Casilico: A versatile CRISPR package for in silico CRISPR RNA designing

Background: The efficiency of CRISPR-Cas system is highly depends on well-designed CRISPR RNA(crRNA). To facilitate the use of various types of CRISPR-Cas systems, there is a need for development ofcomputational tools to design crRNAs which cover different CRISPR-Cas systems with off-target analysiscapability. Numerous crRNA design tools have been developed but nearly all of them are dedicated to designcrRNA for genome editing. Hence, we developed a tool matching the needs of both beginners and experts,named Casilico, which was inspired by the limitations of the current gRNA design tools for designingcrRNAs for Cas۱۲, Cas۱۳, and Cas۱۴ CRISPR-Cas systems.Materials and Methods: Using a list of important features such as mismatch tolerance rules, selfcomplementarity,GC content, frequency of cleaving base around the target site, target accessibility andprotospacer flanking site or protospacer adjacent motif requirement, Casilico searches all potential crRNAsin a user-input sequence. Considering these features, help users to rank all crRNAs for a sequence and makean informed decision about whether a crRNA is suited for an experiment or not. Our tool is sufficientlyflexible to tune some key parameters governing the design of crRNA and identification of off-targets, led toincreases the chances of successful CRISPR-Cas experiments.Results, and Conclusion: Casilico outperforms previous crRNA design tools in the following respects: ۱)supporting any reference genome/transcriptome for which a FASTA file is available; ۲) designing crRNAsthat simultaneously target multiple sequences through conserved region detection among a set of sequences;۳) considering new CRISPR-Cas subtypes; ۴) reporting a list of different features for each candidate crRNA,which can help the user to select the best one. Casilico was successfully applied to design crRNAs fordifferent genes in SARS-CoV-۲ genome, as some of the crRNAs have been experimentally tested in theprevious studies.

Keywords:

Cas۱۳ , Cas۱۲ , Cas۱۴ , Guide RNA , CRISPR Cas