US 11,884,924 B2
Dual strand nucleic acid-guided nickase editing
Brian Chaikind, Boulder, CO (US); and Aamir Mir, Boulder, CO (US)
Assigned to Inscripta, Inc., Pleasanton, CA (US)
Filed by Inscripta, Inc., Boulder, CO (US)
Filed on Feb. 14, 2022, as Appl. No. 17/671,571.
Claims priority of provisional application 63/231,229, filed on Aug. 9, 2021.
Claims priority of provisional application 63/150,060, filed on Feb. 16, 2021.
Prior Publication US 2022/0275377 A1, Sep. 1, 2022
Int. Cl. C12N 15/62 (2006.01); C12N 5/071 (2010.01); C12N 9/22 (2006.01); C12N 15/11 (2006.01); C12N 15/90 (2006.01); C12N 15/86 (2006.01)
CPC C12N 15/62 (2013.01) [C12N 5/0687 (2013.01); C12N 9/22 (2013.01); C12N 15/11 (2013.01); C12N 15/86 (2013.01); C12N 15/907 (2013.01); C12Y 301/21004 (2013.01); C12N 2310/20 (2017.05); C12N 2740/15043 (2013.01); C12N 2800/40 (2013.01); C12N 2800/80 (2013.01)] 17 Claims
 
1. A method for performing nucleic acid-guided nickase/reverse transcriptase fusion editing in a target locus in a genome of a live cell utilizing two CREATE fusion (CF) editing cassettes comprising:
(a) providing the live cell comprising the target locus;
(b) providing a nucleic acid-guided nuclease/reverse transcriptase fusion enzyme;
(c) providing a first CF editing cassette and a second CF editing cassette, wherein the first CF editing cassette comprises a first region of complementarity to a first strand of the target locus, and wherein the second CF editing cassette comprises a second region of complementarity to a second strand of the target locus, wherein the first CF editing cassette comprises a nucleic acid molecule encoding from 5′ to 3′:
(i) a first CF guide RNA (gRNA) comprising a 5′ to 3′ spacer region and a scaffold region; and
(ii) a first repair template comprising first post-edit homology (PEH) region, a first edit, a first nick-to-edit region, and a first primer binding site (PBS) region; and
wherein the second CF editing cassette comprises a nucleic acid molecule encoding from 5′ to 3′:
(iii) a second CFgRNA comprising a 5′ to 3′ spacer region and a scaffold region; and
(iv) a second repair template comprising second PEH region, a second edit, a second nick-to-edit region, and a second PBS region;
(d) providing conditions to allow the nucleic acid-guided nuclease/reverse transcriptase fusion enzyme and the first CF editing cassette to bind to the first strand of the target locus, and the nucleic acid-guided nuclease/reverse transcriptase fusion enzyme and the second CF editing cassette to bind to the second strand of the target locus; and
(e) allowing the nucleic acid-guided nuclease/reverse transcriptase fusion enzyme and the first CF editing cassette to generate an insertion, deletion, or substitution of 1 or more nucleotides in the first strand of the target locus based on the first repair template and allowing the nucleic acid-guided nuclease/reversed transcriptase fusion enzyme and the second CF editing cassette to generate an insertion, deletion, or substitution of 1 or more nucleotides in the second strand of the target locus based on the second repair template.