Abstract
SummaryBase editing (BE) is a promising genome engineering tool for modifying DNA or RNA and has been widely used in various microorganisms as well as eukaryotic cells. Despite the proximal protospacer adjacent motif (PAM) is critical to the targeting range and off-target effect of BE, there is still lack of a specific approach to analyze the PAM pattern in BE systems. Here, we developed a base editing-coupled survival screening method. Using dCas9 from Streptococcus pyogenes (SpdCas9) and its variants xdCas9 3.7 and dCas9 NG as example, their PAM patterns in BE systems were extensively characterized using the NNNN PAM library with high sensitivity. In addition to the typical PAM recognition features, we observed more unique PAMs exhibiting BE activity. These PAM patterns will boost the finding of potential off-target editing event arising from non-canonical PAMs and provide the guidelines for PAM usage in the BE system.
Highlights
Base editing (BE) is an important genome editing technique that harnesses catalytically impaired dead Cas9 or nicking Cas9-guided nucleotide modification enzymes to modify of specific bases (Komor et al, 2016; Nishida et al, 2016)
In addition to the typical protospacer adjacent motif (PAM) recognition features, we observed more unique PAMs exhibiting BE activity. These PAM patterns will boost the finding of potential off-target editing event arising from non-canonical PAMs and provide the guidelines for PAM usage in the BE system
In contrast to Cas9 endonuclease-mediated homologous recombination (Zhou et al, 2019b), the BE system based on dead Cas9 (dCas9) does not cause DNA double-strand breaks (DSBs), which are highly genotoxic for the host, and is regarded as a more promising genome editing tool for applications in basic research and gene therapy (Mention et al, 2019; Rees and Liu, 2018)
Summary
Base editing (BE) is an important genome editing technique that harnesses catalytically impaired dead Cas (dCas9) or nicking Cas (nCas9)-guided nucleotide modification enzymes to modify of specific bases (Komor et al, 2016; Nishida et al, 2016). In contrast to Cas endonuclease-mediated homologous recombination (Zhou et al, 2019b), the BE system based on dCas does not cause DNA double-strand breaks (DSBs), which are highly genotoxic for the host, and is regarded as a more promising genome editing tool for applications in basic research and gene therapy (Mention et al, 2019; Rees and Liu, 2018). Several non-canonical PAMs including NAG, NGA, and NCG have been reported to support cleavage of DNA by SpCas, albeit less efficiently than the canonical NGG PAM (Hu et al, 2019; Jiang et al, 2013). Researchers generally used the canonical NGG PAM in the SpdCas9-based BE system It is currently unknown whether the BE system can use other non-canonical PAMs and result in off-target editing. It is very essential to comprehensively characterize the usefulness of various PAMs and evaluate the off-target risk in the BE system
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