Improved LbCas12a variants with altered PAM specificities further broaden the genome targeting range of Cas12a nucleases

Tóth, Eszter [Tóth, Eszter (molekuláris biológia), author]; Varga, Éva [Varga, Éva (Géntechnológia, b...), author] Institute of Biochemistry; Institute of Enzymology (RCNS); Doctoral School of Multidisciplinary Medicine (SZTE / DI); Kulcsár, Péter István [Kulcsár, Péter István (Molekuláris biológia), author] Institute of Biochemistry; Doctoral School of Multidisciplinary Medicine (SZTE / DI); Kocsis-Jutka, Virág; Krausz, Sarah Laura [Krausz, Sarah (géntechnológia), author] School of PhD Studies (SU); Nyeste, Antal [Nyeste, Antal (Biológus), author] Institute of Biochemistry; Welker, Zsombor; Huszár, Krisztina [Huszár, Krisztina (molekuláris biológia), author] Institute of Enzymology (RCNS); Ligeti, Zoltán [Ligeti, Zoltán Mihály (Biológia), author] Institute of Biochemistry; Doctoral School of Multidisciplinary Medicine (SZTE / DI); Tálas, András [Tálas, András (Molekuláris Biológus), author] Institute of Enzymology (RCNS); School of PhD Studies (SU); Welker, Ervin ✉ [Welker, Ervin (Fehérje feltekeredés), author] Institute of Biochemistry; Institute of Enzymology (RCNS)

English Article (Journal Article) Scientific
Published: NUCLEIC ACIDS RESEARCH 0305-1048 1362-4962 48 (7) pp. 3722-3733 2020
  • SJR Scopus - Genetics: D1
The widespread use of Cas12a (formerly Cpf1) nucleases for genome engineering is limited by their requirement for a rather long TTTV protospacer adjacent motif (PAM) sequence. Here we have aimed to loosen these PAM constraints and have generated new PAM mutant variants of the four Cas12a orthologs that are active in mammalian and plant cells, by combining the mutations of their corresponding RR and RVR variants with altered PAM specificities. LbCas12a-RVRR showing the highest activity was selected for an in-depth characterization of its PAM preferences in mammalian cells, using a plasmid-based assay. The consensus PAM sequence of LbCas12a-RVRR resembles a TNTN motif, but also includes TACV, TTCV CTCV and CCCV. The D156R mutation in improved LbCas12a (impLbCas12a) was found to further increase the activity of that variant in a PAM-dependent manner. Due to the overlapping but still different PAM preferences of impLbCas12a and the recently reported enAsCas12a variant, they complement each other to provide increased efficiency for genome editing and transcriptome modulating applications.
Citation styles: IEEEACMAPAChicagoHarvardCSLCopyPrint
2026-10-10 16:21