Genome-Wide Prediction of Highly Specific Guide RNA Spacers for CRISPR-Cas9-Mediated Genome Editing in Model Plants and Major Crops

摘要

Dear Editor, RNA-guided genome editing (RGE) using the Streptococcus pyogenes CRISPR-Cas9 system (Jinek et al.,2012;Cong et al.,2013;Mali et al.,2013b) is emerging as a simple and highly efficient tool for genome editing in many organisms.The Cas9 nuclease can be programmed by dual or single guide RNA (gRNA) to cut target DNA at specific sites,thereby introducing precise mutations by error-prone non-homologousend-joining repairing or by incorporating foreign DNAs via homologous recombination between target site and donor DNA.The gRNA-Cas9 complex recognizes targets based on the complementarity between one strand of targeted DNA (referred as protospacer) and the 5'-end leading sequence of gRNA (referred to as gRNA spacer) that is approximately 20 base pairs (bp) long (Figure 1A).Besides gRNA-DNA pairing,a protospacer-adjacent motif (PAM) following the paired region in the DNA is also required for Cas9 cleavage.Recent studies reveal that Cas9 could cut the PAM-containing DNA sites that imperfectly match gRNA spacer sequences,resulting in genome editing at undesired positions.