A Genome Mining Toolbox for CRISPR-associated Cas9 orthologues

CRISPR-associated protein system can facilitate accurate and targeted editing of genomes. RNAguided 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 targetedregions. Therefore, concise mutations are introduced bY homologous recombination or non- homologous end-joining repairing. Although the CRISPR / Cas9 is a powerful system, alternativestrategies have been developed to decrease the risk of off-target activities such as using Cas9orthologs. Additionally, only one activity can be mediated by the Cas9 protein fromStreptococcus pyogenes (Sp) at different target regions. In other words, it is unable tosimultaneously mediate a different activityY at other targets. Besides SpCas9, some orthologousCRISPR-Cas9 systems from different species have been discovered and utilized for genomeediting. Therefore, in order for wider application of CRISPR-Case, new and applicable Casproteins, with different requirements for PAM sequence, gRNA length, and tracrRNA and crRNA1
sequences, have nameged from several species as a new tool for effective genome editing.Consequently, discovery of various potential Cas9 proteins becomes a major concern forCRISPR/Cas-mediated genome editing technology to develop straightforward bioinformaticstools with high ifficiencY and versatility to facilitate the artificial design of possible gRNAs.Hence, we aimed to create an application tool to perform high-throughput detection of targetsites based on the specific sequence and length of PAM followed by a constant length of targetsite for not only common Streptococcus pyogenes (SpCas9) but also for other CRISPR-Cassystems. 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, acomplete list of all possible gRNAs along with their useful information can be provided forvarious potential Cas9 orthologs. Such progress expands the toolbox of genome editing and thepossibilities for the site-specific genome engineering.