Compositions, systems, and methods for genome editing
This disclosure provides compositions, recombinant expression constructs, and engineered systems that include a polynucleotide including or encoding a Cas12a tracrRNA, and methods for their use. The materials and methods of the disclosure are especially suited to sequence-specific genome editing of eukaryotic genomic sequences.
1 . An engineered system comprising:
(a) a Cas12a nuclease, or one or more nucleotide sequences encoding the Cas12a nuclease; and
(b) at least one engineered Cas12a crRNA designed to form a complex with the Cas12a nuclease and comprising at least one direct repeat or fragment thereof and a spacer sequence designed to hybridize with a target sequence in a eukaryotic cell, or one or more nucleotide sequences encoding the at least one engineered Cas12a crRNA polynucleotide; and
(c) a Cas12a tracrRNA comprising a sequence selected from the group consisting of SEQ ID NOs: 1, 3, 6, 139-146, 149-169, 223-225 and 227-239.
2 . The engineered system of claim 1 , wherein the Cas12a nuclease is:
(a) a Cas 12a nuclease identified from the genome of a bacterial species selected from the group consisting of Francisella tularensis 1, Prevotella albensis, Lachnospiraceae bacterium MC2017 1, Butyrivibrio proteoclasticus , Peregrinibacteria bacterium GW2011 GWA2 33 10, Parcubacteria bacterium GW2011 GWC2 44 17, Smithella sp. SCADC, Acidaminococcus sp. BV3L6 , Lachnospiraceae bacterium MA2020 , Candidatus Methanoplasma termitum, Eubacterium eligens, Moraxella bovoculi 237 , Leptospira inadai, Lachnospiraceae bacterium ND2006, Porphyromonas crevioricanis 3, Prevotella disiens and Porphyromonas macacae ; or
(b) a Cas12a nuclease selected from the group consisting of LbCas12a, AsCas12a, FnCas12a, and a deactivated Cas12a nuclease; or
(c) a Cas12a nuclease having at least 80% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 11, 107-138, 172, 204-221, and 222; or
(d) a Cas12a nuclease variant selected from the group consisting of (1) a variant LbCpf1 that differs from a wild-type LbCpf1 sequence (SEQ ID NO:135) in containing at least one point mutation selected from the group consisting of G309P, Y312F, M474I, D523N, Q531K, C930A, D937N, V954F/Q, M975L, A984E, 1994L, T1006K, I1014V, V1055N/D, L1065F/Y, Y1180F, V1209G, I1229L, V801I, Y802I, D850A, E943A, and D1198A; (2) a variant AsCpf1 that differs from a wild-type AsCpf1 sequence (SEQ ID NO: 134) in containing at least one point mutation selected from the group consisting of L320P, V980A, Q987N, T1004F/Q, K1035E, T1057K, D1107N, F1117Y, N1291L, D908A, E993A, and D1263A; (3) a variant FnCpf1 that differs from a wild-type FnCpf1 sequence (SEQ ID NO:172) in containing at least one point mutation selected from the group consisting of 1339P, L342F, Q588K, F1017Q, K1047E, N1118D, F1128Y, L867I, D917A, E1006A, and D1255A; and (4) a variant ObCsm1 sequence that differs from a wildtype ObCsm1 sequence (SEQ ID NO:137) in containing at least one point mutation selected from the group consisting of K382N, Q393K, S925A, E933N, K943F/Q, K972L, K1064G, P670I, N671I, D727A, E939A, and D1053A.
3 . The engineered system of claim 2 , wherein the Cas12a tracrRNA is provided:
(a) as DNA encoding a first RNA molecule comprising the Cas12a tracrRNA and comprising a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety; or
(b) as a functional RNA molecule comprising the Cas12a tracrRNA and a functional RNA moiety, or as a polynucleotide encoding the functional RNA molecule; or
(c) by a DNA expression system comprising (i) DNA sequence for a first promoter; and (ii) operably linked and heterologous to the first promoter, DNA encoding a first RNA molecule comprising a Cas12a tracrRNA and comprising a restriction endonuclease cleavage site that is suitable for insertion of a sequence coding for a functional RNA moiety; and (iii) optionally, a terminator 3′ to and operably linked to the DNA encoding the first RNA molecule; or
(d) by a DNA expression system comprising: (i) DNA sequence for a first promoter; and (ii) operably linked and heterologous to the first promoter, a DNA encoding a functional RNA molecule comprising a Cas12a tracrRNA and a functional RNA moiety; and (iii) optionally, a terminator 3′ to and operably linked to the DNA encoding a functional RNA molecule; or
(e) as a Cas12a tracrRNA synthesized in vitro; or
(f) as a Cas12a tracrRNA obtained from a naturally occurring source.
4 . The engineered system of claim 1 , wherein the Cas12a tracrRNA is provided:
(a) in a molar excess, relative to the amount of Cas12a nuclease; or
(b) in a molar amount that is at least 10-fold greater than the molar amount of the Cas12a nuclease.
5 . A delivery particle or delivery liquid comprising the engineered system of claim 1 .
6 . A method of modifying a target sequence in a locus of interest of a eukaryotic cell comprising delivering the engineered system of claim 5 to the locus of interest, wherein the spacer sequence hybridizes with the target sequence, whereby modification of the locus of interest occurs.
7 . A method of producing a eukaryotic cell comprising a modification of a locus of interest comprising delivering the engineered system of claim 6 to a locus of interest, wherein the spacer sequence hybridizes with a target sequence located in the locus of interest of the eukaryotic cell and whereby the engineered system introduces the modification into the locus of interest.
8 . The method of claim 7 , further comprising isolating the eukaryotic cell comprising the modification of a locus of interest.
9 . The method of claim 7 , further comprising propagating the eukaryotic cell comprising the modification of the locus of interest.
10 . The method of claim 7 , further comprising introducing the eukaryotic cell comprising a modification of a locus of interest into a subject, optionally wherein the subject is a human or non-human mammal.
11 . The method of claim 7 , wherein the engineered system is delivered to a locus of interest in an unmodified eukaryotic cell obtained from a subject and wherein the eukaryotic cell comprising the modification of the locus of interest is introduced into the subject, optionally wherein the subject is a human or non-human mammal.
12 . The method of claim 7 , further comprising regenerating the eukaryotic cell comprising a modification of a locus of interest into an organism comprising the modification of the locus of interest.
13 . The method of claim 12 , wherein the organism is a plant, optionally wherein the plant is a maize, soybean, wheat, rice, cotton, or Brassica sp. plant, and optionally further comprising propagating the plant or harvesting seed therefrom.
14 . The method of claim 7 , wherein the modification is selected from the group consisting of an insertion of one or more nucleotides, a deletion of one or more nucleotides, or a substitution of one or more nucleotides.
15 . The method of claim 7 , wherein the eukaryotic cell is a non-human mammalian cell, a plant cell, or a fungal cell.
16 . The method of claim 7 , wherein the eukaryotic cell is an isolated cell in an in vitro axenic culture.
17 . The method of claim 16 , wherein the isolated cell is a human cell.