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

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

doi:10.1016/j.omtn.2021.02.012

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

Mol Ther Nucleic Acids. 2021 Feb 18:24:40-53. doi: 10.1016/j.omtn.2021.02.012. eCollection 2021 Jun 4.

Authors

Affiliations

¹ Department of Immunology and Microbiology, The Scripps Research Institute, Jupiter, FL 33458, USA.
² X-ray Crystallography Core, The Scripps Research Institute, Jupiter, FL 33458, USA.
³ Department of Microbiology, Perelman School of Medicine, University of Pennsylvania, Philadelphia, PA 19104, USA.
⁴ Genomics Core, The Scripps Research Institute, Jupiter, FL 33458, USA.
⁵ Bioinformatics and Statistics Core, The Scripps Research Institute, Jupiter, FL 33458, USA.

Abstract

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.

Keywords: CRISPR; Cas12a; Lb2Cas12a; genome editing; hemophilia; homology-directed repair; non-homologous end-joining; protein engineering.

Abstract

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