Ctnnb1 gene editing agents and uses thereof

Abstract

The present invention relates to gene editing agents (e.g., CRISPR / Cas) that target mutated oncogenes (e.g., a mutated oncogene a cancer is addicted to) and uses thereof (e.g., for inactivation of mutated oncogenes and / or treatment cancer). In some embodiments, the mutated oncogene is a mutated CTNNB1. In some embodiments, the cancer is hepatocellular carcinoma.

Description

[0001] CTNNB1 GENE EDITING AGENTS AND USES THEREOF

[0002] RELATED APPLICATION

[0003] This application claims the benefit under 35 U.S.C. § 119(e) to U.S. Provisional Application No. 63 / 613,962 filed on December 22, 2023, entitled “CTNNB1 GENE EDITING AGENTS AND USES THEREOF”, the entire contents of which are incorporated herein by reference.

[0004] REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0005] The content of the electronic sequence listing (J035670001WO00-SEQ-ZJG.xml; Size: 316,080 bytes; and Date of Creation: December 19, 2024) is herein incorporated by reference in its entirety.

[0006] BACKGROUND

[0007] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR- associated (Cas) genes, collectively known as CRISPR-Cas or CRISPR / Cas systems, are adaptive immune systems in archaea and bacteria that defend particular species against foreign genetic elements. CRISPR-Cas systems have been demonstrated to be suitable for repurposing for gene editing, including in eukaryotic cells.

[0008] SUMMARY

[0009] Mutations (e.g., gain-of-function mutations) in a gene (e.g., a human gene), can lead to various diseases (e.g., human diseases) such as cancer and genetic disorders. Such mutations (e.g., gain-of-function mutations) may be exploited as targets of therapeutic agents for disease(s) associated with the mutations. For example, gene editing agents (e.g., the CRISPR / Cas system) can be designed to specifically target the mutations to inactivate (e.g., knockdown or knockout) a mutated gene, and / or to correct the mutation (e.g., restore the mutated sequence to wild- type sequence).

[0010] Cancer cells may contain multiple genetic and epigenetic abnormalities. In some instances, mutations in a single oncogene can lead to cancer, and the growth and survival of the cancer can often be impaired by the inactivation of such mutated oncogene, despite the involvement of multiple genetic and epigenetic abnormalities in the process of cancer development (“oncogene addiction”).

[0011] Strategies for inactivating such mutated oncogenes, e.g., by knocking it down or knocking it out using gene editing agents (e.g., the CRISPR / Cas system) may be therapeutically efficacious for cancer (e.g., cancer addicted to a mutated oncogene). Sitespecific gene editing agents targeting the sequence(s) containing the mutation(s) in the oncogenes may be engineered such that unintended editing in normal cells that do not contain the mutation are reduced and / or minimized. In some aspects, gene editing agents and methods described herein are for targeting gain-of-function mutations in genes (e.g., oncogenes) and for treating cancers addicted to the mutated oncogenes with minimized off target effect and low toxicity to normal cells.

[0012] Deregulated Wnt / p-catenin signaling is one of the main genetic alterations in human hepatocellular carcinoma (HCC). Comprehensive genomic analyses have revealed that gain-of- function mutations of CTNNB1, which encodes P-catenin, occur frequently in HCC. Over 20% of HCC contain CTNNB1 mutations. Human HCCs with activation of the Wnt / p-catenin pathway demonstrate unique gene expression patterns and pathological features. Activated Wnt / p-catenin synergizes with multiple signaling cascades to drive HCC formation, and it functions through its downstream effectors. Human HCC harboring activating CTNNB1 mutation also show dependency (addiction) to the mutated CTNNB1. Compositions and methods described herein that target mutated CTNNB1 for inactivation via gene editing were demonstrated herein to be efficacious in inhibiting the growth of HCC harboring CTNNB1 with the targeted mutation.

[0013] Some aspects of the present disclosure provide guide RNAs (gRNAs) comprising a crRNA targeting a sequence of CTNNB1 , wherein the sequence of CTNNB1 comprises a mutation associated with cancer. In some embodiments, the mutation is a gain-of-function mutation. In some embodiments, the cancer is addicted to the CTNNB1 comprising the mutation.

[0014] In some embodiments, the sequence of CTNNBlis double- stranded and comprises a complementary strand and a target strand. In some embodiments, the crRNA comprises a spacer comprising a region of complementarity to the complementary strand. In some embodiments, the mutation is a single nucleotide mutation. In some embodiments, the mutation is any one of the mutations listed in Table 1. In some embodiments, the single nucleotide mutation results in an amino acid substitution in a P-catenin protein encoded by CTNNB1. In some embodiments, the amino acid substitution in the P-catenin protein is D32G, D32V, D32N, D32Y, S33C, S33F, S33P, S33Y, G34E, G34V, I35S, H36P, S37F, S37C, S37A, T41A, T41I, S45P, S45F, S45Y, L337I, C621Y, S33A, S37P, R589Q, G34R, K337T, E336K, R537Q, S45C, D32H, or K337I. In some embodiments, the spacer is 17-20 nucleotides in length. In some embodiments, the spacer comprises the nucleotide sequence of any one of SEQ ID NOs: 14-82 or 227-255. In some embodiments, a protospacer adjacent motif (PAM) is located in the target strand and at the 3’ side of a target sequence. In some embodiments, the PAM is of a 1-20 nucleotides distance to the mutation. In some embodiments, the mutation creates a PAM. In some embodiments, the crRNA further comprises a direct repeat component. In some embodiments, the gRNA further comprises a tracrRNA.

[0015] Complexes comprising any one of the gRNAs described herein and an RNA-guided nuclease are also provided.

[0016] Further provided herein are nucleic acid molecules comprising a nucleotide sequence encoding any one of the gRNAs described herein. In some embodiments, the nucleic acid molecule further comprises a nucleotide sequence encoding an RNA-guided nuclease. In some embodiments, the nucleotide sequence encoding the gRNA and / or the nucleotide sequence encoding the RNA-guided nuclease is operably linked to a promoter. In some embodiments, the nucleic acid molecule is a vector, optionally wherein the nucleic acid molecule is a lentiviral vector.

[0017] Cells comprising any one of the gRNAs, complexes, nucleic acid molecules described herein are also provided. In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a hepatocyte, hepatocellular adenoma, or a hepatocellular carcinoma cell.

[0018] Compositions comprising a nucleic acid molecule comprising a nucleotide sequence encoding an RNA-guided nuclease, and any one of the gRNAs described herein are also provided. In some embodiments, the nucleic acid molecule is a vector or an mRNA molecule. In some embodiments, the composition is formulated in a lipid nanoparticle (LNP).

[0019] In some embodiments, the RNA-guided nuclease is Cas9.

[0020] Other aspects of the present disclosure provide methods of cleaving a target nucleic acid in a cell, the method comprising contacting the cell with any one of the complexes, the nucleic acid molecules, or the compositions described herein, wherein the target nucleic acid is a mutated CTNNB1. Further provided herein are methods of reducing the expression and / or activity of a mutated CTNNB1 in a cell, the method comprising contacting the cell any one of the complexes, the nucleic acid molecules, or the compositions described herein. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is a hepatocellular carcinoma cell. In some embodiments, the cell is a hepatocellular adenoma cell. In some embodiments, the cell is in vitro (e.g., cultured). In some embodiments, the cell is in vivo (e.g., in a subject). Further provided herein are methods of reducing the expression and / or activity of a mutated CTNNB1 in a subject, the method comprising administering to the subject any one of the complexes, the nucleic acid molecules, or the compositions described herein.

[0021] Further provided herein are method of treating cancer (e.g., hepatocellular carcinoma or hepatocellular adenoma) in a subject, the method comprising administering to the subject any one of the complexes, the nucleic acid molecules, or the compositions described herein, wherein the cancer is associated with a mutation of CTNNB1. In some embodiments, the hepatocellular carcinoma is a primary cancer. In some embodiments, the hepatocellular carcinoma is metastasized from a primary cancer, optionally wherein the primary cancer is colorectal cancer. In some embodiments, the RNA-guided nuclease introduces a double strand break in CTNNB1, thereby cleaving CTNNB1. In some embodiments, cleavage of CTNNB1 inhibits the growth and / or progression of the cancer (e.g., hepatocellular carcinoma). In some embodiments, the subject is human. In some embodiments, the administration is systemic. In some embodiments, the administration is local.

[0022] BRIEF DESCRIPTION OF THE DRAWINGS

[0023] FIGs. 1A-1B are graphs depicting the results of an experiment to identify a set of oncogenic-mutation targeting Cas9 / crRNA sequences that deplete cancer cells harboring mutations in the CTNNB1 gene. Mean log fold change (“LFC”) of gRNA abundance from Day 2, normalized to the nontargeting gRNA is shown in FIG. 1A. An LFC value of 0 indicates that no cleavage occurred. A gRNA was categorized as off target if it exhibited an LFC less than -0.5 in any cell line without the targeted mutation. Guide RNAs were identified as candidates if they were not off target and depleted the targeted cell line (a cell line with the targeted mutation) with log fold change less than -1. Guide RNAs showing depletion in nontargeted cell lines were removed from the data set, as shown in FIG. IB. Cell lines LS41 IN (no mutation in CTNNB1 and no amino acid substitution in P-catenin), SNU449 (no mutation in CTNNB1 and no amino acid substitution in P-catenin), and SNU398 (harboring S37C substitution in P-catenin), SW1573 (harboring S37F substitution in P-catenin) were tested.

[0024] FIGs. 2A-2B are graphs depicting the results of an experiment testing whether gRNA- 58 (spacer sequence CUGGACUCUGGAAUCCAUUG; SEQ ID NO: 71), which targets a mutation in CTNNB1 that results in a S37C substitution in P-catenin, present in the SNU398 cell line, can deplete cancer cells harboring mutations in the CTNNB1 gene. Mean log fold change (“LFC”) of gRNA abundance is shown in FIG. 2A. The viability of the cell line after exposure to gRNA-58 is shown in FIG. 2B. gRNA-58 selectively reduced cell viability of SNU398 cells, which harbors the targeted S37C substitution in P-catenin.

[0025] FIG. 3 is a graph depicting the results of an experiment to identify a set of oncogenic- mutation targeting Cas9 / crRNA sequences that deplete cancer cells harboring mutations in the CTNNB1 gene. Mean log fold change (“LFC”) of gRNA abundance from Day 2, normalized to the nontargeting gRNA is shown. An LFC value of 0 indicates that no cleavage occurred. A gRNA was categorized as off target if it exhibited an LFC less than -0.5 in any cell line without the targeted mutation. Guide RNAs were identified as candidates if they were not off target and depleted the targeted cell line (a cell line with the targeted mutation) with log fold change less than -1. Cell lines LS411N (no mutation in CTNNB1 and no amino acid substitution in P-catenin), SNU449 (no mutation in CTNNB1 and no amino acid substitution in P-catenin), and SNU398 (harboring S37C substitution in P-catenin), SW1573 (harboring S37F substitution in P-catenin), and NCIH295R (harboring S45P substitution in P-catenin) were tested.

[0026] FIG. 4 is a graph depicting the results of an experiment testing whether gRNAs targeting a mutation in CTNNB1 that results in a S45P substitution in P-catenin, present in the NCIH295R cell line, can deplete cancer cells harboring mutations in the CTNNB1 gene. Mean log fold change (“LFC”) of gRNA abundance is shown. The three S45P targeting gRNAs tested are gRNA-2 (spacer sequence GCUCCUCCUCUGAGUGGUAA; SEQ ID NO: 15); gRNA- 3 (spacer sequence CAGAGGAGGAGCUGUGGUAG; SEQ ID NO: 16); and gRNA-6 (spacer sequence GGAUUGCCUUUACCACUCAG; SEQ ID NO: 19), all of which selectively depleted NCIH295R cells, which harbors the targeted S45P substitution in P- catenin. gRNA-58 was used as a control, which selectively depleted SNU398 cells, which harbors the targeted S37C substitution in P-catenin.

[0027] FIGs. 5A-5G are graphs depicting the results of screening gRNA libraries targeting CTNNB1. The fold change is shown in targeted cell lines, nondependent lines, and other dependent lines for gRNA sequences targeting CTNNB1 amino acid mutations: G34E (gRNAs 48 and 49, FIG. 5A), S33F (gRNAs 67-69, FIG. 5B), S33Y (gRNAs 45-47, FIG. 5C), S37A (gRNAs 61-63, FIG. 5D), S37C (gRNAs 58-60, FIG. 5E), S45F (gRNAs 27-31, FIG. 5F), and S45P (gRNAs 1-6, FIG. 5G).

[0028] FIGs. 6A-6B show CTNNB1 S45P and CTNNB1 S37C genetic POC using somatically engineered HCC mouse models. Micrographs showing livers from mice treated with gRNA targeting CTNNB1 amino acid mutations S45P or S37C are shown in FIG. 6A. Graphs showing the percent (%) weight change and liver weight: body weight (LW:BW) ratio in mice treated with gRNA targeting CTNNB 1 amino acid mutations S45P or S37C are shown in FIG. 6B. Dashed square and circle indicate a mouse with a failed HDT injection. Welch’s t-test was used for statistical analysis.

[0029] FIGs. 7A-7D show the tumor suppression activity of Cas9 mRNA and CTNNB 1 S37C gRNA delivered in a lipid nanoparticle (LNP) in a hepatocellular carcinoma (HCC) mouse model. Graphs showing liver weight:body weight measurements at day 16 (LW:BW) are shown in FIG. 7A. Imaging of livers on day 16 is shown in FIG. 7B. Individual Welch’s t-tests showed significant difference between the endpoint liver weight to body weight ratio of treated and control groups. Micrographs showing H&E staining on liver sections from mice 2-6 for PBS, NTC and LNP1 groups are shown in FIG. 7C. The ImageJ mask to quantify tumor burden is shown on the right side of FIG. 7C. Treatment with Cas9 mRNA and CTNNB 1 S37C gRNA delivered via LNP decreases tumor area relative to controls (NT and PBS) as shown in FIG. 7D. ANOVA with brown-Forsyth multiple comparisons was performed for both LW:BW and H&E analyses.

[0030] DEFINITIONS

[0031] The present invention will be described with respect to particular embodiments, but the invention is not limited thereto but only by the claims. Terms as set forth hereinafter are generally to be understood in their common sense unless indicated otherwise.

[0032] “Oncogene”: As used herein, the term oncogene” refers to a gene that is abnormally expressed or mutated and which under certain conditions is capable of inducing cell transformation, which may result in transforming the cell into a tumor cell and / or causing cancer. In some embodiments, an oncogene can be a gene that is overexpressed in cancer cell (e.g., overexpression due to increase of copy number of the gene) and transmits a signal for proliferation excessively, or a gene in which a mutation occurs which continuously transmit a proliferation signal in the cancer cell. A mutation in a gene (e.g., an oncogene) may include point mutation (e.g., substitution), deletion, addition, insertion, and mutation causing a fusion (e.g., inversion, translocation). Non-limiting examples of oncogenes include: CTNNB1 (encoding P-Catenin), KRAS (encoding KRAS proto-oncogene, GTPase), BRAF (encoding B- Raf proto-oncogene, serine / threonine kinase), EGFR (encoding epidermal growth factor receptor), NRAS (NRAS proto-oncogene, GTPase), PIK3CA (Phosphatidylinositol-4,5- bisphosphate 3-kinase catalytic subunit alpha), and ERBB2 (ERB-B2 receptor tyrosine kinase 2). “Oncogene addiction”: As used herein, the term “oncogene addiction” refers to a phenomenon that cancers (e.g., human cancer), which are typically driven by the progressive accumulation of mutations and epigenetic abnormalities in expression of multiple genes that have highly diverse functions, can be inhibited by reversal of only one or a few of these abnormalities (e.g., a mutated oncogene). Removing an oncogene a cancer is addicted to can be therapeutically effective in treating cancer. Oncogenes different cancers are addicted to have been described, e.g., in Weinstein et al., Cancer Res (2008) 68 (9): 3077-3080, incorporated herein by reference in its entirety.

[0033] “Mutation”: As used herein, the term “mutation” refers to a modification (e.g., substitution, deletion, insertion) of at least one nucleotide or amino acids in a DNA or polypeptide sequence relative to a naturally occurring DNA or polypeptide sequence (e.g., a wild-type DNA or polypeptide sequence such as those listed in Table 1). In some embodiments, a mutation includes, without limitation, point mutation (e.g., substitution), deletion, addition, insertion, and mutation causing a fusion (e.g., inversion, translocation). A mutation may be a missense mutation or a nonsense mutation. A mutation is associated with cancer if there is an increase in the likelihood of developing cancer in the presence of the mutation, relative to when the mutation is absent. The same mutation may be associated with different types of cancers, and a type of cancer may be associated with one or more mutations.

[0034] A mutation may result in a gain-of-function, loss-of function, silence of a gene, or overexpression / activation of a gene. As described herein, a gain-of-function mutation is a type of mutation in which the altered gene product, such as a protein, has new molecular functions, a new pattern of gene expression, and / or new biological effects. As described herein, a loss-of- function mutation is a type of mutation in which the altered gene product does not have the molecular function of the naturally occurring gene product. In some embodiments, a mutation targeted by a gene editing agent described herein is a gain-of-function mutation.

[0035] “CTNNB1”: As used herein, “CTNNB1” refers to the oncogene CTNNB1. “Catenin beta-1”, “P-catenin”, and “beta-catenin” refer to a protein that is encoded by the CTNNB1 gene. P-catenin is a member of the catenin protein family. The domain structure of the P- catenin protein is having a N-terminal intrinsically disordered region, an Armadillo repeat domain (ARM) with armadillo repeats about 40 amino acids long, a transactivator motif(s) and a C-terminal region. P-catenin is involved in regulation and coordination of cell-cell adhesion and gene transcription. In humans, the P-catenin protein is encoded by the CTNNB1 gene and in Drosophila, the homologous protein is called armadillo. P-catenin is a subunit of the cadherin protein complex and acts as an intracellular signal transducer in the Wnt signaling pathway. P-catenin is expressed, at least, in adrenal, appendix, bone marrow, brain, colon, duodenum, endometrium, esophagus, fat, gall bladder, heart, kidney, liver, lung, lymph node, ovary, pancreas, placenta, prostate, salivary gland, skin, small intestine, spleen, stomach, testis, thyroid, and urinary bladder tissues.

[0036] P-catenin is widely expressed in many tissues. Mutations of P-catenin are commonly found in a variety of cancers: in primary hepatocellular carcinoma, colorectal cancer, endometrial cancer, ovarian carcinoma, breast cancer, lung cancer and glioblastoma. It has been estimated that approximately 10% of all tissue samples sequenced from all cancers display mutations in the CTNNB1 gene. MYC-driven lymphomas frequently acquire activating mutations in P-catenin, including a previously unreported mutation in a splice acceptor site. Tumors with these genetic lesions are highly dependent on P-catenin for their survival. See e.g., Choi, Peter S et al., Proceedings of the National Academy of Sciences of the United States of America vol. 111,32 (2014): E3316-24, the entire contents of which are incorporated herein by reference.

[0037] A nonlimiting example of a human CTNNB1 gene sequence is provided in Gene ID: 1499. A nonlimiting example of human CTNNB1 gene sequence is provided in Homo sapiens chromosome 3, GRCh38.pl4 Primary Assembly: NC_000003.12: 41199505..41240443 (SEQ ID NO: 4). Nonlimiting examples of cDNA or mRNA sequences of CTNNB1 are provided in NCBI Accession numbers NM_001904.4, NM_001098209.2, NM_001098210.2, NM_001330729.2. Non-limiting examples of P-catenin amino acid sequences are provided in NCBI accession numbers NP_001091679.1, NP_001091680.1, NP_001317658.1, NP_001895.1.

[0038] Human CTNNB1 gene sequence (NC_000003.12: 41199505..41240443; SEQ ID NO: 4)

[0039] AAGCCTCTCGGTCTGTGGCAGCAGCGTTGGCCCGGCCCCGGGAGCGGAGAGCGAGGGGAGGCGGA

[0040] GACGGAGGAAGGTCTGAGGAGCAGCTTCAGTCCCCGCCGAGCCGCCACCGCAGGTCGAGGACGGTC

[0041] GGACTCCCGCGGCGGGAGGAGCCTGTTCCCCTGAGGTGCTTGGGCGCTCCTTTCCTTATCCTTCCGG

[0042] GGCTGCTCCCGCTTCCTCTCGGAGCCAAACTTCGTAGCAGGCGCGCGGTCCGGGCGGCGGGCTGGGC

[0043] GCAGCCGGGAGGCCTGGGGTTGGGAGCGGGGAGCTCAGGTGGGGGACGGTGAGGGTGGGCCGCGC

[0044] CCGGGGCGCGGAGGGCGGCGGCCGGGCCCGGGTTCCGGTCGCGCTGCCTCTCTGGGGCCCTGGGGG

[0045] CATCGCTTGCGGGGAGGGGGCGCCGCGGGGGCGCGTACAGGAGCCCGGATGGCAGGCGGGGTGGG

[0046] GGTGGGGGTGGGGGTCTGTGGTTTCCGTCCGGGGCTCTGGCCTTGGCCGAGTTTGGGGGAGGGACCC

[0047] GGTGCCTCGGGATGCGCCGGGCCCTGGGTGGGGGGCGGGGTGGGGACGGGGGGCTCCGCCTTCTCA

[0048] GCTCTTGCGGCGAGTTGGGGTTCGGGCGCTGAGGCAGAGACGCCACCCTAAGTCCCATCAGTCCTGG

[0049] GGATCGGACCAGTGGACTTTCTCTTAAGATTTCCTCTTTCATTCTTAAGAATAGAAGTGTTATTATTT

[0050] TTTTTAATGCCCTGGCTATGTGAGTTTGAATCGAAGCAACTTTAAACCTTAGAGCAACTAAACTCTA

[0051] AGTGCAGCGGGTGCGATGCGTCAGTAGGGTGAGCACATAAAAAATCCATGTCTTGCACCTGTATTTT

[0052] AGCGTACTATGCAGGTGAGTGAAAGCAGTGGATAATGTACTGGGAGTCTTATGGATTTATGGTAGTG

[0053] GGTATGAGACCCTGGTGAAATAAGGGGGTGGAGGAAGGCGAAGGTGATGGCTTACTGTTTCTTACC

[0054] AAGTGAACTGCAGGATTCAGCCTCTGACTCAGACCGCTTCGAGAATTTTGTTCGTAGAAATAATTTA

[0055] AATTTATTCAAATAGTTTGATGGCAGCTAAAATTGAATTATAGAGCACGTTTTCTTTTCAGCGGAGT

[0056] GAATTTTTCCTTCGCTCCAAAGCTGGCCAAATGGAATTCAAGCATTGCAACTTCTTTCAGTGTTTTGT CTGGAGAGAGGACTTTGAACCGAGACTTTTCGAAGTTAAGTTCCTATAGCCTGCTTCTGAATCTGCC AAGCTTGAAAGCTTTGGCAGTTGGGTGTATGTAGTTGTTGCCTTCGTTCTCTTCCCTTTTGGAGGGAG CGTTGTCTCCTACTTTGTATCTTCCAGACATCTGTGGTCTTCCCCCCACCCCTCGAGTTTGTGAGTGGT GAATGAAGAAAGACTAGGCTGCTGGTATGCAGAGGTCGGCAAAAGGAAATCGAGGAGTGGTTTTAG TGAAATGAGAGCTTTGTATCATGAATAATGGTGGCTTAGGCTAGACATCAACTTGAAGAGACGGCA GCATTTCCTTTCATAAAGTCTAGGCTAATGTTTTTCAGATCGCTAAGTTGTAGTTTGTCTGGAATTTA GGAAGCCATTTCAGTATTTGTCACTTGGTGAACGAACATTCAATACCTTCAGATGTCTTCGTGTTGAC TTGTATTCATCCTAAGAAATAGTAAATATAGTCTCAAGTGTTATTTATGTTATACTGCTGGTTTATTC TCTGCTTAAATTATTGACATAAATTTCTACTTTGGAGGCTTTTCGTTTGAACTAAGGCTGTGCGGAAT TTATTTTACTTTTATATTTAAATCTTTGAAAAATCTCTGATTAAAAAAAAAGTACCCTTAAAGGTTTG AGGATGTCCTTTCACACCAGACAAAATTTGGTTAATTTGCGCCCAATATTCATTACTTTGACCTAACC TTTGTTCTGAAGGCCGTGTACAAGGACAAGGCCCTGAGATTATTGCAACAGTAACTTGAAAAACTTT CAGAAGTCTATTCTGTAGGATTAAAGGAATGCTGAGACTATTCAAGTTTGAAGTCCTGGGGGTGGGG AAAAATAAAAAACCTGTGCTAGAAAGCTTAGTATAGCATGTAACTTTAGAGTCCTGTGGAGTCCTGA GTCTCCCACAGACCAGAACAGTCATTTAAAAGTTTTCAGGAAAAACCAACTTAAAAAAAAATAAGG TGGCTAATTAAAAAAAAATGAAGCATTTAACAGTGTTCAGGTTTCAGAGTATGGAAGAGGGGTTTTT TAAACTGTTATCTGATTATTTCTTTTACCAACATGATATAGAAAAGTGTATTTCCAGTATTAAAATTT ATCAGACTGAGCTTACTGTTCCTGTTAATGACTGGAATAAAAATTGGCATAAATGAGGGTCTGTATG CTTGTTTTAATAACACCACCACCAAGATAGAAAACGAGGAGGCAAGTTTCTCCAAGGGTATTTTGAA ATGTGTTAGCAAAACTATTGCAGATACTCGTTTTTGTTATAGGGTGAGGTGGGGAGAGGCGCATGCT AAGTATTGTTGAAACTAGGGATGTAGAGAATTAAAAGTTTGAATATAATTATTTTGTAGTTATAAGT AGCAGTGAAATTAAATCTCCTGCAATAGACTATAGAAGTATATTTAGCCAAATGAAACTTCAGTGTT ATTGAAATGAAATAATACATCTGTCCTGTTACAAGATTATTTTTATTTCTCTTGTGGTTTCCTAGCTTC TGATAATCAATAATTGTAGATGAGTAGGTGGTAAGTTTTAAGTTTGTACTTTGAGCTTAGTCGGAAG CATGCTTGACTGCCAACCCGGGGCACAAAGGATGAAGGCTTTTAGAACTGGACAAACTTCTAACAA AAGGTATTTGCAACTCTTTTGTAGTGTGTCATGTTGATTTGTGACATTGTTTTTGAAAATATGTGTTA ACTTAGTTTTCTTGTAGCCCTCTTTTTATTGGAACTGTGGTATCTATTGTTGAAACTGCTTGACTGAG AACATTTTTATACCATAAAAGTAAATAGTAAACATAGCCCAGGAGCGGCTTCTGGTTTGTCCATCGT ATGTAGCCATTGCCTCCTTGTACTCTCATTGAGAAGATACTGATTTGCAGATTCAGTTGTCCTTCTCT AACAGACTATTTATGTAATATTGCAGTTGTGATTGTGATAGGTAAGTGGACCAGTCGGTTAAAATAA ATACTCAGGTTTCACAAAAGGAAAATAATATGATTTGTGTTGATCTAAATGAGTATAGGAGTTAACT CCTATAGTTTTTCATCACTTAAACTCAGGGGAAAGTTCTTTATTTCCTCTGTTTACTTAAGAATGCTG CTTTTGTGTTTCATGCAAGACTGAGCTTGACTCAGTTTGAAACCTAGGCTCATCTGTTGAGGCCTGAA CCCTGCTGTCCTTGAAGTATGCATATAATTTGCTTCCTTCCTAAGGAAAAATAAGCTCTTGAAAGAT AAAGTCAATCACATTAGGAACCCATTTTTAGGGTTTAGCCACTTTTTTTTTTTTTTTTTTTTAACTCAT GGGCATCTCTTCTGTTAAGAGACATTCCCCACTCTCCAAGTTTCCCTCAAGCCTGAAGCAGCAGAGT GAGTAGTGTTGGAGCATGTTTTCATTGCATGCTTGGGTCATGTTGAGTGCCCTCCAGTGGATATAGT ATAATGCTTGTGATTTTTTTTTTTTTAATTCCAAACAAGTTTATGTGGGATATATTTAGGAATAGTTCT GATGAGGGAGAATCAACTAAGAAACCTTTGATTTCTAAAATAATTAATATCATTACTGCTAATTAAA ATACAGGCTTGAGAAAATGTCTTCTCAGCCAATATTTGCAGTAGAAAAGTCGGGAGGTTTTTTAAGG TCACTTTGAGTAGGCAGTTCTGCTTAAATATATCATAATGATAAACCAGAATCTCAGTATAGTACTTT AGGAGGTAAAAGATCATAATATTCAGTTATATTGATGAATTACAGCAACTGAAATTCTCAGAAAAA

