US 12,410,429 B2
Compositions and methods for gene targeting using CRISPR-Cas and transposons
Joseph E. Peters, Ithaca, NY (US)
Assigned to Cornell University, Ithaca, NY (US)
Appl. No. 17/438,427
Filed by Cornell University, Ithaca, NY (US)
PCT Filed Mar. 16, 2020, PCT No. PCT/US2020/022964
§ 371(c)(1), (2) Date Sep. 11, 2021,
PCT Pub. No. WO2020/186262, PCT Pub. Date Sep. 17, 2020.
Claims priority of provisional application 62/882,204, filed on Aug. 2, 2019.
Claims priority of provisional application 62/818,523, filed on Mar. 14, 2019.
Prior Publication US 2022/0145298 A1, May 12, 2022
Int. Cl. C12N 15/113 (2010.01); C07K 14/195 (2006.01); C12N 9/12 (2006.01); C12N 15/10 (2006.01); C12N 15/11 (2006.01); C12N 15/63 (2006.01); C12N 15/90 (2006.01)
CPC C12N 15/113 (2013.01) [C12N 9/1241 (2013.01); C12N 15/102 (2013.01); C12N 15/111 (2013.01); C12N 15/63 (2013.01); C12N 15/90 (2013.01); C12N 2310/20 (2017.05); C12N 2800/90 (2013.01)] 14 Claims
 
1. A method for modifying a chromosome or extrachromosomal element in one or more prokaryotic cells, the method comprising introducing into the one or more prokaryotic cells a TnsA transposon protein that is at least 90% similar to SEQ ID NO:1 but contains an amino acid other than Alanine at position 125 of SEQ ID NO:1; or an expression vector comprising a tnsA gene that encodes and expresses said TnsA transposon protein when introduced into the one or more prokaryotic cells;
wherein the one or more prokaryotic cells also comprise during said modifying:
transposon proteins TnsB, TnsC, TniQ,
Cas proteins Cas8f, Cas5f, Cas7f, Cas6f,
a transposable DNA cargo sequence that is flanked by left and right transposon sequences; and
at least one guide RNA comprising a spacer targeted to a target DNA sequence in the chromosome or the extrachromosomal element, the DNA target sequence comprising a protospacer and a protospacer adjacent motif (PAM) that is 5′ to the protospacer;
wherein the TnsA, TnsB, TnsC, TniQ, Cas8f, Cas5f, Cas7f, Cas6f, and the at least one guide RNA, participate in transposition of the transposable DNA cargo sequence into the chromosome or extrachromosomal element in a location that is 3′ relative to the PAM and the protospacer.