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.
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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.
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