US 11,866,697 B2
Systems, methods, and compositions for targeted nucleic acid editing
Feng Zhang, Cambridge, MA (US); David Benjamin Turitz Cox, Cambridge, MA (US); Jonathan Gootenberg, Cambridge, MA (US); Omar O. Abudayyeh, Cambridge, MA (US); Bernd Zetsche, Cambridge, MA (US); and Jonathan Strecker, Cambridge, MA (US)
Assigned to THE BROAD INSTITUTE, INC., Cambridge, MA (US); MASSACHUSETTS INSTITUTE OF TECHNOLOGY, Cambridge, MA (US); and PRESIDENT AND FELLOWS OF HARVARD COLLEGE, Cambridge, MA (US)
Appl. No. 16/614,549
Filed by THE BROAD INSTITUTE, INC., Cambridge, MA (US); MASSACHUSETTS INSTITUTE OF TECHNOLOGY, Cambridge, MA (US); and PRESIDENT AND FELLOWS OF HARVARD COLLEGE, Cambridge, MA (US)
PCT Filed May 18, 2018, PCT No. PCT/US2018/033394
§ 371(c)(1), (2) Date Nov. 18, 2019,
PCT Pub. No. WO2018/213708, PCT Pub. Date Nov. 22, 2018.
Claims priority of provisional application 62/609,957, filed on Dec. 22, 2017.
Claims priority of provisional application 62/568,133, filed on Oct. 4, 2017.
Claims priority of provisional application 62/561,663, filed on Sep. 21, 2017.
Claims priority of provisional application 62/508,293, filed on May 18, 2017.
Prior Publication US 2020/0283755 A1, Sep. 10, 2020
Int. Cl. C12N 5/10 (2006.01); C12N 9/22 (2006.01); C12N 15/62 (2006.01); C12N 15/10 (2006.01); A61K 48/00 (2006.01); C12N 15/11 (2006.01); C12N 15/79 (2006.01)
CPC C12N 15/102 (2013.01) [A61K 48/00 (2013.01); C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 15/62 (2013.01); C12N 15/79 (2013.01); C12N 2310/533 (2013.01); C12Y 305/04004 (2013.01)] 29 Claims
 
1. A method of modifying an Adenine in a target locus of interest, comprising delivering to said locus:
(a) a Cpf1 nickase protein;
(b) a guide molecule which comprises a guide sequence linked to a direct repeat sequence; and
(c) an adenosine deaminase protein or catalytic domain thereof;
wherein said adenosine deaminase protein or catalytic domain thereof is covalently or non-covalently linked, or is adapted to covalently or non-covalently link after delivery, to said Cpf1 nickase protein or said guide molecule;
wherein said guide molecule forms a complex with said Cpf1 nickase protein and directs said complex to bind a first DNA strand at said target locus of interest, wherein said guide sequence is capable of hybridizing with a target sequence comprising said Adenine within said first DNA strand to form a heteroduplex, wherein said guide sequence comprises a non-pairing Cytosine at a position corresponding to said Adenine resulting in an A-C mismatch in the heteroduplex formed;
wherein said Cpf1 nickase protein nicks a second DNA strand at said target locus of interest displaced by formation of said heteroduplex; and
wherein said adenosine deaminase protein or catalytic domain thereof deaminates said Adenine in said heteroduplex.
 
22. An engineered, non-naturally occurring system suitable for modifying an Adenine in a target locus of interest, comprising
a) a guide molecule which comprises a guide sequence linked to a direct repeat sequence, or a nucleotide sequence encoding said guide molecule;
b) a Cpf1 nickase protein, or a nucleotide sequence encoding said Cpf1 nickase protein; and
c) an adenosine deaminase protein or catalytic domain thereof, or a nucleotide sequence encoding said adenosine deaminase protein or catalytic domain thereof;
wherein said adenosine deaminase protein or catalytic domain thereof is covalently or non-covalently linked to said Cpf1 nickase protein or said guide molecule or is adapted to link thereto after delivery;
wherein said guide sequence is capable of hybridizing with a target sequence comprising an Adenine on a first DNA strand at said target locus to form a heteroduplex, wherein said guide sequence comprises a non-pairing Cytosine at a position corresponding to said Adenine resulting in an A-C mismatch in the heteroduplex formed, and wherein said adenosine deaminase protein or catalytic domain thereof deaminates said Adenine in said heteroduplex; and
wherein said Cpf1 nickase protein is capable of nicking a second DNA strand complementary to said first DNA strand.