Improved Lachnospiraceae bacterium Cas12a (impLbCas12a)

Characteristics

Carries five substitutions on the LbCas12a scaffold — G532R, K538V, Y542R and K595R (the RVRR set) plus D156R — that together produce a PAM consensus resembling TNTN (active on 15 of 16 TNTN combinations, the exception being TCTT) and extend further to NTTV sequences that LbCas12a-RVRR alone does not efficiently target. D156R increases average editing activity approximately 1.3-fold over the LbCas12a-RVRR parent in HEK293 reporter and endogenous-target disruption assays, with the largest single-PAM gains on TACV (1.9-fold) and TATV (1.7-fold). impLbCas12a does not exceed enAsCas12a's activity on NTTV-PAM targets, but exceeds enAsCas12a on RR- and RVR-related PAM sequences.

Applications: Indel formation at TTTV and PAM-divergent targets (TNTN, NTTV) in HEK293 cells that LbCas12a-WT cannot access. Used together with enAsCas12a to cover a combined target space broader than either nuclease alone, since their PAM preferences overlap only partially. A catalytically dead impLbCas12a fused to ten tandem SunTag repeats activates transcription at the SPRN gene promoter from canonical or PAM-divergent target sites, reaching activation comparable to a wild-type-based SunTag system.

Limitations: Carries the LbCas12a-RVRR mutation set, which cleaves more off-target sites than LbCas12a-WT at six of seven GUIDE-seq-profiled TTTV-PAM targets; D156R's effect on off-targets was not separately tested. Activity on the TCTT PAM is negligible, and impLbCas12a does not exceed enAsCas12a on NTTV-PAM targets, so the two are typically paired rather than substituted for one another.

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