PAM-altered Lachnospiraceae bacterium Cas12a variant combining the RVR and RR substitution sets (LbCas12a-RVRR)

Characteristics

Combines four substitutions - G532R, K538V, Y542R and K595R - the same mutation set found at the analogous positions of AsCas12a-RVR and AsCas12a-RR; this combination abolishes nuclease activity entirely in AsCas12a and FnCas12a, but LbCas12a-RVRR retains considerable activity. Active on 15 of the 16 possible TNTN PAM sequences, the sole exception being TCTT, with highest activity on TTTA, and additionally active on TACC, TTCC and CTCC PAMs. Like wild-type LbCas12a, cleavage proceeds through a single RuvC-like domain, producing a staggered double-strand break with a 5' overhang. At endogenous targets, indel formation matches LbCas12a-WT on TTTV PAMs and significantly exceeds it on TTTT, TGTV and TGTT PAMs.

Applications: Genome editing at TNTN-PAM sites inaccessible to wild-type LbCas12a, validated by indel formation at endogenous human targets in HEK293 cells. Substituted into a catalytically dead cytidine base editor (dLbCas12a-RVRR-BE) in place of dLbCas12a-WT, preserving C-to-T editing efficiency at TTTV-PAM targets while extending editing to TNTN-PAM targets.

Limitations: Retains only moderate activity on TCTV PAMs and negligible activity on TCTT. GUIDE-seq on seven endogenous TTTV-PAM targets finds more off-target cleavage sites than LbCas12a-WT at six of the seven, with off-targets diverging from canonical TTTV at all three thymidine positions. impLbCas12a (adds D156R) reaches roughly 1.3-fold higher activity and broader NTTV access than this variant alone.

References

1. Tóth et al. (2020). Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases. Nucleic Acids Res - Tóth 2020 LbCas12a
2. Zetsche et al. (2015). Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. Cell - Zetsche 2015 Cas12a