Methods and compositions for genome editing in non-dividing cells
Disclosed herein are homology-independent targeted integration methods of integrating an exogenous DNA sequence into a genome of a non-dividing cell and compositions for such methods. Methods herein comprise contacting the non-dividing cell with a composition comprising a targeting construct comprising the exogenous DNA sequence and a targeting sequence, a complementary strand oligonucleotide homologous to the targeting sequence, and a nuclease, thereby altering the genome of the non-dividing cell.
1. A method of integrating an exogenous DNA sequence into a target nucleic acid sequence in a genome of a non-dividing cell comprising contacting the non-dividing cell with
a targeting construct comprising the exogenous DNA sequence and a target sequence entirely in reverse orientation with respect to the exogenous DNA sequence,
a complementary strand oligonucleotide homologous to the target sequence, and
a nuclease capable of producing double stranded DNA blunt end cuts,
wherein the exogenous DNA sequence comprises at least one nucleotide difference compared to the genome, and both the target sequence and the target nucleic acid sequence in the genome are recognized and cut by the nuclease, and
wherein the target nucleic acid sequence in the genome is no longer present once the exogenous DNA sequence has been integrated into the genome of the non-dividing cell in the correct orientation.
2. The method of claim 1 , wherein the exogenous DNA sequence corrects a mutation in the genome of the non-dividing cell.
3. The method of claim 1 , wherein the exogenous DNA sequence causes a mutation in the genome of the non-dividing cell.
4. The method of claim 3 , wherein the mutation is selected from a missense mutation, a nonsense mutation, a silent mutation, an insertion, and a deletion.
5. The method of claim 1 , wherein the nuclease is a CRISPR Cas9 nuclease.
6. The method of claim 1 , wherein the non-dividing cell comprises a terminally differentiated cell or a quiescent stem cell.
7. The method of claim 1 , wherein the targeting construct, the complementary strand oligonucleotide, and a polynucleotide encoding the nuclease are contained in a non-viral or a viral vector, and the viral vector is selected from the group consisting of a lentivirus, a retrovirus, an adenovirus, and an adeno-associated virus.
8. The method of claim 1 , wherein the non-dividing cell is selected from one or more of lymphocytes, monocytes, neutrophils, eosinophils, basophils, endothelial cells, epithelial cells, hepatocytes, osteocytes, platelets, adipocytes, cardiomyocytes, neurons, retinal cells, smooth muscle cells, skeletal muscle cells, spermatocytes, oocytes, and pancreas beta cells.
9. A method of treating a genetic disease in a subject in need thereof, wherein the genetic disease results from a mutated gene or fragment thereof having at least one changed nucleotide compared to a wild-type gene or fragment thereof, the method comprising:
contacting at least one cell of the subject with a composition comprising:
a targeting construct comprising a DNA sequence homologous to the wild-type gene or fragment thereof and a target sequence entirely in reverse orientation with respect to the DNA sequence homologous to the wild-type gene or fragment thereof,
a complementary strand oligonucleotide homologous to the target sequence, and
a nuclease capable of producing double stranded DNA blunt end cuts,
wherein both the target sequence and a target nucleic acid sequence in the subject's genome are recognized and cut by the nuclease,
wherein the mutated gene or fragment thereof is replaced with the DNA sequence homologous to the wild-type gene or fragment thereof,
wherein the target nucleic acid sequence is no longer present once the mutated gene or fragment thereof is replaced with the wild-type gene or fragment thereof in the correct orientation.
10. The method of claim 9 , wherein the mutated gene or fragment thereof comprises a mutation selected from the group consisting of a missense mutation, a nonsense mutation, a silent mutation, an insertion, and a deletion.
11. The method claim 9 , wherein the nuclease is a CRISPR Cas9.
12. A method of altering a DNA sequence, comprising contacting the DNA sequence with:
a targeting construct comprising an exogenous DNA sequence and a target sequence entirely in reverse orientation with respect to the exogenous DNA sequence,
a complementary strand oligonucleotide homologous to the target sequences, and
a nuclease capable of producing double stranded DNA blunt end cuts,
wherein the exogenous DNA sequence comprises at least one nucleotide difference compared to the DNA sequence, and both the target sequence and a target nucleic acid sequence in the DNA sequence are recognized and cut by the nuclease,
wherein the target nucleic acid sequence in the DNA sequence is no longer present once the exogenous DNA sequence has been integrated into the DNA sequence in the correct orientation.
13. The method of claim 1 wherein the target nucleic acid sequence in the genome is within Mertk.
14. The method of claim 1 wherein the target nucleic acid sequence in the genome is within LMNA.
15. The method of claim 1 wherein there are at least two copies of the targeting sequence entirely in reverse orientation with respect to the exogenous DNA sequence within the targeting construct.