Granted Patent
B2
US 12,649,928 · App. 16/884,327 · Granted Jun 9, 2026
RNA-guided human genome engineering
Inventors:
George M. Church (Brookline, MA); Prashant G. Mali (La Jolla, CA); Luhan Yang (Somerville, MA)
Assignee:
President and Fellows of Harvard College
C12N15/85C12N9/22C12N15/01C12N15/10C12N15/102C12N15/1024C12N15/63C12N15/81C12N15/8201C12N15/87C12N15/90C12N15/907C12N2310/20C12N2800/80C12N2810/55C12Y301/00
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Abstract
A method of altering a eukaryotic cell is provided including transfecting the eukaryotic cell with a nucleic acid encoding RNA complementary to genomic DNA of the eukaryotic cell, transfecting the eukaryotic cell with a nucleic acid encoding an enzyme that interacts with the RNA and cleaves the genomic DNA in a site specific manner, wherein the cell expresses the RNA and the enzyme, the RNA binds to complementary genomic DNA and the enzyme cleaves the genomic DNA in a site specific manner.
Claims (36)
1 . An RNA-guided system for use in a eukaryotic cell comprising
(1) a guide RNA sequence or a first nucleic acid sequence encoding the guide RNA sequence and
(2) a Cas9 nickase or a nuclease null Cas9, or a second nucleic acid sequence encoding the Cas9 nickase or the nuclease null Cas9,
wherein the guide RNA sequence is a crRNA-tracrRNA fusion transcript comprising the sequence
(SEQ ID NO: 46)
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA
CUUGAAAAAGUGGCACCGAGUCGGUGC.
2 . A method of altering a eukaryotic cell comprising
providing the eukaryotic cell with a guide RNA sequence or a first nucleic acid sequence encoding the guide RNA sequence, wherein the guide RNA sequence comprises a spacer sequence complementary to genomic DNA of the eukaryotic cell, and a scaffold sequence,
providing the eukaryotic cell with a Cas9 nickase, or a second nucleic acid sequence encoding the Cas9 nickase,
wherein the guide RNA sequence binds to complementary genomic DNA and the Cas9 nickase nicks the complementary genomic DNA, and wherein the guide RNA sequence is a crRNA-tracrRNA fusion transcript comprising the sequence
(SEQ ID NO: 46)
GUUUUAGAGCUAGAAAUAGCAAGUUAAAAUAAGGCUAGUCCGUUAUCAA
CUUGAAAAAGUGGCACCGAGUCGGUGC.
3 . The RNA-guided system of claim 1 , wherein the guide RNA sequence has a secondary structure comprising a first hairpin and a second 3′ hairpin.
4 . The RNA-guided system of claim 1 , further comprising a regulatory element operable in the eukaryotic cell operably linked to the first nucleic acid sequence encoding the guide RNA sequence.
5 . The RNA-guided system of claim 1 , further comprising a human U6 polymerase III promoter operably linked to the first nucleic acid sequence encoding the guide RNA sequence.
6 . The RNA-guided system of claim 1 , wherein the second nucleic acid sequence encoding the Cas9 nickase or the nuclease null Cas9 is a human codon optimized nucleic acid.
7 . The RNA-guided system of claim 1 , wherein the second nucleic acid sequence encoding the Cas9 nickase or the nuclease null Cas9 is a human codon optimized nucleic acid that further comprises a nucleic acid sequence encoding a nuclear localization signal.
8 . The RNA-guided system of claim 1 , wherein the second nucleic acid sequence encoding the Cas9 nickase or the nuclease null Cas9 is a human codon optimized nucleic acid that further comprises a nucleic acid sequence encoding a C-terminal nuclear localization signal.
9 . The RNA-guided system of claim 1 , wherein the second nucleic acid sequence encoding the Cas9 nickase or the nuclease null Cas9 is a human codon optimized nucleic acid that further comprises a nucleic acid sequence encoding a C-terminal SV40 nuclear localization signal.
10 . The RNA-guided system of claim 1 , wherein the second nucleic acid sequence encoding the Cas9 nickase or the nuclease null Cas9 is a human codon optimized nucleic acid comprising a regulatory element operable in the eukaryotic cell.
11 . The RNA-guided system of claim 1 , wherein the second nucleic acid sequence encoding the Cas9 nickase or the nuclease null Cas9 is a human codon optimized nucleic acid comprising a cytomegalovirus (CMV) promoter.
12 . The RNA-guided system of claim 1 , wherein the Cas9 nickase is an S. pyogenes Cas9 nickase.
13 . The RNA-guided system of claim 1 , wherein the nuclease null Cas9 is an S. pyogenes nuclease null Cas9.
14 . The method of claim 2 , wherein the guide RNA sequence has a secondary structure comprising a first hairpin and a second 3′ hairpin.
15 . The method of claim 2 , wherein the first nucleic acid sequence encoding the guide RNA sequence further comprises a regulatory element operable in the eukaryotic cell.
16 . The method of claim 2 , wherein the first nucleic acid sequence encoding the guide RNA sequence further comprises a human U6 polymerase III promoter.
17 . The method of claim 2 , wherein the second nucleic acid sequence encoding the Cas9 nickase is a human codon optimized nucleic acid.
18 . The method of claim 2 , wherein the second nucleic acid sequence encoding the Cas9 nickase is a human codon optimized nucleic acid that further comprises a nucleic acid sequence encoding a nuclear localization signal.
19 . The method of claim 2 , wherein the second nucleic acid sequence encoding the Cas9 nickase is a human codon optimized nucleic acid that further comprises a nucleic acid sequence encoding a C-terminal nuclear localization signal.
20 . The method of claim 2 , wherein the second nucleic acid sequence encoding the Cas9 nickase is a human codon optimized nucleic acid that further comprises a nucleic acid sequence encoding a C-terminal SV40 nuclear localization signal.
21 . The method of claim 2 , wherein the second nucleic acid sequence encoding the Cas9 nickase is a human codon optimized nucleic acid comprising a regulatory element operable in the eukaryotic cell.
22 . The method of claim 2 , wherein the second nucleic acid sequence encoding the Cas9 nickase is a human codon optimized nucleic acid comprising a cytomegalovirus (CMV) promoter.
23 . The method of claim 2 , wherein the Cas9 nickase is an S. pyogenes Cas9 nickase.
24 . The method of claim 2 , wherein the eukaryotic cell is a human cell.
Assignments (1)
ASSIGNMENT OF ASSIGNOR'S INTEREST
Recorded May 6, 2022
From: CHURCH, GEORGE M.; MALI, PRASHANT; YANG, LUHAN
To: PRESIDENT AND FELLOWS OF HARVARD COLLEGE
Reel/Frame 059843/0397 →
Continuity (6)
Continuation
14653144
Provisional Application
61779169
· Mar 13, 2013
Provisional Application
61738355
· Dec 17, 2012
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