US2022042047A1PendingUtilityA1

Compositions and methods for modifying a target nucleic acid

61
Assignee: UNIV CALIFORNIAPriority: Dec 2, 2015Filed: Jul 30, 2021Published: Feb 10, 2022
Est. expiryDec 2, 2035(~9.4 yrs left)· nominal 20-yr term from priority
Inventors:Jacob CornChristopher D. Richardson
C12N 15/113C12N 15/11C12N 15/907C12N 9/22C12N 15/102A61K 31/7088C12N 2310/20C12N 9/222
61
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Claims

Abstract

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.

Claims

exact text as granted — not AI-modified
What is claimed is: 
     
         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   (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  or  2 , wherein the Cas9 protein comprises a functional HNH domain and cleaves at least the target strand of genomic DNA. 
     
     
         4 . The method according to any of  claims 1 - 3 , wherein the donor DNA molecule is single stranded. 
     
     
         5 . The method according to any of  claims 1 - 4 , wherein the 5′ homology arm of the donor DNA molecule is 40 to 200 nucleotides in length. 
     
     
         6 . The method according to any of  claims 1 - 5 , wherein the donor DNA molecule comprises a heterologous nucleotide sequence, between the 5′ and 3′ homology arms, that is incorporated into the genomic DNA. 
     
     
         7 . The method according to any of  claims 1 - 6 , wherein the donor DNA molecule comprises one or more synthetic modifications selected from: a base modification, a sugar modification, and a backbone modification. 
     
     
         8 . The method according to  claim 7 , wherein the donor DNA molecule comprises a phosphorothioate linkage. 
     
     
         9 . A system for editing genomic DNA in a eukaryotic cell, the system comprising:
 (a) a Cas9 guide RNA, or one or more nucleic acids encoding said Cas9 guide RNA, wherein the Cas9 guide RNA comprises a guide sequence that is complementary to a target sequence of a target strand of genomic DNA of a eukaryotic cell; and   (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.   
     
     
         10 . The system of  claim 7 , further comprising a Cas9 protein or a nucleic acid encoding said Cas9 protein. 
     
     
         11 . The system of  claim 10 , wherein the Cas9 protein comprises a functional RuvC domain 
     
     
         12 . The system of  claim 10  or  11 , wherein the Cas9 protein comprises a functional HNH domain. 
     
     
         13 . The system of any of  claims 9 - 12 , wherein the donor DNA molecule is single stranded. 
     
     
         14 . The system of any of  claims 9 - 13 , wherein the 5′ homology arm of the donor DNA molecule is 40 to 200 nucleotides in length. 
     
     
         15 . The system of any of  claims 9 - 14 , wherein the donor DNA molecule comprises nucleotide sequence, between the 5′ and 3′ homology arms, that is heterologous to said genomic DNA. 
     
     
         16 . The system of any of  claims 9 - 15 , wherein the donor DNA molecule comprises one or more synthetic modifications selected from: a base modification, a sugar modification, and a backbone modification. 
     
     
         17 . The system of  claim 16 , wherein the donor DNA molecule comprises a phosphorothioate linkage. 
     
     
         18 . A method of editing a target genomic DNA of a eukaryotic cell, the method comprising introducing into the eukaryotic cell:
 (a) a dead Cas9 (dCas9) protein, or a nucleic acid encoding said dCas9 protein, wherein the dCas9 protein does not cleave the target genomic DNA;   (b) 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 genomic DNA; and   (c) a corresponding double stranded or single stranded donor DNA template molecule comprising at least 10 consecutive nucleotides of said target sequence,   wherein the dCas9 protein forms a complex with the Cas9 guide RNA thereby guiding the dCas9 protein to said target sequence, and wherein a nucleotide sequence of the donor DNA molecule is incorporated into the genomic DNA.   
     
     
         19 . The method according to  claim 18 , wherein the donor DNA template molecule is an asymmetric 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. 
     
     
         20 . The method according to  claim 19 , wherein the 5′ homology arm is 40 to 200 nucleotides in length. 
     
     
         21 . The method according to any of  claims 18 - 20 , wherein the donor DNA template molecule is single stranded. 
     
     
         22 . The method according to any of  claims 18 - 21 , wherein the donor DNA template molecule comprises a heterologous nucleotide sequence that is incorporated into the genomic DNA. 
     
     
         23 . The method according to any of  claims 18 - 22 , wherein said introducing comprises introducing into the cell two or more Cas9 guide RNAs, or one or more nucleic acids encoding said two or more Cas9 guide RNAs, wherein the two or more Cas9 guide RNAs hybridize to target sequences that do not overlap with one another and are each separated from one another by 1-100 nucleotides. 
     
