US 12,331,292 B2
RNA-guided DNA integration using Tn7-like transposons
Samuel Henry Sternberg, New York, NY (US); and Sanne Eveline Klompe, New York, NY (US)
Assigned to The Trustees of Columbia University in the City of New York, New York, NY (US)
Filed by The Trustees of Columbia University in the City of New York, New York, NY (US)
Filed on Jun. 26, 2020, as Appl. No. 16/913,299.
Application 16/913,299 is a continuation of application No. 16/812,138, filed on Mar. 6, 2020, granted, now 10,947,534.
Claims priority of provisional application 62/902,171, filed on Sep. 18, 2019.
Claims priority of provisional application 62/884,600, filed on Aug. 8, 2019.
Claims priority of provisional application 62/875,772, filed on Jul. 18, 2019.
Claims priority of provisional application 62/873,455, filed on Jul. 12, 2019.
Claims priority of provisional application 62/866,270, filed on Jun. 25, 2019.
Claims priority of provisional application 62/855,814, filed on May 31, 2019.
Claims priority of provisional application 62/845,218, filed on May 8, 2019.
Claims priority of provisional application 62/822,544, filed on Mar. 22, 2019.
Claims priority of provisional application 62/815,187, filed on Mar. 7, 2019.
Prior Publication US 2020/0325474 A1, Oct. 15, 2020
Int. Cl. C12N 15/113 (2010.01); C12N 9/22 (2006.01); C12N 15/10 (2006.01); C12N 15/63 (2006.01); C12N 15/90 (2006.01)
CPC C12N 15/113 (2013.01) [C12N 9/22 (2013.01); C12N 15/102 (2013.01); C12N 15/63 (2013.01); C12N 15/902 (2013.01); C12N 2310/20 (2017.05)] 9 Claims
 
1. A method for RNA-guided DNA integration comprising:
introducing into a cell: i) an engineered CRISPR-Cas system, and/or one or more vectors encoding the engineered CRISPR-Cas system, ii) an engineered transposon system, and/or one or more vectors encoding the engineered transposon system, and iii) a donor sequence comprising a cargo nucleic acid sequence and first and second transposon end sequences,
wherein, when one or more vectors are employed, the CRISPR-Cas system and the transposon system are on the same or different vector(s),
wherein the cell comprises a nucleic acid sequence with a target site,
wherein the CRISPR-Cas system comprises: (a) at least one Cas protein, and (b) a guide RNA (gRNA),
wherein the engineered CRISPR-Cas system is derived from a Type I CRISPR-Cas system,
wherein the CRISPR-Cas system binds to the target site,
wherein the donor sequence is integrated on the PAM-distal side of the target site, and
wherein the engineered transposon system and the engineered CRISPR-Cas system are derived from the same species.