RNA-guided gene editing and gene regulation
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.
1. A DNA targeting system for deleting exon 51 of a dystrophin gene comprising Cas9 and at least one guide RNA (gRNA), wherein the at least one gRNA comprises a polynucleotide sequence corresponding to SEQ ID NO: 65, SEQ ID NO: 679, or SEQ ID NO: 69.
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 composition for genome editing in a muscle of a subject 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.
6. The composition of claim 5 , wherein the modified AAV vector has enhanced cardiac and skeletal muscle tissue tropism.
7. The DNA targeting system of claim 1 , wherein the DNA targeting system comprises:
a gRNA comprising a polynucleotide sequence corresponding to SEQ ID NO: 65 and a gRNA comprising a polynucleotide sequence corresponding to SEQ ID NO: 69; or
a gRNA comprising a polynucleotide sequence corresponding to SEQ ID NO: 679 and a gRNA comprising a polynucleotide sequence corresponding to SEQ ID NO: 69.
8. 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 7 .
9. The method of claim 8 , wherein the subject is suffering from Duchenne muscular dystrophy.
10. 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 7 .