US 12,460,200 B2
Methods and compositions comprising CRISPR-Cpf1 and paired guide CRISPR RNAs for programmable genomic deletions
Neville Espi Sanjana, New York, NY (US)
Assigned to NEW YORK GENOME CENTER, INC., New York, NY (US); and NEW YORK UNIVERSITY, New York, NY (US)
Filed by NEW YORK GENOME CENTER, INC., New York, NY (US); and NEW YORK UNIVERSITY, New York, NY (US)
Filed on Mar. 2, 2020, as Appl. No. 16/806,197.
Application 16/806,197 is a continuation of application No. PCT/US2018/048767, filed on Aug. 30, 2018.
Claims priority of provisional application 62/552,816, filed on Aug. 31, 2017.
Prior Publication US 2020/0208141 A1, Jul. 2, 2020
Int. Cl. C12N 15/10 (2006.01); C12N 15/86 (2006.01)
CPC C12N 15/1082 (2013.01) [C12N 15/86 (2013.01); C12N 2310/20 (2017.05); C12N 2750/14141 (2013.01)] 20 Claims
 
1. An in vitro method comprising:
(a) transducing a mammalian cell with one or more lentiviral vectors, each vector comprising
(i) a nucleic acid sequence encoding a Cpf1 (Cas12a) protein in operative association with an RNA pol II promoter which controls expression thereof, in a mammalian cell; and
(ii) a flipped CRISPR RNA (crRNA) array comprising at least two spacers,
wherein each spacer encodes an RNA guide, wherein each guide hybridizes to a unique sequence located 3 from a T-rich protospacer-adjacent motif (PAM) in a contiguous region of the genome or a chromosome of a mammalian cell, said array in operative association with an RNA pol III promoter, wherein the RNA pol II promoter and the RNA pol III promoter are independent from each other and are present in opposing orientations with respect to each other; and
(b) culturing said transduced cells, wherein in the cultured cells, the Cpf1 (Cas12a) creates a deletion comprising the chromosome or genome between cleavage sites located downstream of the PAM, thereby providing a plurality of transduced cell cultures, each cell culture comprising said deletion.