Bioinformatic Tools in CRISPR/Cas Platform

CRISPR/Cas has emerged as a game-changing technology for genome editing with widespread applications ranging from human therapeutics to engineering bacterial genomes for beneficial purposes to editing plant genomes for agricultural purposes. Successful genome editing through CRISPR/Cas relies on two components: an appropriate Cas endonuclease and a 20-base-pair (bp), singleguide RNA (sgRNA). CRISPR/Cas is currently favored as a genome editing technique due to its simple design rules and efficient editing capabilities that do not necessarily involve adding any foreign DNA at the target site. Cas endonucleases can be programmed to target any site in the genome by changing the gRNA sequence, highlighting the importance of gRNA design for increased specificity and efficiency, and reduced off-targeting in CRISPR/Cas genome editing. The rapid rise in CRISPR/Cas genome editing and associated applications has led to the development of numerous computational tools for effective sgRNA design. In this chapter, we discuss the essentials of gRNA design and provide an overview of the design process. In addition to summarizing factors which affect gRNA specificity and CRISPR cleavage efficiency, we discuss predictions of target efficiency and off-target detection algorithms. Finally, we describe the application-specific (knockout, activation, repression, base editing, and RNA editing) requirements of gRNA design and different tools to facilitate gRNA design.