hybridizes to the target gene and the Cas endonuclease cleaves the gene. Each identified gene is, in some embodiments, individually contacted with the target gene. In some embodiments, it may be advantageous to contact the target gene at the same time, or substantially the same time, with more than one of the identified gRNAs.

[0118] For each gRNA tested, a percent rate of exon skipping is determined, in addition to, in some embodiments, determining which exon(s) of a target gene are skipped. Effective inducement of exon skipping by an identified gRNA results in, e.g., at least a 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% rate of exon skipping of one or more exons in the target gene.

[0119] (e) correlating a phenotypic change in a protein expressed by the target gene with the percent rate of exon skipping generated by each of the one or more gRNAs.

[0120] Expressed proteins containing exon-skipped regions are then assessed for desirable or undesirable phenotypic changes as compared to the wild-type and / or natural variant(s) of the targeted gene. In some embodiments, phenotypic changes that result in a rate of at least a 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%,34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%,49%, 50%, 51%, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%,64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%,79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% increase in the phenotype as compared to the wild-type and / or natural variant(s) of the targeted gene.

[0121] Phenotype information includes any observable trait determined or contributed by a genotype. Phenotype information may include the physical appearance, biological development, and / or behavior. In some embodiments, phenotype information may include the presence or absence of a particular observable trait. In some embodiments, phenotype information includes the ratio of a subject’s phenotype information to a population of phenotype information. The population of phenotype information may be derived from any of the sources described therein, such as databases or experimental procedures.

[0122] Phenotypes may be assessed using a variety of methods known in the art, including clinical examination and routine laboratory tests. Laboratory tests may include both macroscopic and microscopic methods, molecular methods, radiographic methods such as X-rays, biochemical methods, immunohistochemical methods and others.

[0123] Certain aspects of the present disclosure are for methods of inducing exon skipping in a target gene comprising: (a) identifying one or more gRNAs that bind near or in an exon splicing enhancer (ESE) or an exon splicing silencer (ESS) in the target gene; (b) contacting the target gene with one of the gRNAs identified in step (a) and a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, whereby the gRNA hybridizes to the target gene and the CRISPR- associated endonuclease cleaves the gene; (c) repeating step (b) with another gRNA of step (a) until all gRNAs of step (a) have been contacted with the target gene; (d) determining a percent rate of exon skipping generated by each of the one or more gRNAs; (e) correlating a phenotypic change in a protein expressed by an exon-skipped gene with the percent rate of exon skipping generated by each of the one or more gRNAs; and (f) administering to a subject the gRNA of step (d) determined to have at least a 10% rate of inducing exon skipping and at least a 10% rate of phenotypic change.

[0124] In some embodiments, a therapeutically effective amount of a pharmaceutical composition comprising a Cas endonuclease and one or more guide RNA(s) that generates one or more correlated phenotypic outcome(s) is administered to a subject, with such exon skipping and correlated phenotypic outcome as determined by a method of the present disclosure.

[0125] The pharmaceutical compositions described herein may be administered to a subject in an amount sufficient to induce exon skipping in a target gene resulting in one or more correlated phenotypic outcome(s) at a rate of at least 10%, 1 1 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%,29%, 30%, 31%, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%,44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%,59%, 60%, 61%, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%,74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%. In some embodiments, the pharmaceutical composition induces exon skipping in the target gene at a rate of at least 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%,35%, 36%, 37%, 38%, 39%, 40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%,50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%,65%, 66%, 67%, 68%, 69%, 70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%,80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%,95%, 96%, 97%, 98%, 99%, or 100%.

[0126] In some embodiments, the methods disclosed here are utilized to control heterogeneity of a phenotypic outcome comprising: (a) identifying one or more gRNAs that bind near or in an exon splicing enhancer (ESE) or an exon splicing silencer (ESS) in the target gene; (b) contacting the target gene with one of the gRNAs identified in step (a) and a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)- associated endonuclease, whereby the gRNA hybridizes to the target gene and the CRISPR-associated endonuclease cleaves the gene; (c) repeating step (b) with another gRNA of step (a) until all gRNAs of step (a) have been contacted with thetarget gene; (d) determining a percent rate of exon skipping generated by each of the one or more gRNAs; (e) correlating a desirable phenotypic outcome generated by a protein expressed by an exon-skipped gene with the percent rate of exon skipping generated by each of the one or more gRNAs; and (f) administering to a subject the gRNA of step (d) determined to have at least a 10% rate of inducing exon skipping and correlates with an at least 10% change in phenotypic heterogeneity.

