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Kleinstiver, Benjamin P; Sousa, Alexander A; Walton, Russell T; Tak, Y Esther; Hsu, Jonathan Y; Clement, Kendell; Welch, Moira M; Horng, Joy E; Malagon-Lopez, Jose; Scarfò, Irene; Maus, Marcela V; Pinello, Luca; Aryee, Martin J; Joung, J Keith
Nature biotechnology, 03/2019, Letnik: 37, Številka: 3Journal Article
Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM) . 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 a twofold 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 variants 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.
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JCR | SNIP | JCR | SNIP | JCR | SNIP | JCR | SNIP |
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Vir: Osebne bibliografije
in: SICRIS
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