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Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.

ABSTRACT: 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 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 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.

SUBMITTER: Kleinstiver BP

PROVIDER: S-EPMC6401248 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature

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Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.

Kleinstiver Benjamin P BP Sousa Alexander A AA Walton Russell T RT Tak Y Esther YE Hsu Jonathan Y JY Clement Kendell K Welch Moira M MM Horng Joy E JE Malagon-Lopez Jose J Scarfò Irene I Maus Marcela V MV Pinello Luca L Aryee Martin J MJ Joung J Keith JK

Nature biotechnology 20190211 3

Broad use of CRISPR-Cas12a (formerly Cpf1) nucleases<sup>1</sup> has been hindered by the requirement for an extended TTTV protospacer adjacent motif (PAM)<sup>2</sup>. 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 wi ...[more]

PMID: 30742127

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Project description:BackgroundCRISPR-Cas12a (formerly Cpf1) is an RNA-guided endonuclease with distinct features that have expanded genome editing capabilities. Cas12a-mediated genome editing is temperature sensitive in plants, but a lack of a comprehensive understanding on Cas12a temperature sensitivity in plant cells has hampered effective application of Cas12a nucleases in plant genome editing.ResultsWe compared AsCas12a, FnCas12a, and LbCas12a for their editing efficiencies and non-homologous end joining (NHEJ) repair profiles at four different temperatures in rice. We found that AsCas12a is more sensitive to temperature and that it requires a temperature of over 28 °C for high activity. Each Cas12a nuclease exhibited distinct indel mutation profiles which were not affected by temperatures. For the first time, we successfully applied AsCas12a for generating rice mutants with high frequencies up to 93% among T0 lines. We next pursued editing in the dicot model plant Arabidopsis, for which Cas12a-based genome editing has not been previously demonstrated. While LbCas12a barely showed any editing activity at 22 °C, its editing activity was rescued by growing the transgenic plants at 29 °C. With an early high-temperature treatment regime, we successfully achieved germline editing at the two target genes, GL2 and TT4, in Arabidopsis transgenic lines. We then used high-temperature treatment to improve Cas12a-mediated genome editing in maize. By growing LbCas12a T0 maize lines at 28 °C, we obtained Cas12a-edited mutants at frequencies up to 100% in the T1 generation. Finally, we demonstrated DNA binding of Cas12a was not abolished at lower temperatures by using a dCas12a-SRDX-based transcriptional repression system in Arabidopsis.ConclusionOur study demonstrates the use of high-temperature regimes to achieve high editing efficiencies with Cas12a systems in rice, Arabidopsis, and maize and sheds light on the mechanism of temperature sensitivity for Cas12a in plants.

Dataset Information

Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.

Publications

Engineered CRISPR-Cas12a variants with increased activities and improved targeting ranges for gene, epigenetic and base editing.

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