The highly conserved Sal1 encodes a bifunctional enzyme with inositol polyphosphate-1-phosphatase and 3′ (2′), 5′-bisphosphate nucleotidase activity and has been shown to alter abiotic stress tolerance in plants when disrupted.
These unique target sites can then be output as before. The target sites which pass this comparison test and the subsequent test for location within exon sequences are confirmed unique target sites. The speed of comparison depends on a mismatch count variable (MM count), which ensures that the comparison is stopped (“End”) as soon as there are more than 2 mismatches (identities are indicated by “*”). Each target site is then compared to all identified target sites in these sequences. The search for unique target sites is accomplished by computing all possible target sites in both orientations in all sequences. Final identified common target sites are then displayed in visual and table formats. If exon sequences are available for a particular sequence (indicated by “?” and dashed lines), each candidate target site in both common and unique sets is checked to ensure that this site lies completely within an exon sequence. Running the target site specification expression on this consensus sequence results in the identification of candidate common target sites. The resulting multiple sequence alignment is read by the program and the consensus sequence is computed. In the second branch of the algorithm, multiple similar sequences are first aligned using the ClustalW2 program. The program can provide output for the sequence and location of identified target sites in visual and table formats. The simple sgRNA search is achieved by running a regular expression (search pattern) for the target site specification on all input sequences in both orientations. Target site specification is common to both branches of the algorithm and consists of a target site length, PAM sequence and its location as well as the sequence of the 5’-dinucleotide and the region where a single mismatch is allowed. The dashed lines to the sequence boxes represent two possible branches of the algorithm: simple CRISPR sgRNA search and a search for common and unique target sites in multiple similar sequences. Input data for this algorithm consist of a sgRNA target site specification and sequence data. Search algorithm for sgRNA target sites in individual and multiple similar sequences.