Efficient Transcriptional Gene Repression by Type V-A CRISPR-Cpf1 from Eubacterium eligens
Efficient Transcriptional Gene Repression by Type V-A CRISPR-Cpf1 from Eubacterium eligens
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DOI：
10.1021/acssynbio.6b00368
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发表时间：
2017-07-01
影响因子：
4.7
通讯作者：
Lee, Seung-Goo
中科院分区：
文献类型：
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作者：
Kim, Seong Keun;Kim, Haseong;Lee, Seung-Goo
聚类规则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是应用最广泛的CRISPRi基因调控蛋白。在这里，我们提出了基于V-A型CRISPR-Cas内切酶cpf1的CRISPRi。我们构建了一个l -鼠李糖诱导的CRISPRi系统，该系统使用了真菌菌dna失活的Cpf1 (EedCpf1)，并将其与化脓性链球菌催化失活的Cas9 （SpdCas9）的性能进行了比较。与SpdCas9相比，EedCpf1靶向模板链时比靶向5'非翻译区或编码DNA序列的非模板链时表现出更强的基因抑制作用。EedCpf1在靶向启动子时没有表现出链偏置，并优先使用5'-TTTV-3' （V = A， G或C）原间隔子邻近基序。利用episomal和染色体基因靶点证明了基于eedcpf1的CRISPRi系统的多重抑制。我们的发现将指导有效的eedcpf1介导的CRISPRi遗传控制
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