TABLE 4.
Principles of crRNA designing for type V-A/B/F1 CRISPR-Cas system.
| Rule no | Parameters | |
|---|---|---|
| Positional rules | ||
| 1 | Perfect base pairing with target regiona | |
| 1–1 | Allowing single and consecutive or nonconsecutive double mismatches into the spacer | |
| Type V-A | Central (seed) region or base pairs 1 to 6 LbCas12a target are intolerant to single and consecutive or nonconsecutive double mismatches | |
| Type V-B | Single and consecutive or nonconsecutive double mismatches at any positions of AapCas12b target are acceptable | |
| Type V-F1 | Central (seed) region or base pairs 9 to 16 Cas14a target are intolerant to single and consecutive or nonconsecutive double mismatches | |
| 2 | Protospacer adjacent motif (PAM; CRISPR motif) requirement | |
| PAM sequence of LbCas12a and AapCas12b for target DNA recognition is 5′-TTTV-3′ and 5′-TTN-3′, respectively, located upstream of the target sequence. PAM is not necessary for Cas14a | ||
| 3 | GC content | |
| Thermodynamics rules | ||
| 4 | Target site accessibility | |
| Not having any stable secondary structures in target region. This feature is important in type V-F1 that targets ssDNA. | ||
| 5 | Self-complementarity | |
| BLAST rules | ||
| 6 | Searching off-target effects in target organism | |
| 7 | Searching off-target effects in other organisms | |
a
G-U wobble base pairing is allowable.