[0057] AATTAATGAAAATGTGAATTGTCAATTTGTCTAAAATCATTCACAGAGTAAAACATAAGTGCTCAAC TTGATTATATTAGGAAATAGATAGAAATAAAGGTAATTGAGCCAGTGTATGTGACCTAAAATATAAT GCCCTTAGTGACCATAGGGTTGGTCTCATTTGTACATAGTGGTGGGCCATGATGAACTGTGTTTTGCC CTTTGAATTTTTCCTTAAAAAGCTTTCTCTAGGCTCCTATGTTCATGGTTTTTCTGTTAGTAATATTAT TTTCTGAAAATCCATGTTTCAAATCAGAATCTAATTAGCAACAGGAATGAAGCTTATTCTAAATTAG TTTTTGGAAGTTAAACGGTCAGCATATGGAAATTTTTCAGGGTTTAGATTTTTAAAAATTTGTTTTTC AGAATATGTTGCTGGAATGAAAACGTTAGCGTAGGGACGGAAAATGACACTTACCAGTGATTGCTT TACTTTGCCTGTGGAATTCAGTGTAATTTTGTGGAAACATTGGTATATGATTTTTTACTACTTAAGAA ATGTATTGCTATAGTTAGGGTTTTTTTTTTTTTAAAGGCAAGAATGCCTCAAGTGCTTTATGTGAATG ATTATTTCAGGATGGATTAAATATTCCTCCATCAAGGACCATACTTGTAAATCAGTGATTTCCAAGTT GGTGCTTAGTATTTACAGCATTTACTGTCTATAAGCTTCTGTTCTGATTTTTCAAGAGTTTTCTGAGA AATGAGAGTAGGCTTAAAAGTTCTTTGAAAAATTATGTACATACAACTTACTGAAAAAAATTGCTAC CGGGGACTTAATTTGTCTCTTGAAATGGGCTACTTGCCTTCATTAATGTAGCATACTACAATTTGATG TTCAAGATATGTTACTAAGAATAAGATCGCTTTCAGAAGCCTTATATAGGATTGGTCTTACTACATT GTAGTGGGAATGGCTACTCAAATGTCTCCAGGGCCAGTTAGGTATTGGGTAAATGGGACCATGCAG ACTATTAAAAATTGAAGTGCACATGAAGCAGCCAGTCATAAGCAGCTCCAGCCACTGTGTGGGAAT ATAGTTTATGTTGCCAGATCATCTGATTTCTTTCCCCTAAGTGGGAAATCCAGATCAATGTACATCTC TTGATTTGCAAGTGTTGGTGAACAAAATTCATATTTTAAGATGCTGTATTCAGCACAAATTAAATAC ACTTATTTGCTGAATACTGCCAGTTTGTCCCTCTGCAGTAGTACCATTTGAAGTACAGTGTTTTCATA ATGATTCTGTGAAATGACTGGTTCTGTGAATGTACATAATTTAGCAGATAACATTGTTAAATTATTA GGTTTGTATTTATTTAGGCACTTGGGAAATGCCTTGTGTCAATTGATTATAGATTAGGAGCTTAAAA GCAAGATTTATATTATCAACTTATTTGTGAAGACTGGGAAACCCACATTTTTAAAGTTAGGAATTAA GATGGCCAGGTTCAAGGAAAAGGGGGAGAAGTAACTTTCTTATTACTCAACCATCTTAAATAGAGTT CTTTAAGTGTATTTTTAAGAGGTCTCAAAACTTAATCTGAAGGGACGTCAAATGCTGGACAAATTCT GTGTATACAACTCAAGTCAGCCCCCAATTTTACTGGTCTTTAAATCATGTCCTTTTTACCAGAAGTTT GCATTTCTAAGCTAAACTATTACTGTTAGACTAGATCCAAAACTTAAAAACAGTTTAGGTAATTAAA AATTAATTGAATATAAACGTTTTACTTAAATTAATGGCAAATGGCTTTTTGGCCAATTTAAGTTTATG TAGGCAGTTAAATCGATTTTGGTTAAATCTTTTGCTGCTAACAAGGTATTTCCAGATTTTGAAAAGTG GGGTGGCCTGGTGCCTGTAGTACCAGCACTTTGGGAGGCTGGGGAGGGTGGATCACCTGAGGTCAG GAGTTCGAGACTAGCCTGGCCGACGTGGTGAATACAAAAATTAGCCAGGCATGGTGGCAGGTGCCT GTGATCCCAGCTGCTTGGAAGTCTGAAGCATGAGAATTGCTTGAACCTGGGAAGCGGAGGTTGCAG TGAGCTGAGATCACGCCACTGCACTCCAGCTGGGGCAACAGAGCGAGACTCCATCTCAAGAAAGAA AAGTGGGGTGTTTAGTCTTCAAACTCCGTGTTTAAGTGACTGGAGTGAAAATGTAAATCATAGGCCG GTGTTGGTTTAAAAAGCATCATCTGAAAATAATGCTGTAGTCTGCAATTATTTTTATTACGATACGAT GGTGTAAAATACAAGCAGATCAGTGAACCATTCATGAAACATTAATCCTAAAGGCGTCTCACCCCA AGTCTATCCCACAATCTCCATGAGACTTCGTGGAACCACTGTAAAGTTTCTTGTGTAATATCCCAGA AGTTTCCTACCTCTGGTATCTTTTGAACTTGTTGAAAAGGCTTTTCCACCCCCTCTTTATGATGGTTTG AAGAGTGTGAACATCTGAATGATGCTGGGGTGAAACTGCTTCATAACACTTCCATTTTCTCCCCTATT TATTTCCATATTTTTATTTTTTCACTAATATCCCCACGGTTTTACTTCTGTTTTAGTAATTCACATGTTG CTGGACTAATTCTTTTTAACTGACTTGTAACAGATATGTTAAACCGTTTAAAACTTGGGGGGTATTTT TAACCTACTTTAAGTTAGTTCAAGTTAATCAGTCTACATGGCATATAAACCTTATGATTAATAAATCT TAAATGCTGGTAGCTGAGTTGGAAGCCAAAGACGTACAAAAAAGCTGAAGTGTTAGGTTTAGTGTG ATAAGCTTCTCTTACTAACAGGGTTTTGTAATAGCAGAAATAGATATATGCATATATATGTGCATAT ATATAGCATACCTTATTGGATGTCCATATAAAAATGTGTAAGAAGTTAAATTTACTGCAAAATTTCT TGGGAGTGCAATTTGAAGATGATCTTAAGTGGTGATAGTAGTTTGCTACACTGGGGGATAGTTGTTG CAAACTGCTCCTAATTTTCCTTTACTGTGAAGTAAACTGAACAGCTGTAATAGGGATTAGGAACTGT ACTCCCTCTCTCTCTTTTTTAAGTATAATTAAGTGGTTTTGGGGTAAGGGTGTAGGGAGTGAGTGTCT TAT TG TA TTA TG CT AT AT TT CG CC AA ATA GT TA AC AT CA TG TTA GT GG AA GA GTG CTC TC CA CC AA AT GG TT GA GTA AA GG TTG CG AA TG TG CA CA CC CA TGA TG GG TG AA GT GT ACT AT AG GG TA GA CA TGT GGGTAGACCCTTAAATTCCTTTCTGAGCCATTGAAAGAATGTCCTCAAACTTCGCTTATACTTTATAG TTCATTTAGATACAAAAGTTACAAACTGAATGCTATTTAGGAAACGTAATACACTGACATACCGCTC TTTAAATAGATTATAAATTTAGTATATCAATTTTCTGGCATTTTGCTGAATTTTATTGTTTAGTTTTCA AGCCCAACTATCTTGTTACTTTGTATATCGTAGTTGTCCCCCGTTGATCACTGTTTCCTGCTTAATTGT GCTGTCGTTTTTCCTGGGTCCTGATTCAGAGTGTCAGCATTCTGTTCCCCATAGAATAAGAAGAGGCT AGAAAGTTTACAGATGAGATATCTAGGAATGCCAGAAGATCAGGGGTCACCGTTGAGGCAGAGTAA TTAATTATGGTTAAAATGGTGTTGCTGATAAGTGGGTGCTGGGAAATAATTAAAATTTGATTTTTTA GAAGAATACTTCTCATGCTTGAAGAGCGCCCTCATTATATGCTAAAGGGCCTCAGGTTTTTCCTTATT GCCATTATGCTGCAGATTCTATTACATTTGTCTGAAAAGATCTAAGACAGAAGGGCTGTTTAATACC TTCCCTTTTCTCCTGAACTTCCCCTCTCCTCTCCCCCATCAGGAGCTAAGTAGGAACCCCTTCACCTT GTTACCATCAGATTTCATCAATGGTCTGTCTTTACAATGAAGGAAGTAGTACTGCATTCTGGGCAGA GGCCAGTCCTGAGGCATGCCTTTTCAAGGACATTGTTACTTTAGTTACACTGGCTCTTCTGTTTTAAC TCTTATCCCCCAGACTCTAATCCTGTTGCTTTTTTTGGTCCCCATCTCCCACCTTTCATCATCTGAAAT CCATTCATTGTAACTTCTGGAACTCAGTCGTTAGAAAATCCTTTATATTCTCAATCTTGTGAATGTTC

[0058] CTTTCTTTCTTATTCCAGCTGTAACCTAGCCTTCTCCCCAAGAATGCTACTTCCCTTGCAGCTCTCTCA AGTGGTGAATTTTTCCCTTCTTGCACACCTTATAACACTGAACTAGGAGGTGTGTGGACTAAATGTCT GCTTTTGTTCCTTATTGTCACTTCTTGACCTTTATTTTCCAAAACTTCAAGCTTTGACTTTCATGTGAT CAAATTATACCACCCACTGCCTGTCTTTATTTCAAGCACCTGCAAACCTTCCTGGGTCATTCACATCC TTCTTTGTTCACTTCATTAGCTCTTGGCTCATTGTCACTGTCTCTTATTTCTGTCATAATTCTTGGTGA CATCAGTATCTATGTAGAGCAATACTAGTGAAGATGTGGTCTGGTAACTGTTACCTGTATGAATTAA GATAAGGAGTTATGCCAGAATATAAGTCACCTGTGTCACTAAGTTTACTGTTTAGCTTACTTTTTTTG TAGCAAGATTTTGATGAAGGACGCAATATGTTGATTTACAGTCTGGTACAAATTTTGATGTAGAAGA TGCTTCCAATATCCTGGTCTCTTAGTTCCTTGATTTCTTCTCCAGTGATCTTATTTTCTACCCTAACTC AACTACATATTCCCATTGTCATATCCTAGAATATTTTGTCTTTTATCTGTAACTCTGCTCTCTTCCCCC AATCTCATTTCAAGCATCCCACTTTCTAATTCCTCTAGTAAATACGTCAGTTCCAACAGCCCATCAAT CCCATTGGGACCTACAGTTTATCTATCCAAGCTTTTCCCTGTTCCTCACCCTCACTTCTATACAGCTG AAGTTTCATACTGAATTATAATCACTTTCTCGTATACACGTTTAACAATCTTGTCCCTCCCTGGCTTC ATGCCCAGTGATCTCTTGTATCTATGACCATGTCCTTTATCTTCTCCTCTGTCACTGGATGAACTGTA GCCTTCCAAGATAAGGCCACTCAGTTCATTTGTACAGCAGATTCCATCCCCTCTTGCTCTCAAGAATA TTACTGTGGTATCTCTCTTTTCTTGTCTCTACTGGCTCTTTCCATGAGCAAACATGGTATTATCCCATT ACAAAAAAAATTTTTTCTCCGTCTCTCCTTCCACTCACCACCTCAGTCTCTGCTTCTCTTTCCCGCAAA ATAACCTTGAAAAATTGCTTTATGTACTCCCGTTTTCTTTTGAACCCCTGCCAGTGACCACCACGTTA

[0059] TAAATTTGTAGTTGTCATCTCACTTAATCTGTTAGTAGTATTTGGCACCATTGCTACAGTTGCTTGAA

[0060] ATGCCTTTTCATTGGTTTCCAGGCCACCATGTCTGTTAGCAGCTTTTCCTCTTACTTCACTAGCATTTC

[0061] CTTCTTTGTTTTTTCTGTTATCTTTCTGACCTCTGTTGGAGTGGCTGAAGGTTTAGTCCTTGAATCTTT

[0062] TTTTGTTGTGCATATTTACTCCAGTATCATAGCTTTATACAGATGGTATTTACATCTGTTTGCTAACG

[0063] ATTTCCAAATTGGTATCCTTAAACTGGTATCCAGCTATTTTTTGGTCAGCATTTTGGATGTCTAAGAA

[0064] GCTTCTCAAACTAAACTGACCTCCCGGTTTTCCCCAAAGCTGCATCTTAGTCTTTTCCGAAATGCAAT

[0065] TCTGTCTTTCCAGTTACCTAGCTTAAAAGCTTGCAGTTCTTGACTCATCTTTCTCTCATACCACGTATC

[0066] TGAATTCTCTCTGCAAAAAATTGTCTGTTCTCCCTTCAGAATAAAGTCACGTGTCATTTTATGATGGG

[0067] GATACATTCAGAAATGCGTCATTAGGAGATAATCATGGTTGTGTGAACATCAGAGTATACATAGAC

[0068] AAACCTAGATGGTATAGCCTACTACACATCTAGGCTATATGGTGTGGCCAATTACTATGATGAATAC

[0069] TGTAGGTAATTGTAACATAAAGGTAGGTATTTTTATCTAAACGTATTGAAACATAGAAAAAGTACAG

[0070] TAAAAAATATGGTATCAAAAATAAAAAATGGTACAACTGTATAAGGCAGTTGTGATGAATGGAGCT

[0071] TGCAGGATATGTTGCTCTGGGTGAGTCAGCGAGTGACGATTGAGGGAACGTGAAAGATGTGGGACA

[0072] TCACTGTACACTACTGTAGACTTTATAAACACTGTACACTTGGGCTACACTACATTTTTGTAAGGTTT

[0073] TAAAAGACTTTTTTCTATAATAAACCTTAAATTACTGTCACTTTTTTACTTTATGAATTCTTAATTTTT

[0074] TAAACGTTTTCACTCTTGTAATAACACGTAGCTTAAAACATACATTGTACAGCTGTACAAAAATTTTC

[0075] TTTATATCTTTATAAGCTTTTTTATATTTTTAAAATTACTTTTTACCTTTTAGCTTTTTTGTTGAAAAAC

[0076] TAAGACATGGGCCAGGCGCGGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCTGAGGCAGGC

[0077] GGATCACGAGGTCAGGAGATAAGAGACCATCCTGGCTAACATGGTGAAACCCCGTCTCTACTAAAA

[0078] ATACAAAAAATTAGCCGGGCGTGGTGGCGGGCACCTGTAGTCCGAGCTACTTGGGAGGCTGAGGCA

[0079] GGAGAATGGCGTGAACCCAGGAGGCGGAGTTTGCAGTGAGCCGAGATAGCGCCACTGCACTCCAGT

[0080] CTGGGCGACAGAGCGGAAACTCCGTCTCAAAAAAAAACAAACAAAAAACTAAGACATGAACACAT

[0081] TAGCCTAGGCCTACAGAGGGTCAGGATCATCAGTATCACTGTATTTCCATCTCCACATCTTGTCCTTC

[0082] TGGAATGTCTTCAGAGGCAGTAAACATAAATGGAGCTGCCACCTCCTGTGATAACAGTGCCTTCTGG

[0083] AATACCTCTTGAAGGACCTACCTGTGGCTGTTTTATAGTTAACTTTTTTTTTTTAAGAAGTAACAGAA

[0084] GGAGTACACTCTAATGATAAAAAGTATAGTAAGTACATAAACCTGTAACAATCATTATCATTATCAA

[0085] GTGTCATGTACTGGACATAACTGTATATGCTATACTTTTTTTTTTTGAGATGGCATCTCACTCTGTCA

[0086] CCCAGGCTGGAGTGCAGTGGTGCGAGGATAGCTCACTGTAACCTCAGACTCCTGGGCTCAAGTGATC

[0087] CTCCTACCTCAGCCTCCCAAGTAGCTGGGACTACACCAGGCACCCCACCATGCCTGGCTAATTAAAA

[0088] AAAATTTTTTGTAGAGACAGGGTCTCACTCTGTTGCCAGGGCTGGCCTTGAATTCCTGGCATCAAGT

[0089] AATCCTCCCACTTTGGCCTCACAAAGTGCGAGGATTACAGGTAAGAGCCACCATGTCTGGCCCACTG

[0090] TACTTTTATACAACTGAAGCACAGTAAACCTACTGTGGTTTCGTTTACACCAGCATCACCACAAACA

[0091] CCATGAGTAGAACATTGTGCTGCGACGTTAACGATGGCTACAACATCACTAGGTGATAGGAATTTTT

[0092] CAGCTCCATTATAATCTTATGAGACCACTGTTGTATGTGCAGTTCATCATCCACTGAAATGTCCTTAT

[0093] GTGATGCATGTCTTCATATCCAAAAATATTAATCATTTCTCACTGAAGCCATGCCATGCCATGCCATC

[0094] TTTTGCCTGTATTATTATTTTTCAGCTTTTATTTTAGATTCAGGGTGTACATGTGCAGGTTTGTTAGAA

[0095] AGAGTATATCGTATGATGCTGAAGTTTGGGATACAGTTGAACCAGTCACCCAGGTAGTGAGCATAGT

[0096] ACTCAATAGATAACGTTCTAACATTACTCCTCCTTCCCTCCCTGTTCTTGTCTCTGTCTATTGTATCTT

[0097] TATGTCCATGTGTACCAAATGTTTAGCTCATTCTTGTGAGAACATGTGGCATTTGATTTTGTTTCTGT

[0098] GTTAATTTGCTTACAATAAATAGTCTCCAGCTGCATCCACATTGCTACAAAGGACATGATTTTGTTCT

[0099] TTTTTATAGGCTGCATCATATTCCATGGTGTATAGGTACCACATTTTCTTGATCCAGTCTACCGTTCA

[0100] TGGGCATTTGGGTTGATTGTATCTTTGCTATTATGGATGGCTTTTGCCTATATTATTGGAAAGGCCTT

[0101] CTAACTGGTGTCCCTGCTTACACCGTTTTCCCCCTTAAATGTGTTTTCAACATGGTAGCCAGAGTAAC

[0102] CCTTTTTATAACAATAAATCGTGTAACTTTTTTGTTCAGAAACTTACAGGGCTTACCATTTCATTCAG

[0103] TAAAAGCTCAAGCTCCTGTATAGTCAGACCATATCCTTCATCACCTGTTACTTTTCTCCTCTGACTCT

[0104] TCAGCCTTTTTGTTTTTCCTCAAACTGATGAAGCCTTCATGGCTGATGTCAGATGTTTTGCCCATTGA

[0105] GATCTTCCTTGTTGACTCAGTTGCACTTGGTCATATGATTTTCATTTATTTGGGGTATCTAATCATAAT

[0106] CTGAAAGTTGGCTACTTATTTTTACCCCTTTGAGGGTCCTTGCCCTGTTTTTGTATCCCTGATAGCGG

[0107] GACAGCCAGATATCTGGAACTTACAGGTGTTCAATAAAGTTTTGTTGAATGAATATTCTGGAATCAC

[0108] CCAACCTTTTTTTTCCCCTCCACTTATTTTTCTTCTCCCTTTCACGGCCTGAAAGATGTCCTATGTATA

[0109] TGGTTCCACTTATCACTCTCATCCCAGTTTGTGATATACTATTCCATTATATTACTATTATTAATACAA

[0110] TTCCATTGAACTTGCTCTTGCTGACTTCACCACTGGACCTACATGTTGGCCAAATGGATACTTTATAA

[0111] TTTTAGTCTTGACCCCTGCCTTTGGCACATTTCTTACCTCTAGCACAGCACTGTCCAGTAATCCACAC

[0112] TTTCTGAGACAGTGGAAATGTTCAGTATCTGTGCTGTTCAGTTGGTAGCAACCAGCTACCCATGCCT

[0113] ATTAAACATTTGAAATGTGGCTGTGTGACTAGTGGCAATTATGTTGGAGAGTACAGTTTTAGAAACT

[0114] CCTGTTTTTCTTACATGGCACTACATTTAGTATCACAATCTAATTGTGCAAGCCAGATAGGTAGGAGT

[0115] CATCTTTATTCCTGTTATTTAATTTTTCTCATCTACTATATCCAGTTCATCACATCAACAGCGCCTGTT

[0116] GTTTCTACCTCCTAAATATTTCTTTAGTCTAACTACTACTTGTCCCTAGTGCCACCACCATCTATCAGC

[0117] TGGAATATTGCTATAGCTGCCTTACAGGTTTCCCTTCTTTCCTGTTCTCTTCTAGTTTTTTGAATTTTA

[0118] GTCAGCACGAGATTTTAAAAACTCAAATAAGATTGTGTTATTCACCTGCTTAAAACCTTTCATGACTT TCAGTGTCACGTAGAACAGAAAACACTTTTCTTACCAAAGGCTAGAGAGCTCTACGTGATCTGGCTA

[0119] TTTTTAACGTTTCATTGCACTCACCCTTTTCCTCTATAATCAAACTACTCTGATCTCAAGGGTTAGTTC

[0120] TTGAAAGATGATCATGTTCTTTAATGACTTTAGGTTTTTGTGTGTTATTTTCTATTTCTGGGATGTTTA

[0121] TTCTCTGTTCCTTACATGCTGGCCCTTTTGCATCCTTCTTCAGGTCTCAGCTTACATGTTACCTTCAAG

[0122] AAGCCTTTGACCACTCTAAGTGGGCCCTTCCTTCCACTTCTGCTGTGTAATCCCACTCCCTTCTCCCA

[0123] CTTGTTAATTAGTTACATACTTTTTTGTAATTGTTTATTTGGTTGCTGTCTCCCTCTCAAGAATGCAGG

[0124] GACCATGTCTGCATTCTGCAGTAATCACTACTGCACACCCAGAATCTATTACAGATCCTGGCATGTA

[0125] GCTGATGCATAAATATTTGTTGAATGAAAGTCTGTACATTGTATTTATGCTATTGGTATTGCTATGAC

[0126] CTGAAACTAAAAGGAGTTGTGGAAAAGATTTCTTATGGAACAGAAATATCCCTTTTGATTAATATCA

[0127] CAATCTCGTAAATTGAGAAAACAAAAAAATATATACTACTGGAGCATTCATGTATAGTTGGAGATTA

[0128] TGACTCATTTATTGGTGTGTTTTTGGACTCAGAACAAAGATGAGGGAATATTCCTTAAAGCTCTGTAT

[0129] TGAAATAACGAAAAGCAGTCACATTTTAATAATAGAAGCTTCCTAGCTTACTCTTTCTGTAATCTTCT

[0130] TTTCCTAAATGTAAGAGAGCCTCATAATTATGAGGCTTATTACTAGAGTAAGGCTGTCAAAGGCAGC

[0131] AAAATGTCTTTCTGTTTGGAAGAATAACATAAACTTGACATGTATGGTGGGGGACAGAAGGTTTCAA

[0132] AAGTTTAAGAATCTGTGTTGTCTTAACAAATAGATGCTTCTCAAGGAGCTTACGCTAGTGGTTACTCT

[0133] GTCCAGTCAGGGTTTTTTCTTCTTTAACTTGGGTTCATTTCCTGATGGCACACATGAAGTTTGGATCA

[0134] TATGGTTTGACTTTAGCTATGGTCCTTAGCTATGGGGAGCAGCATCAGCGACCTGTGACATGTAAAT

[0135] TAAAAATACAATGCCAGGGCCCTTCCCCAGCCCCTCTGATAGAGAACCTCTTGGCCATCTGTATTTTT

[0136] AGATGTTCCAGGTTAGTCTGATTAACACCCTTGGTTAAGAACCATTGGGAGGATCTGATTGCCAGTT

[0137] TAAGGGGACCTTCAAGCCTGTAGGTCTTTATAGTTAAAAAAAAAAAAAGATTTTAAAAATCATGCAT

[0138] ATGTTGTGGCTGAATTCTGGTTTAGCACATACTGCTTTTAATGGCCTGAAATGTTTTTCCCAAATAAA

[0139] TTGTCTTGTTATAGCTTTCATGTGTGATTTGGTCCAGCTTCTTGTTTTGAAGATACTTACGGGGGGGA

[0140] ACACTTTGTGATTTCTCTTAGTAACATATTAACCCACTTAAAAACCCTTTCTATTACAGGTCTTCACA

[0141] TTTAGGCTTAATGTGCTTAATTCAAATGTAAAAATACACCTGCCTTTGTTCTCAGTGAAAGTATGTAA

[0142] TAAATAAATGAGGGGTTGGCAAACTACTGCCCACCATCTGTTTTTTTATGGCCTATGAACTAAGAAT

[0143] CGTTTTGGATAGCTAAAAAAAAAAATCAAAAGGATAATTATTTTGTGACGTGAAAATTATATGAAAT

[0144] TCAAATTTCAGTTTCTGTGAATGAAGTTTTAATGGAACACAGCCATCCATGCTTATGTAAGTGTGCAT

[0145] ATTCTCTGGCTGTTTTCACTGCAATAGCAGAGTTGAGTAGTTGTGACAAAGAGTTTATGGCCCACAA

[0146] AACCTAAAATATTTACTTTCTGATGCTTTACAGAAAAAGTTTCCTGAACCTTATTCTAGCTATATGTT

[0147] GTTCATAAATGAATCTTTCGTGGTTCTGAAGGCATTTAAGAATCTCTTAGGTTATAAATTGGCTGGGC

[0148] GCAGTGGCTCACGCCTGTAATCCCAGCACTTTGGGAGGCCGAGGCTGGTGGATCACGAGGGCAGGA

[0149] GTTCAAGATCAGCCTAGCCAAGATGGTGAAACCCTGTCTCCATTAAAAAAAAAAAAAAAAAAAAAA

[0150] AAATAGCTGGGGTTGGTGGTGGGCAGTAATCCCAGCTACTCGGGAGGCTGAGGCAGAGAATTGCTT

[0151] AAACCCAGGAGGCGGAGGATGCAGTGAGCCAAGATCGCGCCACTGCACTCCAGCCTGGGCAACAG

[0152] AGTGAAACACCATCTCAAAAAAAAAAAAAAAAAAAAAACACTCTTAGGTTATAAATAATTGTTGTT

[0153] AGCTCTCCAAGCCTCCATATTACATTTTGTGTGTTCTCCTGTTCACATTTTGAGCATTTTATTTTTTAT

[0154] TAGCACATTCAGTTCATCAGGTATTTAAGAGCTTAATATATGCCAAAGCATATATTAAGCGAGAAGC

[0155] TGTTTCTAAATGTACTGTCTCAGCCCTCACAGAGTTCACTTCATTAGGCTCTTTAAAATTTCTTTCTTT

[0156] AAAAGGTCAGCGTGCTGGTATAGTGGGGAAGGGAAACTCTTACAACACGTCGAGTAGAGGAAGGTT

[0157] ATCATTATGGGATATAATTTGGAAGTCATTGAGTACCTGCCATTAATTCTGCCTGTAGTCTGAATGTA

[0158] GAGATTAACATGTAGAAACTTTTTTGAAATAAAATCTTCAATTTCTTTGGCATATCTAGTACTGTCTA

[0159] GCTAGGCATATAGTCAAAGTATGGTGTATATTTCAAGTATTAAAAGTTTTTTTGGGCTGTAGTCACTG

[0160] TTGAAAGGATATAGTTCTTTACTATTACATGTGATACCTTTATATAAAATTGGCTAACCCCTGTCTTT

[0161] CATTTATCTGCAACACTGACTGTTACCAGTTGTCTCTAACTTTGGTATGGGGGGTGGAAATATGATTA

[0162] GATTGAAAGGGTACATGACTGAGCCACAAGCAGACCTGGATTTGAATTTTAACTGAACGGTTTATTA

[0163] GCTATTCTTACATTAATACTGCTAATCAGTTTTCTTGTGATATGAGGAATGATGTCTTCTTTATGAGG

[0164] TTGCTAGGAAGATTCAATGAGATAACATACTAGGCTCAGAACTGAAGTTGCTAGGAATTTAATTATG

[0165] CTACCTTGTTAAAGTATGTCAAAGGCAGAATTCAGTGTTTAGCTGATACCACAAGGCAGTATCCTAA

[0166] AATTATGCTGTAAAAGATATAAAGATGCTGTAAGTGACTCAGAAACCTAGTGACTTTGTAATGCAGT

[0167] TGATTCTTAGAATACTGTCACTTTAACAGAATAGGAGCTAGGAATGAAGAAATAGTTATTAAATTAC

[0168] TAAAATAGAAAATTTATTGACACATGTAAAGTGACATTTGCTTAAATATTGAAAAATTTGTAGTACT

[0169] ATTTCCTTGCTTTAGAAAACATTGGTTACCACTTTTTTTATTTATAGCAGTTTGTTTTTGCCTTGAGGC

[0170] AAGATGGTTGACTGAGTAGTTGCCACATTTCTTTTGTACAAAGTCCATTTCATAGGCCATCTAGCTTT

[0171] TATGCTTAGAAACATTTCCTTAACGTTATATTTCAGTATTTGGCTAACCTATATAGGGTTAAATTATA

[0172] TAGGCTAACTTCTCGGACAGATATTTCTAATAATTTATGTATTTGGTTCTGCAAATGTATGCAAAAAT

[0173] ATATGTACAAAGGTATGCAGATGCCTTGCATACTTGATATATGTTAAATTTTTTTTAATGTAGACCTT

[0174] TTTCGTTCTCTTTAATGACTATATGGTATTCCACCATCCCCCGCTCACCTGGACAACTACAGTAACCT

[0175] CCTAAATGGTGTTTCTACTTTGCTATTGCCCCTTATTGTCTTTTTTCCCCTTTATAGCTGCTGGAGTGA

[0176] ATTTTAGAAAGCCTAAGTCATACATCACATTGCTTCATGGGCATCCCAGTACACTTTGGATTTTATTT TACATCCTTACTGATCTGATTCTCATCTCTGTCTCTTCATGGTTCTCTGCCTTCTAGTTACACTGGTGA CCTTTCAAAACCTTTACCACATTGAGTTCATTCCTTACTTTTCACTCTTTCTCTGCCTGGAGTGTTCTG CCCCATCTTTACGTGGCCAGCTGCTCCTCCTCTGATGAAATGTCTCTTCCTCACAGGCCTTCCCTGAC

