Compositions and methods for modifying a target nucleic acid
View Patent ↗The present disclosure provides a system for editing genomic DNA, the system comprising an asymmetric donor DNA template; and methods of editing genomic DNA involving use of an asymmetric donor DNA template. The present disclosure provides a system for editing genomic DNA, the system comprising a Cas9 polypeptide with reduced enzymatic activity; and methods of editing genomic DNA involving use of a Cas9 polypeptide with reduced enzymatic activity.
1. A method of editing genomic DNA of a eukaryotic cell, wherein the genomic DNA comprises a target strand and a non-target strand, the method comprising introducing into the cell:
(a) a Cas9 guide RNA, or one or more nucleic acids encoding said Cas9 guide RNA, wherein the Cas9 guide RNA hybridizes to a target sequence of the target strand of the genomic DNA;
(b) an asymmetric double stranded or single stranded donor DNA molecule comprising a 5′ homology arm and a 3′ homology arm, wherein the 3′ homology arm is 20 to 50 nucleotides in length, is shorter than the 5′ homology arm, and comprises at least 10 consecutive nucleotides of said target sequence, and wherein the ratio of the length of the 5′ homology arm to the 3′ homology arm is from 2.5:1 to 5:1; and
(c) a Cas9 protein or a nucleic acid encoding said Cas9 protein,
wherein (i) the Cas9 protein forms a complex with the Cas9 guide RNA thereby guiding the Cas9 protein to said target sequence, (ii) the 3′ homology arm of the donor DNA molecule hybridizes to the non-target strand of the genomic DNA, and (iii) a nucleotide sequence of the donor DNA molecule is incorporated into the genomic DNA.
2. The method according to claim 1 , wherein the Cas9 protein comprises a functional RuvC domain and cleaves at least the non-target strand of genomic DNA.
3. The method according to claim 1 , wherein the Cas9 protein comprises a functional HNH domain and cleaves at least the target strand of genomic DNA.
4. The method according to claim 1 , wherein the 5′ homology arm of the donor DNA molecule is 50 to 200 nucleotides in length.
5. The method according to claim 1 , wherein the donor DNA molecule comprises a heterologous nucleotide sequence, between the 5′ and 3′ homology arms, that is incorporated into the genomic DNA.
6. The method according to claim 1 , wherein the donor DNA molecule comprises one or more synthetic modifications selected from: a base modification, a sugar modification, and a backbone modification.
7. The method according to claim 1 , wherein the non-target strand comprises a protospacer-adjacent motif (PAM), wherein the 3′ homology arm comprises at least 10 consecutive nucleotides that are complementary to a nucleotide sequence in the PAM-distal non-target strand, and wherein the 5′ homology arm does not comprise at least 10 consecutive nucleotides that are complementary to a nucleotide sequence in the PAM-distal non-target strand.