“A Genome Mining Toolbox for CRISPR-associated Cas9 orthologues”

CRISPR-associated protein system can facilitate accurate and targeted editing of genomes. RNA-guided Cas9 can be employed as an efficient genome editing tool in human, plants, and animals. The Cas9 nuclease can be programmed by guide RNA (gRNA) in order to cut DNA at targeted regions. Therefore, concise mutations are introduced by homologous recombination or non-homologous end-joining repairing. Although the CRISPR/Cas9 is a powerful system, alternative strategies have been developed to decrease the risk of off-target activities such as using Cas9 orthologs. Additionally, only one activity can be mediated by the Cas9 protein from Streptococcus pyogenes (Sp) at different target regions. In other words, it is unable to simultaneously mediate a different activity at other targets. Besides SpCas9, some orthologous CRISPR–Cas9 systems from different species have been discovered and utilized for genome editing. Therefore, in order for wider application of CRISPR-Cas, new and applicable Cas proteins, with different requirements for PAM sequence, gRNA length, and tracrRNA and crRNA sequences, have emerged from several species as a new tool for effective genome editing. Consequently, discovery of various potential Cas9 proteins becomes a major concern for CRISPR/Cas-mediated genome editing technology to develop straightforward bioinformatics tools with high efficiency and versatility to facilitate the artificial design of possible gRNAs. Hence, we aimed to create an application tool to perform high-throughput detection of target sites based on the specific sequence and length of PAM followed by a constant length of target site for not only common Streptococcus pyogenes (SpCas9) but also for other CRISPR-Cas systems. To this end, different search modes for gRNA detection were applied including i) coding strand searching, ii) anti-coding strand searching, iii) both strand searching. Also, a complete list of all possible gRNAs along with their useful information can be provided for various potential Cas9 orthologs. Such progress expands the toolbox of genome editing and the possibilities for the site-specific genome engineering.