[0177] CACCCACTAGAGTAGCACATCTTCTACCTCATAAACTTGTTTATTAGTATTTCTTACTCTAAATTTTCT

[0178] TTTAAATTGCTTAATTCCCTAACAGTAGAATATAAGCTTCACTGTATGTATGATCTTGTTGACTCTCT

[0179] TACTCATTGTTATTGTAATACCAGTAACAAAGGGTGTTTAAAATTTGTTCAGTGGGTGAATATATGTT

[0180] CCATTTAATGGATAAATTATTTTTTATTCAGTCTCCTGTTGATGGACATTTGAATAATTTCCATCTTTT

[0181] TCTCTATGAATGCCTCACTTGGCATGCTTCTGACAGTATTGCCACAGAATACATTTCTGTTATAAAAA

[0182] TTGAATTTTTAAGTCAAAGGGTAGTTACACTTTAATGGATAGTGGCAGCTTACTATCAAAAGTTTCT

[0183] GCTAGTTTCACCATATCCTTATTAGCAGTAGATATTATCAATCTTTTCAATCTTTGCCAATCTGATAA

[0184] GCAAAAAGTAAATGGGTTTAAACATCCTTTGTATATATTCATTGCTCACTTTATGTTTTTCCTTTGAA

[0185] ATGTTATTTCTTGTTCTTTCCCTGCAGTATGATTCTTTCTTTTTTTGACTTGTTCCCAGTTTTTTGTGTA

[0186] CTATGGATATTAGCCTTTAATTATGTTACGGATGTTCTAGTATGTTATTTTTTGAATTACTTCAAATGT

[0187] GATTTGTTGCTCAGATTTTAAAAACTACATACACAAATTATCTCATGTTTCCCTTTTTGGTTTCAATTT

[0188] CGACTCATGCTTAATCAGTTCATCGATTGGGCATGGTTTTATTCTTAATATATACCCGTATTTTATCTC

[0189] ATTTTATTTTTTTACGTGTAAATATTTGGTGAATATAGGTTTAATTTTAATGTAAAATAAGGATGAAA

[0190] AATGATAGTTGGAATTACAAGCCCATTTCTCCTAATACTTTTAATCAAGTAATCCACTAATTGAAAT

[0191] ATTACCTTCTTCATTTATGAAATTGCCACATTATATCTGGGTGTTTTTCTGCCTACTACAGTCTCTTAC

[0192] CCATTTCTTTCCTAATAATACAATACTTGAATTGCTGTGGTTGTTGATTTATAATGTTATCTTAATGAT

[0193] AACATTATAAATGTGATGGAACTGGTTCCTCCTTATAGTTCTTCTTAAATCAAGAACAAGACATATC

[0194] TTCCCATTTACTCTCGTATGTATCTCATTTTACTGTTATGAATGAAATCTGTCCTATTTGTGTATAGGA

[0195] AAATAGTTTTTGTATGTAATTGTGATATGGCCAGTTTTATTAAAAATTTGGTTAAACTAAGAGTTGTT

[0196] TTCTGTTCAGCCTTATCATACTATAAAATCCACATAAAATGGGTATAAAAGTGTCGCAGGACACTGG

[0197] GCTCAGATGATTCTCCCACCTCAGCTTCCCAAGTAGCTGGGACTACAGCGGCATATGCCACCACACC

[0198] CAGCCAATTTTTAAATAAGTTTTAAAAATAGTATTTTTAGTAGAGACAGGGTTTCACCATGTTGCCC

[0199] AGGCTGGTCTTGAACTCCTGGACTCAGACAATCCACCTGCCTTGGCTTCCCAAAGTGTTGGGATTAC

[0200] AGGTGTGAGCCACCACACCTTGCCGAATTGCAGCCATATTTAATACTTTTTTCCATCCTATTCCCTTT

[0201] GCTGCCCCCAGGCCTCCTGTATTGATAGCCCGCTATTAAGAAGCTAGTGTATATTCTTTGCATACTTT

[0202] TACTTCATAAACTATATGAAGCATTGTTCTGTTTTTTAACTTAATTGGTATAAAATTATATTTTGGAA

[0203] ATTCAGTATATTCTGTGAAAATTATTTAGAAAATGTGCCTCTGAGATAAAGCCTATTCAGGATGTAT

[0204] CTTAAAGGAGATAGCTGTGCTTTAACATTATCAGTCTTTTTGGCTGCTTATGTTAATATAAGTTGGAG

[0205] AAAAACAGTCTGCTTTTTGTGATAATATGTTCTTGGAGATGGAGTGAAAGATTGTTTAAAAACATTG

[0206] TCTTTTTTTTCCCCTGAAGTACCAGTATTTATTTTAGGATTATGTTACTGATCAAAGATGCTGTGTGG

[0207] AGTTACTCATTGGTGAGACTAACAATAAATCACACATGCAAAGGATGTTACCATAATCTAATTATTT

[0208] TAAACAGTAAAATTATATTCTAAGACATCCAGTTGGCCTATATGTGCTATATCAATGACTATCAAGG

[0209] GGCTTTTTATGTATACTGTATACATGTACTTCACAAAAATATAAAAGGATGACATCAAAAATCTGGC

[0210] AAGCCAAAAGCCTACATTACATGTAGCAAATAAATAAGCATATGAACTTATTGGAATTTAAAACCCT

[0211] GTAGGATGGGCGGGTGATGGTATGTATGTTAGATGTGTGGACATATCTATTAAAAGTTGTGTCAGAT

[0212] AACAGCTGGTGCTGACAAGCCCTTGGTAAGATGGCAGCATGTTCAATATGTTCTGTGAAAATTATCT

[0213] CAGTTTATGATCTGTCAGTATTGTGGAGCTATGCATGAAAGGACTTAAAATTCTTACCCTTAAACTC

[0214] AGTAACAGTGTTTCTAGAACTTCTGGTGATATGGGAAATTAAGAGAATTATTTATATGCAAAGGTGT

[0215] TTATTGCAGCATTGTTGGAATAATAGACAAAATGGGGAAGAACAAGCTCAGAATGGAGGAGGTAGC

[0216] TTATAGTATAGACATACGATACAATCCAGATGATAATATTTTATAATAGTCTTCACAAGGAATTTTA

[0217] TATTTTTATTTTTAAAAATACATAGCAGTGAGTTTAATATACCAAACATACCAAAATGTCATCATTTA

[0218] CTGTGTGGTGGACTCATATGATGGAGATGATAAATAAAAATATTAATTTATTTGAGGCATATATTTA

[0219] TGGCTGAGGAAGGAAGACAGTTATGAAGAACAGCTCATTCTGGAAACATACTAATTTTTCCCAGCC

[0220] ATAAAGAGATTTCCTATTTCTTTTTTTTTTCCATTTACCTTCTGTTTCCTACCTGAGAAGATTTCATAC

[0221] TTCTAATAACCATTTGTGTACCTATTTAAAGACAGTACCAAAGGCATACATTTTAGTGTTTGGAGGA

[0222] CCAAGGGTCATTTGATGTTTGATGCTTATTGACTATTCGAGGATGACAAGACACCTTGAGAACACAC

[0223] ACACCCACACCCACACCCACACCCTCACCCACCCACCCCACCCCCCTCCCCGAAGAAAGCTGTGAAG

[0224] GAAGAAAGCAGAAAAGAACCTGGAGTGAGTTGTAACTTAAAATGTTAGTGTTGCATGAAGTGTGTT

[0225] AAAACAGGAAGATTTGAGGAAATTGCATACATTTTCTAGATGGCAAAGTATTACTGGTGACAGTTA

[0226] ATGAAAATGCATATGCATGTGTTTTTAGATTTACAAATTTTACTAAGAACTTTTTAAAAATCCCTGAA

[0227] GGTGTATCAAAAGTTTATCATGCTTATGAAATAGAGTAGCACTTTCTAACTTTAAAACGGGGAATAA

[0228] TTCTTTGGATCTTGATTATTGGAAAAGTGAATTATGAATTGCTAGTATAAAACTGTGGTTTTAAAATA

[0229] TGTCTGCTTTATATTTTTATGTAGCAGATTTACTCCTAGTTAATAATACTCAAACTTACTGAAAACTA

[0230] AGGTAATTAAGATAATTCTGTCCTGATGGGAAGAGGAAAAATAACTTCAGTGTGAAATCTATTATAT

[0231] ATTAGTTGTGGCAAGATTTCTCCCATTGACTTTGACTGGAGACATTTATAGGGTTAAAATCGGAAAT

[0232] AGCACGGTGAATTTTGAAGTATCCTTGTAGTTGGAAAGAGTATTATGTTCATATTGCCAAAAAAAAG

[0233] ATGCATGGATGCATTAGACTGGATGGAAAATACATGAGAAGTTGGCTAGCCCCCTCTTTGTCAAAAC

[0234] ATCACTTGGTGGTGATAAAGCTGTTGGAAAACACAGCATTCTAATGTAGTCTGTAGTTTAATGATAA

[0235] TCTGTGTCTTGAAACATTTAGCGTAGTACTTATACAAACCTAGATGGCATAGTGTACTGCATGCCTA

[0236] GCCTATATAGTATAGCCTGTTGCTTCTAGGGTGTAAAGCTGTATAGCGTGTTACTATAGGCAGTTGA AACAGTGGTATTTATGTATCCTTTTTTTTTTTTTTAAATTCTTTTAAGAGACAGGGTCTTGCTCTGTTG CCCAGGCTGGATGCATTGGTGTGATCATAGCTCACTATAACCTTGAACTCCTAAGTGATCCTCTTTGC CTCAGCCTCCCCAGTGGCTAGGACTACAGGCACATACTACCACACCTGGCTAATTTTTAACATTTTTT TGTAGAGATGGAATTTCGCTGTGTTGTCCAGGCTGGTCTTGGAACTCTTGTGCTGCAGCAATCCACC CGCCTCCCAAAGTGTTAGAATTACAAGCCACTTCGCCTGGCTTGTTTACCTAAACATAGAAAAGATC CAGTAAAAATACAGAATTAAAATCTTGTGGGGCCACTGTAGCATATGTAGTCCATCTTGACTGAAAT GTCCTTATGCAGTGCATGATTGTACTTCATAATTTTTAAGCACTCCTCCCTCTTGATTGGTACTTAGT GGATTTTATCATTTTTGTTTCTTCATAATTCTTTCTGAAATGTCTACTGGTTGGACCTTTGATCTCCTG AATTGATCGTGATTTCTTCTGTTGTATTTTTTGTCTTTGTCATTTTTTTGTACTCTAGGCAGTTTTCTCA ATTTTAGTTTCTATTCAACTTTTTGTTTTTATTTATTCTCTCCAGTATTTATGGAGATACTAAATTGAA GTGTTCTGTTTCTCTCTCCACCCTATCCCTAGTTTCAAGTTTTATCTCAGTTTCTATGGAGTCAGTTTT TTCGTTGCTTTAAAAAAAAATTTTCCTGAAGTGATTGGTAAGTTTTGGCTAATTGGGAGCACTAGAA TTGGGCCCTTAATGGTTGGCAGGGTGTGGTGGAGGAGAGACAGCCCTTAGTCCAAAGGCTCAGGCC AGAAAAAGAAAGAGGAAGGCTTTCCTTTTCCTTTCCGGAGCAGGGTTCTGCCCTAGGTCTTGCTTGG CAGTCTATTTGATTTCTTTAGCAGTTAATGCTCAGTTTTTTGGCATATGTGGATCTGCCTCCAGAGCA GGTACAAGGTGAGTGAGTCTATGCTGTTACCTAATTAGATCCCCATTTCTACCCTTTGTTTTTACTTC TCTATCTACTGATAGGTTTTTACCCTCCTTCACCTCATAGGGTTGCAGTGAAGAGCAAGATGAATTTT TATTTATGTTGCATAAATTTTAAAAGCTAAAAAATATATATGTAATGTTGGGAAGTCCCAGTGTACA AATGGCTATTGTAAATTTGGAACATGAACTTGCTTTTTTCCATTGTAAAAATGAAATCATTATAAATT GCGGTCAAGTTACTAGGTCAGCCCACACAGAGTTTACCCAGTAATATGCGTAAATGTTTTGCCTTTG

[0237] CATCAACAACAAGGAAAAACAGTACTATAAAAAAATGTTCCTGGAAGCCGGATGTATCAAAGCACT TCTGAAATAGCTATATAGCCTATAGACATGACCAGTTGGTTTCTGAGTCTGTTGACATTGGCCAAAG GAGAAGCTCAGTGTAGAACATGTTTGGAGTCTCCTTTTGCAGAAATACATTGGAGGCTGGAGTGGG GAACCAATTTTTCAGAAAGGTGGTGAAGTAGTTACATAGCCACTCTTTTAAAGACAGTCAAAAGATA GAAACTAAGGCCAGGTGTTGGCTCACATCTGAGATAGGAAAATCACTTGAACCTGGGAGGCGGAGG TTGCAGTGAGCCCAGTATGCACCTCTGCACTCCAGCCTGGTTTGGCAAGAGACCAAAACTCTGTCTC AAAAAAAAACAAAACATAGTTCACACTTAAATATTTTATTCCATATCTTTACATACCCAATATGTTA ATTTATAGTTCAAGATGAACTTGTTTGGGACAGATTTTGTAATAAAGGAAATCGTGTTATTAGAAAT ATCTAGAGGCCATGAGCCCTTAAACTGTTCTAATTTGCAAGTAGTTCCCTGTGTGATGCAGTTTTTTT CAATATTGCACAATAAAGGCAAAATACGGACAAATTAGATGATAAGATTTATATAAATTTTTAAAAT ATTGATCAAAATATGTATCCATATTGGTAATATTTGTATTTATAATAAATCATTGCTGTAAATTTGAA CTTAGAAAAATTTTACTAATAAAGGTGCTTTTGTGTTGCAAACTTTCATTTGAAAAGTAATTTTTCTT TGTACCAAAAAATCTAAAATTCGCTATTCTAGTCACCAAAATTTGCTTTATGAAAAATAATTTTTGAT GGCACTATATCAGAAAACAACTTGTTAAAGAAAATGTGGAGTTTTTAAAATCCCACTGTACCTCTGT TATCCAAAGGGGATCTGTGAATTTTTCTGTGAAAGGTTAAAAAAGGAGAGACCTTTAGGAATTCAG AGAGCAGCTGATTTTTGAATAGTGTTTTCCCCTCCCTGGCTTTTATTATTACAACTCTGTGCTTTTTCA TCACCATCCTGAATATCTATAATTAATATTTATACTATTAATAAAAAGACATTTTTGGTAAGGAGGA GTTTTCACTGAAGTTCAGCAGTGATGGAGCTGTGGTTGAGGTGTCTGGAGGAGACCATGAGGTCTGC GTTTCACTAACCTGGTAAAAGAGGATATGGGTTTTTTTTGTGGGTGTAATAGTGACATTTAACAGGT ATCCCAGTGACTTAGGAGTATTAATCAAGCTAAATTTAAATCCTAATGACTTTTGATTAACTTTTTTT

[0238] AGGGTATTTGAAGTATACCATACAACTGTTTTGAAAATCCAGCGTGGACAATGGCTACTCAAGGTTT GTGTCATTAAATCTTTAGTTACTGAATTGGGGCTCTGCTTCGTTGCCATTAAGCCAGTCTGGCTGAGA TCCCCCTGCTTTCCTCTCTCCCTGCTTACTTGTCAGGCTACCTTTTGCTCCATTTTCTGCTCACTCCTCC TAATGGCTTGGTGAAATAGCAAACAAGCCACCAGCAGGAATCTAGTCTGGATGACTGCTTCTGGAG CCTGGATGCAGTACCATTCTTCCACTGATTCAGTGAGTAACTGTTAGGTGGTTCCCTAAGGGATTAG GTATTTCATCACTGAGCTAACCCTGGCTATCATTCTGCTTTTCTTGGCTGTCTTTCAGATTTGACTTTA TTTCTAAAAATATTTCAATGGGTCATATCACAGATTCTTTTTTTTTAAATTAAAGTAACATTTCCAAT CTACTAATGCTAATACTGTTTCGTATTTATAGCTGATTTGATGGAGTTGGACATGGCCATGGAACCA GACAGAAAAGCGGCTGTTAGTCACTGGCAGCAACAGTCTTACCTGGACTCTGGAATCCATTCTGGTG CCACTACCACAGCTCCTTCTCTGAGTGGTAAAGGCAATCCTGAGGAAGAGGATGTGGATACCTCCCA AGTCCTGTATGAGTGGGAACAGGGATTTTCTCAGTCCTTCACTCAAGAACAAGTAGCTGGTAAGAGT ATTATTTTTCATTGCCTTACTGAAAGTCAGAATGCAGTTTTGAGAACTAAAAAGTTAGTGTATAATA GTTTAAATAAAATGTTGTGGTGAAGAAAAGAGAGTAATAGCAATGTCACTTTTACCATTTAGGATAG CAAATACTTAGGTAAATGCTGAACTGTGGATAGTGAGTGTTGAATTAACCTTTTCCAGATATTGATG GACAGTATGCAATGACTCGAGCTCAGAGGGTACGAGCTGCTATGTTCCCTGAGACATTAGATGAGG GCATGCAGATCCCATCTACACAGTTTGATGCTGCTCATCCCACTAATGTCCAGCGTTTGGCTGAACC ATCACAGATGCTGAAACATGCAGTTGTAAACTTGATTAACTATCAAGATGATGCAGAACTTGCCACA CGTGCAATCCCTGAACTGACAAAACTGCTAAATGACGAGGACCAGGTAAGCAATGACATAGCTAGC TTTTTAGTCTGCTTTGAAGTAAATGCTCAAGGGGAGTAGTTTCAGAATGTCTACCCAATACCAGTAC TTGAAAACTAACGATGTTTCTGAATTCCTGTATTACAGGTGGTGGTTAATAAGGCTGCAGTTATGGT

[0239] CCATCAGCTTTCTAAAAAGGAAGCTTCCAGACACGCTATCATGCGTTCTCCTCAGATGGTGTCTGCT ATTGTACGTACCATGCAGAATACAAATGATGTAGAAACAGCTCGTTGTACCGCTGGGACCTTGCATA

[0240] ACCTTTCCCATCATCGTGAGGGCTTACTGGCCATCTTTAAGTCTGGAGGCATTCCTGCCCTGGTGAAA

[0241] ATGCTTGGGTAAGAAAACATGTCAGAATGCTTGAAGCTAAAAAGTAGAAGAGTATACTCACAATAT

[0242] TTCTGATGAGGCTTTTTTCTTCTTCCCAGTTCACCAGTGGATTCTGTGTTGTTTTATGCCATTACAACT

[0243] CTCCACAACCTTTTATTACATCAAGAAGGAGCTAAAATGGCAGTGCGTTTAGCTGGTGGGCTGCAGA

[0244] AAATGGTTGCCTTGCTCAACAAAACAAATGTTAAATTCTTGGCTATTACGACAGACTGCCTTCAAAT

[0245] TTTAGCTTATGGCAACCAAGAAAGCAAGGTAAGAGAATTATTCTTTATGTGGTTTTCATGGAGCATT

[0246] GGACACCTCCAGTGTCATGTCATTCCATGCAGTGTTCCTAACCTTTTTGGCACCAGGGACCAGTTTCG

[0247] TGGAAAACAGTTTTTCCATGAATGGGTTGTGGGAATGGTTTCTGGATGACACCATTCCACCTCAGAT

[0248] AATCAGGCATTAGATTCTCATAGGGAGCGTGCAGCCTAGATCCCTCGCATGTGCAGTCCACACTAGG

[0249] GTTTCTACTCCTATGAGACTCTCATGGTGCAGTTGATCTGACAGGAGGTAGAGCTCAAGCCAGGTAA

[0250] TGCTCGCTCACCTGCCACTTACCTCCTGCTGTGCAGCCCAGTTCATTTCTGTTCTTTTAAATTTTTGAG

[0251] TTTCCATATGTAAAGCACTATGCGAAGTAGTAGGGATATGGTAGGCAAGCTTCTCTTCACACTTTTG

[0252] TTCTTAGGTGGGATGTAGATGTTGGGAATAATAACCTAATATTTAATTTGTGTAGTGGGAAGAAGTG

[0253] GGGCTATGAGGGCACATAACACAAGTTGAAACTGACTCTTTTTGAGGGTTCAAGGAGACCTCTTGGA

[0254] GGAAGTGATAGTTGAGTTCAGTGTTCAAGGATGAGAAGGGATTCACTAGGTGAAGGTTAGGTGAGA

[0255] AAACAACATCTTTGAAACGAAGGAAGGAGATGGAAAGTTTTGGGAATTTAAGAAATACTAATAGTA

[0256] AGGAGGAAGAAAGGTTTGAGGTGAGGCTATTGAGATAGACTTAGCAGATCTCATAGGGCTTTGTAG

[0257] AGCATGTTTAAAAGCACAATGGGAAATTTCAGCAGAAGCCTGAAATGATGAAATTTGTTTTTAGAA

[0258] AATTGGGGCAGTGTTGAAAGGGAAGATATACAGGGAATGAAAGGACAAGCATGAATGATCATTTTA

[0259] TGGTATCTGTTTTTAAGGTGGATATAATTAGGAAAATTAAAGGGCCAAATGATGAGGAGTTAAGTGC

[0260] CAGTTCTGGTTCAAATTTTCAGTGAATCAGTTTTGATATAACTTTCATCTTAGGGCATTACTCTTGCC

[0261] TACCAACATAGTTTCTAAATTTTTTTCTTTTGGTGTGATCACTGTGGGAAGAAGGAAATTGGGCCCA

[0262] AACTGATACATTGTTTGGAGGACTGGGATGTCTGAATTTGAGTGGAATGCTTTAAAAGGACAAGTTG

[0263] GATAGGGCCCCAGTATGGGGGTCTGAGTGATGGGGTCCAGGAATACATTTAGGTCCAATGGCAAGC

[0264] TGGCTGAAATTCTTGTATAATAAAATAGGTTGGTAATATGGCTCTTCTCAGACATGTGATCAAGATT

[0265] CCTTGACTAACAAGATATATATATATATCTTTCTAGCTCATCATACTGGCTAGTGGTGGACCCCAAG

[0266] CTTTAGTAAATATAATGAGGACCTATACTTACGAAAAACTACTGTGGACCACAAGCAGAGTGCTGA

[0267] AGGTGCTATCTGTCTGCTCTAGTAATAAGCCGGCTATTGTAGAAGCTGGTAAGTATATGTATCTATTC

[0268] TGAGTCTTGTGTATAGCATCTGCAGTTCTAATTAGATTACTTTTCTTAGGAAAAGGTGGTAGAACTTT

[0269] AACTACTGAAAATAAATGGTCCTATTCAGTTTGCAGCCAAGATTTACATTCAGAGTACCTGTCATCT

[0270] GGATTGTAGCTAAATATTTAAGGCTAGTTTAGGTAGAGTTCTTATTATCCATCAAAAATGATGGCAT

[0271] ATGTTTTGCTTAATAAAATTTGTTTGTAATTTCAGTTTTGAGTAAACCTAAGATTTGCTAACAGAGCT

[0272] GTGAATTTATAGGAGAAAAGACAAATTCTAATATAGTACAGTTTTATGTAAAGTGATTGCTTTATTA

[0273] GTAGATGCTCATGAGCAGTTTTTGTTTTGTTTTAACTTTTAGGTTCCGGGTAATGTGCAGGCTTGTTA

[0274] TATAGGTAAATTGCATGTCACAGGGGTTTCGTGTGCAGATTATTTTGTCACCCAGGCAGTAAGTATT

[0275] GTACCCAATAGGTAGTTTTTCAGTTCTTTACCTCCCACCCGTAAGTAGGCCCCAGTGTCTGTTGTTCC

[0276] CTTCTTTGTGCCCGTGTGTACTCAGTGTTTACCTCCCACTTATAAGTGAGAACATGTGGTATTTGGTT

[0277] TTCTATTCCTATGTTAGTTTGCTTAGGATAATGGCCTCCAGCTCCATCCATGTTGCTGAGGAAGACAT

[0278] CTTGGTATTTTTTTATGGCTGCTTAGTATTCCATAGTATATATGTACCACATTTTCTTTATCTAGTCTA

[0279] CCATTGATGGGCATTTAGGTTAATTCCATATCTTTGCTATTGTGAATAATGCTGCAGTGAACATATGC

[0280] ATGCATGTGTCTTTATGGTAAAAAGATTTCTTTTTCTTTGGGCATATACCTAATAATAGGATTGCTGG

[0281] ATTGAATGGTAATTCTGTCAGGTTTTTTGAGAAATCACCAAATTGCTTTCCACAATGGCTGAACTAAT

[0282] TTACTTTCCCACCAGCAGTGTATAAGCATTCTCTTTTCTCAGCAACCTCACCAGCATCTGTCATTTTTT

[0283] GACTTTTTATTAGTAGCCATTCTAACTGGTGTGAGACGGTATCTCATTGTGGTTTTGATTTGCATTTCT

[0284] CTAATGATCAGTGATGTCGAGCTTTTCTTCATATGTTTCTTGGCCACTTGTATGTCTTCTTTTGAAAAG

[0285] TGTCTGTTCATGTCCTTTGCCCACTTTTTAATGGGGTTGTTCTTTTTTGCTTGTTAATTTAAGTTTATTG

[0286] TAAACTCTGGATATTAGACCTTTGTCAGATGCATAGTTTGCCAGTACTTTCTCCCATGCCAGTACTTT

[0287] CTCCCATTCTGTAGGTTGTCTGTTTACTCTGTTGATTTCTTTTGCTGCGCAGAAGCTCTTTATACTGTC

[0288] CCATTTGTCAGTTTTTGTTTTTGTTGCAACTTCTCTTGGCATCTTCGTCATGAAATCTTTGCCAGGTCT

[0289] TATGTCCAGAATGGTATTTCCTAGGTTATCTTGCAGAGTTTTTACAGTTTTAAGTTTTATATTTAAGTC

[0290] TTTAATCCATTCTGAGTTGATTTTTGTACATCATGTAAGGATGGGGTGCAGTTTCAATCTTGGATGTG

[0291] GCTAGCCAGTTATCCCAGCACCATTTATTGAATAGGGAGTCCTTTCCCCATTGCTTGTTTTTGTTTAC

[0292] TTGTTAGGTGTGCGGCCTAACTTCTGGGCTTTCTTTTCTGTTCCATTGGTCTCTGTGTCTGTTTGTATA

[0293] CCAGTACCATGCTGTGATTGTAACCTTGTATTAACAGTATAGCTTGAAGTTGGGTAAAGTGATTCCT

[0294] CCAGTTTTGTTCTTTTTGCTTAGGATTGCCTTGGCTATTCAGGCTCTTTTTTGGGTTCATATGAATTTT

[0295] TAAATAGTTTTTTTTTAATTATGTGAAGAATGCCATTGGTAGTTTGGTAGGAATAGCATTGAATCTGT

[0296] GAATTGCTTTGGGCTGTATGGCCATTTTAACAATATTGATTCTTCCTGCCATGAAATAGAATGTTTTT

[0297] TCATTTGTTGGTGTCATCTCTGATTTCTTTGAGCAGTGTTTTTTGTAATTCTCATTGTAGAGATCTTTC

[0298] ACCTCCCTGGTTAGTTGTATTCCTAGGTATTTTATTCTTTTTGTGGCTTTGGTAAATGGGATTGCATTC

[0299] TTGATTTGGCTTGCAGCTTGGATGTTGTTGGTGTCTAGAAATGCTTCTGACTTTTGTACATTGATTTTT ATATCCTGAAACTTTGCTGAAGTTTATTGGATCAAGGAGCTTTTGGGCAGAGATTATGGGGTTTTCT

[0300] AGGTATAGAATCATATTGTTTGCAAACAGACTTCCTATTTGGATGCATTTTCTTTCTCTTGCCTGATT

[0301] A

[0302] AT TG TTA TG TC TA TG CT TG AT TT TT CT TG GC AC GC TT AG TA TT TA TTT ATC AT GG AT AA GT GT CC AT ACA CTG TT AG TA AA TT GA TG GA GT TG AT CC TG TT TC GT TA AA TG AT TA TT GG TA GG TAA TA GA TC TA G

