Patent ID: 11884924
Assignee: INSCRIPTA, INC.
Field: Biotechnology (Chemistry)
Classification: CPC C | IPC C

Claim 0:
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.