Extended Data Fig. 4: KNIT editing enables repeated editing to increase insertion efficiency.
From: Efficient and precise programmable DNA knock-in without double-strand breaks
a, Schematic of repeated insertions at a specific locus via a second (2nd) transfection of Cas9 or KE1. Cas9 generates indels that hinder the original sgRNA binding during the 2nd transfection. b,c, Representative flow cytometry analysis (b) and percentages of EGFP+ HEK293T cells (c) following Cas9- or KE1-mediated EGFP-P2A insertion into the ACTB locus after the first (1st) or second (2nd) transfection. Control: HEK293T cells treated with KE1 and non-targeting sgRNA. d,e, Representative flow cytometry analysis (d) and the percentages of EGFP+ HEK293T cells (e) following Cas9- or KE1-mediated P2A-EGFP insertion at the H2B locus after the first (1st) or second (2nd) transfection. Control: HEK293T cells treated with KE1 and non-targeting sgRNA. f, Cas9-induced off-target insertion sites (left) and bar graph (right) showing off-target insertion efficiencies following repeated editing with Cas9 or KE1 for EGFP-P2A insertion at the ACTB locus. OT1-OT4: Cas9-mediated off-target insertion sites identified by genome-wide analysis, based on a previously reported 1% threshold. Red letters indicate sgRNA mismatch sites within the off-target DNA sequences. See Extended Data Fig. 3d for method details. For c, e, and f, bars show mean ± s.d.; dots show individual values; n = 3 biological replicates. P values: unpaired two-tailed Student’s t-test.