[0303] GCAATTGGGGAAAAGAATAGATGGTTTGTACTAGGGCCTCTTGGGTTCTGTGTGTGTGTGTGTGTGT

[0304] GTGTGTGTGTGTCATGAAAACAGTTACTTTTTAGCTACCAAGCATTTTTTCTCCTTTCAGTAACCCAC

[0305] CTAACAACATTTACTCAGAATTTCAAAGCAAGCTTCAAATCAGTATTGAAAGAAGGAAAAATATAA

[0306] AGGCATTTAATGGAAGAAAATGTTGGGAATAAAGTATAGGGCTGGCAACACTTACTTTTCTCACTTA

[0307] TTGAGAGTAATTTTACTTGGGAATTTATGAGAGAGAAAGACATTATGATTGCTCCAGGTAACTACTG

[0308] GCAGAGGAACCATAGTCTTGGGGATAGACAAATGTGGCTGAGTTCATATAGAATGAGGGGATGGGA

[0309] TGTAAATTCTGTCAGCTGTTCCAGCAGTAACCTGTAATGTAGGCTAAAAATACAGATTTTGAGATTT

[0310] ATTTAATCAGAATCCCTGGAGTGTTAATTTTTATATCAAGATCTCATAGTGTTTTATTTGAAGTGACA

[0311] GGGAGGTCTGTAGATAGCTGGACATGTATGGGACTGGAAGCTTAGGAATCTTTAAGTTCTTCCAGGT

[0312] TATTCTTATGTTCATTTGTTTATTCTGAAAATAGCATCTAATGTATTTTAAGAAATGGAATAGGCACA

[0313] TAGTATACATTGGGTAACACAACAGATAGGGTCCCCGTGCTTAATTCTTAGTCTTGTGAAGGTGACA

[0314] AAAATACTTAAAAATATGTGATCCTAAATTAGAATGAGTGTTATGGGAGAAATGACAGCAAATAGT

[0315] GATGAGAATTAATGGGGAGGGGAATTGTCTAGATGAGAGGGAAAAGGTCTCCTTGAAAAGGGGAT

[0316] GTTAAGTGGGACTGCAGGATGAGAGGGAACCGTCTCTTGTCTATATGAGAAGTGAGGGTTAAACGT

[0317] TTTCCAGGTAGAGAAAAGGAACACCATGTGCTATGTCTTAGAACCAGGGATATCCAGTCTTTTGGCT

[0318] TCCCTGGGCCACATTGGAAGAAGAATAATTGTCTTGGGCTACACACCAAATACACTAATGATAGCTG

[0319] ATGAGCTAAAACAAAAAAAAATTGCAAAAGAATCTCATAATGTTTAAGAAAGTTTACGAATTTGTG

[0320] TTGGGCTACATTCAGAGCTGTCCTAGGCCATGTGGCCCATGGGCTGCAGGTTGGACAAGCTTGCCTT

[0321] AGAAGGAAAGAGATTGGTCAGGCACGGTGGCTCACGCCTGTAATTCCAGCACTTTGGGAGGCTGAG

[0322] GTGGGCGGATCATGAGGTCAGGAGATCGAGACCAGCCTGGCTAACACAGTGAAACCCCATCTCTAC

[0323] TAAAAATACAAAAAGTTAGCCGGGCGTGGTGGCAGGCGCCTGTAGTCCCAGCTACTTGGGAGGCTG

[0324] AGGCAGGAGAATGGTGTGAACCCGGGAGGCGGAGCTTGCAGTGAGCTGAGATAGCGCCACTGCACT

[0325] TCAGCCTGGGCGACAGAGTGAGACTCTATCTCAAAAAAAAAAAAAAGGGAAAGAGATTGTGGAGA

[0326] TCCAGGTGCTGAAGAGAAGGTCTGCATAAACAGAACTTAGTAATGAGGTGGATGGCCTGGTATGAG

[0327] GTTGAGGTTAGGTAAGCAGAGCCATAACATGCAGGACTTTCTAGGTTCCTATAAGATAGTTACTACT

[0328] CATGGAGTTTATTCATGCTTTATTCCAGCTTTGGAGCCATAGATACAGAATACTTTGGTCAGTTTGGA

[0329] AGGCTAGGTGGGATCCAAATTCTAAACGGTTCCTCAGGGTTATACTAAAGTATTTCTATTATCTTAA

[0330] AAGGATGCTGAGACACTTTCGATGGTTGTTTATCAATAGCAAAGCATCACAGTGGTGTGTTTAAAAT

[0331] ATTAATAATAGCATTGTATAGATTAACAGTTTGAATGACCAAAAGCTAGAAGACCAGACTACTGAG

[0332] ATGTTACAGGCTTTTAGGAATGAAATAGTTTGCTTTTAGAACTCAATAGCAAAGGGCAGATGTCTGA

[0333] GATGCCTGAAAGAATCATAGAATGTAATAATATAGGAGCTAAGGGAGCAACCAAAAACGGTTTGTG

[0334] GAGGGGACAACATTGGTACCATGAAGATAAATGGAACCCTCAGAAGGCATCCTTAATTTTTGAACA

[0335] TAATAATTTAAGAAGCTGACTTAAAGTGACTTAAAAGGTCAGTAGGTAGCTGGAAATGTATGATACT

[0336] AGAATGCAAGAGAGGCAGGCTAGAGATTTGGAAGTTTCCCTCTTAGTATATAGGGGTAAGGGCAGC

[0337] AGGGAAGGGGAGGTAGAGGTGCCACAGAGTCATCTGTATGGGACTTTTTTTTTTACCCTAGAACTGC

[0338] TGAATCAGAATGTGTGTGTTTTAAAGTCTCTGTAGGCCATTCTGATGGACATCTGGGGTTAAAATCC

[0339] ATTCTCTTAGAGTTAATAGTTATGTAAAGGGAGGGAATGAAGTCTTAAAGAGGGGAAAGAAGGTAG

[0340] TCATTTCACAAATACTGAGCATCCTGATCATCAGTCTTACGCAGATCATTCTATTAGTAGCTGGAGCT

[0341] ACTATGAAAAAGGAACCCAACAGAGGTGATCTTTGTCTTGTAGGGAAAGTGGAGTAACTTACACTA

[0342] TGAAGGAGAAGTGCAGGGTACCATAAGAATTACAGCAGATAGACCTCATCTGAGGAAATAAAACA

[0343] GACCCGAAAGATGAAGGAGACAAGGAAAAGTATCTCTTACTGCATTCAGAAGTGATTTAAGTTGAA

[0344] GATGGATGAGCGAAGTTAATCTACTATGTGGGCATTGGGCTTCCATTTATACTCCTTTGCCAGAGTA

[0345] AATGTCCCCCATTTAAGGGTCCTAAAGGATGGAAGATTGTAAACCTTGGAACACATGTTTTGTAGTC

[0346] AGTGAATTGTATAAAGTCCCTGACAGTAAGTGTTTTCATGCCGTCTTTCTGGATTGTTCTTACCCCAG

[0347] GAATTTACCTAGCTTCTTTAGGTCTTTAGTCAGATGTCACCTTCACAGTGAGGTGACCTAATTATCTA

[0348] TTTAAAATCGCAGCCCCACTCCATTATTTTTCTCCATAGCCCTTTAATATCATCTGACATACTGTATG

[0349] GTTTTAGTTTATTGTATATTTTTCTGCCTCTTCCAACTAGATCATAAATTCTGAGGGTAGGAACTTCT

[0350] GAATATTTTTGTTCACTGGTCTATCTGCAGCTCAGAACAGGACCTGGTACTGAATAAATATTTTTGAA

[0351] ATGATTGAATGGATGAAAAGAAATGAGTAATAAGAATATTACCTAAGGGGGACAGTGGAGATAAC

[0352] AAAGGCTTTTTCGGCTTAGGAAAGGAACAGTAGCTATTTGAGAGTTTGTCACTAGTGAGGTGAACTG

[0353] GCAAAGTGAAGGAAACTGAGCAACATTCTAGAAAATGAGAGGAAATCAAATACTTAGGTGAAAGG

[0354] AAGTAAACTCTGGAAATACAGAAGGACACCTCCTAAGGCTAGAACAGATATTTAGGATTGATAGGC

[0355] ACTTCTAGCTAATGACTAGGGCCTTATATCCTTTTTAATTTTCTAGGTGGAATGCAAGCTTTAGGACT

[0356] TCACCTGACAGATCCAAGTCAACGTCTTGTTCAGAACTGTCTTTGGACTCTCAGGAATCTTTCAGATG

[0357] CTGCAACTAAACAGGTAAATTCTGAGTAAACTGGTGCCATGGGAATAGAGTCAAGATGAGTATGTG

[0358] CTTGTACTGACCATCTGTTTTTATCTCCATAGGAAGGGATGGAAGGTCTCCTTGGGACTCTTGTTCAG

[0359] CTTCTGGGTTCAGATGATATAAATGTGGTCACCTGTGCAGCTGGAATTCTTTCTAACCTCACTTGCAA TAATTATAAGAACAAGATGATGGTCTGCCAAGTGGGTGGTATAGAGGCTCTTGTGCGTACTGTCCTT CGGGCTGGTGACAGGGAAGACATCACTGAGCCTGCCATCTGTGCTCTTCGTCATCTGACCAGCCGAC ACCAAGAAGCAGAGATGGCCCAGAATGCAGTTCGCCTTCACTATGGACTACCAGTTGTGGTTAAGCT CTTACACCCACCATCCCACTGGCCTCTGATAAAGGTAAATTGTCAAAGTAGAATTTACCTTTGTTGC AGAATTGAAAATGAAGCATCTCTAGCTGTTGGATGGCTGTCTAAGCATAGTGATCAATAAGTAGGA

[0360] ATTGTATTCCTTAGTAAGTAGGAAGTATGGCTGCGATAGGGGTAAGATTCTGAAATGTTTGTGTAGT CAGAACTACTTTTAGTTGATACCAATAGATTTAGTGTGGTGGGAATTTTAGGGTAAGAAAATGATTT

[0361] TGTTGAGTTGTATGCCAGTTCTTCCTTCTGTTTTTCAGGCTACTGTTGGATTGATTCGAAATCTTGCCC TTTGTCCCGCAAATCATGCACCTTTGCGTGAGCAGGGTGCCATTCCACGACTAGTTCAGTTGCTTGTT CGTGCACATCAGGATACCCAGCGCCGTACGTCCATGGGTGGGACACAGCAGCAATTTGTGGTAGGT AAATTCTTACAGTGATACCTGGCTATCTAAAAGGAATGCATAAATCCAAAGGATCCTGAACTTCTTT CTTTGGTCATTGGTTCCCCCCATCCGTCTTCCTGAAGAGCTAATGACAAAGTAAATAAATAAATAAT

[0362] TACACATTTCTATGGCTGCAGAGAAAATAAGGCATAGTGTGGCCCCAGTGATATTTCCTTGGACACG TCCTTCACATGGTCAGTCTTACAAAGGTTGGGTTAGGTGTTTCATAAAGTGTTCTCATTTAATTTACA CAAAGGCCCACTTCCTTAGGAAGAGGTAGAGTCATAATTTGAGATCAAATCTGTGTAATTTCAGAGC CTCTTACCCTTGCCTCATCATGCATTTTGACTATAAATATTTAGCAGTCCGTTTTATTATCTTTTCTGT

[0363] GAGTTAAACTTTTTTCATGGACCTAAGAATATTCAGAAATAAGTAGTAGCATTTCTGTACTCTTAACC ACAAAAATCTCAACCTGAAGCTTTGATACAAAGTTTGTGTCTTAAAAGTAGCTTCATTAAAAGTATA GTCTAATGACATTTCTGATTTCTCAGACTTTAAGACCTTATTAGGTTAGTTTAGAAAACAAAGATGG AGCCTACCAGAACAGATGTTAGGAATCTCATTTTGCTGGTTGCTTTGTGTATGTACTCATATTGGGGC TTTGGCTTTCTTCATTTATTACTGTTGGTATTGGCCCATCTCCATGAGGTGACTTAATAGAACGTTGA

[0364] GGGCACCTTTTATTTTAAATCTCTTTTCTAGGAAGAAGAGAGTTTTTGTGTCCTTGTAAGAATCAAGT TATTTATAAAAGCTGCTAAATGTAGCAGAATAATAACCCCTTTTAAAACTCAAATCCAGAAACAGGA GAAACAGATGGTACTTACATATTGCAAAAGCTATCTTCCTTCTATACATGAGGCTGTCAGCTGAATA GTCTTGGAAGAGTGAGGAGTGAATTTTTCTGCTGGCAACTCGGTTAGTTTTAGCAGTTGGTGCTAAA ACTTGGCAAAGTTTTCACCAAATACATGGAAGATATACAAAAATAGAGGGGGCATGTAAAAGAAAA

[0365] ACGTTGACATAGTCTGAGCATTACTTTCTCATCTTCTCTTTTTATATACCTTTTACCCAGAATGATTGG TGCCCTTACTGTAGGAAAGTTGTCTTTGGGATTCAGCGCTGTATGGAAGCTCTGTTGCACTGTGTATG GGGGAGGGGTGCTGCTTTGAATTAGTGCTGCCAGGAGGCCTCTTTTCAGTGACATTCAAGTTAATGG AATCCTTCTTCCTTCCTGAACTAATTGCAAGTTACGGGGAACTTCGGGTATATAATGTAAATAATTAC AGTCTAATAATTGTTCCTCAAACTTTACAGAGGAGAATGCCCTGTTTGTTAACCATGTTTCTTTTGGC

[0366] AGGAGGGGGTCCGCATGGAAGAAATAGTTGAAGGTTGTACCGGAGCCCTTCACATCCTAGCTCGGG ATGTTCACAACCGAATTGTTATCAGAGGACTAAATACCATTCCATTGTTTGTGCAGGTATGTTTTAAG TGAAGTGTTCTAGGTTTTATGTCCATAAAATTTCCAGATTGTAATGACTAATAACATTTCAGAAAATT AGGGACCATAATAGGGTTACCAACATTTAATTTTATGAAAATTCCCTACATTTTTTGGTCAGTAAGA GAAACATTGAGACTTGAGAAGAGGGAGGAGATTTCACATTTCACTTTTATGGGTGCCTAGAGGGGA

[0367] GAGCTGACCTGGGCTGCCAGAGGCAGGGCATAGACCCCCAACCAATTCTGGGTTTTCCAAATCTTAG ATCAGTTAGAGCTGCCTCTGAAGAAAGGGTTTATAGCTAAAAAATATTATGGAAATCCAGTGCTCCA GAGCATTAAACACCCCAAGACATAAAATTCAGAGAATATTATTTACTACAGTGTGAATGCCTCTTGC ACTCTGAATTGGGAATGTTTGCACCACAGTGGGGGGCTTGCCATGTTTTAGCTTTAGATTTAATTAG GTTTTGTTTGTGTTTTCTCCTTAGCTGCTTTATTCTCCCATTGAAAACATCCAAAGAGTAGCTGCAGG

[0368] GGTCCTCTGTGAACTTGCTCAGGACAAGGAAGCTGCAGAAGCTATTGAAGCTGAGGGAGCCACAGC TCCTCTGACAGAGTTACTTCACTCTAGGAATGAAGGTGTGGGTAAGTAAAAAGGAACCAAAGCCTTT AGCAGATGTGTACATTGAAGTCTCAGTTTTTCCTCAAGGGCCTTTTTCTCCTTGTCTCTTAGCGACAT ATGCAGCTGCTGTTTTGTTCCGAATGTCTGAGGACAAGCCACAAGATTACAAGAAACGGCTTTCAGT TGAGCTGACCAGCTCTCTCTTCAGAACAGAGCCAATGGCTTGGAATGAGGTAGGGAAATGTGAGCA

[0369] GTTATTTATCTGGTAGTTTCCTAGAGCAGGTATGGCAGCTTGTTCTTTCCTCTCAAAACACTTAGTAC ACATTCATTTGCATTGATGTTTCCCTGGCTTGAGTATTTCTTCTTTATGCTGTCTAGCAACTGCTCTGA

[0370] GGAAGAACTATAATACAAGCTTTAAAGAGTCTGTTCAGAATCATTACAAATAAGTTGTGTTATTTAA AATTATAATTCATAAGGGAGAAAGATGAAAAATGTTACCAGATTAAAGAAGATTTTTCAAAAGGAT GTAAGGAAAGAGGCAGTGTTAAACACTGTTAAGAGGACAGTTTATCAGTATTTTTTACTAAACTTTA

[0371] ATAAAACTTTTCTATTTGAATTTCTGCTATGAATTTTTCTTCAGCATTTGTCCTCAGTACAGGTGGTTC CTTGAAACATTGTTTCTAATAAAACTAGAACATCCTGATATTTTATCCATTCTATAGAGATCATTGAT GGTACACAGACATACAGTGGATTATGTTTGTTGAGTGAATGGAAAGAGAGATTGTTAGGTTTACAAC GATGCAGCTCTTGAGACCGGAGTTTAAGATCAGCCTGGGCAACATAGTGAAACCCCATCTTTAGCTG GGCATGGAGATGGATGCCTATAGTCCTAGCTACTGGGGAGACGGGGGCAGGAGGATTGCTTGAACC

[0372] CAGGAGTTAACAGACTGCACTCAGTGACAGAGCCAGACTCCAACACAAAAAAAAAAAAAAAAAAA AAGCAAATTACCAGTGAGTAGTGTGTTACTTGGGTTTTTAATAGGCATCTTATTAACATGTTCCAACT TGAGCCCTTAACTTTCTCCACCTACCCCCTTCCACAAACCTGTTTTCACTGTCTTCTCTGTCTTAGTTA ATGTCAGCTTTGTCTGTCCAGCTGCTCAGGCTAAAACTTTTCTTTCATATAACACATCCTATCAGCAG CTCCTGTTTGTGGGTAGGCATTTTGCCTTTTTTTTTTTTTTTTTTTTTAAACTGCTATATCTCTAGCATG TAGAACAGTGCCTGGCAGCACATAATAGGTGCTTAATATAATATTTGTTGAAAGAACAAGTCAGTG

[0373] AGTATTTTTAATGTGAGGTGCAAAGAGAAAAAAAAATGTATCTTTGAGGTGTGGAGTTTTGAAGAA

[0374] CTTCCATTTTCTAAGCATTTGTGTAATGTTGGAGTTACTTGTTCCTTTTGTAATCTGAAAGTATGCTTT

[0375] AAAAAAAATTAGTGTACTTTTGAGAATTTTCATTTTGCTTTCTATTCTTCCTTGCTTTGTGCATGTTTA

[0376] TCTAGACTGCTGATCTTGGACTTGATATTGGTGCCCAGGGAGAACCCCTTGGATATCGCCAGGATGG

[0377] TATGTGTCTCATATTTCTCGATTAACTCCAGATCAAGCTAAAGTTCTAAAACTTTTATCAGAAGAGCC

[0378] GGTTTGCTCATCTGGGAAACCAGTGTTGGCAGAAAAGTAGTGGCTTCAATTAAAAGCAGTTCTTAAA

[0379] TTCCAGTCAGCAACAGTATCTTTAATGGAGCACAGGGAATTCAGAGCCACACAATGAGTAGCAGTA

[0380] GGATTACACCACCAACAAATACATGCTACTGCTAGGCCTCTGCAGTGCAGGATGTTACAATTTACCT

[0381] GGCTTTTTATTCTCTTTTTGGCCAGAGGACTCATAATACCTTTGTCTACAAGCTACCCAAGGAAGATA

[0382] GGAAAACTCCTGTTTCTAGGCTCAGATCTCGGGTGGGTTTTTACATAGTTGCATTATCATCAGGGTTT

[0383] TCTTGAAAAGCTAATTTAAATCTGGGTAATGAACATGGAGGATGGCATAGACCACTAACAATTATA

[0384] ACTGTCTTACATTTATAACCGCATCTGCTTCTACCTAATTATGAAACCACTAAAGCGCAGATTCTTAC

[0385] TGTGAGAAATAACATGTCAACCCTAAGATAAAATATGTTGAGGTTTCATGGAAATAGTGCCTTTCCT

[0386] TAGTACTTTTGTGGGTGTCACTTGGCCTTTTTGTCAAGATAGATTACACCTGCCAGACCTCATTATTG

[0387] TCTTAATCCTCCTTCCCATGACTTCTCACTGCCTAGGTGGTCACACAGTAGATTCCTGCTTCTTCTCCT

[0388] CGGGAACCCCAAGTCTCTTGACAGGGGTAAATGCAGAGTGTTCAGGGTTAGACTAATGATGTGACT

[0389] AGGCCCTGCTGGTGTGCCTGTCTGATGGAAATAGATGTTATTTGTGTAGTCTCATGGGTGGCCTGGC

[0390] ACTGAGTAATTACTTGGCTAAAGAAAGCTGGAGGTTGAAGAGGCTAGAAAGCGTTGTTTTCTGACA

[0391] AGTTTGCTGCTGAACTTTGGATGCCCTAACCTCAGTGTTAACGTCTATGTCTGCTTCTCTCCTCTCTCT

[0392] TTTGCCTTCCTTCTTGCCTATTTTGTTGACACCCTGACTCTTCTAGATCCTAGCTATCGTTCTTTTCACT

[0393] CTGGTGGATATGGCCAGGATGCCTTGGGTATGGACCCCATGATGGAACATGAGATGGGTGGCCACC

[0394] ACCCTGGTGCTGACTATCCAGTTGATGGGCTGCCAGATCTGGGGCATGCCCAGGACCTCATGGATGG

[0395] GCTGCCTCCAGGTGACAGCAATCAGCTGGCCTGGTTTGATACTGACCTGTAAATCATCCTTTAGGTA

[0396] AGAAGTTTTAAAAAGCCAGTTTGGGTAAAATACTTTTACTCTGCCTACAGAACTTCAGAAAGACTTG

[0397] GTTGGTAGGGTGGGAGTGGTTTAGGCTATTTGTAAATCTGCCACAAAAACAGGTATATACTTTGAAA

[0398] GGAGATGTCTTGGAACATTGGAATGTTCTCAGATTTCTGGTTGTTATGTGATCATGTGTGGAAGTTAT

[0399] TAACTTTAATGTTTTTTGCCACAGCTTTTGCAACTTAATACTCAAATGAGTAACATTTGCTGTTTTAA

[0400] ACATTAATAGCAGCCTTTCTCTCTTTATACAGCTGTATTGTCTGAACTTGCATTGTGATTGGCCTGTA

[0401] GAGTTGCTGAGAGGGCTCGAGGGGTGGGCTGGTATCTCAGAAAGTGCCTGACACACTAACCAAGCT

[0402] GAGTTTCCTATGGGAACAATTGAAGTAAACTTTTTGTTCTGGTCCTTTTTGGTCGAGGAGTAACAATA

[0403] CAAATGGATTTTGGGAGTGACTCAAGAAGTGAAGAATGCACAAGAATGGATCACAAGATGGAATTT

[0404] ATCAAACCCTAGCCTTGCTTGTTAAATTTTTTTTTTTTTTTTTTTAAGAATATCTGTAATGGTACTGAC

[0405] TTTGCTTGCTTTGAAGTAGCTCTTTTTTTTTTTTTTTTTTTTTTTTTGCAGTAACTGTTTTTTAAGTCTCT

[0406] CGTAGTGTTAAGTTATAGTGAATACTGCTACAGCAATTTCTAATTTTTAAGAATTGAGTAATGGTGT

[0407] AGAACACTAATTCATAATCACTCTAATTAATTGTAATCTGAATAAAGTGTAACAATTGTGTAGCCTT

[0408] TTTGTATAAAATAGACAAATAGAAAATGGTCCAATTAGTTTCCTTTTTAATATGCTTAAAATAAGCA

[0409] GGTGGATCTATTTCATGTTTTTGATCAAAAACTATTTGGGATATGTATGGGTAGGGTAAATCAGTAA

[0410] GAGGTGTTATTTGGAACCTTGTTTTGGACAGTTTACCAGTTGCCTTTTATCCCAAAGTTGTTGTAACC

[0411] TGCTGTGATACGATGCTTCAAGAGAAAATGCGGTTATAAAAAATGGTTCAGAATTAAACTTTTAATT

[0412] CATTC

[0413] Human CTNNB1 cDNA sequence (NM_001098209.2; SEQ ID NO: 5; mRNA sequence is

[0414] SEQ ID NO: 5 with all the T’s replaced by U’s)

[0415] AAGCCTCTCGGTCTGTGGCAGCAGCGTTGGCCCGGCCCCGGGAGCGGAGAGCGAGGGGAGGCGGA

[0416] GACGGAGGAAGGTCTGAGGAGCAGCTTCAGTCCCCGCCGAGCCGCCACCGCAGGTCGAGGACGGTC

[0417] GGACTCCCGCGGCGGGAGGAGCCTGTTCCCCTGAGGGTATTTGAAGTATACCATACAACTGTTTTGA

[0418] AAATCCAGCGTGGACAATGGCTACTCAAGCTGATTTGATGGAGTTGGACATGGCCATGGAACCAGA

[0419] CAGAAAAGCGGCTGTTAGTCACTGGCAGCAACAGTCTTACCTGGACTCTGGAATCCATTCTGGTGCC

[0420] ACTACCACAGCTCCTTCTCTGAGTGGTAAAGGCAATCCTGAGGAAGAGGATGTGGATACCTCCCAA

[0421] GTCCTGTATGAGTGGGAACAGGGATTTTCTCAGTCCTTCACTCAAGAACAAGTAGCTGATATTGATG

[0422] GACAGTATGCAATGACTCGAGCTCAGAGGGTACGAGCTGCTATGTTCCCTGAGACATTAGATGAGG

[0423] GCATGCAGATCCCATCTACACAGTTTGATGCTGCTCATCCCACTAATGTCCAGCGTTTGGCTGAACC

[0424] ATCACAGATGCTGAAACATGCAGTTGTAAACTTGATTAACTATCAAGATGATGCAGAACTTGCCACA

[0425] CGTGCAATCCCTGAACTGACAAAACTGCTAAATGACGAGGACCAGGTGGTGGTTAATAAGGCTGCA

[0426] GTTATGGTCCATCAGCTTTCTAAAAAGGAAGCTTCCAGACACGCTATCATGCGTTCTCCTCAGATGG

[0427] TGTCTGCTATTGTACGTACCATGCAGAATACAAATGATGTAGAAACAGCTCGTTGTACCGCTGGGAC

[0428] CTTGCATAACCTTTCCCATCATCGTGAGGGCTTACTGGCCATCTTTAAGTCTGGAGGCATTCCTGCCC

[0429] TGGTGAAAATGCTTGGTTCACCAGTGGATTCTGTGTTGTTTTATGCCATTACAACTCTCCACAACCTT TTATTACATCAAGAAGGAGCTAAAATGGCAGTGCGTTTAGCTGGTGGGCTGCAGAAAATGGTTGCCT

[0430] TGCTCAACAAAACAAATGTTAAATTCTTGGCTATTACGACAGACTGCCTTCAAATTTTAGCTTATGG

[0431] CAACCAAGAAAGCAAGCTCATCATACTGGCTAGTGGTGGACCCCAAGCTTTAGTAAATATAATGAG

[0432] GACCTATACTTACGAAAAACTACTGTGGACCACAAGCAGAGTGCTGAAGGTGCTATCTGTCTGCTCT

[0433] AGTAATAAGCCGGCTATTGTAGAAGCTGGTGGAATGCAAGCTTTAGGACTTCACCTGACAGATCCA

[0434] AGTCAACGTCTTGTTCAGAACTGTCTTTGGACTCTCAGGAATCTTTCAGATGCTGCAACTAAACAGG

[0435] AAGGGATGGAAGGTCTCCTTGGGACTCTTGTTCAGCTTCTGGGTTCAGATGATATAAATGTGGTCAC

[0436] CTGTGCAGCTGGAATTCTTTCTAACCTCACTTGCAATAATTATAAGAACAAGATGATGGTCTGCCAA

[0437] GTGGGTGGTATAGAGGCTCTTGTGCGTACTGTCCTTCGGGCTGGTGACAGGGAAGACATCACTGAGC

[0438] CTGCCATCTGTGCTCTTCGTCATCTGACCAGCCGACACCAAGAAGCAGAGATGGCCCAGAATGCAGT

[0439] TCGCCTTCACTATGGACTACCAGTTGTGGTTAAGCTCTTACACCCACCATCCCACTGGCCTCTGATAA

[0440] AGGCTACTGTTGGATTGATTCGAAATCTTGCCCTTTGTCCCGCAAATCATGCACCTTTGCGTGAGCAG

[0441] GGTGCCATTCCACGACTAGTTCAGTTGCTTGTTCGTGCACATCAGGATACCCAGCGCCGTACGTCCA

[0442] TGGGTGGGACACAGCAGCAATTTGTGGAGGGGGTCCGCATGGAAGAAATAGTTGAAGGTTGTACCG

[0443] GAGCCCTTCACATCCTAGCTCGGGATGTTCACAACCGAATTGTTATCAGAGGACTAAATACCATTCC

[0444] ATTGTTTGTGCAGCTGCTTTATTCTCCCATTGAAAACATCCAAAGAGTAGCTGCAGGGGTCCTCTGTG

[0445] AACTTGCTCAGGACAAGGAAGCTGCAGAAGCTATTGAAGCTGAGGGAGCCACAGCTCCTCTGACAG

[0446] AGTTACTTCACTCTAGGAATGAAGGTGTGGCGACATATGCAGCTGCTGTTTTGTTCCGAATGTCTGA

[0447] GGACAAGCCACAAGATTACAAGAAACGGCTTTCAGTTGAGCTGACCAGCTCTCTCTTCAGAACAGA

[0448] GCCAATGGCTTGGAATGAGACTGCTGATCTTGGACTTGATATTGGTGCCCAGGGAGAACCCCTTGGA

[0449] TATCGCCAGGATGATCCTAGCTATCGTTCTTTTCACTCTGGTGGATATGGCCAGGATGCCTTGGGTAT

[0450] GGACCCCATGATGGAACATGAGATGGGTGGCCACCACCCTGGTGCTGACTATCCAGTTGATGGGCT

[0451] GCCAGATCTGGGGCATGCCCAGGACCTCATGGATGGGCTGCCTCCAGGTGACAGCAATCAGCTGGC

[0452] CTGGTTTGATACTGACCTGTAAATCATCCTTTAGCTGTATTGTCTGAACTTGCATTGTGATTGGCCTG

[0453] TAGAGTTGCTGAGAGGGCTCGAGGGGTGGGCTGGTATCTCAGAAAGTGCCTGACACACTAACCAAG

[0454] CTGAGTTTCCTATGGGAACAATTGAAGTAAACTTTTTGTTCTGGTCCTTTTTGGTCGAGGAGTAACAA

[0455] TACAAATGGATTTTGGGAGTGACTCAAGAAGTGAAGAATGCACAAGAATGGATCACAAGATGGAAT

[0456] TTATCAAACCCTAGCCTTGCTTGTTAAATTTTTTTTTTTTTTTTTTTAAGAATATCTGTAATGGTACTG

[0457] ACTTTGCTTGCTTTGAAGTAGCTCTTTTTTTTTTTTTTTTTTTTTTTTTGCAGTAACTGTTTTTTAAGTCT

[0458] CTCGTAGTGTTAAGTTATAGTGAATACTGCTACAGCAATTTCTAATTTTTAAGAATTGAGTAATGGTG

[0459] TAGAACACTAATTCATAATCACTCTAATTAATTGTAATCTGAATAAAGTGTAACAATTGTGTAGCCT

[0460] TTTTGTATAAAATAGACAAATAGAAAATGGTCCAATTAGTTTCCTTTTTAATATGCTTAAAATAAGC

[0461] AGGTGGATCTATTTCATGTTTTTGATCAAAAACTATTTGGGATATGTATGGGTAGGGTAAATCAGTA

[0462] AGAGGTGTTATTTGGAACCTTGTTTTGGACAGTTTACCAGTTGCCTTTTATCCCAAAGTTGTTGTAAC

[0463] CTGCTGTGATACGATGCTTCAAGAGAAAATGCGGTTATAAAAAATGGTTCAGAATTAAACTTTTAAT

[0464] Human P-catenin amino acid sequence (Accession No. NP_001091679.1; SEQ ID NO: 3)

[0465] MATQADLMELDMAMEPDRKAAVSHWQQQSYLDSGIHSGATTTAPSLSGKGNPEEED

[0466] VDTSQVLYEWEQGFSQSFTQEQVADIDGQYAMTRAQRVRAAMFPETLDEGMQIPSTQ

[0467] FDAAHPTNVQRLAEPSQMLKHAVVNLINYQDDAELATRAIPELTKLLNDEDQVVVNK

[0468] AAVMVHQLSKKEASRHAIMRSPQMVSAIVRTMQNTNDVETARCTAGTLHNLSHHRE

[0469] GLLAIFKSGGIPALVKMLGSPVDSVLFYAITTLHNLLLHQEGAKMAVRLAGGLQKMV

[0470] ALLNKTNVKFLAITTDCLQILAYGNQESKLIILASGGPQALVNIMRTYTYEKLLWTTSR

[0471] VLKVLSVCSSNKPAIVEAGGMQALGLHLTDPSQRLVQNCLWTLRNLSDAATKQEGM

[0472] EGLLGTLVQLLGSDDINVVTCAAGILSNLTCNNYKNKMMVCQVGGIEALVRTVLRAG

[0473] DREDITEPAICALRHLTSRHQEAEMAQNAVRLHYGLPVVVKLLHPPSHWPLIKATVGL

[0474] IRNLALCPANHAPLREQGAIPRLVQLLVRAHQDTQRRTSMGGTQQQFVEGVRMEEIV

[0475] EGCTGALHILARDVHNRIVIRGLNTIPLFVQLLYSPIENIQRVAAGVLCELAQDKEAAE

[0476] AIEAEGATAPLTELLHSRNEGVATYAAAVLFRMSEDKPQDYKKRLSVELTSSLFRTEP MAWNETADLGLDIGAQGEPLGYRQDDPSYRSFHSGGYGQDALGMDPMMEHEMGGH HPGADYPVDGLPDLGHAQDLMDGLPPGDSNQLAWFDTDL

[0477] “Clustered regularly interspaced short palindromic repeats (CRISPR)”: As used herein, the term “clustered regularly interspaced short palindromic repeats” or “CRISPR” refers to a segment of nucleotides or polynucleotides (e.g., DNA) that contain a number of short repeating sequences referred to as “repeats”, which are separated by “spacer” sequences, originally found in archaea and bacteria. In archaea or bacteria species that naturally contain the CRISPR system, the CRISPR sequences are derived from DNA fragments of bacteriophages that had previously infected the prokaryote. They are used to detect and destroy DNA from similar bacteriophages during subsequent infections. Hence these sequences play a key role in the antiviral or anti-phage defense system of prokaryotes and provide a form of acquired immunity. The CRISPR system is found in approximately 50% of sequenced bacterial genomes and nearly 90% of sequenced archaea. When the CRISPR sequence is expressed, the “repeats” can form secondary structures (e.g., hairpins) and / or comprise unstructured singlestranded sequences. The repeats usually occur in clusters and frequently diverge between species. The repeats are regularly interspaced with “spacers,” resulting in a repeat- spacerrepeat locus architecture. A spacer-repeat unit encodes a crisprRNA (crRNA), which is processed into a mature form of the spacer-repeat unit.