[0127] In some embodiments, phenotypic heterogeneity can be controlled (e.g., reduced as compared to untreated systems) by administering to a subject pharmaceutical compositions comprising a Cas endonuclease and one or more guide RNA(s) identified by a method of the present disclosure that induces desirable splice variants of the target gene to thereby alter (or more specifically control) the phenotypic heterogeneity of a target gene and / or the cells and / or systems related thereto.

[0128] The pharmaceutical compositions described herein may be administered to a subject in an amount sufficient to control phenotypic heterogeneity at a rate of at least 10%, 11 %, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21 %, 22%, 23%, 24%,25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%,40%, 41 %, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%,55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%,70%, 71 %, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%,85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% reduction as compared to an untreated target. In some embodiments, the pharmaceutical composition induces exon skipping in the target gene at a rate of at least 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, 21%, 22%, 23%, 24%, 25%, 26%, 27%, 28%, 29%, 30%, 31 %, 32%, 33%, 34%, 35%, 36%, 37%, 38%, 39%, 40%, 41%, 42%, 43%, 44%, 45%, 46%, 47%, 48%, 49%, 50%, 51 %, 52%, 53%, 54%, 55%, 56%, 57%, 58%, 59%, 60%, 61 %, 62%, 63%, 64%, 65%, 66%, 67%, 68%, 69%, 70%, 71%, 72%, 73%, 74%, 75%, 76%, 77%, 78%, 79%, 80%, 81 %, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%.EXAMPLES

[0129] Example 1 : Characterization of CRISPR-induced exon skipping

[0130] The exon skipping events occur because the indels created in the clonal cell lines are within regions that are essential for efficient RNA splicing, therefore the degree of disrupting these regions causes exon skipping.

[0131] Figure 1 provides an overview of Applicant’s broad-based CRISPR / Cas9 gene editing strategy. A single guide RNA (gRNA) designated (1 ) (SEQ ID NO:1 ), was used to target exon 2, which encodes the Neh2 domain of the protein. A separate pair of gRNAs designated (2) (SEQ ID NO:2) and (3) (SEQ ID NO:3) was used to target exon 4, which encodes the first half of the Neh5 domain. A dual gRNA approach was also used where both gRNAs (2) and (3) were simultaneously transfected to generate large fragment deletions within exon 4. By targeting two nonadjacent exons of NRF2, one can analyze the consequence of molecular rearrangements induced by CRISPR / Cas activity.

[0132] Applicant sought to examine if the genetic disruption of NRF2 would influence the types of mRNA populations in the targeted cell (Figure 2). The first column lists the clonal identification number with the associated gRNA used to target NRF2. The second column lists the genomic NRF2 sequence of each clone and the allele-specific indel patterns induced by the gRNA listed. The normal genomic sequence is listed before each set of clones with a depiction of the gRNA sequence, the PAM, and the cleavage site represented by the vertical line. The third column lists the population of mRNA transcripts arising from each clonal cell line generated by the action of CRISPR- directed gene editing. For example, an indel (i.e., -2) listed in the third column indicates that this indel, initially identified by genomic sequencing, is also apparent in the mRNA transcript population. In most of the clonal cell lines, the second transcript population may arise from an exon skipping event, likely induced by ESEs2, which are highlighted.

[0133] The genetic analysis of the knockout of NRF2 - specifically the genotype of clones 1 -17, 2-16 and 2-23, is presented in Figure 2A. These clones were created by targeting exon 2 with a single gRNA (gRNA (1 )). The genomic indels for each clone were deconvoluted by the deconvolution software program DECODR3through paired-end analysis, which utilizes both forward and reverse sequencing of each clone. DECODR presents the indel distribution of each clone from raw sequence data and the percentages correspond to the indels for each allele. In this case, most of the clones present with three alleles, as the A549 cell line is known to be hypo-triploid4.Applicant’s genetic analysis indicates that clone 1 -17 contains a two base pair deletion at the target site across all three alleles causing a frameshift mutation that forms stop codons downstream. Clone 2-16 bears a single base pair deletion upstream of the target site also inducing a frameshift that forms stop codons further downstream. In contrast, clone 2-23 contains a single allelic disruption through a two base pair deletion at the target site causing a frameshift mutation and forming stop codons downstream. The other two alleles of the NRF2 gene in clone 2-23 are wild type. Clones 1 -17 and 2- 16 each harbor two different transcript populations, the first reflecting the CRISPR- induced alterations present in the genomic DNA and the second population showing exon skipping. Interestingly, despite containing a genomic indel on a single allele in addition to two wildtype alleles at the NRF2 locus, clone 2-23 contains only a single wildtype transcript population. The cleavage site of gRNA (1 ) falls between the first two nucleotides (G - C) of the fifth ESE site in exon 2. The location of each ESE relative to the CRISPR target site seems to play a key role in the exon skipping events.

