Non-human animals having a hexanucleotide repeat expansion in a c9orf72 locus

Abstract

A non-human animal (e.g., a rodent) model for diseases associated with a C9ORF72 heterologous hexanucleotide repeat expansion sequence is provided, which non-human animal comprises a heterologous hexanucleotide repeat (GGGGCC) in an endogenous C9ORF72 locus. A non-human animal disclosed herein comprising a heterologous hexanucleotide repeat expansion sequence comprising at least one instance, e.g., repeat, of a hexanucleotide (GGGGCC) sequence may further exhibit a characteristic and/or phenotype associated with one or more neurodegenerative disorders (e.g., amyotrophic lateral sclerosis (ALS) and/or frontotemporal dementia (FTD), etc.). Methods of identifying therapeutic candidates that may be used to prevent, delay or treat one or more neurodegenerative (e.g., amyotrophic lateral sclerosis (ALS, also referred to as Lou Gehrig's disease) and frontotemporal dementia (FTD)) are also provided.

1 . A non-human animal or non-human animal cell comprising in its genome a heterologous hexanucleotide repeat expansion sequence inserted at an endogenous C9orf7 2 locus, wherein the heterologous hexanucleotide repeat expansion sequence comprises at least one repeat of the hexanucleotide sequence set forth as SEQ ID NO: 1. 
     
     
         2 .- 32 . (canceled) 
     
     
         33 . A method of identifying a therapeutic candidate for the treatment of a disease or condition associated with the presence of a hexanucleotide repeat expansion sequence, the method comprising
 (a) administering a candidate agent to a non-human animal or a non-human animal cell comprising a C9orf72 locus genetically modified to comprise a hexanucleotide repeat expansion sequence comprising at least one repeat of the hexanucleotide sequence set forth as SEQ ID NO:1;   (b) performing one or more assays to determine if the candidate agent has an effect on one or more signs, symptoms and/or conditions associated with the disease or condition; and   (c) identifying the candidate agent that has an effect on the one or more signs, symptoms and/or conditions associated with the disease or condition as the therapeutic candidate.   
     
     
         34 .- 40 . (canceled) 
     
     
         41 . A host cell comprising a heterologous hexanucleotide repeat expansion sequence. 
     
     
         42 . The host cell of  claim 41 , wherein the host cell is a bacterial cell. 
     
     
         43 . A CRISPR/Cas system comprising a Cas protein and/or one or more gRNA, wherein the one or more gRNA is encoded by a DNA comprising a sequence selected from the group consisting of SEQ ID NO:38, SEQ ID NO:39, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, SEQ ID NO:48, SEQ ID NO:49; SEQ ID NO:50, and a combination thereof. 
     
     
         44 . The CRISPR/Cas system of  claim 43 , wherein the one or more gRNA comprises a first, second and third gRNA, wherein the first gRNA is encoded by a DNA comprising the sequence set forth as SEQ ID NO: 39, wherein the second gRNA is encoded by a DNA comprising the sequence set forth as SEQ ID NO: 44, and wherein the third gRNA is encoded by a DNA comprising the sequence set forth as SEQ ID NO: 50. 
     
     
         45 . The CRISPR/Cas system of  claim 44 , further comprising a fourth gRNA encoded by a DNA comprising the sequence set forth as SEQ ID NO: 47. 
     
     
         46 . The CRISPR/Cas system of  claim 45 , further comprising a fifth, sixth, and seventh gRNA, wherein the fifth gRNA is encoded by a DNA comprising the sequence set forth as SEQ ID NO: 46, the sixth gRNA is encoded by a DNA comprising the sequence set forth as SEQ ID NO: 48, and the seventh gRNA is encoded by a DNA comprising the sequence set forth as SEQ ID NO: 49. 
     
     
         47 . The CRISPR/Cas system of  claim 42 , wherein the gRNA comprises a tracrRNA encoding by a DNA comprising a sequence set forth as SEQ ID NO:63, 64 or 65. 
     
     
         48 . The CRISPR/Cas system of  claim 47 , wherein the tracrRNA is encoded by a DNA comprising the sequence set forth as SEQ ID NO:63. 
     
     
         49 . The CRISPR/Cas system of  claim 47 , wherein the tracrRNA is encoded by a DNA comprising the sequence set forth as SEQ ID NO:64. 
     
     
         50 . The CRISPR/Cas system of  claim 47 , wherein the tracrRNA is encoded by a DNA comprising the sequence set forth as SEQ ID NO:65. 
     
     
         51 . The CRISPR/Cas system of  claim 43 , further comprising an expression construct, wherein the expression construct comprises a nucleic acid encoding the Cas protein and/or DNA encoding the at least one gRNA, and wherein the expression construct optionally further comprises a drug resistance gene.