[0478] “RNA guided nuclease”: As used herein, an “RNA guided nuclease” refers to a polypeptide or complex of polypeptides having RNA and DNA binding activity and DNA cleavage activity (e.g., endonuclease activity), or a DNA-binding subunit of such a complex, wherein the DNA binding activity is sequence- specific and depends on the sequence of an RNA bound by the polypeptide (“RNA guided”). Nonlimiting examples of RNA guided nucleases include Cas nucleases. A “Cas nuclease” or “Cas protein” as used herein, refers to Cas nucleases that are able to cleave one or both strands of a double stranded target nucleic acid (e.g., double stranded DNA such as a gene) at a specific location determined by an RNA bound to the Cas nuclease. Cas nucleases can be divided into Class I (type I, III, and IV) and Class II (type II, V, and VI). In some embodiments, Cas nucleases include, without limitation, a Csm or Cmr complex of a type III CRISPR system, the CaslO, Csml, or Cmr2 subunit thereof, a Cascade complex of a type I CRISPR system, the CasIII subunit thereof, and Class II Cas nucleases. In some embodiments, an RNA guided nuclease is a Class I Cas nuclease. In some embodiments, an RNA guided nuclease is a Class II Cas nuclease, which is a single- chain polypeptide with RNA guided nuclease activity (e.g., endonuclease activity). A nonlimiting example of a Class II Cas nuclease is the Cas9 nuclease.

[0479] “CRISPR associated protein 9 (Cas9)”: As used herein, the term “Cas9” refers to a wild-type Cas9 nuclease and any functional fragment or variants thereof. Cas9 recognizes a short motif in the CRISPR repeat sequences (the protospacer adjacent motif or PAM) to help distinguish self versus non-self sequences. Gene editing using CRISPR / Cas system is accomplished using a guide RNA (gRNA) and an RNA-guided nuclease that functions to introduce a double stranded break at a target nucleotide or polynucleotide (e.g., DNA) sequence. RNA-guided nucleases (e.g., Cas9) use RNA:DNA hybridization to determine target DNA cleavage sites, and can cleave, in principle, any sequence specified by the guide RNA. Methods of using RNA-programmable nucleases, such as Cas9, for site-specific cleavage (e.g., to modify a genome) are known in the art (see e.g., Cong et al., Science 339, 819-823 (2013); Mali et al., Science 339, 823-826 (2013); Hwang et al., Nature biotechnology 31, 227-229 (2013); Jinek et al., eLife 2, e00471 (2013); Dicarlo et al., Nucleic acids research (2013); Jiang et al., Nature biotechnology 31, 233-239 (2013); the entire contents of each of which are incorporated herein by reference).

[0480] Cas9 nuclease sequences and structures are known to those of skill in the art (see, e.g., Ferretti et al., Proc. Natl. Acad. Sci. U.S.A. 98:4658-4663(2001); Deltcheva E et al., Nature 471:602-607(2011); and Jinek et al., Science 337:816-821(2012), the entire contents of each of which are incorporated herein by reference).

[0481] Cas9 orthologs have been identified in various species, including, but not limited to, Streptococcus pyogenes (NCBI cDNA Reference Sequence: NC_002737.2:854751-858857; Protein Reference Sequence: WP_010922251.1), Streptococcus thermophilus (NCBI cDNA Reference Sequence: NZ_LR822015.1: 623743-627129; Protein Reference Sequence: WP_059257345.1), and Staphylococcus aureus (NCBI Protein Reference Sequence: WP_306789577.1; WP_ 166732469.1). Cas9 orthologs have also been identified in various other species, including, without limitation, Prevotella intermedia (NCBI cDNA Reference Sequence: NZ_CP019300.1: 607993- 612135; Protein Reference Sequence: WP_028906301.1); Streptococcus iniae (NCBI cDNA Reference Sequence: NZ_JH930418.1: 361106-365212; Protein Reference Sequence: WP_003099269.1); Streptococcus thermophilus NCBI cDNA Reference Sequence: NZ_LR822015.1: 623743-627129; Protein Reference Sequence: WP_059257345.1), Streptococcus pyogenes (NCBI cDNA Reference Sequence: NC_002737.2:854751-858857; Protein Reference Sequence: WP_010922251.1), o Neisseria meningitidis (NCBI cDNA Reference Sequence: NZ_CP021520.1: 84774-88022; Protein Reference Sequence: WP_002229869.1). Additional suitable Cas9 nucleases and sequences will be apparent to those of skill in the art based on this disclosure, and such Cas9 nucleases and sequences include Cas9 sequences from the organisms and loci disclosed in Chylinski et al., (2013) RNA Biology 10:5, 726-737; the entire contents of which are incorporated herein by reference.

[0482] In some embodiments, a Cas9 nuclease suitable for use in compositions and methods described herein comprises a HNH or HNH-like nuclease domain, and a RuvC or RuvC-like nuclease domain. When the target nucleic acid is a double stranded nucleic acid (e.g., double stranded DNA), HNH or HNH-like nuclease domains can cleave the non-target strand (also referred to herein as the complementary strand), and the RuvC or RuvC-like nuclease domain cleaves the target strand (i.e., the strand where the PAM is located).

[0483] A wild- type Cas9 nuclease can introduce double- strand breaks in a target nucleic acid, e.g., a gene such as an oncogene. The double-strand break can stimulate a cell’s endogenous DNA-repair pathways (e.g., homology-dependent repair (HDR), non-homologous end joining (NHEJ), alternative non-homologous end joining (A-NHEJ), microhomology-mediated end joining (MMEJ)). NHEJ can repair cleaved target nucleic acid without the need for a homologous template. This can sometimes result in small deletions or insertions (indels) in a target nucleic acid at the site of cleavage, and can lead to disruption or alteration of gene expression, and / or inactivation of a gene. For the purposes of the present disclosure, no DNA template is provided for HDR. NHEJ and MMEJ occur at low frequency or efficiency such that the double strand break introduced into a target nucleic acid (e.g., a gene such as an oncogene) effectively knocks down or knocks out the targeted gene.

[0484] “Guide RNA (gRNA)”: As used herein, the term “guide RNA (gRNA)” refers to an RNA molecule that facilitates the targeting of a nuclease (e.g., an RNA guided nuclease such as a Cas9 nuclease) described herein to a target sequence (e.g., a sequence of an oncogene). In the native Type II CRISPR / Cas system, Cas9 is guided to its target sites with the aid of two RNAs: the crisprRNA (crRNA) which defines the genomic target for Cas9, and the transactivating CRISPR RNA (tracrRNA) which acts as a scaffold linking the crRNA to Cas9 and facilitates processing of mature crRNAs from pre-crRNAs derived from CRISPR arrays. In most systems used for CRISPR-mediated genome editing, these two small RNAs have been condensed into one RNA sequence known as the guide RNA (gRNA) or single guide RNA (sgRNA). In the present disclosure, the terms gRNA and sgRNA are used interchangeably.

[0485] A crRNA comprises a spacer that is involved in targeting a target nucleic acid. For example, in the naturally occurring form in prokaryotes, the spacer of a crRNA targets a foreign invader nucleic acid (e.g., from a bacteriophage). For the purposes of the present disclosure, a spacer of a crRNA targets (e.g., specifically binds to or is complementary to) a complementary sequence in a gene (e.g., an oncogene). In some embodiments, a spacer of a crRNA targets (e.g., specifically binds to or is complementary to) a sequence containing a mutation (e.g., a mutation in a mutated oncogene). In some embodiments, a spacer of a crRNA is 17-20 nucleotides in length (e.g., 17, 18, 19, or 20 nucleotides in length). In some embodiments, a spacer of a crRNA is 20 nucleotides in length. Spacers shorter than 17 nucleotides or longer than 20 nucleotides are also contemplated. A spacer can be located at the 5' or 3' end of the crRNA. In some embodiments, a spacer is located at the 5’ end of the crRNA. In some embodiments, a gRNA described herein comprises a crRNA targeting a target nucleic acid (e.g., a sequence of an oncogene). In some embodiments, a gRNA described herein comprises a crRNA comprising a spacer comprising a region of complementary to a target nucleic acid (e.g., a sequence of an oncogene).

[0486] In some embodiments, the crRNA further comprises a direct repeat component. In some embodiments, a “direct repeat” or “repeat” component is 12-22 (e.g., 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22) nucleotides in length. Direct repeats shorter than 12 nucleotides or longer than 22 nucleotides are also contemplated. In some embodiments, a direct repeat component is located on the 3’ side of the spacer in a crRNA. In some embodiments, a direct repeat component is located on the 5’ side of the spacer in a crRNA.

[0487] In some embodiments, a gRNA described herein comprises a crRNA, and further comprises a tracrRNA. A tracrRNA refers to an RNA including a sequence that forms a structure required for a CRISPR-associated protein to bind to a specified target nucleic acid. In some embodiments, the tracrRNA base pairs with the crRNA to form a functional gRNA. The tracrRNA acts as a binding scaffold for an RNA guided nuclease (e.g., Cas9). Nonlimiting examples of tracrRNA sequences, may be found for example in, W02016100951, and in Hsu et al., Nat Biotechnol. 2013 Sep; 31(9): 827-832, the contents of each which are incorporated herein in their entirety.

[0488] A tracrRNA sequence can have a length of from about 6 nucleotides to about 150 nucleotides. For example, a tracrRNA sequence can have a length of from about 6 nt to about 50 nt, from about 6 nt to about 40 nt, from about 6 nt to about 30 nt, from about 6 nt to about 25 nt, from about 6 nt to about 20 nt, from about 6 nt to about 15 nt, from about 8 nt to about 40 nt, from about 8 nt to about 30 nt, from about 8 nt to about 25 nt, from about 8 nt to about 20 nt or from about 8 nt to about 15 nt, from about 15 nt to about 150 nt, from about 15 nt to about 130 nt, from about 15 nt to about 100 nt, from about 15 nt to about 80 nt, from about 15 nt to about 50 nt, from about 15 nt to about 40 nt, from about 15 nt to about 30 nt or from about 15 nt to about 25 nt. In some embodiments, a tracrRNA sequence has a length of approximately 14 nucleotides. A tracrRNA sequence can be at least about 60% identical to a reference tracrRNA sequence (e.g., wild type tracrRNA sequence from .S'. pyogenes) over a stretch of at least 6, 7, or 8 contiguous nucleotides. For example, a tracrRNA sequence can be at least about 60% identical, at least about 65% identical, at least about 70% identical, at least about 75% identical, at least about 80% identical, at least about 85% identical, at least about 90%) identical, at least about 95% identical, at least about 98% identical, at least about 99% identical, or 100% identical, to a reference tracrRNA sequence (e.g., wild type tracrRNA sequence from S. pyogenes) over a stretch of at least 6, 7, or 8 contiguous nucleotides.

[0489] A tracrRNA sequence can comprise more than one duplexed region (e.g., hairpin, hybridized region). A tracrRNA sequence can comprise two duplexed regions. A tracrRNA may comprise a secondary structure. A tracrRNA may contain more than one secondary structure. In some embodiments, a tracrRNA sequence may comprise a first secondary structure and a second secondary structure and a first secondary structure comprises more nucleotides than a second secondary structure. In some embodiments, a tracrRNA may comprise a first secondary structure, a second secondary structure, and a third secondary structure and said first secondary structure comprises less nucleotides than said second secondary structure and said second secondary structure comprises more nucleotides than said third secondary structure. The number of secondary structures and corresponding nucleotide lengths is not particularly limited.

[0490] Nonlimiting examples of tracrRNA sequences that may be used in a gRNA described herein include: GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG AAAAAGUGGCACCGAGUCGGUGCUUU (SEQ ID NO: 10); GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCA (SEQ ID NO: 11);

[0491] GUUGGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUC AACUUGAAAAAGUGGCACCGAGUCGGUGCUUUUUUU (SEQ ID NO: 12);

[0492] GGAACCAUUCAAAACAGCAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAAC UUGAAAAAGUGGCACCGAGUCGGUGC (SEQ ID NO: 13).

[0493] GUUUUAGAGCUAGAAAUAGCAAGUUAAAAU (SEQ ID NO: 314).

[0494] In some embodiments, a tracrRNA of a gRNA described herein comprises a nucleotide sequence that is at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100%) identical to the nucleotide sequence of any one of SEQ ID NOs: 10-13 and 314. In some embodiments, a tracrRNA of a gRNA described herein comprises a nucleotide sequence that is at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100%) identical to the nucleotide sequence of SEQ ID NO: 10. In some embodiments, a tracrRNA of a gRNA described herein comprises a nucleotide sequence that is at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, or 100%) identical to the nucleotide sequence of SEQ ID NO: 314.

[0495] In some embodiments, any one of the gRNAs described herein may comprise a nucleobase sequence as provided herein (e.g., in Table 2) and one or more chemical modifications (e.g., a chemically modified nucleoside comprising a chemical modification in the base and / or the ribose of a nucleoside, and / or a chemical modification in one or more intemuleoside linkages). Suitable chemically modified nucleosides include, without limitation, a 2’-modified nucleoside such as a 2’ -deoxy-nucleoside (DNA), a 2’-O-methyl (2’-O-Me) modified nucleoside, a 2’-F modified nucleoside, a 2’-O-methoxyethyl (2’ -MOE) modified nucleoside, a LNA, ENA, cET. In some embodiments, a modified intemucleoside linkage is a phosphorothioate internucleoside linkage.

[0496] “Protospacer adjacent motif (PAM)”: As used herein, the term “protospacer adjacent motif’ or “PAM” refers to a DNA sequence adjacent to a target sequence (e.g., a target sequence in an oncogene) to which a guide RNA or a complex comprising a guide RNA (e.g., a guide RNA targeting a mutated oncogene) and an RNA-guided nuclease (e.g., Cas9 nuclease) specifically binds. In the case of a double- stranded target nucleic acid, the guide RNA binds to a complementary strand (the strand that comprises a sequence complementary to the spacer of the crRNA and hybridizes to the spacer), and a PAM sequence as described herein is present in the non-complementary strand (the strand that does not hybridize to the spacer, also referred to herein as the “target strand”). As used herein, the term “adjacent” includes instances in which a guide RNA or a complex comprising a guide RNA (e.g., a guide RNA targeting a mutated oncogene) and an RNA-guided nuclease (e.g., Cas9 nuclease) specifically binds, interacts, or associates with a target sequence that is immediately adjacent to a PAM. In such instances, there are no nucleotides between the target sequence and the PAM. The term “adjacent” also includes instances in which there are a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides between the target sequence, to which a guide RNA or a complex comprising a guide RNA (e.g., a guide RNA targeting a mutated oncogene) and an RNA- guided nuclease (e.g., Cas9 nuclease) specifically binds, and the PAM. It is to be understood that if a target nucleic acid is a double stranded DNA molecule (e.g., a gene), either the positive (+) strand (the strand that contains the coding sequence for a gene product such as protein or RNA)) or the negative (-) strand (the strand that contains the reverse complementary sequence of the coding sequence for a gene product such as a protein or RNA) may be a target strand. In some embodiments, the target strand is the positive (+) strand and the PAM is located on the negative (+) strand. In some embodiments, the target strand is the negative (-) strand and the PAM is located on the positive (-) strand.

[0497] In some embodiments, a PAM sequence may be 2-6 (e.g., 2-6, 2-5, 2-4, 2-3, 3-6, 3-5, 3-4, 4-6, 4-5, 5-6) nucleotides in length. In some embodiments, a PAM sequence may be 2, 3, 4, 5, or 6 nucleotides in length. In some embodiments, a PAM sequence is a PAM sequence for a Cas9 nuclease. In some embodiments, a PAM sequence may be 3 nucleotides in length. In some embodiments, a PAM sequence comprises a nucleobase sequence of “NGG” (5’ to 3’), wherein “N” represents any nucleobase and “G” represents the nucleobase guanine. In some embodiments, a PAM sequence may be 5 or 6 nucleotides in length. In some embodiments, a PAM sequence comprises the nucleobase sequence of “NNGRR” or “NNGRRN,” wherein the “N” represents any nucleobase, “G” represents the nucleobase guanine, and “R” represents a purine (guanine (G) or adenine (A)). Nonlimiting examples of PAM sequences for Cas9 nuclease may be found in Fonfara et al, Nucleic Acids Research, 42: 2577-2590 (2014), and WO2018154387, the contents of each of which are incorporated herein by reference.

[0498] “Target”: As used herein, the terms “target” or “targeting” refers to the ability of a guide RNA (e.g., through the spacer of the crRNA of the guide RNA) or a complex comprising an RNA guided nuclease and a guide RNA, to bind to a specific target nucleic acid and not to other nucleic acids that do not have the same sequence as the target nucleic acid.

[0499] “Target nucleic acid”: As used herein, the term “target nucleic acid” or “target sequence” refers to a specific nucleic acid sequence that specifically binds to a guide RNA or a complex comprising an RNA guided nuclease and a guide RNA described herein. In some embodiments, the target nucleic acid is or includes a gene (e.g., an oncogene). In some embodiments, a target sequence targeted by a guide RNA comprises a mutation, relative to a wild-type sequence. In some embodiments, the target nucleic acid is single-stranded. In some embodiments, the target nucleic acid is double-stranded.

[0500] “Target strand”: As used herein, the term “target strand” refers to a strand in a double stranded target nucleic acid on which a PAM is located. A target strand described herein does not contain a sequence complementary to a spacer of a gRNA and does not hybridize with the spacer. A target strand, once bound by a Cas9 nuclease, can be cleaved by the RuvC domain of the Cas9 nuclease. Correspondingly, the strand that hybridizes with a spacer and does not contain a PAM is referred to herein as the “non-target strand” or “complementary strand” interchangeably.

[0501] “Target sequence”: As used herein, the term “target sequence” refers to a sequence in the target strand that is upstream (one the 5’ side) of a PAM (e.g., immediately upstream or a small number (e.g., 1, 2, 3, 4, or 5) of nucleotides upstream). In some embodiments, a target sequence described herein is complementary to a sequence a spacer is complementary to in the complementary strand. In other words, in some embodiments, a target sequence (DNA) described herein correspond to the sequence a spacer (RNA), wherein each of the thymine (T) of the target sequence is a uridine (U) in the spacer. In some embodiments, a target sequence described herein further comprises a PAM sequence.

[0502] “Complementary strand”: As used herein, the term “complementary strand” refers to a strand in a double stranded target nucleic acid that comprises a sequence complementary to the spacer and hybridizes with the spacer. A PAM for the spacer is not located in a complementary strand. One skilled in the art would appreciate that the terms “target strand” and “complementary strand” are in relation to each specific spacer. In other words, for different spacers targeting a same target nucleic acid (e.g., a gene such as an oncogene), a target strand for one spacer may be the complementary strand for another spacer, and vice versa.

[0503] “Specifically binds”: As used herein, the term “specifically binds” refers to the ability of a molecule to bind to a binding partner with a degree of affinity or avidity that enables the molecule to be used to distinguish the binding partner from an appropriate control in a binding assay or other binding context. For example, a spacer described here specifically bind to a target sequence when the spacer, the crRNA comprising the space, a guide RNA comprising the spacer, and a complex comprising an RNA guided nuclease and such guide RNA binds to the intended target sequence with higher affinity than a control sequence that is different from the target sequence.

[0504] “Activity”: As used herein, the term “activity” refers to a biological activity. In some embodiments, activity includes enzymatic activity, e.g., catalytic ability of an effector. For example, activity can include nuclease activity (e.g., the nuclease activity of an RNA-guided nuclease such as Cas9). In some embodiments, activity of a gene refers to biological effect(s) such gene or gene product may have in a biological system (e.g., in a cell or a subject). For example, activity can refer to biological effects of an oncogene (e.g., a mutated oncogene) in tumorigenesis. “Administering”: As used herein, the terms “administering” or “administration” means to provide an agent (e.g., a gene editing agent as described herein) to a subject in a manner that is physiologically and / or (e.g., and) pharmacologically useful (e.g., to treat a disease or condition in the subject). In some embodiments, administration can be systemic. In some embodiments, administration can be local. In some embodiments, administration can be direct delivery to the selected cell, tissue, organ, or system of a subject via oral, inhalation, intraocular, intravenous including facial vein injection and retroorbital injection, intracerebroventricular (ICV), intracistema magna (ICM) injection, intramuscular, intrathecal, intracranial, subcutaneous, intradermal, intratumoral, and other parental routes of administration. Routes of administration may be combined, if desired. In some embodiments, any one of the nucleic acid molecules and compositions comprising such described herein can be administered via any route that is appropriate for the present disclosure. Administration may be continuous or intermittent, depending, for example, upon the recipient's physiological condition, whether the purpose of the administration is therapeutic, and other factors known to skilled practitioners. The administration may be essentially continuous over a preselected period of time or may be in a series of spaced doses.

[0505] “Approximately”: As used herein, the term “approximately” or “about,” as applied to one or more values of interest, refers to a value that is similar to a stated reference value. In some embodiments, the term “approximately” or “about” refers to a range of values that fall within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than or less than) of the stated reference value unless otherwise stated or otherwise evident from the context (except where such number would exceed 100% of a possible value).

[0506] “Complementary”: As used herein, the term “complementary” refers to the capacity for precise pairing between two nucleobases or two sets of nucleobases. In particular, complementary is a term that characterizes an extent of hydrogen bond pairing that brings about binding between two nucleobases or two sets of nucleobases. For example, if a nucleobase at one position of an oligonucleotide (e.g., a gRNA) is capable of hydrogen bonding with a nucleobase at the corresponding position of a target nucleic acid (e.g., a gene or an mRNA), then the two nucleobases are considered to be complementary to each other at that position. Base pairings may include both canonical Watson-Crick base pairing and non- Watson-Crick base pairing (e.g., Wobble base pairing and Hoogsteen base pairing). For example, in some embodiments, for complementary base pairings, adenosine-type bases (A) are complementary to thymidine-type bases (T) or uracil-type bases (U), that cytosine-type bases (C) are complementary to guanosine-type bases (G), and that universal bases such as 3- nitropyrrole or 5-nitroindole can hybridize to and are considered complementary to any A, C, U, or T. Inosine (I) has also been considered in the art to be a universal base and is considered complementary to any A, C, U or T. In another example, if an oligonucleotide (e.g., a gRNA) comprises a set of nucleobases (e.g., 6, 7, 8, 9, 10 or more consecutive nucleobases) capable of hydrogen bonding with a set of nucleobases nucleobase (e.g., 6, 7, 8, 9, 10 or more consecutive nucleobases) of a target nucleic acid (e.g., a gene or an mRNA), then the oligonucleotide (e.g., a gRNA) is considered to be complementary to the target nucleic acid (e.g., a gene or an mRNA). Two complementary nucleic acid molecules are able to non-covalently bind under appropriate temperature and solution ionic strength conditions.

[0507] For clarity, for the purposes of the present disclosure, in some embodiments, for an oligonucleotide (e.g., a gRNA) to be considered complementary to a target nucleic acid (e.g., a gene or an mRNA), full (i.e., 100%) complementarity is not required. For example, in some embodiments, one or more (e.g., one, two, or more) mismatches and / or additional sequences may be present in either the oligonucleotide (e.g., tRNA) or the target nucleic acid (e.g., a gene or an mRNA) as long as the two molecules are sufficiently complementary to hybridize and form a duplex.

[0508] In some embodiments, a spacer of a guide RNA described herein is complementary to a target nucleic acid (e.g., the complementary strand of a double stranded target nucleic acid) if the spacer comprises at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the target nucleic acid (e.g., the complementary strand of a double stranded target nucleic acid). As used herein, the term “substantially complementary” refers to a polynucleotide (e.g., a spacer of a crRNA) that has a certain level of complementarity to a target nucleic acid (e.g., the complementary strand of a double stranded target nucleic acid). In some embodiments, the level of complementarity is such that the polynucleotide (e.g., a spacer of a crRNA) can hybridize to the target nucleic acid molecule (e.g., the complementary strand of a double stranded target nucleic acid) with sufficient affinity to permit an effector polypeptide (e.g., Cas9) that is complexed with the polynucleotide to act (e.g., cleave) on the target nucleic acid (e.g., to introduce a double strand break). In some embodiments, a spacer that is substantially complementary to a target nucleic acid (e.g., the complementary strand of a double stranded target nucleic acid) has less than 100% complementarity to the target sequence. In some embodiments, a spacer that is substantially complementary to a target nucleic acid molecule (e.g., the complementary strand of a double stranded target nucleic acid) has at least about 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% complementarity to the target nucleic acid (e.g., the complementary strand of a double stranded target nucleic acid). In some embodiments, a spacer that is substantially complementary to a target nucleic acid (e.g., the complementary strand of a double stranded target nucleic acid) has less than 100% complementarity to the target sequence. In some embodiments, a spacer that is substantially complementary to a target nucleic acid (e.g., the complementary strand of a double stranded target nucleic acid) has no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 mismatches to the target nucleic acid (e.g., the complementary strand of a double stranded target nucleic acid). In some embodiments, a spacer is fully (100%) complementary to a target nucleic acid (e.g., the complementary strand of a double stranded target nucleic acid) across the length of the spacer. It is to be understood that all complementarity or level of complementarity described herein is across the region of complementarity of any spacer. In some embodiments, the region of complementarity is across the entire length of a spacer.

[0509] “Region of complementarity”: As used herein, the term “region of complementarity” refers to a nucleotide sequence, e.g., of an oligonucleotide, that is sufficiently complementary to a cognate nucleotide sequence, e.g., of a target nucleic acid (e.g., the complementary strand of a double stranded target nucleic acid), such that the two nucleotide sequences are capable of annealing to one another under physiological conditions (e.g., in a cell). In some embodiments, a region of complementarity is fully complementary to a cognate nucleotide sequence of target nucleic acid (e.g., the complementary strand of a double stranded target nucleic acid). However, in some embodiments, a region of complementarity is partially complementary to a cognate nucleotide sequence of target nucleic acid (e.g., at least 80%, 90%, 95% or 99% complementarity). In some embodiments, a region of complementarity contains 1, 2, 3, or 4 mismatches compared with a cognate nucleotide sequence of a target nucleic acid (e.g., the complementary strand of a double stranded target nucleic acid).

[0510] “Complex”: As used herein, the term “complex” refers to a grouping of two or more molecules, wherein the one or more molecules associate with each other via non-covalent bonding (e.g., hydrogen bonding or hydrophobic interaction) or covalent linkages. In some embodiments, the complex comprises a polypeptide and a nucleic acid molecule interacting with (e.g., binding to, coming into contact with, adhering to) one another. As used herein, the term “complex” can refer to a grouping of a guide RNA (gRNA) and a polypeptide (e.g., a Cas9 polypeptide). As used herein, in some embodiments, the term “complex” can refer to a grouping of an RNA guide, a polypeptide, and a target sequence. As used herein, the term “complex” can refer to a grouping of a gRNA targeting a mutated oncogene and a Cas9 nuclease.