[0134] Since Applicant mapped the genetic outcomes of CRIS PR-directed gene editing at the genomic and transcript level, Applicant sought to visualize exon skipping activity at the protein level. As shown in Figure 2A, wildtype NRF2 (A549 cells) is visible, migrating between 95 kDa to 1 10 kDa, consistent with previous observations5. Two bands appear at approximately 90 kDa, which reflect what has been widely described as nuclear (top band) and cytoplasmic NRF2 (lower band)6-9. Lane 2 contains whole cell lysate from clone 1 -17, the clonal cell line which contains a population of transcripts with exon 2 and 3 skipping as well as transcripts with the two base pair deletion. As predicted, there is no visible protein migrating at 90 kDa because the transcript does not produce a protein, however the band migrating at approximately 75 kDa represents the shortened transcript. In lane 3, the 90 kDa band is absent, again, indicating that the transcript with the single base pair deletion fails to produce a protein;the lower band seen in this lane reflects the exon 2 skipped transcript that Applicant identified as part of the mRNA transcript population. In the fourth lane, whole cell lysate from clone 2-23 reveals a faint but visible band at 90 kDa and an associated band below it, like the first lane with A549 parental cells. Clone 2-23 harbors two wildtype alleles and a single mutated allele; hence, Applicant expect wildtype protein to be produced.

[0135] To evaluate the universality of the exon skipping phenomena, Applicant utilized a dual system of gRNA (2) and gRNA (3) (Figure 2B) which aims to disrupt exon 4 thereby establishing another family of clonal variants. The dual guide RNA approach was designed to remove a 103 base pair fragment from the middle of exon 4. Each clone reveals a diversity of genomic signatures including the loss of three ESE sites. Of the ten clones characterized, only clone 33 displayed exon skipping of exon 4. Both clones 33 and 21 contain 101 bp deletions, yet only clone 33 exhibits exon skipping. Taken together, Applicant’s data suggest the outcomes of genetic knockout, even at the single nucleotide level due to nonhomologous end-joining activity (resection) can induce or suppress exon skipping.

[0136] Further protein analysis of the genetically engineered clonal isolates is presented in Figure 2B. The first lane, after the protein ladder, reflects the appearance of wildtype NRF2 (95 kDa) generated from A549 parental whole cell lysate. In the second lane is whole cell lysate from clone 15 with a visible band at 90 kDa. This clone contains a 1 , 103, 102 base pair deletion in the genomic DNA which is reflected in the mRNA transcript. The 1 and 103 base pair deletion is frameshifting and is likely being degraded. However, the 102 base pair deletion is in-frame and most likely being transcribed into mutant protein that is being visualized in the western blot. The 102 base pair deletion is a loss of 34 amino acids which would result in protein about 4 kDa smaller in size. The same concept applies to the fourth lane which contains whole cell lysate of Clone 32. Clone 32 contains a 103, 104, and 102 base pair deletion seen in the genomic DNA and reflected in the mRNA transcript. The 103 and 104 base pair deletion is frameshifting and again likely being degraded, whereas the 102-base pair, like in clone 15, is in frame and being expressed as a mutant protein. In the third laneis whole cell lysate from clone 21 which contains a 1 , 103 and 101 bp deletion in the gDNA and is reflected in the mRNA. In this instance, all three indels induce frameshifting and likely the reason for the complete knockout of NRF2. In the fifth lane is whole cell lysate for clone 33 which contains a homozygous 101 base pair deletion in the genomic DNA leading to exon 4 skipping in the mRNA. Since the 101 base pair deletion is frameshifting, the protein that is being picked up in the western blot is the exon 4 skipped protein. Exon 4 skipping is a loss of 192 base pair or 64 amino acids resulting in a protein about 7 kDa smaller in size. Based on the protein expression pattern in these four clones, each clonal cell line produces allele-specific.

