Department of Cell Biology, Institute of Life Science, Kurume University, Asahi-machi 67, Kurume, Fukuoka 830-0011, Japan.
G3 (Bethesda). 2021 Apr 15;11(4). doi: 10.1093/g3journal/jkab051.
Controllable and reversible transcriptional repression is an essential method to study gene functions. A systematic knock-down method using catalytically inactive Cas9 (dCas9) was originally established in bacteria. dCas9 forms a ribonucleoprotein with a small guide RNA and uses it to recognize a specific DNA sequence via Watson-Crick base-pairing. When specifically bound to a targeted DNA, dCas9 impairs RNA polymerase activity and represses transcription of that target gene. This technology, CRISPRi, has been implemented in several organisms, but not in Schizosaccharomyces pombe using dCas9. Here, we provide a plasmid that expresses dCas9 and sgRNA in fission yeast. With this plasmid, CRISPRi repressed endogenous gene transcription by as much as 87%. This transcriptional repression method is controllable, reversible, and efficient enough to alter cellular phenotypes. Here, we offer a CRISPRi method to choose proper targeting sequences for transcriptional repression in fission yeast. Implementation of CRISPRi will help to reveal gene functions and to develop tools based on dCas9 technology in S. pombe.
转录抑制的可控性和可逆性是研究基因功能的重要方法。最初在细菌中建立了一种使用无催化活性 Cas9（dCas9）的系统敲低方法。dCas9 与小向导 RNA 形成核糖核蛋白，并通过 Watson-Crick 碱基配对识别特定的 DNA 序列。当特异性结合到靶 DNA 时，dCas9 会损害 RNA 聚合酶活性并抑制靶基因的转录。这项名为 CRISPRi 的技术已在几种生物中得到实施，但在裂殖酵母中尚未使用 dCas9 实施。在这里，我们提供了一种在裂殖酵母中表达 dCas9 和 sgRNA 的质粒。利用该质粒，CRISPRi 可抑制内源基因转录高达 87%。这种转录抑制方法具有可控性、可逆性和足够的高效性，可以改变细胞表型。在这里，我们提供了一种在裂殖酵母中进行转录抑制的 CRISPRi 方法，以选择合适的靶向序列。CRISPRi 的实施将有助于揭示基因功能，并为基于 dCas9 技术的工具在 S. pombe 中的开发提供帮助。