[0511] “Cancer”: As used herein, the terms “cancer” or “tumor” are used interchangeably and refer to a group of diseases involving abnormal cell growth with the potential to invade or spread to other parts of the body, and benign tumors, which do not spread. The term cancer encompasses primary cancer and cancer that is metastatic cancer. The term cancer includes solid tumors and liquid tumors.

[0512] “Decrease”: As used herein, the terms “decrease”, “reduce”, “reduction”, “inhibit” or “inhibition” are all used herein generally to mean a decrease by a statistically significant amount. However, for avoidance of doubt, “decrease”, “reduced”, “reduction”, “inhibit” or “inhibition” means a decrease by at least 5% as compared to a reference level, for example a decrease by at least about 10%, or at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or at least about 95%, or at least about 98% or at least about 99%, or more as compared to a reference level.

[0513] “Gene”: As used herein, the term “gene” has its meaning as understood in the art. It will be appreciated by those of ordinary skill in the art that the term “gene” may include gene regulatory sequences (e.g., promoters, enhancers, etc.) and / or intron sequences. It will further be appreciated that definitions of gene include references to nucleic acids that do not encode proteins but rather encode functional RNA molecules such as RNAi agents, ribozymes, tRNAs, etc. For the purpose of clarity, it should be noted that, as used in the present application, the term “gene” generally refers to a portion of a nucleic acid that encodes a protein; the term may optionally encompass regulatory sequences, as will be clear from context to those of ordinary skill in the art. This definition is not intended to exclude application of the term “gene” to non- protein-coding expression units but rather to clarify that, in most cases, the term as used in this document refers to a protein-coding nucleic acid. In some embodiments, the term “gene” may be a mutated gene.

[0514] “Increase”: As used herein, the terms “increased”, “increase” or “enhance” or “activate” or “elevated” are all used herein to generally mean an increase by a statically significant amount; for the avoidance of any doubt, the terms “increased”, “increase” or “enhance” or “activate” or “elevated” means a statistically significant increase, such as an increase of at least 5% as compared to a reference level, for example an increase of at least about 10%, at least about 20%, or at least about 30%, or at least about 40%, or at least about 50%, or at least about 60%, or at least about 70%, or at least about 80%, or at least about 90% or up to and including a 100% increase or any increase between 5-100% as compared to a reference level, or at least about a 2-fold, or at least about a 3-fold, or at least about a 4-fold, or at least about a 5-fold or at least about a 10-fold increase, or any increase between 2-fold and 10-fold or greater as compared to a reference level.

[0515] “Nucleic acid”: As used herein, the terms “nucleic acid” and “nucleic acid molecule” refer to a compound comprising a nucleobase and an acidic moiety, e.g., a nucleoside, a nucleotide, or a polymer of nucleotides. Typically, polymeric nucleic acids, e.g., nucleic acid molecules comprising three or more nucleotides are linear molecules, in which adjacent nucleotides are linked to each other via a phosphodiester linkage. In some embodiments, “nucleic acid” refers to individual nucleic acid residues (e.g., nucleotides and / or nucleosides). In some embodiments, “nucleic acid” refers to an oligonucleotide chain comprising three or more individual nucleotide residues. As used herein, the terms “oligonucleotide” and “polynucleotide” can be used interchangeably to refer to a polymer of nucleotides (e.g., a string of at least three nucleotides). In some embodiments, “nucleic acid” encompasses single or double-stranded RNA as well as single and / or double-stranded DNA. Nucleic acids may be naturally occurring, for example, in the context of a genome, a gene, a transcript, an mRNA, tRNA, rRNA, siRNA, snRNA, gRNA, a plasmid, cosmid, chromosome, chromatid, or other naturally occurring nucleic acid molecule. On the other hand, a nucleic acid molecule may be a non-naturally occurring molecule, e.g., a recombinant DNA or RNA, an artificial chromosome, an engineered genome, or fragment thereof, or a synthetic DNA, RNA, DNA / RNA hybrid, or including non-naturally occurring nucleotides or nucleosides. Furthermore, the terms “nucleic acid,” “DNA,” “RNA,” and / or similar terms include nucleic acid analogs, i.e., analogs having other than a phosphodiester backbone. Nucleic acids can be purified from natural sources, produced using recombinant expression systems and optionally purified, chemically synthesized, etc. Where appropriate, e.g., in the case of chemically synthesized molecules, nucleic acids can comprise nucleoside analogs such as analogs having chemically modified bases or sugars, and backbone modifications. A nucleic acid sequence is presented herein in the 5’ to 3’ direction unless otherwise indicated. In some embodiments, a nucleic acid is or comprises natural nucleosides (e.g., adenosine, thymidine, guanosine, cytidine, uridine, deoxyadenosine, deoxythymidine, deoxy guanosine, and deoxycytidine); nucleoside analogs; and / or modified phosphate groups.

[0516] “Protein”: As used herein, the terms “protein,” “peptide,” and “polypeptide” are used interchangeably, and refer to a polymer of amino acid residues linked together by peptide (amide) bonds. The terms refer to a protein, peptide, or polypeptide of any size, structure, or function. Typically, a protein, peptide, or polypeptide will be at least three amino acids long. A protein, peptide, or polypeptide may refer to an individual protein or a collection of proteins. One or more of the amino acids in a protein, peptide, or polypeptide may be modified, for example, by the addition of a chemical entity such as a carbohydrate group, a hydroxyl group, a phosphate group, a famesyl group, an isofarnesyl group, a fatty acid group, a linker for conjugation, functionalization, or other modification, etc. A protein, peptide, or polypeptide may also be a single molecule or may be a multi-molecular complex. A protein, peptide, or polypeptide may be just a fragment of a naturally occurring protein or peptide. A protein, peptide, or polypeptide may be naturally occurring, recombinant, or synthetic, or any combination thereof. The term “fusion protein” as used herein refers to a hybrid polypeptide which comprises protein domains from at least two different proteins. One protein may be located at the amino-terminal (N-terminal) portion of the fusion protein or at the carboxyterminal (C-terminal) protein thus forming an “amino-terminal fusion protein” or a “carboxyterminal fusion protein,” respectively. Any of the proteins provided herein may be produced by any method known in the art. For example, the proteins provided herein may be produced via recombinant protein expression and purification, which is especially suited for fusion proteins comprising a peptide linker. Methods for recombinant protein expression and purification are well known, and include those described by Green and Sambrook, Molecular Cloning: A Laboratory Manual (4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012)), the entire contents of which are incorporated herein by reference.

[0517] “Lipid nanoparticle (LNP)”: As used herein, the term “lipid nanoparticle” or “LNP” refers to a vesicle formed by one or more lipid components. Lipid nanoparticles are typically used as carriers for nucleic acid delivery in the context of pharmaceutical development. They work by fusing with a cellular membrane and repositioning its lipid structure to deliver a drug or active pharmaceutical ingredient (API). Generally, lipid nanoparticle compositions for such delivery may contain synthetic ionizable or cationic lipids, phospholipids (e.g., compounds having a phosphatidylcholine group), cholesterol, and a polyethylene glycol (PEG) lipid; however, these compositions may also include other lipids. The sum composition of lipids typically dictates the surface characteristics and thus the protein (opsonization) content in biological systems thus driving biodistribution and cell uptake properties.

[0518] “Messenger RNA (mRNA)”: As used herein, the term “messenger RNA” or the term “mRNA” refers to any polynucleotide which encodes a polypeptide of interest and which is capable of being translated to produce the encoded polypeptide of interest in vitro, in vivo, in situ or ex vivo. In some embodiments, mRNA includes at least a coding region, a 5' UTR, and a 3' UTR.

[0519] “Promoter”: As used herein, a “promoter” refers to a nucleotide sequence that is capable of controlling the expression of a coding sequence or gene. Promoters are generally located 5” of the sequence that they regulate. Promoters may be derived in their entirety from a native gene, or be composed of different elements derived from promoters found in nature. When operably linked to a polynucleotide (e.g., a polynucleotide encoding a gene product such as a protein or RNA), a promoter determines the point and frequency of initiation of transcription of the polynucleotide, thus influencing the strength of expression of said polynucleotide. The term “operably linked” refers to the association of nucleic acid sequences on a single nucleic acid fragment so that the function of one of the sequences is affected by another. For example, a regulatory DNA sequence is said to be “operably linked to” or “associated with” a DNA sequence that codes for an RNA or a polypeptide if the two sequences are situated such that the regulatory DNA sequence affects expression of the coding DNA sequence (i.e., that the coding sequence or functional RNA is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. Regulatory sequences that may be operably linked to a polynucleotide (e.g., a polynucleotide encoding a gene product such as a protein or RNA) include, without limitation, promoters, repressor binding sites, activator binding sites, enhancers, and terminators.

[0520] “Sequencer identity”: As used herein “sequence identity” refers to the extent to which two optimally aligned polynucleotide or peptide sequences are invariant throughout a window of alignment of components, e.g., nucleotides or amino acids. An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Optimal alignment of sequences for aligning a comparison window are well known to those skilled in the art and may be conducted by tools such as the local homology algorithm of Smith and Waterman, the homology alignment algorithm of Needleman and Wunsch, the search for similarity method of Pearson and Lipman, and preferably by computerized implementations of these algorithms such as GAP, BESTFIT, FASTA, and TFASTA available as part of the GCG® Wisconsin Package® (Accelrys Inc., Burlington, MA). An “identity fraction” for aligned segments of a test sequence and a reference sequence is the number of identical components which are shared by the two aligned sequences divided by the total number of components in the reference sequence segment, i.e., the entire reference sequence or a smaller defined part of the reference sequence. Percent sequence identity is represented as the identity fraction multiplied by 100. The comparison of one or more sequences may be to a full-length sequence or a portion thereof, or to a longer sequence. As used herein, two sequences are substantially identical when the percent sequence identity between the two sequences is at least about 70%, at least about 80%, at least about 85%, at least about 90%, or even greater, such as about 98%, about 99%. Two sequences are identical when the percent sequence identity between the two sequences is 100%.

[0521] “Subject”: As used herein, the term “subject” refers to a mammal. In some embodiments, a subject is non-human primate, or rodent. In some embodiments, a subject is a human. In some embodiments, a subject is a patient, e.g., a human patient that has or is suspected of having a disease. In some embodiments, the subject is a human patient who has or is suspected of having cancer.

[0522] “Treat”: As used herein, the terms “treat” and “treatment” refer to both therapeutic treatment and measures that can alleviate symptoms or provide some benefit to a subject, wherein the object is to prevent or slow down (lessen) an undesired physiological change or disorder, such as the growth, development or spread of a disease (e.g., cancer). Beneficial or desired clinical results include, but are not limited to, alleviation of symptoms, diminishment of extent of disease, stabilized (i.e., not worsening) state of disease, delay or slowing of disease progression, amelioration or palliation of the disease state, and remission (whether partial or total), whether detectable or undetectable. In some embodiments, “treatment” can also mean prolonging survival as compared to expected survival if not receiving treatment. In some embodiments, “treatment” can also mean delaying progression of disease as compared to the progression of disease if not receiving treatment. Those in need of treatment include those already with a condition, disease or disorder, or those suspected to or susceptible to have a condition, disease or disorder.

[0523] “Therapeutically effective amount”: As used herein, the term “therapeutically effective amount” means an amount of a compound of the present invention that (i) treats the particular disease, condition, or disorder, (ii) attenuates, ameliorates, or eliminates one or more symptoms of the particular disease, condition, or disorder, or (iii) prevents or delays the onset of one or more symptoms of the particular disease, condition, or disorder described herein. In the case of cancer, the therapeutically effective amount of the drug may reduce the number of cancer cells; reduce the tumor size; inhibit (i.e., slow to some extent and preferably stop) cancer cell infiltration into peripheral organs; inhibit (i.e., slow to some extent and preferably stop) tumor metastasis; inhibit, to some extent, tumor growth; and / or relieve to some extent one or more of the symptoms associated with the cancer. To the extent the drug may prevent growth and / or kill existing cancer cells, it may be cytostatic and / or cytotoxic. For cancer therapy, efficacy can be measured, for example, by assessing the time to disease progression (TTP) and / or determining the response rate (RR).

[0524] “Upstream / downstream”: As used herein, the terms “upstream” and “downstream,” when referring to relative positions within a single stranded nucleic acid (DNA or RNA), upstream refers to the 5’ side of a reference position or sequence, and downstream refers to the 3’ side of a reference position or sequence. For example, a first sequence is upstream of a second sequence when the 3’ end of the first sequence occurs before the 5’ end of the second sequence. A first sequence is downstream of a second sequence when the 5’ end of the first sequence occurs after the 3’ end of the second sequence. The terms “upstream” and “downstream,” when referring to relative positions within a double stranded nucleic acid (e.g., a gene), relate to the 5’ to 3’ direction, respectively, in which RNA transcription of the gene occurs.

[0525] “Variant”: As used herein, the term “variant” refers to a nucleotide sequence or an amino acid sequence that is different from the reference nucleotide or polypeptide by one or more nucleotides or amino acids, respectively. For example, a variant may differ from a reference sequence by one or more substitutions, deletions, and / or additions. In some embodiments, a variant is a “functional variant” which retains some or all of the ability of the reference nucleotide or polypeptide. In some embodiments, a variant encompasses a functional variant comprising a nucleotide or amino acid sequence at least 75%, at least 76%, at least 77%, at least 78%, at least 79%, at least 80%, at least 81%, at least 82%, at least 83%, at least

[0526] 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least

[0527] 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least

[0528] 98%, at least 99%, and even 100% identical to a reference nucleotide or amino acid sequence, respectively.

[0529] “Vector”: As used herein, the terms “plasmid,” “vector,” and “cassette” are to be given their respective ordinary and customary meanings to a person of ordinary skill in the art, and are used without limitation to refer to an extra chromosomal element often carrying genes which are not part of the central metabolism of the cell, and usually in the form of circular double-stranded DNA molecules. Such elements may be autonomously replicating sequences, genome integrating sequences, phage or nucleotide sequences, linear or circular, of a single- or double-stranded DNA or RNA, derived from any source, in which a number of nucleotide sequences have been joined or recombined into a unique construction which is capable of introducing a promoter fragment and DNA sequence for a selected gene product along with appropriate 3' untranslated sequence into a cell.

[0530] “Lentiviral vector”: As used herein, the term “lentiviral vector” refers to a vector for gene delivery that were originally derived from the human immunodeficiency virus type- 1 (HIV-1) lentivirus. Lentiviral vectors are defective for replication, and thus considered relatively safe, but are capable of stably integrating into the genomic DNA of a broad range of dividing and nondividing mammalian cell types. The ability to stably integrate at semi-random genomic positions make lentiviral vectors a unique and ideal tool for studying stochastic variation in gene expression.

[0531] DETAILED DESCRIPTION

[0532] Oncogene Addiction

[0533] Mutations (e.g., gain-of-function mutations) in a gene (e.g., a human gene), can lead to various diseases (e.g., human diseases) such as cancer and genetic disorders. Such mutations (e.g., gain-of-function mutations) may be exploited as targets of therapeutic agents for disease(s) associated with the mutations. For example, gene editing agents (e.g., the CRISPR / Cas system) can be designed to specifically target the mutations to inactivate (e.g., knockdown or knockout) a mutated gene, and / or to correct the mutation (e.g., restore the mutated sequence to wild- type sequence).

[0534] Cancer cells may contain multiple genetic and epigenetic abnormalities. In some instances, mutations in a single oncogene can lead to cancer, and the growth and survival of the cancer can often be impaired by the inactivation of such mutated oncogene, despite the involvement of multiple genetic and epigenetic abnormalities in the process of cancer development (“oncogene addiction”).

[0535] The identification of cancer cells in multiple tumor types that have oncogene addiction and are highly sensitive to the targeted inhibition of single driver mutations has resulted in the development of several molecularly targeted therapeutics. For example, the BCR-ABL kinase inhibitor imatinib for the treatment of chronic myelogenous leukemia (CML), the EGFR kinase inhibitor gefitinib for the treatment of non-small cell lung cancer (NSCLC), and the BRAF kinase inhibitor vemurafenib for the treatment of advanced melanoma. Although these oncogene-targeted agents have provided clinical responses, many patients ultimately experience a recurrence of their disease due to the development of drug resistance. New and improved therapies targeting oncogene addiction remain needed. Deregulated Wnt / p-catenin signaling is one of the main genetic alterations in human hepatocellular carcinoma (HCC). Comprehensive genomic analyses have revealed that gain-of- function mutation of CTNNB1, which encodes P-catenin, occur frequently in HCC. Over 20% of HCC contain CTNNB1 mutations. Human HCCs with activation of the Wnt / p-catenin pathway demonstrate unique gene expression patterns and pathological features. Activated Wnt / p-catenin synergizes with multiple signaling cascades to drive HCC formation, and it functions through its downstream effectors. Human HCC harboring activating CTNNB1 mutation also show dependency (addiction) to the mutated CTNNB1. Compositions and methods described herein that target mutated CTNNB1 for inactivation via gene editing were demonstrated herein to be efficacious in inhibiting the growth of HCC harboring CTNNB1 with the targeted mutation.

[0536] The present disclosure, in some aspects, provides strategies for inactivating mutated oncogenes (e.g., mutated CTNNBI), e.g., by knocking it down or knocking it out using gene editing agents (e.g., the CRISPR / Cas system). Such strategies may be therapeutically efficacious for cancer (e.g., cancer addicted to a mutated oncogene such as hepatocellular carcinoma (HCC) addicted to a mutated CTNNBI). Site-specific gene editing agents targeting the sequence(s) containing the mutation(s) in an oncogene (e.g., CTNNBI) may be engineered such that untended editing in normal cells that do not contain the mutation are reduced and / or minimized. In some aspects, gene editing agents and methods described herein are for targeting gain-of-function mutations in genes (e.g., an oncogene such as CTNNBI) and for treating cancers addicted to the mutated oncogenes (e.g., HCC) with minimized off target effect and low toxicity to normal cells.

[0537] Non-limiting examples of mutations in a mutated CTNNBI gene that may be targeted (e.g., for inactivation) using a composition or method described herein, and amino acid substitutions caused by mutations in the CTNNBI gene are provided in Table 1.

[0538] Table 1. Nonlimiting examples of targeted mutations in CTNNBI gene

[0539] Gene Editing Agents

[0540] Some aspects of the present disclosure provide gene editing agents that target mutations (e.g., gain-of-function mutations) in genes. In some embodiments, the gain-of-function mutations are associated with disease or disorders such as cancer or genetic diseases. In some embodiments, gene editing agents described herein target mutations (e.g., gain-of-function mutations in genes) in an oncogene (e.g., CTNNBI). In some embodiments, gene editing agents described herein can induce a double strand break (DSB) in a mutated oncogene (e.g., CTNNBI), resulting in reduced (e.g., reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more) expression and / or activity of the oncogene (e.g., CTNNBI in a cell (e.g., cancer cell such as an HCC cell), relative to the level of expression and / or activity of the mutated oncogene in a cell (e.g., a cancer cell) without the gene editing agents. In some embodiments, gene editing agents described herein can induce a double strand break (DSB) in a mutated oncogene (e.g., CTNNBI), thereby inactivating or substantially inactivating (e.g., at least at least 80%, at least 90%, at least 95% at least 98%, or at least 99% reduction of expression and / or activity) the mutated oncogene (e.g., CTNNBI). In some embodiments, gene editing agents described herein inactivate a mutated oncogene ((e.g., CTNNBI), result in cancer cell (e.g., HCC cell) death, and are efficacious in treating cancer (e.g., a cancer addicted to the oncogene being inactivated, such as HCC).

[0541] Gene editing agents suitable for use in accordance with the present disclosure include known agents capable of making site-specific gene editing, e.g., without limitation, sitespecific recombinases, zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and RNA guided nucleases (e.g., a CRISPR / Cas system).

[0542] Zinc-finger nucleases (ZFNs) and their uses for gene editing are known in the art, e.g., described in Carroll et al., Genetics. 2011 Aug; 188(4): 773-782, the entire contents of which are incorporated herein by reference.

[0543] Transcription activator-like effector nucleases (TALENs) and their uses for gene editing are known in the art, e.g., described in Becker et al., Gene and Genome Editing, Volume 2, December 2021, 100007, the entire contents of which are incorporated herein by reference.

[0544] CRISPR / Cas System

[0545] In some embodiments, gene editing agents described herein are CRISPR / Cas systems comprising an RNA guided nuclease (e.g., Cas9), and a guide RNA (gRNA), wherein the gRNA targets a mutated oncogene (e.g., CTNNBI) and the RNA guided nuclease induces a double stranded break in the mutated oncogene (e.g., CTNNBI), thus reducing (e.g., reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, at least 90%, at least 95% or more) expression and / or activity of the mutated oncogene (e.g., CTNNBI), and / or inactivating or substantially inactivating (e.g., at least at least 80%, at least 90%, at least 95% at least 98%, or at least 99% reduction of expression and / or activity) the mutated oncogene (e.g., CTNNBI).

[0546] As such, some aspects of the present disclosure provide spacers of gRNAs that target a sequence of an oncogene (e.g., CTNNBI), wherein the sequence of the oncogene comprises a mutation associated with cancer (e.g., a gain-of-function mutation). In some embodiments, a gain-of-function mutation in CTNNBI is associated with hepatocellular carcinoma (HCC).

[0547] The present disclosure further provides crRNAs comprising a spacer that targets a sequence of an oncogene (e.g., CTNNBI), wherein the sequence of the oncogene comprises a mutation associated with cancer (e.g., a gain-of-function mutation). In some embodiments, a crRNA described herein comprises such a spacer and further comprises a direct repeat component (e.g., at the 3’ or 5’ side of the spacer).

[0548] The present disclosure further provides guide RNAs (gRNAs) comprising a crRNA comprising a spacer that targets a sequence of an oncogene (e.g., CTNNB1), wherein the sequence of the oncogene comprises a mutation associated with cancer (e.g., a gain-of-function mutation). In some embodiments, a crRNA described herein comprises such a spacer and further comprises a direct repeat component (e.g., at the 3’ or 5’ side of the spacer). In some embodiments, a gRNA described herein further comprises a tracrRNA (e.g., a tracrRNA known in the art or provided herein, such as the tracrRNA comprising the nucleotide sequence of any one of SEQ ID NOs: 10-13).

[0549] In some embodiments, a spacer described herein is fully complementary or substantially complementary to a target nucleic acid (e.g., the complementary strand of a double stranded target nucleic acid) across the length of a region of complementarity in the spacer. In some embodiments, the target nucleic acid is any one of the mutated oncogenes described herein (e.g., an oncogene listed in Table 1). All complementarity and level of complementarity between a spacer and a target nucleic acid described herein is across the length of a region of complementarity in the spacer.

[0550] In some embodiments, a spacer described herein comprises a region of complementarity of a target nucleic acid (e.g., a sequence of CTNNB1 comprising the mutation). In some embodiments, the mutation is a single nucleotide mutation (e.g., an A to T mutation, A to C mutation, A to G mutation, T to C mutation, T to G mutation, T to A mutation, G to C mutation, G to A mutation, G to T mutation, C to G mutation, C to T mutation, or C to A mutation). In some embodiments, the mutation (e.g., single nucleotide mutation) results in an amino acid substitution in a protein encoded by the oncogene. Nonlimiting examples of amino acid substitutions resulting from mutations (e.g., single nucleotide mutations) in oncogenes are provided in Table 1. It is to be understood that when a mutation (e.g., single nucleotide mutation) in the positive (+) strand of a gene is present, there is a corresponding mutation (e.g., single nucleotide change) in the negative (-) strand at the position complementary to the mutated position in the positive (+) strand. When a “mutation” is referred to herein, the term encompasses the changes in the positive (+) strand, the negative (-) strand, or in both strands.

[0551] In some embodiments, a target nucleic acid (e.g., a sequence of CTNNBI) comprising a mutation (e.g., a mutation listed in Table 1) that is targeted by a spacer is double stranded, which comprises a complementary strand and a target strand, each comprising the mutation. In some embodiments, a positive (+) strand of a double stranded sequence of an oncogene (e.g., CTNNBI) is the complementary strand, and the negative (-) strand of double stranded sequence of an oncogene (e.g., CTNNBI) is the target strand. In some embodiments, a positive (+) strand of a double stranded sequence of an oncogene (e.g., CTNNBI) is the target strand, and the negative (-) strand of double stranded sequence of an oncogene (e.g., CTNNBI) is the complementary strand. As used herein, a “positive (+) strand” of a double stranded sequence refers to the strand that comprises the sequence encoding the gene product (e.g., protein or RNA), a “negative (-) strand” refers to the strand that comprises the sequence complementary to the sequence encoding the gene product (e.g., protein or RNA).

[0552] A spacer described herein comprises a region of complementarity to the complementary strand (e.g., either the positive (+) strand or the negative (-) strand). In some embodiments, spacers of the present disclosure specifically target a mutated sequence in an oncogene by containing nucleotide(s) complementary to the mutated positions in the region of complementarity (such nucleotide(s) is referred to herein as “mutation targeting position(s)”). Spacers comprising the mutation-targeting position have reduced or no binding affinity to a wild type sequence without the mutation, thereby specifically targeting cells and / or sequences in which the mutation is present. The mutation targeting position may be present anywhere in the region of complementary of a spacer, provided that a PAM is present adjacent to the mutation (e.g., either on the positive (+) strand or on the negative (-) strand) in an oncogene.

[0553] In some embodiments, a spacer described herein comprises a region of complementarity to the complementary strand, wherein the complementary strand is the positive (+) strand of the oncogene such as CTNNBI (in this instance, the PAM is located in the negative (-) strand, which is the target strand). In such instances, the region of complementary of a spacer comprises mutation targeting positions (e.g., one mutation targeting position) complementary to the mutated position in the positive (+) strand of the oncogene such as CTNNBI. In such instances, in some embodiments, the spacer comprises a nucleotide sequence that is substantially identical (e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical) to the nucleotide sequence of the negative (-) strand that is complementary to the sequence in the positive (+) strand that comprises the mutation.

[0554] In some embodiments, a spacer described herein comprises a region of complementarity to the complementary strand, wherein the complementary strand is the negative (-) strand of the sequence of the oncogene such as CTNNBI (in this instance, the PAM is located in the positive (+) strand, which is the target strand). In some embodiments, the region of complementary of a spacer comprises mutation targeting positions (e.g., one mutation targeting) complementary to the mutated position in the negative (-) strand of the oncogene such as CTNNB1. In some embodiments, the spacer comprises a nucleotide sequence that is substantially identical (e.g., at least 80%, at least 90%, at least 95%, at least 99%, or 100% identical) to the nucleotide sequence of the positive (+) strand that is complementary to the sequence in the negative (-) strand that comprises the mutation.

[0555] In some embodiments, a spacer described herein is 15-22 nucleotides in length. For example, a spacer may be 15, 16, 17. 18, 19, 20, 21, or 22 nucleotides in length. In some embodiments, a spacer described herein is 20 nucleotides in length. In some embodiments, a region of complementarity of a spacer described herein is 15-22 nucleotides in length. For example, a region of complementarity of a spacer may be 15, 16, 17. 18, 19, 20, 21, or 22 nucleotides in length. In some embodiments, a region of complementarity of a spacer described herein is 20 nucleotides in length.

[0556] As described herein, in some embodiments, a spacer comprises a mutation targeting position (e.g., a nucleotide that is complementary to a single nucleotide mutation in a complementary strand). In some embodiments, for a given mutation (e.g., CTNNB1 mutations listed in Table 1), one or more spacer sequences may be designed, depending on the presence of a PAM on either the positive (+) strand or the negative (-) strand, and / or the location of the PAM relative to the location of the mutation in the target nucleic acid. In some embodiments, more than one PAM is present in the vicinity of a mutation, and multiple spacers targeting the mutation can be designed. Different spacers targeting the same mutation may differ in nucleotide sequences, and / or differ in the location of the mutation targeting position in the spacer. In some embodiments, a mutation targeting position of a spacer may be located at any position of the spacer, e.g., at nucleotide position 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 of a spacer.

[0557] PAMs that may be used in accordance with the present disclosure include, without limitation, PAMs recognized by a Cas9 nuclease, such as “NGG,” “NNGRR,” or “NNGRRN,” in which the “N” represents any nucleobase, “G” represents the nucleobase guanine, and “R” represents a purine (guanine (G) or adenine (A)). In some embodiments, a PAM is located in the target strand, and is at the 3’ side of a target sequence as described herein. A PAM may be present at a position of different distance to the mutation position in the target sequence. As used herein, a “distance” between a PAM and a mutation position (e.g., position of a single nucleotide mutation) in a target sequence is determined by setting the position of the 5’end nucleotide of the PAM as position 0, and count upstream (i.e., towards the 5’ direction of the target sequence) until the mutation position is reached. The “count” of the mutation position is the “distance” between a PAM and a mutation position. In some embodiments, for a spacer described herein, the distance between a PAM in a target sequence of the spacer and the mutation position in the target sequence is between 1-20 nucleotides (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nucleotides).

[0558] In some embodiments, a mutation (e.g., a mutation in CTNNBT) results in a PAM that is not present in a wild type sequence, enabling specific targeting of the mutation by gene editing agents described herein such as a CRISPR / Cas system. The PAM resulted from the mutation may be present on the positive (+) strand or the negative (-) strand of the oncogene. In some embodiments, a mutation (e.g., a mutation in CTNNBI) results in a PAM in the positive (+) strand that is not present in a wild type sequence. In this case, the target sequence is upstream (on the 5’ side) of the PAM in the positive (+) strand and the spacer contains no mutation targeting position. In some embodiments, a mutation (e.g., a mutation in an oncogene) results in a PAM in the negative (-) strand that is not present in a wild type sequence. In this case, the target sequence is upstream (on the 5’ side) of the PAM in the negative (-) strand and the spacer contains no mutation targeting position. One skilled in the art would appreciate that “upstream (one the 5’ side)” in the negative (-) strand means downstream in the direction of gene transcription. When a PAM is created by a mutation, the distance between a PAM and the mutation position may be -1, -2, or -3 nucleotides (i.e., counting downstream (towards the 3’ direction) from the 5’ end nucleotide of the PAM, which is position 0).