[0137] Applicant was able to confirm the presence of similar rearrangements in the NRF2 gene in a separate cell line, H1703, with a different gRNA (4) (SEQ ID NO:4) (Figure 3). Clonal cell lines were fully characterized as previously described for genomic indel signature (Figure 3A & B), transcript (Figure 3B) and protein (Figure 3C) contributions. Utilizing different gRNAs to achieve exon skipping, associated with CRISPR-directed gene editing, demonstrates the broad-based nature of this metabolic activity.

[0138] Example 2: Phenotypic impact of CRISPR-induced exon skipping

[0139] The discovery and characterization of transcript populations in CRISPR-directed NRF2 knockout cell lines led us to ask whether such molecular change at the DNA and RNA level would affect the function of NRF2. One of its key functions is to protect cells against excess stress by activating genes involved in cytoprotective pathways. As such, NRF2 enables resistance to chemotherapy, and therefore disabling NRF2 should reduce resistance and elevate chemo-sensitivity10-12. Figure 4A depicts the different splice variants achieved by targeting NRF2 using CRISPR in A549 cells.

[0140] To evaluate changes in chemoresistance in a variety of clonal cell lines, Applicant utilized the MTS viability assay, a colorimetric method for measuring metabolically active and viable cells in proliferation, cytotoxicity and chemosensitivity assay. Figure 4B displays the relative cell proliferation of A549s and the derivative clones, 1 -17, 2-16, 2-23, 15, 21 , 32 and 33, when treated with various concentrationsof cisplatin. Two separate experiments were conducted using each clone in quadruplet for a total of eight data points collected for each concentration of each clone. Exon 2 and 3 skipping causes the loss of the functional Neh2 and Neh4 domain, respectively. The loss of these domains but not the Neh4 domain is impairing the functionality of NRF2, therefore increasing chemosensitivity of these cells as compared to the A549 parental cells which contain wildtype NRF2. Cells with exon 2 skipping do exhibit some increased chemosensitivity due to lower NRF2 expression, potentially caused by side effects of the CRIS PR-directed gene editing reaction itself or unknown functions of the Neh2 domain. Exon 4 skipping displays heightened sensitivity to cisplatin at even low concentrations, indicating the importance of the domain present within the exon, similar to cells lacking NRF2 completely (NRF2 null). Cells with truncated NRF2 (NRF2 trunc) do show sensitivity similar to exon 2&3 skipped cells. This data presents a genetically diverse population of cells targeted with CRISPR to disrupt NRF2. Each clonal cell line presents a unique molecular outcome that dictates its response to chemotherapy.

[0141] Example 3: CRISPR-induced exon skipping in NRF2

[0142] Once exon skipping events were characterized and assessed for phenotypic changes, we wanted to assess the exon skipping frequency in a population of CRISPR targeted cells. Different cancer background cells were utilized (KYSE - esophageal squamous cancer, FADU - head and neck squamous cancer) with three different gRNAs targeting exon 2 (sg4, SEQ ID NO:4) or exon 4 (sg76 SEQ ID NO:5 or sg83 SEQ ID NO:6). Cells were transfected with CRISPR gRNA and spCas9 mRNA using CRISPRmax (ThermoFisher), following manufacturer’s protocol. Cells were collected 72 hours after transfection and RNA was isolated and converted to cDNA. The NRF2 gene was amplified from cDNA using specific primers to amplify the whole transcript to assess the presence of splice variants (5utr FWD SEQ ID NO:7; ex5 REV SEQ ID NO:8) by agarose gel electrophoresis (Figure 5). Targeting both exon 2 and exon 4 in both cell lines produces alternate splice variants as shown by the multiple bands present in each lane as opposed to the unedited samples in lane 1 and 5.

