Performance of the Cas9 nickase system in Drosophila melanogaster.
Bottom Line: Mutations converting Cas9 to a DNA nickase have great potential for reducing off-target effects in vitro.Interestingly, Cas9 nickase greatly reduces the ability to generate mutants with one sgRNA, suggesting that the application of Cas9 nickase and sgRNA pairs can almost avoid off-target effects when generating indel mutants.However, Cas9(D10A) is not as effective as Cas9 in replacing the entire coding sequence of piwi with two sgRNAs.
Affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.Show MeSH
Mentions: Because Cas9D10A can efficiently generate indel mutants through the NHEJ pathway, we then evaluated the efficiency of Cas9D10A in HDR. Wild-type Cas9 nuclease had been successfully applied in generating mutants through HDR (Baena-Lopez et al. 2013; Gratz et al. 2014; Yu et al. 2014; Xue et al. 2014). Ideally, complete loss-of-function mutants need to dispose of entire coding sequences. To precisely replace the entire coding sequence, the approach we developed involved the transgenic Cas9 flies, two sgRNAs, and a plasmid DNA donor with a selective marker (Materials and Methods; Figure 3A).
Affiliation: Gene Regulatory Lab, School of Medicine, Tsinghua University, Beijing 100084, China.