Fig. 4

Reducing the length of replaced sequence surrounding DSB improves HDR and reduces NHEJ. a Schematic illustration of the replaced sequence (RS) in pD-mNEonGreen-sg-RS1-39 bp-HA300-300 bp (same as pD-mNEonGreen-HA300- 300 bp) donor. Before integration of the insert by HDR, 1 bp in the left arm (in purple) and 39 bp on the right arm (in pink) need to be replaced. b Schematic illustration of the replaced sequence in pD-mNEonGreen-sg-RS1-0 bp-HA300-300 bp. Compared to the former donor, the homology in the right arm in this donor extends to the cut site on genomic DNA, making the replaced sequence to be 0 bp on the right side. c Effects of RS length on HDR efficiency. n = 3; error bars represent S.E.M. **P ≤0.01, by Student’s paired t-test. d Summary of Sanger sequencing results. e Distribution of different knockin patterns when using the two donors. f qPCR analysis of donor plasmid backbone-forward insertion. n = 3; error bars represent S.E.M. *P ≤ 0.05, by Student’s paired t-test