[0143] Example 4: Consequence of indel profile on exon skipping events

[0144] To assess the hierarchy of splicing motifs present within exon 2 of NRF2, two different gRNAs were compared for the frequency of exon skipping events (Figure 6A). gRNA (4) (SEQ ID NO:4) and R34G gRNA (SEQ ID NO:9) were transfected into cells, clonal cells were further expanded and characterized. Figure 6B and 6C present the genomic DNA analysis by Sanger sequencing. Figure 6B presents two clonal cell lines from targeting with gRNA (4). The indels that occur in these clones remove 1 base from an ESE motif (as shown in the orange box. This indel creates a new splice variant as characterized using previously described methods. Figure 6C presents six clonal cell lines from targeting with R34G gRNA. The indels that occur in each clone disrupts both ESE motifs as seen in the orange boxes as dashes. However, these indels do not create new splice variants. The indel pattern that occurs as a result of targeting the second ESE repairs the first ESE; therefore, there is a hierarchy of ESEs that drive exon skipping to occur - in this case, the first ESE is driving exon skipping.

[0145] Example 5: CRISPR-induced exon skipping in EGFR

[0146] In addition to CRISPR induced exon skipping in NRF2, we wanted to assess the exon skipping frequency in a population of cells CRISPR targeted to an alternative gene within an ESE. A lung adenocarcinoma cell line, PC9, was utilized for CRISPR targeting of EGFR. A gRNA was designed to target exon 19 of EGFR as shown in Figure 7A represented by Ex19 del2 gRNA1 (SEQ ID NO:10). Cells were transfected with CRISPR gRNA and purified spCas9 protein precomplexed as an RNP, following Lonza’s Nucleofector 4d protocol. Cells were collected 72 hours after transfection and genomic DNA and RNA were isolated. The genomic DNA was used as a template for amplification of the EGFR gene for next generation sequencing and CRISPResso216analysis (Figure 7B) using specific primers EGFR Ex19 NGS FWD (SEQ ID NO:11 ) and EGFR Ex19 NGS REV (SEQ ID NO:12). The sequencing data demonstrates that the resulting indels from CRISPR / Cas9 targeting at this site have a wide spectrum, many of which delete the ESE sequence either partially or completely. The RNA was converted to cDNA and the EGFR gene was amplified from the cDNA using specificprimers EGFR cDNA FWD1 (SEQ ID N0:13) and EGFR cDNA REV1 (SEQ ID N0:14) to amplify the sequence between exon 15 and exon 21 to assess the presence of splice variants by agarose gel electrophoresis (Figure 70). When PC9 cells, either WT (designated WT-1 or WT-2) or a PC9 clonal derivative cell line (designated 6-1 and 6- 2) are targeted with Ex19 del2 gRNA1 / Cas9 RNP, exon skipping is evident as compared to the PBS treated samples. Targeting the EGFR exon 19 results in alternative splicing outcomes in the edited cells.

[0147] Example 6 (Prophetic): Assessing exon skipping efficiency in vitro as it correlates to a phenotypic change

[0148] To assess the exon skipping efficiency of various other genes, guide RNAs will be designed for exons that encode functional protein domains of the target gene. The guide RNA and Cas9 designed to target various ESEs or ESSs will be transfected into cells using previously described methods and alternative splicing will be assessed. Clonal cells will be expanded containing unique splice variants in order to test the phenotypic change. The ESE or ESS showing exon skipping as a result of CRISPR targeting will be further optimized by characterizing and improving the efficiency of the guide RNA or location of targeting within the region of the ESE or ESS. Once optimized, cells will be transfected and the population of cells after targeting will be assessed for exon skipping frequency. Cells will be assessed for the change in phenotypic outcome to assess the functional disruption of the gene.

[0149] Example 7 (Prophetic): Nuclease-agnostic induced exon skipping for phenotypic changes

[0150] Various nucleases (SpCas9, SaCas9, Cas12a, TALENs) will be tested to characterize the frequency of ESE or ESS disruption after nuclear enzymatic activity and indel creation. Nucleases will be designed and tested as previously described.

[0151] Example 8 (Prophetic): CRISPR-induced exon skipping in KLKB1

[0152] To assess CRISPR-induced exon skipping in a genetic disease background, gRNAs will be designed for targeting KLKB1 exons in ESE / ESS regions that encode functional domains that lead to activation of hereditary angioedema (HAE) while avoiding domains that have alternative functions. These gRNAs will be transfected into cells using previously described methods and alternative splicing will be assessed. Clonal cells will be expanded containing unique splice variants in order to test the phenotypic change. The ESE or ESS showing exon skipping as a result of CRISPR targeting will be further optimized by characterizing and improving the efficiency of the guide RNA or location of targeting within the region of the ESE or ESS. Once optimized, cells will be transfected and the population of cells after targeting will be assessed for exon skipping frequency. Cells will be assessed for the change in phenotypic outcome to assess the functional disruption of the gene.

