Fig. 2

Effect of ssODN homology length on insertion efficiency at the AAVS1 locus. K562 cells were nucleofected with 4 μg of pCS2-Cas9-IRES-GFP-polyA-gRNA (AAVS1) co-expression vector and 0.3 nmol of ssODN donors with different lengths. Cells were harvested 2-day post nucleofection, and the GFP-positive cells were sorted. Genomic DNA was isolated and 100 ng DNA was used for PCR amplification with a P2P4 primer pair (a), or P2P4 and F1R1 primer pairs (b). The numbers on the top of the gel images represent the homology length in nucleotides of a ssODN donor. A 20-mer donor has two 10-base homology arms. Each ssODN contains an EcoRI site between the homology arms. The DNA sequence of each ssODN is shown in Supplementary Table 1. M: DNA Marker. NC: PCR control; CON1: normal cells; CON2: cells transfected with pCS2-Cas9-IRES-GFP-polyA-gRNA (AAVS1) co-expression vector only; CON3: cells transfected with ssODN donor (80 nucleotides) only; 20–80: cells transfected with pCS2-Cas9-IRES-GFP-polyA-gRNA (AAVS1) co-expression vector plus various lengths of ssODNA donors. Arrows indicate the predicted amplified fragments