Nucleases
Programmable nucleases are enzymes engineered to cut DNA at specific sequences, enabling targeted genome modification. Selection depends on the length of DNA recognition sequence, cutting mechanism (blunt vs. staggered ends), size constraints for delivery vehicles, and compatibility with different guide RNA architectures.
| Name | Size (kb) | PAM | Origin |
|---|---|---|---|
| Al3Cas12f | — | TTTV |
Alistipes sp. |
| Al3Cas12f RKK | — | TTTV |
Alistipes sp. (engineered) |
| AsCas12a | — | TTTV |
Acidaminococcus sp. BV3L6 type V CRISPR-Cas system |
| AsCas12f | — | 5'-NTTR (R = A or G) |
Acidibacillus sulfuroxidans |
| Cas12a | — | TTTV |
Lachnospiraceae bacterium ND2006 type V CRISPR-Cas system |
| Cas12f | — | TTTR (T-rich) |
Uncultivated archaea and bacteria; class 2 type V-F CRISPR-Cas systems |
| Cas13a RNA-Targeting | — | H (3' protospacer flanking site, non-G nucleotide — not a DNA PAM) |
Leptotrichia shahii type VI-A CRISPR-Cas system |
| Cas9 D10A Nickase | — | NGG |
Engineered from Streptococcus pyogenes Cas9 (D10A) |
| CasMINI | — | TTTR |
Engineered from Un1Cas12f1 by protein mutagenesis and gRNA scaffold redesign |
| dCas9 Catalytically Dead | — | NGG |
Engineered from Streptococcus pyogenes Cas9 (D10A/H840A) |
| enAsCas12a | — | TTYN, VTTV, TRTV (also recognizes canonical TTTV) |
Engineered from Acidaminococcus sp. AsCas12a (E174R/S542R/K548R) |
| eSpCas9 | — | NGG |
Engineered from Streptococcus pyogenes Cas9 by alanine substitution |
| eSpCas9(1.1) | — | NGG |
Engineered from Streptococcus pyogenes Cas9 (K848A/K1003A/R1060A) |
| hfCas12Max | — | TTN |
Engineered from xCas12i via intermediate Cas12Max (N243R) |
| impLbCas12a | — | TNTN, broadened further to NTTV |
Engineered from LbCas12a-RVRR (+ D156R) |
| LbCas12a-RVRR | — | TNTN |
Engineered from LbCas12a by transferring AsCas12a-RVR/RR PAM substitutions |
| NmeCas9 | — | NNNNGATT |
Neisseria meningitidis type II-C CRISPR-Cas system |
| OpenCRISPR-1 | — | NGG |
Synthetic: designed by large language models trained on natural CRISPR operons |
| OsCas12f | — | YTTH |
Oscillibacter sp. type V-F CRISPR-Cas system |
| RhCas12f | — | NCCD |
Ruminiclostridium herbifermentans, via bioinformatic ortholog screening |
| SaCas9 | — | NNGRRT |
Staphylococcus aureus type II CRISPR-Cas system |
| SaCas9-KKH | — | NNNRRT |
Engineered from Staphylococcus aureus Cas9 (E782K/N968K/R1015H) |
| SpCas9 | — | NGG |
Streptococcus pyogenes type II CRISPR-Cas system |
| SpCas9-HF1 | — | NGG |
Engineered from Streptococcus pyogenes Cas9 (N497A/R661A/Q695A/Q926A) |
| St1Cas9 | — | NNAGAAW |
Streptococcus thermophilus CRISPR1 locus (strain LMD-9) |
| xCas9 | — | NG (NGG, NGA, NGT, NGC), GAA, GAT, CAA |
Phage-assisted continuous evolution (PACE) from Streptococcus pyogenes Cas9 |