[0153] Example 9: CRISPR-induced exon skipping in exon 4 of NRF2 by SpCas9

[0154] To assess the hierarchy of splicing motifs present within exon 4 of NRF2, six different gRNAs (Figure 8A) were compared for the frequency of exon skipping events (Figure 9B). spCas9-13 (SEQ ID:15), gRNA-76 (SEQ ID NO:5), spCas9-21 (SEQ ID NO:16), gRNA-83 (SEQ ID NO:6), spCas9-25 (SEQ ID NO:17), spCas9-26 (SEQ ID NO:18) were transfected into A549 and H1703 cells, respectively, with spCas9 mRNA using MessengerMax reagent. Cells were collected 72 hours post transfection and genomic DNA and RNA was isolated for each sample. NRF2 exon 4 was PCR amplified from the genomic DNA and amplicons were Sanger sequenced and analyzed using DECODR to assess gene editing efficiency (EE%). In the A549 cells, editing efficiency in cells ranged from 71 -88% while in the H1703 cells, it ranged from 0-95%. RNA was converted to cDNA and the cDNA was used as a template for PCR amplification of the NRF2 transcript using primers designed in the 5’ UTR through exon 5 (amplicon size - 798 bp). Amplicons were then run on an agarose gel to assess exon skipping events. A full-length transcript will be 798 bp on the gel whereas an exon 4 skipped transcript will be 606 bp and an exon 3 and 4 skipped transcript will be 516 bp.As shown in the gel, each gRNA produces variable exon skipping evens at different frequencies.[01551 References

[0156] 1 . Banas, K. et al. Exon skipping induced by CRISPR-directed gene editing regulates the response to chemotherapy in non-small cell lung carcinoma cells. Gene Ther (2022) doi : 10.1038 / s41434-022-00324-7.

[0157] 2. Tuladhar, R. et al. CRISPR-Cas9-based mutagenesis frequently provokes on-target mRNA misregulation. Nat Commun 10, 1 -10 (2019).

[0158] 3. Bloh, K. et al. Deconvolution of Complex DNA Repair (DECODR): Establishing a Novel Deconvolution Algorithm for Comprehensive Analysis of CRISPR- Edited Sanger Sequencing Data. CRISPR J crispr.2020.0022 (2021 ) doi :10.1089 / crispr.2020.0022.

[0159] 4. Korrodi-Gregorio, L., Soto-Cerrato, V., Vitorino, R., Fardilha, M. & Perez- Tomas, R. From proteomic analysis to potential therapeutic targets: Functional profile of two lung cancer cell lines, A549 and SW900, widely studied in pre-clinical research. PLoS One VI , 165973 (2016).

[0160] 5. Lau, A., Tian, W., Whitman, S. A. & Zhang, D. D. The predicted molecular weight of Nrf2: It is what it is not. Antioxidants and Redox Signaling 18, 91 -93 (2013).

[0161] 6. Shinjo, T. et al. Propofol induces nuclear localization of Nrf2 under conditions of oxidative stress in cardiac H9c2 cells. PLoS ONE vol. 13 Preprint at https: / / doi.Org / 10.1371 / journal. pone.0196191 (2018).

[0162] 7. Kemmerer, Z. A., Ader, N. R., Mulroy, S. S. & Eggler, A. L. Comparison of human Nrf2 antibodies: A tale of two proteins. Toxicology Letters 238, 83-89 (2015).

[0163] 8. Tian, Y. et al. Modification of platinum sensitivity by KEAP1 / NRF2 signals in non-small cell lung cancer. Journal of Hematology and Oncology 9, 83 (2016).

[0164] 9. Apopa, P. L., He, X. & Ma, Q. Phosphorylation of Nrf2 in the transcription activation domain by casein kinase 2 (CK2) is critical for the nuclear translocation and transcription activation function of Nrf2 in IMR-32 neuroblastoma cells. Journal of

[0165] 10. Bialk, P., Wang, Y., Banas, K. & Kmiec, E. B. Functional Gene Knockout of NRF2 Increases Chemosensitivity of Human Lung Cancer A549 Cells in Vitro and in a Xenograft Mouse Model. Mol Ther Oncolytics 11 , 75-89 (2018).

