SpCas9-HF1 enhances accuracy of cell cycle-dependent genome editing by increasing HDR efficiency, and by reducing off-target effects and indel rates

Figures

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  Figure 1. Comparison of HDR efficiency and on- and off-target mutation rates among SpCas9, AcrIIA4-Cdt1-T2A-SpCas9, and SpCas9 variants at the EMX1 site (A) Schematic representation of this study. Synergistic, precise genome editing by combining high-fidelity SpCas9 variants and anti-CRISPR mediated activity control. (B) On- and off-target sequences used in experiments. (C and D) Evaluations of HDR efficiency in the first (C) and the second (D) screenings. (E and F) Evaluations of on- and off-target mutation rates in the first (E) and the second (F) screenings. HDR efficiency was evaluated using HindIII digestion for PCR products amplified from target sites in extracted genomic DNA. HDR efficiency was calculated using the following formula: 100 × ((b + c)/(a + b + c)), where “a” is the integrated intensity of undigested PCR product, and “b” and “c” are integrated intensities of each cleavage product. On- and off-target mutation rates were analyzed by TIDE, utilizing Sanger sequencing data of PCR products amplified from target sites in extracted genomic DNA. On-target mutations include mutagenesis by a-EJ and NHEJ. Samples are identified by names under graph bars. IIA4CDTeSpCas9 means…

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  Figure 2. Comparison of HDR efficiency and on- and off-target mutation rates among SpCas9, eSpCas9, AcrIIA4-Cdt1-T2A-eSpCas9, AcrIIA5-Cdt1-T2A-eSpCas9, SpCas9-HF1, AcrIIA4-Cdt1-T2A-SpCas9-HF1, AcrIIA5-Cdt1-T2A-SpCas9-HF1, LZ3 Cas9, AcrIIA4-Cdt1-T2A-LZ3 Cas9, and AcrIIA5-Cdt1-T2A LZ3 Cas9 at the AAVS1 (A and D), EMX1 (B and E), and VEGFA (C and F) sites. Samples are identified by names under graph bars. IIA4CDT or IIA5CDT and SpCas9 variant names mean co-expression of AcrIIA4-Cdt1 or AcrIIA5-Cdt1 with indicated SpCas9 variants. HDR efficiency was evaluated using HindIII digestion for PCR products amplified from target sites in extracted genomic DNA. HDR efficiency was calculated with the following formula: 100 × ((b + c)/(a + b + c)), where “a” is the integrated intensity of undigested PCR product, and “b” and “c” are integrated intensities of each cleavage product. On- and off-target mutation rates were analyzed with TIDE utilizing Sanger sequencing data of PCR products amplified from target sites in extracted genomic DNA. On-target mutations include mutagenesis by a-EJ and NHEJ. n = 4 for AAVS1 and EMX1 target samples. n = 3 for VEGFA target samples. N.D., not determined. Significance in…