Kim Seong Keun, Kim Haseong, Ahn Woo-Chan, Park Kwang-Hyun, Woo Eui-Jeon, Lee Dae-Hee, Lee Seung-Goo
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.
ACS Synth Biol. 2017 Jul 21;6(7):1273-1282. doi: 10.1021/acssynbio.6b00368. Epub 2017 Apr 11.
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.
成簇规律间隔短回文重复序列干扰（CRISPRi）是一种新兴的人工基因调控技术。II型CRISPR-Cas核酸内切酶Cas9是CRISPRi基因调控中使用最广泛的蛋白质。在此，我们展示了基于V-A型CRISPR-Cas核酸内切酶Cpf1的CRISPRi。我们构建了一种由来自优杆菌（Eubacterium eligens）的失活DNA酶的Cpf1（EedCpf1）组成的鼠李糖诱导型CRISPRi系统，并将其性能与化脓性链球菌（Streptococcus pyogenes）的催化失活Cas9（SpdCas9）进行了比较。与SpdCas9不同，当EedCpf1靶向模板链时，其基因抑制作用比靶向5'非翻译区或编码DNA序列的非模板链时更强。当靶向启动子时，EedCpf1没有链偏好性，并且优先使用5'-TTTV-3'（V = A、G或C）原间隔相邻基序。使用附加型和染色体基因靶点证明了基于EedCpf1的CRISPRi系统的多重抑制作用。我们的研究结果将指导高效的EedCpf1介导的CRISPRi基因控制。