[0559] Table 2. Non-limiting examples of spacers for gRNAs targeting CTNNBI mutations

[0560]

[0561]

[0562]

[0563]

[0564]

[0565]

[0566] In some embodiments, a spacer described herein targets a target sequence in CTNNB1 (e.g., a sequence in the target strand), wherein the target sequence comprises the nucleobase sequence of any one of SEQ ID NOs: 86-154 or 256-284. In some embodiments, a spacer described herein comprises a region of complementarity that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) complementary to a sequence in the complementary strand of CTNNB 1 that is the reverse complementary sequence of a target sequence as set forth in any one of 86-154 or 256-284.

[0567] In some embodiments, a target sequence in CTNNB 1 includes a PAM. In some embodiments, a spacer described herein targets a target sequence in CTNNB 1 (e.g., a sequence in the target strand), wherein the target sequence comprises the nucleobase sequence of any one of SEQ ID NOs: 158-226 or 285-313. In some embodiments, a spacer described herein comprises a region of complementarity that is at least 80% (e.g., at least 80%, at least 85%, at least 90%, at least 95%, at least 98%, at least 99%, or 100%) complementary to a sequence in the complementary strand of CTNNB 1 that is the reverse complementary sequence of a target sequence as set forth in any one of SEQ ID NOs: 158-226 or 285-313.

[0568] In some embodiments, a spacer described herein comprises a nucleobase sequence that contains no more than 5, no more than 4, no more than 3, no more than 2, or no more than 1 different nucleobases than a spacer sequence as set forth in any one of SEQ ID NOs: 14-82 or 227-255. In some embodiments, a spacer described herein comprises the nucleobase sequence of any one of SEQ ID NOs: 14-82 or 227-255.

[0569] In some embodiments, any one of the spacers described herein is part of a crRNA. In some embodiments, a crRNA comprises any one of the spacers described herein (e.g., spacers listed in Table 2, any one of SEQ ID NOs: 14-82 or 227-255), and further comprises a direct repeat component.

[0570] In some embodiments, any one of the crRNA described herein is part of a guide RNA. In some embodiments, a guide RNA comprises any one of the crRNA described herein, and further comprise a tracrRNA (e.g., a tracrRNA comprising a nucleotide sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 10-13 and 314).

[0571] In some embodiments, a guide RNA comprises any one of the crRNA described herein, and further comprise a tracrRNA (e.g., a tracrRNA comprising a nucleotide sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 10). In some embodiments, a guide RNA comprises any one of the crRNA described herein, and further comprise a tracrRNA (e.g., a tracrRNA comprising a nucleotide sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 314).

[0572] In some embodiments, a gRNA described comprises a crRNA comprising any one of the spacers described herein (e.g., spacers listed in Table 2, any one of SEQ ID NOs: 14-82 or 227-255), and further comprise a tracrRNA (e.g., a tracrRNA comprising a nucleotide sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to any one of SEQ ID NOs: 10-13 and 314).

[0573] In some embodiments, a gRNA described comprises a crRNA comprising any one of the spacers described herein (e.g., spacers listed in Table 2, any one of SEQ ID NOs: 14-82 or 227-255), and further comprise a tracrRNA (e.g., a tracrRNA comprising a nucleotide sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 10).

[0574] In some embodiments, a gRNA described comprises a crRNA comprising any one of the spacers described herein (e.g., spacers listed in Table 2, any one of SEQ ID NOs: 14-82 or 227-255), and further comprise a tracrRNA (e.g., a tracrRNA comprising a nucleotide sequence at least 70%, at least 80%, at least 90%, at least 95%, or 100% identical to SEQ ID NO: 314).

[0575] A guide RNA described herein can be complexed with an RNA guided nuclease (e.g., a Cas9 nuclease) for gene editing. Cas9 nucleases suitable for use in gene editing (e.g., for inducing a double strand break and / or for inactivation of a gene) are provided herein and / or known in the art, e.g., as described in Ferretti et al., Proc. Natl. Acad. Sci. U.S.A. 98:4658- 4663(2001); Deltcheva et al., Nature 471:602-607(2011); and Jenik et al., Science 337:816- 821(2012), the entire contents of each of which are incorporated herein by reference.

[0576] In some embodiments, a Cas9 nuclease that may be used in accordance with the present disclosure may be a wild type Cas9 nuclease from Streptococcus pyogenes, Streptococcus thermophilus, Staphylococcus aureus, Corynebacterium ulcerans, Corynebacterium diphtheriae, Spiroplasma syrphidicola, Prevotella intermedia, Spiroplasma taiwanense, Streptococcus iniae, Belliella baltica, Psychroflexus torquisl, Streptococcus thermophilus, Listeria innocua, Campylobacter jejuni, or Neisseria meningitidis, and any functional fragments or variants thereof. In some embodiments, a Cas9 nuclease that may be used in accordance with the present disclosure is a wild type Cas9 nuclease from Streptococcus pyogenes (NCBI cDNA Reference Sequence: NC_002737.2:854751-858857; Protein Reference Sequence: WP_010922251.1), and any functional fragments or variants thereof. As a non-limiting example, the nucleotide and amino acid sequences of the wild type Cas9 nuclease from Streptococcus pyogenes are provided below.

[0577] Streptococcus pyogenes Cas9 nucleotide sequence (NC_002737.2:854751-858857; SEQ ID

[0578] NO: 1)

[0579] ATGGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGG

[0580] CGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAA

[0581] TACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTG

[0582] GAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACAC

[0583] GTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAA

[0584] GTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAA

[0585] GAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATG

[0586] AGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTGATAAA

[0587] GCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCAT

[0588] TTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTAT

[0589] CCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGT

[0590] GGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAG

[0591] AAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTC

[0592] ATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAA

[0593] GATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATT

[0594] GGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAG

[0595] ATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCC

[0596] CTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTT

[0597] AAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATC

[0598] AATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATT

[0599] TTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGG

[0600] TGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTC

[0601] TATTCCCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAG

[0602] ACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTT

[0603] CGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGAT

[0604] GACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGAT

[0605] AAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCT

[0606] TCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTT

[0607] ATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATT TCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAA

[0608] AAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGA

[0609] TAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACC

[0610] ATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGA

[0611] AGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGA

[0612] TTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACA

[0613] GCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATG

[0614] GTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGT

[0615] TTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGA

[0616] AGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATT

[0617] GCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGT

[0618] TGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATT

[0619] GAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAG

[0620] CGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAG

[0621] AGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTC

[0622] CAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTG

[0623] ATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGAC

[0624] AATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAA

[0625] GTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAA

[0626] GTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGA

[0627] GTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATC

[0628] ACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAA

[0629] ATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCT

[0630] GACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCA

[0631] TGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATC

[0632] CAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAA

[0633] ATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTT

[0634] ACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATT

[0635] CGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATA

[0636] AAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATT

[0637] GTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAA

[0638] AAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATA

[0639] TGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGG

[0640] AAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAAT TATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGA

[0641] TATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGA

[0642] GTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGA

[0643] AATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTA

[0644] TGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAG

[0645] CAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCG

[0646] TGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATA

[0647] GAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGAC

[0648] GAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAAC

[0649] GATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACT

[0650] GGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACTGA

[0651] Streptococcus pyogenes Cas9 amino acid sequence (WP_010922251.1; SEQ ID NO: 2)

[0652] MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGET

[0653] AEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERH

[0654] PIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLN

[0655] PDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKK

[0656] NGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLA

[0657] AKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFF

[0658] DQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIP

[0659] HQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSE

[0660] ETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVK

[0661] YVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDR

[0662] FNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDK

[0663] VMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTF

[0664] KEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIE

[0665] MARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNG

[0666] RDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVV

[0667] KKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQ

[0668] ILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNA

[0669] VVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTE

[0670] ITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKE

[0671] SILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGI

[0672] TIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNE LALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILAD

[0673] ANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVL

[0674] DATLIHQSITGLYETRIDLSQLGGD

[0675] In some embodiments, a Cas9 nuclease used in accordance with the present disclosure comprises an amino acid sequence that is at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 2. In some embodiments, a Cas9 nuclease used in accordance with the present disclosure comprises an amino acid sequence that is at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 2 and is functional. In some embodiments, a Cas9 nuclease used in accordance with the present disclosure comprises or consists of the amino acid sequence of SEQ ID NO: 2.

[0676] In some embodiments, a Cas9 nuclease used in accordance with the present disclosure is encoded by a nucleotide sequence that is at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 1. In some embodiments, a Cas9 nuclease used in accordance with the present disclosure is encoded by a nucleotide sequence that is at least 70% (e.g., at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100%) identical to SEQ ID NO: 1, and is functional. In some embodiments, a Cas9 nuclease used in accordance with the present disclosure is encoded by the nucleotide sequence of SEQ ID NO: 2.

[0677] In some embodiments, Cas9 variants with improved activity (e.g., higher efficiency in cleaving a target nucleic acid / or reduced off target effect) may be used. Such Cas9 variants have been described in the art, e.g., in Huang et al., Cells, vol. 11,14 2186. 13 Jul. 2022, doi:10.3390 / cellsl l l42186. Examples of Cas9 variants with improved activity that may be used in accordance with the present disclosure, include, but are not limited to, SpCas9- D1135E, eSpCas9(1.0), eSpCas9(l.l), SpCas9-HFl, HypaCas9, HiFi Cas9, xCas9-3.6, xCas9-

[0678] 3.7, Sniper-Cas9, evoCas9, SpartaCas, LZ3 Cas9, miCas9, SuperFi-Cas9.

[0679] In some embodiments, any one of the gRNAs described herein is complexed with a Cas9 nuclease to form a complex (e.g., a ribonucleoprotein complex). In some embodiments, complexation of a gRNA and Cas9 nuclease occurs at a temperature lower than about any one of 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26 °C, 27°C, 28°C, 29°C, 30°C, 31°C, 32°C, 33°C,

[0680] 34°C, 35°C, 36°C, 37°C, 38°C, 39°C, 40°C, 41°C, 42°C, 43°C, 44°C, 45°C, 50°C, or 55°C. In some embodiments, a gRNA does not dissociate from the Cas9 nuclease at about 37°C over an incubation period of at least about any one of lOmins, 15mins, 20mins, 25mins, 30mins, 35mins, 40mins, 45mins, 50mins, 55mins, Ihr, 2hr, 3hr, 4hr, or more hours.

[0681] In some embodiments, any one of the gRNA and Cas9 nuclease described herein are complexed in a complexation buffer. In some embodiments, a Cas9 nuclease is stored in a buffer that is replaced with a complexation buffer to form a complex with the gRNA. In some embodiments, a Cas9 nuclease is stored in a complexation buffer.

[0682] In some embodiments, the complexation buffer has a pH in a range of about 7.3 to 8.6. In some embodiments, the pH of the complexation buffer is about 7.3. In some embodiments, the pH of the complexation buffer is about 7.4. In some embodiments, the pH of the complexation buffer is about 7.5. In some embodiments, the pH of the complexation buffer is about 7.6. In some embodiments, the pH of the complexation buffer is about 7.7. In some embodiments, the pH of the complexation buffer is about 7.8. In some embodiments, the pH of the complexation buffer is about 7.9. In some embodiments, the pH of the complexation buffer is about 8.0. In some embodiments, the pH of the complexation buffer is about 8.1. In some embodiments, the pH of the complexation buffer is about 8.2. In some embodiments, the pH of the complexation buffer is about 8.3. In some embodiments, the pH of the complexation buffer is about 8.4. In some embodiments, the pH of the complexation buffer is about 8.5. In some embodiments, the pH of the complexation buffer is about 8.6.

[0683] In some embodiments, a Cas9 nuclease can be overexpressed and complexed with a gRNA in a host cell prior to purification as described herein. In some embodiments, mRNA or DNA encoding a Cas9 nuclease is introduced into a cell so that the Cas9 nuclease is expressed in the cell. In some embodiments, a gRNA is also introduced into the cell, whether simultaneously, separately, or sequentially from a single mRNA or DNA construct, such that the ribonucleoprotein complex is formed in the cell.

[0684] Nucleic Acids and Vectors

[0685] The present disclosure further provides nucleic acid molecules comprising a nucleotide sequence encoding any one of the spacers, crRNAs, or gRNAs, and / or RNA guided nucleases (e.g., Cas9 nuclease) described herein.

[0686] In some embodiments, a nucleic acid molecule comprises a nucleotide sequence encoding a gRNA described herein. In some embodiments, the nucleic acid molecule further comprises a promoter operably linked to the nucleotide sequence encoding the gRNA. In some embodiments, a nucleotide acid molecule comprises a nucleotide sequence encoding a gRNA described herein, wherein the nucleotide sequence is operably linked to a promoter, and optionally further operably linked to one or more (e.g., 1, 2, 3, or more) regulatory sequences (e.g., enhancers, transcriptional terminators, etc.).

[0687] In some embodiments, a nucleic acid molecule comprises a first nucleotide sequence encoding a gRNA described herein, and further comprises a second nucleotide sequence encoding an RNA guided nuclease (e.g., a Cas9 nuclease). In some embodiments, a nucleic acid molecule a nucleic acid molecule comprises a first nucleotide sequence encoding a gRNA described herein, and further comprises a second nucleotide sequence encoding an RNA guided nuclease (e.g., a Cas9 nuclease), wherein the first nucleotide sequence is operably linked to a promoter, and the second nucleotide sequence is operably linked to a promoter, and each nucleotide sequence is optionally further operably linked to one or more (e.g., 1, 2, 3, or more) regulatory sequences (e.g., enhancers, transcriptional terminators, etc.). In some embodiments, a nucleic acid molecule a nucleic acid molecule comprises a first nucleotide sequence encoding a gRNA described herein, and further comprises a second nucleotide sequence encoding an RNA guided nuclease (e.g., a Cas9 nuclease), wherein the first nucleotide sequence and the second nucleotide sequence is each operably linked to a separate promoter. In some embodiments, a nucleic acid molecule a nucleic acid molecule comprises a first nucleotide sequence encoding a gRNA described herein, and further comprises a second nucleotide sequence encoding an RNA guided nuclease (e.g., a Cas9 nuclease), wherein the first nucleotide sequence and the second nucleotide sequence are operably linked to one single promoter (i.e., encoding a polycistronic transcript). In some embodiments, a nucleic acid molecule a nucleic acid molecule comprises a first nucleotide sequence encoding a gRNA described herein, and further comprises a second nucleotide sequence encoding an RNA guided nuclease (e.g., a Cas9 nuclease), wherein the first nucleotide sequence and the second nucleotide sequence is each operably linked to a separate promoter.

[0688] In some embodiments, the promoter is a constitutive promoter. In some embodiments, the promoter is an inducible promoter. In some embodiments, promoters may be between 35- 105 (e.g., 50-60) base pairs in size. The promoter to drive expression of the sequence encoding the gRNA and / or RNA guided nuclease (e.g., Cas9 nuclease) to be delivered can be any desired promoter (including native promoter sequences), selected by known considerations, such as the level of expression of a nucleic acid functionally linked to the promoter and the cell type in which the vector is to be used. In some embodiments, the promoter may be reduced- sequence or re-configured promoters. In some embodiments, any one of the nucleotide sequences encoding an RNA guided nuclease (e.g., Cas9 nuclease) further comprises coding sequences for a nuclear localization signal and / or signal peptides.

[0689] In some embodiments, any one of the nucleic acid molecules described herein is DNA (e.g., an expression cassette, a viral vector, a nonviral vector, a plasmid, a nanoplasmid, a DNA fragment, a DNA thread, or a closed end DNA thread). In some embodiments, the nucleic acid molecule is RNA. In some embodiments, the vector is a viral vector. In some embodiments, the vector is a lentiviral vector. Use of the lentiviral vector advantageously permits transduction of cells that are dividing, as well as cells that are quiescent (i.e., non-dividing). Methods of delivering lentiviral vectors, or delivery of a CRISPR / Cas9 system using a lentiviral vector are well known in the art. For example, see W02006089001A2, W02008099148A1, WO2017197038A1, the contents of which are incorporated herein in their entirety.

[0690] Cells

[0691] Further provided herein are cells comprising any one of the spacers, crRNAs, gRNAs, an RNA guided nuclease (e.g., Cas9 nuclease), a complex comprising a gRNA and an RNA guided nuclease (e.g., Cas9 nuclease), or a nucleic acid molecule described herein. Administration to a cell can be accomplished by any means, including simply contacting the cell. In some embodiments, the cell is in vitro (e.g., cultured cells). Methods of introducing nucleic acids or a ribonucleoprotein complexes into cells cultured in vitro are known in the art, including, without limitation, transfection transduction, transformation, and electroporation. In some embodiments, the cell is in vivo (e.g., in a subject). In some embodiments, any one of the spacers, crRNAs, gRNAs, an RNA guided nuclease (e.g., Cas9 nuclease), a complex comprising a gRNA and an RNA guided nuclease (e.g., Cas9 nuclease), or a nucleic acid molecule described herein can be formulated for administration to a subject.

[0692] In some embodiments, the cell is an isolated cell. In some embodiments, the cell is in cell culture or a co-culture of two or more cell types. In some embodiments, the cell is ex vivo. In some embodiments, the cell is obtained from a living organism and maintained in a cell culture. In some embodiments, the cell is a single-cellular organism.

[0693] In some embodiments, the cell is a prokaryotic cell. In some embodiments, the cell is a bacterial cell or derived from a bacterial cell. In some embodiments, the cell is an archaeal cell or derived from an archaeal cell. In some embodiments, the cell is a eukaryotic cell. In some embodiments, the cell is an animal cell or derived from an animal cell. In some embodiments, the cell is a vertebrate cell or derived from a vertebrate cell. In some embodiments, the cell is a mammalian cell or derived from a mammalian cell. In some embodiments, the cell is a non-human primate cell (e.g., a cynomolgus monkey cell). In some embodiments, the cell is a human cell. In some embodiments, the cell is a rodent cell. In some embodiments, the cell is synthetically made, sometimes termed an artificial cell.

[0694] In some embodiments, the cell is derived from a cell line. A wide variety of cell lines for tissue culture are known in the art. Examples of cell lines include, but are not limited to, 293T, MF7, K562, HeLa, CHO, and transgenic varieties thereof. Cell lines are available from a variety of sources known to those with skill in the art (see, e.g., the American Type Culture Collection (ATCC) (Manassas, Va.)). In some embodiments, the cell is an immortal or immortalized cell.

[0695] In some embodiments, the cell is a primary cell. In some embodiments, the cell is a stem cell such as a totipotent stem cell (e.g., omnipotent), a pluripotent stem cell, a multipotent stem cell, an oligopotent stem cell, or an unipotent stem cell. In some embodiments, the cell is an induced pluripotent stem cell (iPSC) or derived from an iPSC. In some embodiments, the cell is a differentiated cell.

[0696] In some embodiments, the cell is a cancer cell. In some embodiments, the cell is a human cancer cell. In some embodiments, the cell is a hepatocyte. In some embodiments, the cell is a hepatocellular adenoma cell. In some embodiments, the cell is a hepatocellular carcinoma cell.

[0697] Compositions and Methods of Delivery

[0698] Further provided herein are compositions comprising any one of the spacers, crRNAs, gRNAs, an RNA guided nuclease (e.g., Cas9 nuclease), a complex comprising a gRNA and an RNA guided nuclease (e.g., Cas9 nuclease), or a nucleic acid molecule (e.g., lentiviral vector) described herein. In some embodiments, the present disclosure provides a composition comprising a nucleic acid molecule comprising a nucleotide sequence encoding an RNA- guided nuclease (e.g., a Cas9 nuclease), and any one of the gRNAs described herein. In some embodiments, the nucleic acid molecule encoding the RNA-guided nuclease (e.g., a Cas9 nuclease) is an mRNA.

[0699] Compositions, gRNAs, or complexes described herein may be formulated, for example, including a carrier, such as a carrier and / or a polymeric carrier, e.g., a liposome, and delivered by known methods to a cell (e.g., a prokaryotic, eukaryotic, plant, mammalian, etc.). Such methods include, but not limited to, transfection (e.g., lipid-mediated, cationic polymers, calcium phosphate, dendrimers); electroporation or other methods of membrane disruption (e.g., nucleofection), viral delivery (e.g., lentivirus, retrovirus, adenovirus, AAV), microinjection, microprojectile bombardment (“gene gun”), fugene, direct sonic loading, cell squeezing, optical transfection, protoplast fusion, impalefection, magnetofection, exosome- mediated transfer, lipid nanoparticle-mediated transfer, and any combination thereof.

[0700] In some embodiments, the method comprises delivering one or more nucleic acids (e.g., nucleic acids encoding an RNA guided nuclease such as a Cas9 nuclease and a guide RNA etc.), one or more transcripts thereof, and / or a pre-formed Cas9 nuclease / gRNA complex to a cell, where a ternary complex is formed. Exemplary intracellular delivery methods, include, but are not limited to: viruses or virus-like agents; chemical-based transfection methods, such as those using calcium phosphate, dendrimers, liposomes, or cationic polymers (e.g., DEAE-dextran or polyethylenimine); non-chemical methods, such as microinjection, electroporation, cell squeezing, sonoporation, optical transfection, impalefection, protoplast fusion, bacterial conjugation, delivery of plasmids or transposons; particle-based methods, such as using a gene gun, magnectofection or magnet assisted transfection, particle bombardment; and hybrid methods, such as nucleofection. In some embodiments, the present application further provides cells produced by such methods, and organisms (such as animals, plants, or fungi) comprising or produced from such cells.

[0701] In some embodiments, a composition described herein is a pharmaceutical composition. In some embodiments, a pharmaceutical composition may include, as optional ingredients, pharmaceutically acceptable carriers, diluents, solubilizing or emulsifying agents, and salts of the type that are well-known in the art. Specific non-limiting examples of the carriers and / or diluents that are useful in the pharmaceutical formulations of the present invention include water and physiologically acceptable buffered saline solutions such as phosphate buffered saline solutions pH 6.0-9.0 and water.

[0702] In some embodiments, the composition is formulated as in a particle, such as a nanoparticle. In some embodiments, the particle is a liposome or a lipid nanoparticle (LNP). LNPs suitable for delivery of nucleic acids and / or ribonucleoprotein complexes, and lipids suitable for use in such LNPs have been described herein art, e.g., in WO2012135805, WO2017049245, W02018081480, WO2017075531, W02017004143, WO2017173054, W02010144740, W02019191780, W02023003995, U.S. Patent Nos. 6,858,225, 6,815,432, 8,158,601, 8,058,069, 8,822,668, 9,006,417, 9,518,272, 11,246,933, 11,135,312, 11,149,278, 10,961,188, and in Han et al., Nature Communications volume 12, Article number: 7233 (2021), the entire contents of each of which are incorporated herein by reference. In some embodiments, a LNP is used to deliver any one of the gene editing agents described herein to a targeted cell and / or tissue (e.g., cancer cell or tissue). In some embodiments, a LNP is used to deliver any one of the gene editing agents described herein to a liver (e.g., a hepatocyte, hepatocellular adenoma, or a hepatocellular carcinoma cell).

[0703] Methods

[0704] The present disclosure, in some aspects, provides methods of cleaving a target nucleic acid (e.g., a mutated CTNNBI), methods of reducing the expression and / or activity of a mutated oncogene (e.g., CTNNBI) in a cell (e.g., a cancer cell such as a human cancer cell), such methods comprises contacting a cell (e.g., any one of the cells described herein) with a complex comprising any one of the gRNAs described herein and an RNA guided nuclease (e.g., a Cas9 nuclease), one or more nucleic acid molecules encoding a gRNA and an RNA guided nuclease (e.g., a Cas9 nuclease), or a composition (e.g., a composition formulated in an LNP) comprising an RNA guided nuclease (e.g., Cas) / gRNA complex or nucleic acids encoding an RNA guided nuclease (e.g., a Cas9 nuclease) and a gRNA. Any one of the delivery methods described herein or known to one skilled in the art may be used.

[0705] Methods of treating cancer (e.g., a cancer addicted to mutated CTNNBI such as hepatocellular carcinoma) in a subject (e.g., a human subject having cancer or is suspected to have or susceptible to cancer) are also provided, wherein the methods comprise administering to the subject a complex comprising any one of the gRNAs described herein and an RNA guided nuclease (e.g., a Cas9 nuclease), one or more nucleic acid molecules encoding a gRNA and an RNA guided nuclease (e.g., a Cas9 nuclease), or a composition (e.g., a composition formulated in an LNP) comprising an RNA guided nuclease (e.g., Cas) / gRNA complex or nucleic acids encoding an RNA guided nuclease (e.g., a Cas9 nuclease) and a gRNA.

[0706] In some embodiments, a cancer treated using a composition or method described herein is addicted to an oncogene (e.g., a mutated CTNNBI having a mutation provided in Table 1). In some embodiments, the cancer is a solid tumor. In some embodiments, the cancer is a premalignant condition. As described herein, the term “premalignant” refers to a condition where abnormal cells have grown and may develop into cancer. In some embodiments, the premalignant condition is hepatocellular adenoma. In some embodiments, the cancer is hepatocellular carcinoma. In some embodiments, the hepatocellular carcinoma is a primary cancer. In some embodiments, the hepatocellular carcinoma is metastasized from a primary cancer, optionally wherein the primary cancer is colorectal cancer.

[0707] In some embodiments, a composition or method described herein introduces a double strand break in the oncogene (e.g., CTNNBI), thereby cleaving the oncogene (e.g., CTNNBI). In some embodiments, cleavage of the oncogene (e.g., CTNNBI) results in reduced (e.g., reduced by at least 10%, at least 20%, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 90%, or more) expression and / or activity of the mutated oncogene (e.g., CTNNBI), relative to in a cell without a gene editing agent described herein. In some embodiments, cleavage of the oncogene (e.g., CTNNBI) results in inactivation of the mutated oncogene (e.g., CTNNBI). In some embodiments, inactivation of a mutated oncogene a cancer is addicted to (e.g., CTNNBI) leads to cancer cell death, thereby treating the cancer (e.g., HCC). In some embodiments, a subject is a mammal. In some embodiments, a subject is human. In some embodiments, a subject is a rodent or a non-human primate (e.g., cynomolgus monkey).

[0708] The administration may be by any appropriate route known in the art including, but not limited to, oral, enteral or parenteral routes, including intravenous, intrathecal, direct intraventricular, intramuscular, subcutaneous, intramedullary, transdermal, airway (aerosol), pulmonary, nasal, intranasal, intraperitoneal, intraocular, rectal, vaginal, transmucosal, intestinal, and topical (including buccal and sublingual) administration.

[0709] Exemplary modes of administration include, but are not limited to, injection, infusion, instillation, inhalation, or ingestion. “Injection” includes, without limitation, intravenous, intramuscular, intraarterial, intrathecal, intraventricular, intracapsular, intraorbital, intracardiac, intradermal, intraperitoneal, transtracheal, subcutaneous, subcuticular, intraarticular, subcapsular, subarachnoid, intraspinal, intra-cerebrospinal, and intrasternal injection and infusion. In preferred embodiments, the compositions are administered by intravenous infusion or injection.

[0710] The agents of the present disclosure may be administered and dosed in accordance with current medical practice, taking into account the clinical condition of the subject, the site and method of administration, the scheduling of administration, the subject's age, sex, body weight and other factors relevant to clinicians of ordinary skill in the art.

[0711] In some embodiments, the agents of the present disclosure are formulated such that they are suitable for extended release. Such extended-release compositions may be conveniently administered to a subject at extended dosing intervals. The agents described herein are administered in effective amounts. An “effective amount” refers to the amount which achieves a desired reaction or a desired effect alone or together with further doses. In the case of treatment of a particular disease or of a particular condition, the desired reaction preferably relates to inhibition of the course of the disease. This comprises slowing down the progress of the disease and, in particular, interrupting or reversing the progress of the disease. The desired reaction in a treatment of a disease or of a condition may also be delay of the onset or a prevention of the onset of said disease or said condition.

[0712] An effective amount of an agent described herein will depend on the condition to be treated, the severeness of the disease, the individual parameters of the patient, including age, physiological condition, size and weight, the duration of treatment, the type of an accompanying therapy (if present), the specific route of administration and similar factors. Accordingly, the doses administered of the agents described herein may depend on various of such parameters. In the case that a reaction in a patient is insufficient with an initial dose, higher doses (or effectively higher doses achieved by a different, more localized route of administration) may be used.

[0713] Kits

[0714] The present disclosure provides kits that can be used, for example, to carry out a method described herein. In some embodiments, the kits include an gRNA and a Cas9 nuclease. In some embodiments, the kits include a polynucleotide that encodes such a Cas9 nuclease, and optionally the polynucleotide is comprised within a vector, e.g., as described herein. In some embodiments, the kits or systems include a polynucleotide that encodes a gRNA disclosed herein. The Cas9 nuclease and the gRNA in a complex (e.g., as a ribonucleoprotein) can be packaged within the same or other vessel within a kit or system or can be packaged in separate vials or other vessels, the contents of which can be mixed prior to use. The kits or systems can additionally include, optionally, a buffer and / or instructions for use of the guide RNA and Cas9 nuclease.

[0715] All references, publications, and all information associated with Accession or Reference numbers for gene, cDNA / mRNA, and protein sequences (e.g., in NCBI or other sequence database) cited herein are hereby incorporated by reference in their entirety. EXAMPLES

[0716] The following examples are provided to further illustrate some embodiments of the present invention but are not intended to limit the scope of the invention; it will be understood by their exemplary nature that other procedures, methodologies, or techniques known to those skilled in the art may alternatively be used.

[0717] Example 1: Cancer cell depletion in cancer cells harboring CTNNB1 mutations

[0718] A screen was conducted to identify gRNAs that target mutations in CTNNB1 oncogene that strongly and specifically depletes cancer cells harboring the targeted mutations in the CTNNB1 gene.

