Rna-guided gene editing and gene regulation

Abstract

Disclosed herein are Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas) 9-based system related compositions and methods of using said CRISPR/Cas9-based system related compositions for altering gene expression and genome engineering. Also disclosed herein are compositions and methods of using said compositions for altering gene expression and genome engineering in muscle, such as skeletal muscle and cardiac muscle.

What is claimed is: 
     
         1 . A DNA targeting system for deleting exon 51 of a dystrophin gene, the system comprising Cas9 and at least one guide RNA (gRNA), wherein the at least one gRNA binds and targets a polynucleotide sequence comprising SEQ ID NO: 67, SEQ ID NO: 70, SEQ ID NO: 100, or SEQ ID NO: 680. 
     
     
         2 . An isolated polynucleotide encoding the DNA targeting system of  claim 1 . 
     
     
         3 . A vector comprising the isolated polynucleotide of  claim 2 . 
     
     
         4 . A cell comprising the isolated polynucleotide of  claim 2 . 
     
     
         5 . A method of treating a subject in need thereof having a mutant dystrophin gene, the method comprising administering to the subject the DNA targeting system of  claim 1 . 
     
     
         6 . The method of  claim 5 , wherein the subject is suffering from Duchenne muscular dystrophy. 
     
     
         7 . A method of correcting a mutant dystrophin gene in a cell, the method comprising administering to a cell containing a mutant dystrophin gene the DNA targeting system of  claim 1 . 
     
     
         8 . A composition for genome editing in a muscle of a subject, the composition comprising a modified adeno-associated virus (AAV) vector and a nucleotide sequence encoding the DNA targeting system of  claim 1 , wherein the muscle is skeletal muscle or cardiac muscle. 
     
     
         9 . The composition of  claim 8 , wherein the modified AAV vector has enhanced cardiac and skeletal muscle tissue tropism.