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Synth Syst Biotechnol. 2018 Nov 19;4(1):1-9. doi: 10.1016/j.synbio.2018.11.002. eCollection 2019 Mar.

Systematically investigating the key features of the DNase deactivated Cpf1 for tunable transcription regulation in prokaryotic cells.

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CAS Key Laboratory of Microbial Physiological and Metabolic Engineering, State Key Laboratory of Microbial Resources, Institute of Microbiology Chinese Academy of Sciences, Beijing, 100101, China.
College of Life Science, University of Science and Technology of China, Hefei, 230027, China.
Center for Quantitative Biology, Peking University, Beijing, 100871, China.
University of Chinese Academy of Science, Beijing, 100149, China.


With a unique crRNA processing capability, the CRISPR associated Cpf1 protein holds great potential for multiplex gene regulation. Unlike the well-studied Cas9 protein, however, conversion of Cpf1 to a transcription regulator and its related properties have not been systematically explored yet. In this study, we investigated the mutation schemes and crRNA requirements for the DNase deactivated Cpf1 (dCpf1). By shortening the direct repeat sequence, we obtained genetically stable crRNA co-transcripts and improved gene repression with multiplex targeting. A screen of diversity-enriched PAM library was designed to investigate the PAM-dependency of gene regulation by dCpf1 from Francisella novicida and Lachnospiraceae bacterium. We found novel PAM patterns that elicited strong or medium gene repressions. Using a computational algorithm, we predicted regulatory outputs for all possible PAM sequences, which spanned a large dynamic range that could be leveraged for regulatory purposes. These newly identified features will facilitate the efficient design of CRISPR-dCpf1 based systems for tunable multiplex gene regulation.

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