Journal
ACS SYNTHETIC BIOLOGY
Volume 6, Issue 7, Pages 1273-1282Publisher
AMER CHEMICAL SOC
DOI: 10.1021/acssynbio.6b00368
Keywords
CRISPRi; deactivated Cpf1; deactivated Cas9; Eubacterium eligens; protospacer adjacent motif Streptococcus pyogenes
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Funding
- Korea Institute of Energy Technology Evaluation and Planning (KETEP) - Ministry of Trade, Industry and Energy (MOTE) arid C1 Gas Refinery Program through the National Research Foundation of Korea (NRF) [20163030091540, NRF-2015M3D3A1A01064875]
- Ministry of Science, ICT & Future Planning (MSIP) of the Republic of Korea
- KRIBB Research Initiative Program
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Clustered regularly interspaced short palindromic repeats interference (CRISPRi) is an emerging technology for artificial gene regulation. Type II CRISPR-Cas endonuclease Cas9 is the most widely used protein for gene regulation with CRISPRi. Here, we present type V-A CRISPR-Cas endonuclease Cpf1-based CRISPRi. We constructed an L-rhamnose-inducible CRISPRi system with DNase-deactivated Cpf1 from Eubacterium eligens (EedCpf1) and compared its performance with catalytically deactivated Cas9 from Streptococcus pyogenes (SpdCas9). In contrast to SpdCas9, EedCpf1 showed stronger gene repression when it was targeted to the template strand than when it was targeted to the nontemplate strand of the 5' untranslated region or coding DNA sequences. EedCpf1 exhibited no strand bias when targeted to the promoter, and preferentially used the 5'-TTTV-3' (V = A, G, or C) protospacer adjacent motif. Multiplex repression of the EedCpf1-based CRISPRi system was demonstrated using episomal and chromosomal gene targets. Our findings will guide an efficient EedCpf1-mediated CRISPRi genetic control
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