     
         24 . The method according to  claim 23 , wherein the two or more Cas9 guide RNAs hybridize to target sequences that overlap with the donor DNA template molecule. 
     
     
         25 . The method according to  claim 23 , wherein said introducing comprises introducing into the cell three or more Cas9 guide RNAs, or one or more nucleic acids encoding said three or more Cas9 guide RNAs, wherein the three or more Cas9 guide RNAs hybridize to target sequences that do not overlap with one another and are each separated from one another by 1-100 nucleotides. 
     
     
         26 . The method according to  claim 25 , wherein the three or more Cas9 guide RNAs hybridize to target sequences that overlap with the donor DNA template molecule. 
     
     
         27 . The method according to  claim 25 , wherein said introducing comprises introducing into the cell four or more Cas9 guide RNAs, or one or more nucleic acids encoding said four or more Cas9 guide RNAs, wherein the four or more Cas9 guide RNAs hybridize to target sequences that do not overlap with one another and are each separated from one another by 1-100 nucleotides. 
     
     
         28 . The method according to  claim 27 , wherein the four or more Cas9 guide RNAs hybridize to target sequences that overlap with the donor DNA template molecule. 
     
     
         29 . The method according to any of  claims 18 - 28 , wherein the donor DNA molecule comprises one or more synthetic modifications selected from: a base modification, a sugar modification, and a backbone modification. 
     
     
         30 . The method according to  claim 29 , wherein the donor DNA molecule comprises a phosphorothioate linkage. 
     
     
         31 . A system for editing genomic DNA in a eukaryotic cell, the system comprising:
 (a) a dead Cas9 (dCas9) protein, or a nucleic acid encoding said dCas9 protein, wherein the dCas9 protein lacks catalytically active RuvC and HNH domains;   (b) a Cas9 guide RNA, or one or more nucleic acids encoding said Cas9 guide RNA, wherein the Cas9 guide RNA comprises a guide sequence that is complementary to a target sequence of a target genomic DNA of a eukaryotic cell; and   (c) a corresponding double stranded or single stranded donor DNA template molecule comprising at least 10 consecutive nucleotides of said target sequence.   
     
     
         32 . The system of  claim 31 , wherein the donor DNA template molecule is an asymmetric 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 the at least 10 consecutive nucleotides of said target sequence. 
     
     
         33 . The system of  claim 32 , wherein the 5′ homology arm of the donor DNA molecule is 40 to 200 nucleotides in length. 
     
     
         34 . The system of any of  claims 31 - 33 , wherein the donor DNA template molecule is single stranded. 
     
     
         35 . The system of any of  claims 31 - 34 , wherein the donor DNA template molecule comprises a nucleotide sequence that is a heterologous to said target genomic DNA. 
     
     
         36 . The system of any of  claims 31 - 35 , wherein the system comprises two or more Cas9 guide RNAs, or one or more nucleic acids encoding said two or more Cas9 guide RNAs, wherein the guide sequences of the two or more Cas9 guide RNAs are complementary to target sequences that do not overlap with one another and are each separated from one another by 1-100 nucleotides. 
     
     
         37 . The system of  claim 36 , wherein the two or more Cas9 guide RNAs are complementary to target sequences that overlap with the donor DNA template molecule. 
     
     
         38 . The system of  claim 36 , wherein the system comprises three or more Cas9 guide RNAs, or one or more nucleic acids encoding said three or more Cas9 guide RNAs, wherein the guide sequences of the three or more Cas9 guide RNAs are complementary to target sequences that do not overlap with one another and are each separated from one another by 1-100 nucleotides. 
     
     
         39 . The system of  claim 38 , wherein the three or more Cas9 guide RNAs are complementary to target sequences that overlap with the donor DNA template molecule. 
     
     
         40 . The system of  claim 38 , wherein the system comprises four or more Cas9 guide RNAs, or one or more nucleic acids encoding said four or more Cas9 guide RNAs, wherein the guide sequences of the four or more Cas9 guide RNAs are complementary to target sequences that do not overlap with one another and are each separated from one another by 1-100 nucleotides. 
     
     
         41 . The system of  claim 40 , wherein the four or more Cas9 guide RNAs are complementary to target sequences that overlap with the donor DNA template molecule. 
     
     
         42 . The system of any of  claims 31 - 41 , wherein the donor DNA molecule comprises one or more synthetic modifications selected from: a base modification, a sugar modification, and a backbone modification. 
     
     
         43 . The system of  claim 42 , wherein the donor DNA molecule comprises a phosphorothioate linkage. 
     
     
         44 . The system of any of  claims 31 - 43 , wherein the system comprises a eukaryotic cell comprising said target genomic DNA.

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