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

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.

Automated summary

CRISPR effector proteins, such as Cas12a/Cpf1, can introduce double-stranded breaks into the mammalian genome to facilitate gene editing. In this study, researchers observed that a novel Cas12a ortholog, Lb2Cas12a, edited mammalian genes with efficiency comparable to well-characterized Cas12a orthologs. By introducing mutations into Lb2Cas12a, the researchers created a variant, Lb2-KY, that exhibited enhanced cleavage efficiency for a range of target sequences beyond those targeted by commonly used Cas12a orthologs. This Lb2-KY variant demonstrated efficient editing of hemoglobin target regions useful for correcting sickle-cell anemia, indicating its potential for modifying clinically relevant targets in the human genome.