Methods and systems for identifying crispr/cas off-target sites

A computer-implemented method for identifying possible cleavage locations of a nucleotide-directed nuclease for testing and selecting the nuclease with the fewest off-target cleavage locations comprising:

• in a computer system,
• comparing a series of query sequences comprising a guide strand sequence and one or more guide strand sequence variants comprising one or more nucleotide insertions, one or more nucleotide deletions, and/or one or more nucleotide substitutions relative to the guide sequence to genomic sequence,
• exhaustively searching the genomic sequence for target cleavage sites matching user-supplied criteria, wherein the criteria comprise number of mismatches, number and/or size of differences in length (bulges), the presence of a protospacer adjacent motif (PAM) suffix, or a combination thereof, compared to the query sequence,
• locating all sites that may be cleaved including both mismatches and bulges,
• assigning a score to the returned target cleavage locations indicative of the predictive likelihood of cleavage at the target cleavage location, wherein target cleavage locations comprising genomic sequences having higher sequence identity to the guide sequence receive a lower score relative to target cleavage locations comprising genomic sequences having lower sequence identity to the guide sequence, wherein increasing numbers of substitutions, deletions, and insertions at the target cleavage location increase the score, and wherein the score is increased more for deletion(s) in the genomic sequence relative to the guide sequence (RNA bulges) than for insertions in the genomic sequence relative to the guide sequence (DNA bulges),
• ranking the target cleavage locations based on their scores,
• selecting the nucleases with the lower likelihood of predictive off-target cleavage sites and
• amplifying and assaying the genomic sequence at the target cleavage site after nuclease treatment to ensure specific genome editing.