Table 1.
Guide RNA features that could influence on-target activity
| Categories | Efficient features | Inefficient features |
|---|---|---|
| Overall nucleotide usage | A count | U, G count |
| A in the middle | GG, GGG count | |
| AG, CA, AC, UA count | UU, GC count | |
| Position-specific nucleotides | G in position 20 | C in position 20 |
| A in position 20 | U in positions 17–20 | |
| G, A in position 19 | G in position 16 | |
| C in position 18 | T in PAM (TGG) | |
| C in position 16 | G in position +1 (NGGG) | |
| C in PAM (CGG) | ||
| Motifs | NGG PAM (esp. CGGH) | poly-N (esp. GGGG) |
| TT, GCC at the 3′ end | ||
| Structural | GC content 40–60% | GC > 80% or GC < 35% |
| High GC content in positions 4–8 | Stable self-folding | |
| High GC content in positions 15–20 | Stable DNA/RNA duplex | |
| Low Tm in the middle | ||
| Accessibility in positions 18–20 | ||
| Epigenetic | Target with open chromatin | Methylated, inaccessible target |
| Miscellaneous | Target near N-terminus of protein | Target introns, 5′/3′ UTRs |
| High Cas9-gRNA concentration | Low Cas9–gRNA concentration |
T m: melting temprature, UTRs: untranslated regions, H: adenine/cytosine/thymine. Adapted from (32).