[0166] 1 1 . Banas, K., Rivera-Torres, N., Bialk, P., Yoo, B. C. & Kmiec, E. B. Kinetics of nuclear uptake and site-specific DNA cleavage during CR IS PR-directed gene editing in solid tumor cells. Molecular Cancer Research 18, 891 -902 (2020).

[0167] 12. Homma, S. et al. Nrf2 Enhances Cell Proliferation and Resistance to Anticancer Drugs in Human Lung Cancer. Clinical Cancer Research 15, 3423-3432 (2009).

[0168] 13. Yeo et al. 2004, Proc. Natl. Acad. Sci. U.S.A. 101 (44):15700-15705.

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[0170] 15. Hovhannisyan and Carstens, 2005, Mol. Cell. Biol. 25(1 ):250-263;Minovitsky et al. 2005, Nucleic Acids Res. 330:714-724

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Claims

CLAIMSWhat is claimed is:1 . A method of identifying a guide RNA (gRNA) that induces exon skipping in a target gene comprising:(a) identifying one or more gRNAs that bind near or in an exon splicing enhancer (ESE) or an exon splicing silencer (ESS) in the target gene;(b) contacting the target gene with one of the gRNAs identified in step (a) and a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, whereby the gRNA hybridizes to the target gene and the CRISPR-associated endonuclease cleaves the gene;(c) repeating step (b) with another gRNA of step (a) until all gRNAs of step (a) have been contacted with the target gene;(d) determining a percent rate of exon skipping generated by each of the one or more gRNAs; and(e) correlating a phenotypic change in a protein expressed by the target gene with the percent rate of exon skipping generated by each of the one or more gRNAs.

2. The method of claim 1 , further comprising after step (e) the step of administering to a subject a gRNA of step (d) determined to have at least a 10% rate of inducing exon skipping that correlates with the phenotypic change of step (e).

3. The method of claim 1 or 2, wherein the one or more gRNAs each bind within 30 bases of the ESE or ESS.

4. A method of inducing exon skipping in a target gene comprising:(a) identifying one or more gRNAs that bind near or in an exon splicing enhancer (ESE) or an exon splicing silencer (ESS) in the target gene;(b) contacting the target gene with one of the gRNAs identified in step (a) and a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, whereby the gRNA hybridizes to the target gene and the CRISPR-associated endonuclease cleaves the gene;(c) repeating step (b) with another gRNA of step (a) until all gRNAs of step(a) have been contacted with the target gene;(d) determining a percent rate of exon skipping generated by each of the one or more gRNAs;(e) correlating a phenotypic change in a protein expressed by an exonskipped gene with the percent rate of exon skipping generated by each of the one or more gRNAs; and(f) administering to a subject the gRNA of step (d) determined to have at least a 10% rate of inducing exon skipping and at least a 10% rate of phenotypic change.

5. The method of claim 4, wherein the one or more gRNAs each bind within 30 bases of the ESE or ESS.

6. A method of inducing desirable splice variants of a target gene to control heterogeneity of a phenotypic outcome comprising:(a) identifying one or more gRNAs that bind near or in an exon splicing enhancer (ESE) or an exon splicing silencer (ESS) in the target gene;(b) contacting the target gene with one of the gRNAs identified in step (a) and a Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-associated endonuclease, whereby the gRNA hybridizes to the target gene and the CRISPR-associated endonuclease cleaves the gene;(c) repeating step (b) with another gRNA of step (a) until all gRNAs of step (a) have been contacted with the target gene;(d) determining a percent rate of exon skipping generated by each of the one or more gRNAs;(e) correlating a desirable phenotypic outcome generated by a protein expressed by an exon-skipped gene with the percent rate of exon skipping generated by each of the one or more gRNAs; and(f) administering to a subject the gRNA of step (d) determined to have at least a 10% rate of inducing exon skipping and correlates with an at least 10% change in phenotypic heterogeneity.

7. The method of claim 6, wherein the one or more gRNAs each bind within 30 bases of the ESE or ESS.

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