title: Efficient Transcriptional Gene Repression by Type V-A CRISPR-Cpf1 from Eubacterium eligens.
abstract: 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.
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB) , Daejeon 34141, Republic of Korea.|Biosystems and Bioengineering Program, University of Science and Technology (UST) , Daejeon 34113, Republic of Korea.
authors.forename: Seong Keun
authors.initials: SK
authors.lastname: Kim
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB) , Daejeon 34141, Republic of Korea.|Biosystems and Bioengineering Program, University of Science and Technology (UST) , Daejeon 34113, Republic of Korea.
authors.forename: Haseong
authors.initials: H
authors.lastname: Kim
authors.affiliation: Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB) , Daejeon 34141, Republic of Korea.
authors.forename: Woo-Chan
authors.initials: WC
authors.lastname: Ahn
authors.affiliation: Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB) , Daejeon 34141, Republic of Korea.
authors.forename: Kwang-Hyun
authors.initials: KH
authors.lastname: Park
authors.affiliation: Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB) , Daejeon 34141, Republic of Korea.|Bio-Analytical Science Program, University of Science and Technology (UST) , Daejeon 34113, Republic of Korea.
authors.forename: Eui-Jeon
authors.initials: EJ
authors.lastname: Woo
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB) , Daejeon 34141, Republic of Korea.|Biosystems and Bioengineering Program, University of Science and Technology (UST) , Daejeon 34113, Republic of Korea.
authors.forename: Dae-Hee
authors.identifier: 0000-0002-0423-9057
authors.initials: DH
authors.lastname: Lee
authors.affiliation: Synthetic Biology and Bioengineering Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB) , Daejeon 34141, Republic of Korea.|Biosystems and Bioengineering Program, University of Science and Technology (UST) , Daejeon 34113, Republic of Korea.
authors.forename: Seung-Goo
authors.initials: SG
authors.lastname: Lee
mesh_terms: D001426:Bacterial Proteins / Q000235:genetics* / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000235:genetics / Q000502:physiology; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D005051:Eubacterium / Q000235:genetics* / Q000378:metabolism*; D013297:Streptococcus pyogenes / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial
keywords: CRISPRi; Eubacterium eligens; Streptococcus pyogenes; deactivated Cas9; deactivated Cpf1; protospacer adjacent motif