Engineered CRISPR–Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing
| Autor: | Jonathan Y. Hsu, Irene Scarfò, Martin J. Aryee, Moira M. Welch, Benjamin P. Kleinstiver, Jose Malagon-Lopez, Russell T. Walton, Kendell Clement, Y. Esther Tak, Marcela V. Maus, Alexander A. Sousa, Joy E. Horng, J. Keith Joung, Luca Pinello |
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| Rok vydání: | 2019 |
| Předmět: |
T-Lymphocytes
Biomedical Engineering Bioengineering Computational biology Biology medicine.disease_cause Applied Microbiology and Biotechnology Article Epigenesis Genetic 03 medical and health sciences 0302 clinical medicine Bacterial Proteins Genome editing medicine Humans CRISPR Acidaminococcus Epigenetics Gene 030304 developmental biology Ribonucleoprotein Gene Editing 0303 health sciences Mutation HEK 293 cells Endonucleases Protospacer adjacent motif HEK293 Cells Ribonucleoproteins Molecular Medicine CRISPR-Cas Systems 030217 neurology & neurosurgery Biotechnology |
| Zdroj: | Nature biotechnology |
| ISSN: | 1546-1696 1087-0156 |
| DOI: | 10.1038/s41587-018-0011-0 |
| Popis: | Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases1 has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)2. To address this limitation, we engineered an enhanced Acidaminococcus sp. Cas12a variant (enAsCas12a) that has a substantially expanded targeting range, enabling targeting of many previously inaccessible PAMs. On average, enAsCas12a exhibits two-fold higher genome editing activity on sites with canonical TTTV PAMs compared to wild-type AsCas12a, and we successfully grafted a subset of mutations from enAsCas12a onto other previously described AsCas12a variants3 to enhance their activities. enAsCas12a improves the efficiency of multiplex gene editing, endogenous gene activation, and C-to-T base editing, and we engineered a high-fidelity version of enAsCas12a (enAsCas12a-HF1) to reduce off-target effects. Both enAsCas12a and enAsCas12a-HF1 function in HEK293T and primary human T cells when delivered as ribonucleoprotein (RNP) complexes. Collectively, enAsCas12a provides an optimized version of Cas12a that should enable wider application of Cas12a enzymes for gene and epigenetic editing. [AU: Revised abstract OK?] |
| Databáze: | OpenAIRE |
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