RESEARCH ARTICLE CRISPR MultiTargeter: A Web Tool to Find Common and Unique CRISPR Single Guide RNA Targets in a Set of Similar Sequences

Genome engineering has been revolutionized by the discovery of clustered regularly inter-spaced palindromic repeats (CRISPR) and CRISPR-associated system genes (Cas) in bacteria. The type IIB Streptococcus pyogenes CRISPR/Cas9 system functions in many species and additional types of CRISPR/Cas systems are under development. In the type II system, expression of CRISPR single guide RNA (sgRNA) targeting a defined sequence and Cas9 generates a sequence-specific nuclease inducing small deletions or insertions. Moreover, knock-in of large DNA inserts has been shown at the sites targeted by sgRNAs and Cas9. Several tools are available for designing sgRNAs that target unique locations in the genome. However, the ability to find sgRNA targets common to several similar se-quences or, by contrast, unique to each of these sequences, would also be advantageous. To provide such a tool for several types of CRISPR/Cas system and many species, we de-veloped the CRISPRMultiTargeter software. Similar DNA sequences in question are dupli-cated genes and sets of exons of different transcripts of a gene. Thus, we implemented a basic sgRNA target search of input sequences for single-sgRNA and two-sgRNA/Cas