A more efficient CRISPR-Cas12a variant derived from Lachnospiraceae bacterium MA2020
| Autor: | Wenhui He, Guocai Zhong, Mai H. Tran, Li Pan, Huihui Mou, Michael Farzan, Ian Stiskin, Kelly R Sheptack, HaJeung Park, Gogce Crynen, Pabalu Karunadharma, Christopher L. Nobles, Tianling Ou, Haimin Wang |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
RM1-950 Computational biology Biology non-homologous end-joining 03 medical and health sciences 0302 clinical medicine Genome editing hemophilia Drug Discovery CRISPR genome editing Guide RNA Gene Trans-activating crRNA Cas12a Effector Cas9 protein engineering Protospacer adjacent motif Lb2Cas12a homology-directed repair 030104 developmental biology 030220 oncology & carcinogenesis Molecular Medicine Original Article Therapeutics. Pharmacology |
| Zdroj: | Molecular Therapy. Nucleic Acids Molecular Therapy: Nucleic Acids, Vol 24, Iss, Pp 40-53 (2021) |
| ISSN: | 2162-2531 |
| Popis: | 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. Graphical Abstract 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. |
| Databáze: | OpenAIRE |
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