[0719] Guide RNA (gRNA) Library design

[0720] A gRNA library was constructed that included a set of gRNAs (Table 2) targeting mutations in the CTNNB1 oncogene. Additionally, 126 gRNAs targeting known essential genes were used as positive controls, 264 gRNAs targeting known nonessential genes were used as negative controls, and 145 nontargeting gRNAs were used. Also, 4 known effective CTNNB1 gRNAs from a previously constructed library (see, e.g., Sanson et al., Nat Commun 9, 5416 (2018)) were used. Finally, 12 gRNAs targeting wild type CTNNB1 at the same loci as the mutation-targeting guides were used.

[0721] The gRNAs targeting CTNNB1 mutations were designed as follows. The twenty most frequent CTNNB1 point mutations in the Cancer Genome Atlas Liver and intrahepatic bile duct cancer (TCGA-LIHC) program were identified from the National Institutes of Health, National Cancer Institute, Genomic Data Commons (GDC) data portal (Gene name: “’CTNNB1” , accessed March 25, 2023). For each of the twenty most frequent CTNNB1 point mutations, all 20 nucleotide (nt) sequences overlapping the CTNNB1 point mutation and terminating in a Cas9 nuclease protospacer-adjacent motif (PAM) sequence “NGG” on either the positive (+) strand or the negative (-) strand were selected as potential mutation-targeting crRNA spacer sequences. Additionally, if the CTNNB1 point mutation created a new PAM sequence, the 20- nt sequence terminating in the new PAM sequence was selected as a spacer. gRNAs were constructed using the crRNA spacer sequences.

[0722] The 12 gRNAs targeting wild type CTNNB1 at the same loci as the mutation-targeting guides were designed as follows. For each PAM sequence present in wildtype CTNNB1, which terminates a CTNNB1 -mutation targeting gRNA, a gRNA targeting wildtype CTNNB1 was also selected as controls.

[0723] Cell line selection

[0724] Four cell lines were selected to be tested. (1) SNU-449, a CTNNB 1 -wildtype, CTNNB1 non-dependent cell line and should show no depletion for any CTNNB 1 -targeting gRNA. (2) LS411N, a CTNNB 1 -wildtype, Adenomatous polyposis coli (“APC”) -mutant, CTNNB1- dependent cell line and should show depletion from the 12 gRNAs targeting wild type CTNNB1, but not the gRNAs targeting CTNNB1 mutations. (3) SNU-398, a CTNNB1 S37C cell line that is expected to show depletion for CTNNB1 S37C-targeting gRNAs and wildtype gRNAs not overlapping the S37 locus. (4) SW1573, a CTNNB 1 S33F cell line that should show depletion for gRNAs targeting the CTNNB1 point mutation S33F and gRNAs targeting wild type sequences that do not overlap the S33 locus.

[0725] Cell lines and cell culture

[0726] To determine the optimal initial cell seeding conditions for cell culture, the 4 cell lines, SNU449, SNU398, SW1573, and LS411N were cultured according to the following. SNU449 and SNU398 cells were cultured using RPMI-1640 (Invitrogen) with 10% heat-inactivated fetal bovine serum (FBS). LS411N cells were cultured using RPMI-1640 with 10% FBS. SW1573 cells were cultured using L-15 Medium (Invitrogen) with 10% FBS. SNU449, SNU398 and LS411N cells were cultured at 37°C incubator with 5% CO2. SW1573 cells were cultured at 37 °C incubator without CO2.

[0727] To determine cell sub-confluency (-80%) in T75 flasks, cells were seeded to T75 flasks with estimated cell seeding for 3 or 4 days with 2 seeding conditions for each cell type. After culturing, cells were collected and counted. The optimal cell seeding condition was based on the normalization of the actual cell sub-confluence and the ideal one to be made. Cell doubling time was calculated based on the initial cell seeding number and collected cell number using Formula 1.

[0728] Doubling

[0729] (Formula

[0730] Puromycin killing test

[0731] Cells from the 4 cell lines, SNU449, SNU398, SW1573, and LS411N, were seeded in 96-well plates with the optimal initial cell seeding for 4-day cell culture determined in “Cell lines and cell culture” . The next day, 10 pl of 1 lx puromycin (Gibco Al 1138-03) of different doses with a series of dilutions were dispensed to the 96-well plates. The cells were incubated for 3 days at 37°C in the incubator. CellTiter-Glo Kit (Promega, G7570) was detected on day 3 post puromycin killing, and the cell survival ratio was calculated according to the values read out.

[0732] Polybrene titration test

[0733] Cells from the 4 cell lines, SNU449, SNU398, SW1573, and LS411N, were seeded in 96-well plates with optimal initial cell seeding for 4-day cell culture. The next day, 10 pl of l lx polybrene (Millipore TR-1003) of different doses with a series of dilutions were dispensed to the 96-well plates. The cells were incubated for 3 days at 37°C in the incubator. CellTiter- Glo Kit (Promega, G7570 was detected on day 3 post polybrene addition, and the cell survival ratio was calculated according to the values read out.

[0734] Lentivirus production

[0735] To produce lentivirus, 3.6 x 107293FT cells were collected for reverse transfection in each T150 flask. For each T150 flask, 15 pg library plasmid (pLentiCRISPRv2), 11.25 pg psPAX2 (Addgene, 12260), 7.5 pg pCMV-VSV-G (Addgene, 8454) and 67.5 pl P3000 diluted in 3 ml Opti-MEM (Gibco, 11058021) were mixed with 100 pL Lipofectamine 3000 (Invitrogen, L3000015) diluted in 3.5 ml Opti-MEM. The mixture was added into 293FT cells and the medium was refreshed 12 hours post transfection. Virus supernatant was collected 48 hours post refreshing medium, centrifugated at 1,800 g at 4°C for lOmin to pellet the cell debris and filtrated with a 0.45 pm low protein binding membrane (Millipore, SE1M003M00). The lentivirus was concentrated by ultra-centrifugation at 25,000 rpm at 4°C for 2 hours, and then resuspended and aliquoted to store at -80°C.

[0736] Lentivirus titration

[0737] To test the lentivirus titer, cells from the 4 cell lines, SNU449, SNU398, SW1573, and LS41 IN, were seeded with optimal cell seeding in a 6-well plate for 3 days and infected by lentivirus at serial dilution with optimal polybrene added the next day. 24 hours post-infection, cells were collected and re-seeded in 96-well plates with 10 replicates for each dose. After overnight incubation, puromycin was added to 5 replicates for 3 -day puromycin selection, and then the cell viability was detected by CellTiter-Glo Kit (Promega, G7570) to determine the virus titer. To optimize the virus titer for the CRISPR screen, cells were then seeded with optimal cell seeding in a T75 flask for 3 days and infected by lentivirus with target multiplicity of infection (MOI) of 0.2, 0.4, and 0.6 and optimal polybrene added the next day. 24 hours postinfection, cells were re-seeded and selected with puromycin following the same procedures as above. The cell viability was detected by CellTiter-Glo Kit (Promega, G7570) 3 days post the puromycin selection.

[0738] CRISPR screen

[0739] At day 0, cells from the 4 cell lines, SNU449, SNU398, SW1573, and LS411N, were seeded in T75 flasks and the gRNA library (pLentiCRISPRv2) was transduced into 4 cell lines with 2 replicates for each cell line the next day. At day 3, cells were re-seeded to T75 flasks for screen and 96-well plates for MOI QC. 4 xlO6cells (library coverage > 6000x) were collected as the reference pellets. After overnight culture, puromycin was added to cells for 3-day selection. At day 7, the cell viability in 96-well plates was detected by CellTiter-Glo Kit (Promega, G7570) for the MOI QC. Cells cultured in T75 flasks were replated and the puromycin was withdrawn. At day 10, cells were replated and seeded in 6-well plates for the Cas9 nuclease activity test. Then the cells were cultured, and the cell passage frequency was kept at twice a week. At day 21, 2 xlO6cells (library coverage > 3000x) were collected as the mid-point pellets. At day 31, 17 days post puromycin selection (>10 doublings), 2 xlO6cells (library coverage > 3000x) were collected as the end-point pellets.

[0740] Cas9 nuclease activity test

[0741] To test the Cas9 nuclease activity, cells from the 4 cell lines, SNU449, SNU398, SW1573, and LS41 IN, were either treated with the gRNA library by transducing cells with the lentivirus or not treated (“parental cells”). Cells were seeded with optimal cell seeding in a 6- well plate for 4 days and infected by the reporter virus, GFP-BFP lentiviral reporter (Addgene #67980), with multiple doses and optimal polybrene added. Cas9 nuclease activity was tested by flow cytometry (BD LSRFortesa) 3 days after reporter virus infection.

[0742] NGS sample preparation

[0743] The genomic DNAs from the 4 cell lines, SNU449, SNU398, SW1573, and LS411N as treated above in the “Cas9 nuclease activity test” were purified using the QIAGEN blood & cell culture DNA mini kit (QIAGEN- 13323) according to the manufacturer’s protocol. To decide the optimal PCR condition, template loading, and PCR cycles were tested for the 1st round of PCR using Q5 Hot Start High-Fidelity 2X polymerase (NEB- M0494L). PCR amplification was conducted using 2 pg of the genomic DNA as the template of 100 pl reaction. The 2nd step of PCR was conducted using the PCR amplicon from the 1st PCR as templates. The amplicons were confirmed by running on a 0.2% agarose gel and sent for the next-generation sequencing (NGS). The primers for the 1st round of PCR are 5'- AATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCG-3' (SEQ ID NO: 6) (Forward) and 5”-TAGGCACCGGATCAATTGCCGAC-3' (SEQ ID NO: 7) (Reverse). The primers for the 2nd round of PCR are 5'-( 1-9 bp of variable length sequences) TCTTGTGGAAAGGACGAAACACCG-3' (SEQ ID NO: 8) (Forward) and 5'-(l-9 bp of variable length sequences) TGTGGGCGATGTGCGCTCT-3' (SEQ ID NO: 9) (Reverse).

[0744] Data Analysis

[0745] Readcounts for each gRNA were extracted by counting numbers of sequences with a fluorescence value for pHRed ofgreater than 30 and containing an exact match to the combination of the tracrRNA GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAACUUG AAAAAGUGGCACCGAGUCGGUGCUUU (SEQ ID NO: 10) and the gRNA. Readcounts for each sample were scaled such that the median counts of each gRNA matched the median of the median of such counts over samples. Samples were grouped by whether they were drawn from round 1 or round 2 of PCR. Within groups, the mean of the day 2 samples was taken as the reference. Then, for each day 20 and day 34 sample, log fold change was computed as log2(sample + 1) - log2(reference + 1)

[0746] Log fold change (“LFC”) was then shifted such that the median of nontargeting gRNAs in each sample was 0, representing log fold change relative to no cutting (FIG. 1A).

[0747] Off target gRNAs were those CTNNB1 mutant-targeting gRNAs that exhibited median log fold change (across replicates and time points) less than -0.5 in any line without the targeted mutation. Cell line information and whether it harbored a targeted mutation is described in Table 3. 25 of the tested gRNAs were so identified and removed from the data, as shown in FIG. IB. gRNAs were identified as successful if they were not off target, and successfully depleted the targeted line with log fold change less than -1. gRNA-58 (spacer sequence CUGGACUCUGGAAUCCAUUG; SEQ ID NO: 71), targeting S37C substitution in P-catenin, met the criteria and is indicated by the dashed rectangle in FIG. IB.

[0748] Table 3: Cell lines harboring targeted CTNNB1 mutations

[0749] Further, a second screen was carried out as described above, which included a cell line NCIH295R, harboring the S45P substitution in P-catenin, in addition to the cell lines listed in Table 3 (FIG. 3). Several guides gRNA-2 (spacer sequence GCUCCUCCUCUGAGUGGUAA; SEQ ID NO: 15), gRNA-3 (spacer sequence CAGAGGAGGAGCUGUGGUAG; SEQ ID NO: 16), and gRNA-6 (spacer sequence GGAUUGCCUUUACCACUCAG; SEQ ID NO: 19) were selected for further testing.

[0750] Example 2: Cancer cell depletion in cancer cells harboring CTNNB1 mutations

[0751] The purpose of this experiment was to determine whether the gRNAs selected for further testing in Example 1, had the ability to deplete cancer cells in cancer cell lines harboring the respective targeted mutations.

[0752] The SNU398 cell line that has a mutation in the CTNNB1 gene that resulted in a S37C substitution in P-catenin, is targeted by gRNA-58 (spacer sequence CUGGACUCUGGAAUCCAUUG; SEQ ID NO: 71). gRNA-58 and cells were prepared as described in Example 1. The log fold change (“LFC”) and relative cell viability were measured as shown in FIGs. 2A and 2B, respectively. gRNA-58 exhibited a log fold change less than -1 and above -0.5 in any cell line without the CTNNB1 point mutation targeted by gRNA-58. The mean viability of the SNU398 cells with gRNA-58 was 42% and 30% at days 20, and 34, respectively.

[0753] These results demonstrate that gRNA-58 successfully depleted the cell line, SNU398, that had the CTNNB1 point mutation targeted by gRNA-58. Additionally, gRNA-58 had little to no off-target effects in other cell lines. Furthermore, gRNA-58 specifically decreased cell viability in the SNU398 cell line.

[0754] The NCIH295R cell line, harboring a mutation in the CTNNB1 gene that resulted in a S45P substitution in P-catenin, is targeted by gRNA-2 (spacer sequence GCUCCUCCUCUGAGUGGUAA; SEQ ID NO: 15), gRNA-3 (spacer sequence CAGAGGAGGAGCUGUGGUAG; SEQ ID NO: 16), and gRNA-6 (spacer sequence GGAUUGCCUUUACCACUCAG; SEQ ID NO: 19). gRNA-2, gRNA-3, and gRNA-6 and cells were prepared as described in Example 1. The log fold change (“LFC”) was measured as shown in FIG. 4. The tested S45P targeting gRNAs selectively depleted NCIH295R cells. gRNA-58 was used as a control, which selectively depleted SNU398 cells, which harbors the targeted S37C substitution in P-catenin. Further experiments are being conducted to further test the activities for these gRNAs.

[0755] Example 3: Screening gRNA libraries

[0756] The performance of the CRISPR-Cas9 system at ablating gene function depends strongly on the specific gRNA chosen, and the most performant gRNAs are difficult to predict in advance. To identify the best gRNA for an oncogenic mutation (i.e., CTNNBF), all possible CRISPR-Cas9 wildtype gRNAs against a given mutation were designed. The number of possible gRNAs was determined by the presence of the PAM recognition sequence, NGG, within 21 nucleotides 5' of the mutated gene locus on either positive or negative strand. In the case that the mutation created a novel PAM, the corresponding gRNA was also designed. PAMs where the mutation fell in the “N” nucleotide of the PAM do not lead to mutant- specific gRNAs and were not used. The number of designed gRNAs per oncogenic CTNNB1 mutation varied from 0 to 10, with a median of 2. A total of 98 CTNNB1 mutation-targeting gRNAs were designed (Table 2).

[0757] To evaluate the performance of gRNAs against their targets, cell lines available from ATCC with a common oncogenic drivers in one of the chosen target oncogenes were identified, and preferably screened in the Cancer Dependency Map with a reported CRISPRGeneEff ectScore in the mutated oncogene of - 1 or lower, indicating strong sensitivity to loss. If an gRNA caused loss of viability in a cell line with the targeted mutation, and only that cell line, the gRNA was potent and selective against the oncogenic mutation. Each gRNA hybridized into this construct consisted of a crRNA (i.e., guide), which was a 20 nucleotide sequence matching targeted region of the genome and the Hsu tracrRNA (GUUUUAGAGCUAGAAAUAGCAAGUUAAAAU; SEQ ID NO: 314). gRNA libraries were constructed for screening. Each construct in the library was constructed with a lentiviral plasmid for transduction, the pLentiCRISPRv2 plasmid, and each cell line (Table 4) was transfected with lentivirus of the appropriate construct. Optimal cell seeding density, screening time, multiplicity of infection (MOI), and concentrations of polybrene (to support cell infection) and puromycin (to select for infected cells) were determined per cell line. Cell lines were cultured in a minimum of four replicates, with two replicates harvested after puromycin selection to provide an initial gRNA abundance estimate. The remaining two replicates were harvested at the designated endpoint. All harvested cells were sequenced for gRNA abundance after PCR amplification of the gRNA. Total reads of each gRNA were counted, and the fold change in relative abundance of each gRNA between the early and late time point was computed. This change was scaled by the median of nontargeting gRNA fold changes, so that a value of 1 indicated no difference in viability compared to a nontargeting gRNA, while a value of 0.5 meant 50% dropout (FIGs. 5A-5G).

[0758] Table 4: Cell lines for screening gRNA libraries The following CTNNB1 gRNA sequences and targeting mutations were tested in the screen: • gRNA sequences targeting CTNNB1 amino acid mutation G34E, SEQ ID NOs: 61 and 62 (FIG. 5A).

[0759] • gRNA sequences targeting CTNNB1 amino acid mutation S33F, SEQ ID NOs: SO- 82 (FIG. 5B).

[0760] • gRNA sequences targeting CTNNB1 amino acid mutation S33Y, SEQ ID NOs: 58- 60 (FIG. 5C).

[0761] • gRNA sequences targeting CTNNB1 amino acid mutation S37A, SEQ ID NOs: 74- 76 (FIG. 5D).

[0762] • gRNA sequences targeting CTNNB1 amino acid mutation S37C, SEQ ID NOs: 71- 73 (FIG. 5E).

[0763] • gRNA sequences targeting CTNNB1 amino acid mutation S45F, SEQ ID NOs: 40- 44 (FIG. 5F).

[0764] • gRNA sequences targeting CTNNB1 amino acid mutation S45P, SEQ ID NOs: 14- 19 (FIG. 5G).

[0765] Example 4: In vivo targeting of CTNNB1 mutations in mice and delivery of gRNA and Cas9 mRNA using lipid nanoparticles

[0766] Engineered. HCC mouse models expression human CTNNB1 S45P and human CTNNB1 S37C mutants

[0767] CTNNB1 mutations are validated drivers of hepatocellular carcinoma (HCC). To confirm this in a murine model of HCC driven by expression of Myc and hCTNNBl mutant, plasmid delivery of Cas9 and gRNA targeting the introduced CTNNB 1 mutant was examined. Hydrodynamic transfection was used to introduce transposable vectors containing 20 pg mutant hCTNNBls45Por hCTNNBls37Cand 10 pg cMyc into FVB / NJ mice (Jackson Laboratory, 001800). This had been shown to be sufficient to cause hepatocyte transformation and induction of HCC in the FVB / NJ mice within 2-4 weeks depending on the dose of transposons delivered (data not shown). In the genetic tumor rescue experiments, 10 pg of pX330 plasmid containing Cas9 and either hCTNNBls45PgRNA, hCTNNBls437CgRNA, or a non-targeting control GAL gRNA was added to the HDT volume. Mice were injected at 6-8 weeks of age, when their body weights were approximately 20-25 grams (g). HDT plasmids were suspended at the noted concentrations in 2 mL of saline and administered via tail-vein injection over 7 seconds. A 10:1 mass ratio of combined HDT plasmids to SB 100 transposase plasmid was used. Animals were sacrificed on day 18 post-HDT. Livers were removed, weighed, and imaged at the time of sacrifice.

[0768] Co-delivery of transposons and plasmid containing Cas9 / gRNA to the target resulted in a reduction of LW:BW (liver weight: body weight), a metric for tumor burden. LW:BW was significantly lower following delivery of LNPs containing gRNAs targeting CTNNB1S37Cand trending towards significantly reduced LW:BW following delivery of LNPs containing gRNA targeting CTNNB1S45P(FIGs. 6A-6B). This data suggests that knockout (KO) of specific CTNNB1 mutations prevents tumor formation, resulting in a reduction in tumor burden.

[0769] Cas9 and gRNAs targeting CTNNB1 S37C mutation exhibits tumor suppressing activity in HCC mouse model

[0770] CTNNB1 mutations are validated drivers of HCC. To determine whether Cas9 and a gRNA targeting hCTNNB 1S37Cwould reduce tumor burden in a murine model of HCC, Cas9 mRNA and gRNA targeting hCTNNB 1S37Cwere formulated in an LNP for delivering into the mice. Hydrodynamic transfection was used to introduce transposable vectors containing 5 pg mutant hCTNNB 1S37Cand 10 pg cMyc into FVB / NJ mice (Jackson Laboratory, 001800). This had been shown to be sufficient to cause hepatocyte transformation and induction of HCC in the FVB / NJ mice within 2-4 weeks depending on the dose of transposons delivered (data not shown). Mice were injected at 6-8 weeks of age, when their body weights were approximately 20-25 g. HDT plasmids were suspended at the noted concentrations in 2 mL of saline and administered via tail-vein injection over 7 seconds. A 10:1 mass ratio of combined HDT plasmids to SB 100 transposase plasmid was used.

[0771] LNPs were formulated as described by Finn, et al., Cell Rep (2018) 22(9):2227-2235, with modifications.

[0772] At day 4 and day 7 post-HDT, LNPs were dosed intravenously (i.v.) at 1.5 mg total RNA per kg body weight in 0.2 mL PBS via the lateral tail vein per animal. Non-targeting guide (sgGAL) and PBS control animals were used as negative controls for all studies. Animals were euthanized by cervical dislocation under isoflurane anesthesia on day 16 post- HDT. Livers were removed, weighed, and imaged at the time of sacrifice.

[0773] Tissues were fixed with 10% formalin at 4°C for greater than 48h and submitted to the UT Southwestern Tissue Management facility for processing and H&E staining. Slides were imaged with a Hamamatsu NanoZoomer S60 slide scanner controlled by NZAcquire 2.1.6.55 software in semi-automatic mode using a 20x objective lens. Raw unprocessed images were exported in NDP view 2 software. The results show that therapeutic delivery of LNPs containing Cas9 mRNA and gRNA targeting CTNNB1S37Cat day 4 and 7 post-HDT resulted in a significant reduction in LW:BW compared to the non-targeting gRNA control (FIGs. 7A-7B), and a significant reduction in tumor burden based on tumor area compared to both PBS and non-targeting gRNA controls following histological quantification of tumor area (FIGs. 7C-7D).

[0774] All publications, patents, and patent applications are incorporated herein by reference. While in the foregoing specification this invention has been described in relation to certain embodiments thereof, and many details have been set forth for purposes of illustration, it will be apparent to those of skill in the art that the invention is susceptible to additional embodiments and that certain details described herein may be varied considerably without departing from the basic principles of the invention.

[0775] The use of the terms “a” and “an” and “the” and similar references in the context of describing the invention are to be construed to cover both the singular and the plural, unless otherwise indicated herein or clearly contradicted by context. The terms “comprising,” “having,” “including,” and “containing” are to be construed as open-ended terms (i.e., meaning” including, but not limited to”) unless otherwise noted. Recitation of ranges of values herein are merely intended to serve as a shorthand method of referring individually to each separate value falling within the range, unless otherwise indicated herein, and each separate value is incorporated into the specification as if it were individually recited herein. All methods described herein can be performed in any suitable order unless otherwise indicated herein or otherwise clearly contradicted by context. The use of any and all examples, or exemplary language (e.g., “such as”) provided herein, is intended merely to better illuminate the invention and does not pose a limitation on the scope of the invention unless otherwise claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention.

[0776] Embodiments of this invention are described herein, including the best mode known to the inventors for carrying out the invention. Variations of those embodiments may become apparent to those of ordinary skill in the art upon reading the foregoing description. The inventors expect skilled artisans to employ such variations as appropriate, and the inventors intend for the invention to be practiced otherwise than as specifically described herein. Accordingly, this invention includes all modifications and equivalents of the subject matter recited in the claims appended hereto as permitted by applicable law. Moreover, any combination of the above-described elements in all possible variations thereof is encompassed by the invention unless otherwise indicated herein or otherwise clearly contradicted by context.

[0777] The entire contents of all of the references (including literature references, issued patents, published patent applications, and co pending patent applications) cited throughout this application are hereby expressly incorporated by reference. Although the foregoing specification and examples fully disclose and enable the present invention, they are not intended to limit the scope of the invention, which is defined by the claims appended hereto.

Claims

CLAIMSWHAT IS CLAIMED IS:

1. A guide RNA (gRNA) comprising a crRNA targeting a sequence of CTNNB1, wherein the sequence of CTNNB1 comprises a mutation associated with cancer, optionally wherein the mutation is a gain-of-function mutation.

2. The gRNA of claim 1, wherein the cancer is addicted to the CTNNB1 comprising the mutation.

3. The gRNA of claim 1 or claim 2, wherein the sequence of CTNNB1 is double-stranded and comprises a complementary strand and a target strand.

4. The gRNA of claim 3, wherein the crRNA comprises a spacer comprising a region of complementarity to the complementary strand.

5. The gRNA of any one of claims 1-4, wherein the mutation is a single nucleotide mutation.

6. The gRNA of any one of claims 1-5, wherein the mutation is any one of the mutations listed in Table 1.

7. The gRNA of claim 5 or claim 6, wherein the single nucleotide mutation results in an amino acid substitution in a P-catenin protein encoded by CTNNB1.

8. The gRNA of claim 7, wherein the amino acid substitution in the P-catenin protein is D32G, D32V, D32N, D32Y, S33C, S33F, S33P, S33Y, G34E, G34V, I35S, H36P, S37F, S37C, S37A, T41A, T41I, S45P, S45F, S45Y, L337I, C621Y, S33A, S37P, R589Q, G34R, K337T, E336K, R537Q, S45C, D32H, or K337I.

9. The gRNA of any one of claims 1-8, wherein the spacer is 17-20 nucleotides in length.

10. The gRNA of any one of claims 4-9, wherein the spacer comprises the nucleotide sequence of any one of SEQ ID NOs: 14-82 or 227-255.

11. The gRNA of any one of claims 1-10, wherein a protospacer adjacent motif (PAM) is located in the target strand and at the 3’ side of a target sequence.

12. The gRNA of claim 11, wherein the PAM is of a 1-20 nucleotides distance to the mutation.

13. The gRNA of any one of claims 1-10, wherein the mutation creates a PAM.

14. The gRNA of any one of claims 1-13, wherein the crRNA further comprises a direct repeat component.

15. The gRNA of any one of claims 1-14, wherein the gRNA further comprises a tracrRNA.

16. A complex comprising an RNA-guided nuclease and the gRNA of any one of claims 1- 15.

17. A nucleic acid molecule comprising a nucleotide sequence encoding the gRNA of any one of claims 1-15.

18. The nucleic acid molecule of claim 17, wherein the nucleic acid molecule further comprises a nucleotide sequence encoding an RNA-guided nuclease.

19. The nucleic acid molecule of claim 18, wherein the nucleotide sequence encoding the gRNA and / or the nucleotide sequence encoding the RNA-guided nuclease is operably linked to a promoter.

20. The nucleic acid molecule of any one of claims 17-19, wherein the nucleic acid molecule is a vector, optionally wherein the nucleic acid molecule is a lentiviral vector.

21. A composition comprising: a nucleic acid molecule comprising a nucleotide sequence encoding an RNA-guided nuclease, and the gRNA of any one of claims 1-15.

22. The composition of claim 21, wherein the nucleic acid molecule is a vector or an mRNA molecule.

23. The composition of claim 21 or claim 22, wherein the composition is formulated in a lipid nanoparticle (LNP).

24. The composition of claim 21, the nucleic acid molecule of any one of claims 17-20, or the composition of any one of claims 21-23, wherein the RNA-guided nuclease is Cas9.

25. A method of cleaving a target nucleic acid in a cell, the method comprising contacting the cell with the complex of claim 16 or claim 24, the nucleic acid molecule of any one of claims 17-20, and 24, the composition of any one of claims 21-24, wherein the targeted nucleic acid is a mutated CTNNB1.

26. A method of reducing the expression and / or activity of a mutated CTNNB1 in a cell, the method comprising contacting the cell with the complex of claim 16 or claim 24, the nucleic acid molecule of any one of claims 17-20, and 24, the composition of any one of claims 21-24.

27. The method of claim 26, wherein the cell is a hepatocyte.

28. The method any one of claims 25-27, wherein the cell is a hepatocellular carcinoma cell or a hepatocellular adenoma cell.

29. The method of any one of claims 25-28, wherein the cell is in vitro.

30. The method of any one of claims 25-28, wherein the cell is in vivo.

31. A method of reducing the expression and / or activity of a mutated CTNNB1 in a subject, the method comprising administering to the subject the complex of claim 16 or claim 24, the nucleic acid molecule of any one of claims 17-20, and 24, the composition of any one of claims 21-24.

32. A method of treating hepatocellular carcinoma in a subject, the method comprising administering to the subject the complex of claim 16 or claim 24, the nucleic acid molecule of any one of claims 17-20, and 24, the composition of any one of claims 21-24.

33. A method of treating hepatocellular adenoma in a subject, the method comprising administering to the subject the complex of claim 16 or claim 24, the nucleic acid molecule of any one of claims 17-20, and 24, the composition of any one of claims 21-24.

34. The method of claim 32, wherein the hepatocellular carcinoma is a primary cancer.

35. The method of claim 32, wherein the hepatocellular carcinoma is metastasized from a primary cancer, optionally wherein the primary cancer is colorectal cancer.

36. The method of any one of claims 25-35, wherein the RNA-guided nuclease introduces a double strand break in CTNNB1, thereby cleaving CTNNB1.

37. The method of claim 36, wherein cleavage of CTNNB1 inhibits the growth and / or progression of the hepatocellular carcinoma.

38. The method of any one of claims 31-35, wherein the subject is human.

39. The method of any one of claims 31-37, wherein the administration is systemic.

40. The method of any one of claims 31-38, wherein the administration is local.

41. A cell comprising the gRNA of any one of claims 1-15, the complex of claim 15 or claim 24, or the nucleic acid molecule of any one of claims 16-19 and 24, optionally wherein the cell is a cancer cell.

42. The cell of claim 40, wherein the cell is a hepatocyte, a hepatocellular adenoma, or a hepatocellular carcinoma cell.

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