DSM Biotechnology Center, Delft 2613 AX, The Netherlands.
Department of Chemistry, University of Hamburg, Hamburg 20146, Germany.
Nucleic Acids Res. 2021 Jul 21;49(13):7775-7790. doi: 10.1093/nar/gkab529.
CRISPR Cas12a is an RNA-programmable endonuclease particularly suitable for gene regulation. This is due to its preference for T-rich PAMs that allows it to more easily target AT-rich promoter sequences, and built-in RNase activity which can process a single CRISPR RNA array encoding multiple spacers into individual guide RNAs (gRNAs), thereby simplifying multiplexed gene regulation. Here, we develop a flexible dCas12a-based CRISPRi system for Saccharomyces cerevisiae and systematically evaluate its design features. This includes the role of the NLS position, use of repression domains, and the position of the gRNA target. Our optimal system is comprised of dCas12a E925A with a single C-terminal NLS and a Mxi1 or a MIG1 repression domain, which enables up to 97% downregulation of a reporter gene. We also extend this system to allow for inducible regulation via an RNAP II-controlled promoter, demonstrate position-dependent effects in crRNA arrays, and use multiplexed regulation to stringently control a heterologous β-carotene pathway. Together these findings offer valuable insights into the design constraints of dCas12a-based CRISPRi and enable new avenues for flexible and efficient gene regulation in S. cerevisiae.
CRISPR Cas12a 是一种 RNA 可编程内切酶，特别适合基因调控。这是因为它对富含 T 的 PAMs 的偏好使其更容易靶向富含 AT 的启动子序列，并且其内置的 RNase 活性可以将单个 CRISPR RNA 阵列中编码多个间隔区的序列处理成单个向导 RNA（gRNA），从而简化了多重基因调控。在这里，我们为酿酒酵母开发了一种灵活的基于 dCas12a 的 CRISPRi 系统，并系统地评估了其设计特点。这包括NLS 位置的作用、抑制结构域的使用以及 gRNA 靶位的位置。我们的最佳系统由具有单个 C 末端 NLS 的 dCas12a E925A 和 Mxi1 或 MIG1 抑制结构域组成，可实现高达 97%的报告基因下调。我们还将此系统扩展到通过 RNA 聚合酶 II 控制的启动子进行诱导性调节，证明了 crRNA 阵列中的位置依赖性效应，并使用多重调节严格控制异源β-胡萝卜素途径。这些发现为基于 dCas12a 的 CRISPRi 的设计约束提供了有价值的见解，并为酿酒酵母中灵活高效的基因调控开辟了新途径。
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