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

• Main Authors: Mai H. Tran, Hajeung Park, Christopher L. Nobles, Pabalu Karunadharma, Li Pan, Guocai Zhong, Haimin Wang, Wenhui He, Tianling Ou, Gogce Crynen, Kelly Sheptack, Ian Stiskin, Huihui Mou, Michael Farzan
• Format: Article
• Language: English
• Published: Elsevier 2021-06-01
• Series: Molecular Therapy: Nucleic Acids
• Subjects: CRISPR; Cas12a; genome editing; protein engineering; Lb2Cas12a; hemophilia
• Online Access: http://www.sciencedirect.com/science/article/pii/S2162253121000470

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.