Extended Data Fig. 5: Comparison of the editing efficiencies of various integration methods at AAVS1 locus.
a, Schematics of various KI methods for targeting an EF1α-EGFP transgene into AAVS1 locus. Traditional HDR donor is a covalent circular plasmid, which harbors long-range (~800 bp) homologous arms flanking the transgene. HMEJ donor is a intracellularly linearized double-stranded DNA template, which harbors short-range (~50 bp) homologous arms flanking the transgene. The donor for the dsCTS-based method is a double-stranded DNA template containing Cas9 target sequences (CTSs) flanking homologous arms on each side. The donor for the ssCTS-based method is a single-stranded DNA template with hybrid CTSs flanked by homologous arms. All aforementioned methods mediate targeted integrations at genomic DSB sites, whereas eePASSIGE, PASTE and PRIME-In mediate targeted integrations at genomic nick sites without introducing DSBs. b, Gel analysis of prepared dsCTS and ssCTS donors. Molecular weight markers (bp) are indicated on the left of the gel image. c, Editing efficiencies of various KI methods for targeting an EF1α-EGFP transgene at AAVS1 locus, as measured by frequencies of EGFP + HEK293T cells 14 days after transfection. Data are from three independent biological replicates. The results are presented as the mean ± SD. Statistical significance was calculated using two-way ANOVA with Dunnett’s multiple comparisons test.
