Figure 1.
Workflow diagram of sgRNAcas9 software.
Finding CRISPR target sites and off-target risk assessment includes 4 basic steps. It is noteworthy that to evaluate off-target effects exaclty, different types of potential off-target cleavage sites (POT) are classified.
Figure 2.
Determine the sequence of the base-pairing region on the sgRNA by the use of different searching mode.
(A) Sense strand searching mode (the direction of sequence is 5′ to 3′). (B) Anti-sense strand searching mode (the direction of sequence is 3′ to 5′). (C) Paired-gRNA searching mode. Notes: different mode of sgRNA binding to target DNA strand are shown. A and B are single strand searching mode, there is also an option for using both strand searching mode (not shown).
Figure 3.
Scheme of classifying off-target cleavage sites with the sgRNAcas9 program.
(A) Different segments of the target sequence. (B) The potential off-target cleavage sites (POT) were classified into three categories by count number and position of mismatches. (C) Distinction of true and false off-targets. (D) Evaluating the degree of risk of the POT. The dangerous degree of POT from low to high is dependent on the number and position of mismatches.
Figure 4.
Workflow and filter criteria for selecting candidate CRISPR target sites (protospacers) with high specificity using sgRNAcas9.
Table 1.
Running time for finding CRISPR target sites and searching off-targets.
Figure 5.
Example of CRISPR on-target sequences found by sgRNAcas9.
(A) The paired-gRNA target sites of Emx1 gene found by sgRNAcas9 program and the paired-gRNA searching mode. (B) Illustration of one pair sgRNA targeting at exon1 of Emx1.
Figure 6.
Example of the classification of potential off-target sequences by sgRNAcas9.
(A) Searching off-target sites for each sgRNA targeting at human Emx1 (hEMX1) gene. (B) Classifying POT by number and position of mismatches into three types. Notes: “seed_ident”, strand for seed region, was to identity to on-target sites (Type I). “region I_ident”, strand for region I identical to on-target site (Type II). “random_0_3M”, strand for regions with 1∼3 mismatched bases randomly distributed on the region I, II and III, but with at least one mismatched base located on the region I (Type III).