Gene editing method using virus
Provided is a method of inserting a polynucleotide sequence into a genome of a cell. The method comprises: generating a double-strand break at a target site of the genome; and introducing into the cell a virus. The virus comprises a nucleic acid comprising the polynucleotide sequence to be inserted or the complementary sequence thereof. The nucleic acid does not comprise a homologous arm or comprises very short (5˜25 bp) homologous arms corresponding to the target site. Also provided herein is a composition for inserting a polynucleotide sequence into a genome of a cell. The composition comprises a site-specific nuclease capable of generating a DNA double-strand break at a target site of the genome and a virus comprising a nucleic acid comprising the polynucleotide sequence or the complementary sequence thereof.
1. A method of inserting a polynucleotide sequence encoding B domain deleted Factor VIII into a genome of a cell, the method comprising:
generating a DNA double-strand break at a target location of the genome via introducing into the cell a composition comprising a CRISPR-associated (Cas) nuclease and a CRISPR-Cas guide RNA directed to the target location; and
introducing into said cell an adeno-associated virus (AAV), wherein said AAV comprises a nucleic acid comprising the polynucleotide sequence encoding B domain deleted Factor VIII or the complementary sequence thereof, wherein the nucleic acid does not comprise a homologous arm corresponding to the target site, and wherein the polynucleotide sequence is flanked by a sequence targeted by the CRISPR-Cas guide RNA.
2. The method of claim 1 , wherein the target site is within a coding region, a safe-harbor locus or a non-coding region.
3. The method of claim 1 , wherein the cell is a human cell.
4. The method of claim 1 , wherein the cell is in vivo.
5. The method of claim 1 , wherein the cell is from a human subject having hemophilia.
6. The method of claim 1 , wherein the target location comprises a sequence of SEQ ID NO: 5.
7. The method of claim 1 , wherein the B domain deleted Factor VIII has a sequence of SEQ ID NO: 1.
8. The method of claim 1 , wherein the sequence encoding the B domain deleted Factor VIII has a sequence of SEQ ID NO: 2.
9. The method of claim 1 , wherein the nucleic acid comprising the polynucleotide sequence encoding the B domain deleted Factor VIII has a sequence of SEQ ID NO: 4.
10. The method of claim 1 , wherein the CRISPR-associated (Cas) nuclease is introduced into the cell via a vector having a sequence of SEQ ID NO: 3.