Dual strand nucleic acid-guided nickase editing

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.