Modified CPF1 guide RNA
View Patent ↗The invention provides a nucleic acid comprising a Cpf1 crRNA, a processing sequence 5′ of the Cpf1 crRNA, and an extension sequence 5′ of the processing sequence. The invention also provides a composition comprising the nucleic acid, a carrier, and optionally Cpf1. Additionally, the invention provides method of genetically modifying a eukaryotic target cell, comprising contacting the eukaryotic target cell with the nucleic acid or the composition to genetically modify a target nucleic acid in the cell.
1 . A nucleic acid comprising
a Cpf1 crRNA, wherein the Cpf1 crRNA comprises a stem-loop domain located 5′ of a targeting sequence;
an extension sequence of at least 6 nucleotides 5′ of the crRNA;
and, optionally, a processing sequence between the crRNA and the extension sequence, wherein the processing sequence is a sequence that is self-cleaved by Cpf1;
wherein the extension sequence does not comprise a processing sequence, an aptamer, or the sequence of the Cpf1 crRNA, and wherein the extension sequence comprises a nucleotide comprising a 2′ deoxy modification.
2 . The nucleic acid of claim 1 , wherein the nucleic acid comprises a processing sequence, and the processing sequence comprises a fragment of a direct repeat sequence of a Cpf1 array, wherein the direct repeat sequence comprises a crRNA sequence portion and a processing portion positioned 5′ of the crRNA sequence portion, and the fragment comprises at least 5 contiguous nucleotides of the processing portion of the direct repeat sequence.
3 . The nucleic acid of claim 2 , wherein the processing sequence comprises a fragment of at least 10 nucleotides of the processing portion of the direct repeat sequence.
4 . The nucleic acid of claim 2 , wherein the processing sequence comprises the entire processing portion of the direct repeat sequence.
5 . The nucleic acid of claim 1 , wherein the extension sequence comprises 10 to 100 nucleotides.
6 . The nucleic acid of claim 1 , wherein the nucleic acid contains only a single Cpf1 crRNA sequence.
7 . The nucleic acid of claim 2 , further comprising a second processing sequence 5′ of the extension sequence, and a second extension sequence 5′ of the second processing sequence.
8 . The nucleic acid of claim 1 , further comprising a donor nucleic acid hybridized or covalently linked thereto, optionally wherein: the donor nucleic acid is covalently linked 5′ of the processing sequence, or 5′ of the extension sequence; the donor nucleic acid is linked to the processing sequence or extension sequence by a linker group; or the donor nucleic acid is hybridized to the extension sequence and/or processing sequence.
9 . The nucleic acid of claim 1 , wherein the extension sequence comprises fewer than about 60 nucleotides.
10 . The nucleic acid of claim 1 , wherein the nucleic acid does not comprise a processing sequence.
11 . The nucleic acid of claim 10 , wherein the nucleic acid further comprises a donor nucleic acid covalently linked 5′ of the extension sequence, optionally by a linker group, or hybridized to the extension sequence.
12 . The nucleic acid of claim 1 , wherein the extension sequence comprises a self-hybridizing sequence, optionally, a semi-stable hairpin structure, a stable hairpin structure, a pseudoknot structure, a G-quadraplex structure, a bulge loop structure, an internal loop structure, a branch loop structure, or a combination thereof.
13 . The nucleic acid of claim 1 , wherein the extension sequence or portion thereof is resistant to nuclease degradation.
14 . The nucleic acid of claim 1 , wherein the extension sequence comprises one or more modified internucleotide bonds.
15 . The nucleic acid of claim 14 , wherein a region of 4 or more contiguous nucleotides of the extension sequence, or the entire extension sequence, has modified internucleotide bonds.
16 . The nucleic acid of claim 14 , wherein the modified internucleotide bonds comprise phosphorothioate, phosphorodithioate, methylphosphonate, phosphoramidate, 2′-O-methyl, 2′-O-methoxyethyl, 2′-fluoro, bridged nucleic acid (BNA), or phosphotriester modified bonds, or a combination thereof, and/or wherein the extension sequence comprises one or more xeno nucleic acids (XNA).
17 . The nucleic acid of claim 1 , wherein the nucleic acid further comprises a biotin and/or avidin or streptavidin molecule attached to the 5′ terminus of the extension sequence.
18 . A composition comprising the nucleic acid of claim 1 and a carrier, and optionally further comprising a Cpf1 protein or nucleic acid encoding the Cpf1 protein.
19 . The composition of claim 18 , wherein the composition is substantially free of a divalent metal ion that promotes Cpf1 cleavage.
20 . The composition of claim 18 , wherein the composition comprises a cationic lipid.
21 . The composition of claim 18 , wherein the nucleic acid is in a liposome or partially or totally encapsulated by a polymer nanoparticle, or attached to a metal or polymer nanoparticle.
22 . A method of genetically modifying a eukaryotic target cell, comprising contacting the eukaryotic target cell with the nucleic acid of claim 1 to genetically modify a target nucleic acid.
23 . The method of claim 22 , wherein the target cell is a mammalian cell, optionally a human cell.
24 . The nucleic acid of claim 1 , wherein the extension sequence comprises no more than 100 nucleotides.
25 . A composition comprising
(a) a nucleic acid comprising a Cpf1 crRNA, wherein the Cpf1 crRNA comprises a stem-loop domain located 5′ of a targeting sequence;
an extension sequence of at least 4 nucleotides and no more than 100 nucleotides positioned 5′ of the crRNA;
and, optionally, a processing sequence between the crRNA and the extension sequence, wherein the processing sequence is a sequence that is self-cleaved by Cpf1;
wherein the extension sequence does not comprise a processing sequence, an aptamer, or the sequence of the Cpf1 crRNA, and wherein the extension sequence comprises a nucleotide comprising a 2′ deoxy modification; and
(b) a liposome, polymer nanoparticle, or metal nanoparticle; wherein the nucleic acid is in the liposome, or partially or totally encapsulated by the polymer nanoparticle, or attached to the metal nanoparticle or polymer nanoparticle.
26 . The composition of claim 25 , wherein the composition comprises a Cpf1 protein, and the nucleic acid and Cpf1 protein is in the liposome or partially or totally encapsulated by the polymer nanoparticle, or attached to the metal or polymer nanoparticle.
27 . The modified guide RNA of claim 1 , wherein the extension sequence comprises 9 to 100 nucleotides.
28 . The modified guide RNA of claim 27 , wherein the extension sequence comprises 9 to 60 nucleotides.
29 . A composition comprising
(a) a nucleic acid comprising a Cpf1 crRNA, wherein the Cpf1 crRNA comprises a stem-loop domain located 5′ of a targeting sequence;
an extension sequence of at least 4 nucleotides and no more than 100 nucleotides positioned 5′ of the crRNA;
and, optionally, a processing sequence between the crRNA and the extension sequence, wherein the processing sequence is a sequence that is self-cleaved by Cpf1;
wherein the extension sequence does not comprise a processing sequence, an aptamer, or the sequence of the Cpf1 crRNA; and
(b) a Cpf1 protein;
the nucleic acid and Cpf1 protein are in a liposome or partially or totally encapsulated by a polymer nanoparticle, or attached to a metal or polymer nanoparticle.