A more efficient CRISPR-Cas12a variant derived from Lachnospiraceae bacterium MA2020

• Published in: Molecular therapy. Nucleic acids Vol. 24; pp. 40 - 53
• Main Authors: Tran, Mai H., Park, Hajeung, Nobles, Christopher L., Karunadharma, Pabalu, Pan, Li, Zhong, Guocai, Wang, Haimin, He, Wenhui, Ou, Tianling, Crynen, Gogce, Sheptack, Kelly, Stiskin, Ian, Mou, Huihui, Farzan, Michael
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 04.06.2021; American Society of Gene & Cell Therapy; Elsevier
• Subjects: Cas12a; CRISPR; genome editing; hemophilia; homology-directed repair; Lb2Cas12a; non-homologous end-joining; Original; protein engineering; CRISPR; genome editing; hemophilia; protein engineering; non-homologous end-joining; Cas12a; Lb2Cas12a; homology-directed repair
• ISSN: 2162-2531; 2162-2531
• DOI: 10.1016/j.omtn.2021.02.012

CRISPR effector proteins introduce double-stranded breaks into the mammalian genome, facilitating gene editing by non-homologous end-joining or homology-directed repair. Unlike the more commonly studied Cas9, the CRISPR effector protein Cas12a/Cpf1 recognizes a T-rich protospacer adjacent motif (PAM) and can process its own CRISPR RNA (crRNA) array, simplifying the use of multiple guide RNAs. We observed that the Cas12a ortholog of Lachnospiraceae bacterium MA2020 (Lb2Cas12a) edited mammalian genes with efficiencies comparable to those of AsCas12a and LbCas12a. Compared to these well-characterized Cas12a orthologs, Lb2Cas12a is smaller and recognizes a narrow set of PAM TTTV. We introduced two mutations into Lb2Cas12a, Q571K and C1003Y, that increased its cleavage efficiency for a range of target sequences beyond those of the commonly used Cas12a orthologs AsCas12a and LbCas12a. In addition to the canonical TTTV PAM, this variant, Lb2-KY, also efficiently cleaved target regions with CTTN PAMs. Finally, we demonstrated that Lb2-KY ribonucleoprotein (RNP) complexes edited two hemoglobin target regions useful for correcting common forms of sickle-cell anemia more efficiently than commercial AsCas12a RNP complexes. Thus, Lb2-KY has distinctive properties useful for modifying a range of clinically relevant targets in the human genome. [Display omitted] CRISPR-Cas12a effector proteins edit mammalian genomes using a T-rich PAM with few off-targets. Tran et al. engineered a Cas12a nuclease from Lachnospiraceae bacterium MA2020. They demonstrated that this variant recognized a broadened PAM and edited mammalian genomes with higher efficiencies than commonly used orthologs, expanding Cas12a utility in genome engineering.