gRNA STABILIZATION IN NUCLEIC ACID-GUIDED NICKASE EDITING
The present disclosure provides compositions of matter, methods and instruments for nucleic acid-guided nickase/reverse transcriptase fusion editing in live cells. Editing efficiency is improved using fusion proteins (e.g., the nickase-RT fusion) that retain certain characteristics of nucleic acid-directed nucleases (e.g., the binding specificity and ability to cleave one or more DNA strands in a targeted manner) combined with reverse transcriptase activity. Editing cassettes are employed, comprising a gRNA and a repair template where the 3′ end of the repair template is protected from degradation.
1 . A CREATE fusion editing cassette for performing nucleic acid-guided nickase/reverse transcriptase fusion editing comprising from 3′ to 5′:
an RNA repair template comprising:
an RNA stabilization moiety;
a linker region;
a primer binding region capable of binding to a nicked target DNA;
a nick-to-edit region; and
a region of post-edit homology;
a gRNA comprising:
a guide sequence; and
a scaffold region.
2 . The CREATE fusion editing cassette of claim 1 , wherein the RNA stabilization moiety is a G quadraplex, an RNA hairpin, an RNA pseudoknot or an exoribonuclease resistant RNA.
3 . The CREATE fusion editing cassette of claim 2 , wherein the RNA stabilization moiety is a G quadraplex.
4 . The CREATE fusion editing cassette of claim 3 , wherein the RNA stabilization moiety is a G quadraplex selected from SEQ ID No: 1; SEQ ID No: 2; SEQ ID No: 3; SEQ ID No: 4; SEQ ID No: 5; SEQ ID No: 6; SEQ ID No: 7; SEQ ID No: 8; SEQ ID No: 9; and SEQ ID No: 10.
5 . The CREATE fusion editing cassette of claim 3 , wherein the RNA stabilization moiety is a G quadraplex selected from SEQ ID No: 11; SEQ ID No: 12; SEQ ID No: 13; SEQ ID No: 14; SEQ ID No: 15; SEQ ID No: 16; SEQ ID No: 17; SEQ ID No: 18; SEQ ID No: 19; and SEQ ID No: 20.
6 . The CREATE fusion editing cassette of claim 3 , wherein the RNA stabilization moiety is a G quadraplex selected from SEQ ID No: 21; SEQ ID No: 22; SEQ ID No: 23; SEQ ID No: 24; SEQ ID No: 25; SEQ ID No: 26; SEQ ID No: 27; SEQ ID No: 28; SEQ ID No: 29; and SEQ ID No: 30.
7 . The CREATE fusion editing cassette of claim 3 , wherein the RNA stabilization moiety is a G quadraplex selected from SEQ ID No: 31; SEQ ID No: 32; SEQ ID No: 33; SEQ ID No: 34; SEQ ID No: 35; SEQ ID No: 36; SEQ ID No: 37; SEQ ID No: 38; SEQ ID No: 39; and SEQ ID No: 40.
8 . The CREATE fusion editing cassette of claim 3 , wherein the RNA stabilization moiety is a G quadraplex selected from SEQ ID No: 41; SEQ ID No: 42; SEQ ID No: 43; SEQ ID No: 44; SEQ ID No: 45; SEQ ID No: 46; SEQ ID No: 47; SEQ ID No: 48; and SEQ ID No: 29.
9 . The CREATE fusion editing cassette of claim 2 , wherein the RNA stabilization moiety is an RNA hairpin.
10 . The CREATE fusion editing cassette of claim 9 , wherein the RNA stabilization moiety is an RNA hairpin selected from SEQ ID No: 50; SEQ ID No: 51; SEQ ID No: 52; SEQ ID No: 53; SEQ ID No: 54; SEQ ID No: 55; SEQ ID No: 65; SEQ ID No: 66; SEQ ID No: 67; SEQ ID No: 68; SEQ ID No: 69; and SEQ ID No: 70.
11 . The CREATE fusion editing cassette of claim 2 , wherein the RNA stabilization moiety is an RNA pseudoknot.
12 . CREATE fusion editing cassette of claim 11 , wherein the RNA stabilization moiety is an RNA pseudoknot selected from SEQ ID No: 50; SEQ ID No: 56; SEQ ID No: 57; SEQ ID No: 58; SEQ ID No: 59; SEQ ID No: 60; SEQ ID No: 61; SEQ ID No: 62; SEQ ID No: 63; and SEQ ID No: 64.
13 . The CREATE fusion editing cassette of claim 2 , wherein the RNA stabilization moiety is an exoribonuclease resistant RNA.
14 . The CREATE fusion editing cassette of claim 13 , wherein the RNA stabilization moiety is an exoribonuclease resistant RNA selected from SEQ ID No: 71; SEQ ID No: 72; and SEQ ID No: 73.
15 . The CREATE fusion editing cassette of claim 1 , wherein the linker region is from 0 to 20 nucleotides in length.
16 . The CREATE fusion editing cassette of claim 1 , wherein the primer binding region is from 0 to 20 nucleotides in length.
17 . The CREATE fusion editing cassette of claim 1 , wherein the nick-to-edit region is from 0 to 20 nucleotides in length.
18 . The CREATE fusion editing cassette of claim 1 , wherein the region of post-edit homology is 3 to 20 nucleotides in length.
19 . The CREATE fusion editing cassette of claim 1 , wherein the guide sequence of the gRNA is capable of hybridizing to a genomic target locus and wherein the scaffold sequence of the gRNA is capable of interacting or complexing with a nucleic acid-guided nuclease.