You can try our package in R, Casilico. It supports Cas12, Cas13, and Cas14 CRISPR-Cas systems. Using a list of important features such as mismatch tolerance rules, self-complementarity, GC content, frequency of cleaving base around the target site, target accessibility and PFS (protospacer flanking site) or PAM (protospacer adjacent motif) requirement, CaSilico searches all potential crRNAs in a user-input sequence.
CaSilico outperforms previous crRNA design tools in the following respects: 1) supporting any reference genome/transcriptome for which a FASTA file is available; 2) designing crRNAs that simultaneously target multiple sequences through conserved region detection among a set of sequences; 3) considering new CRISPR-Cas subtypes; 4) reporting a list of different features for each candidate crRNA, which can help the user to select the best one. Our paper was puplished in Frontiers in Bioengineering and Biotechnology.
Our tool is sufficiently flexible to tune some key parameters governing the design of crRNA and identification of off-targets, which can be led to increases the chances of successful CRISPR-Cas experiments. Given these capabilities, Casilico addresses end-user concerns arising from the use of sophisticated bioinformatics algorithms and has a wide range of potential research applications in different areas especially design of crRNA for pathogen diagnosis. Casilico was successfully applied to design crRNAs for different genes in SARS-CoV-2 genome, as some of the crRNAs have been experimentally tested in the previous studies.