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CRISPR 相关的 V 型蛋白作为控制 S. cerevisiae 合成基因回路中 mRNA 稳定性的工具。

CRISPR-associated type V proteins as a tool for controlling mRNA stability in S. cerevisiae synthetic gene circuits.

机构信息

School of Pharmaceutical Science and Technology, Tianjin University, 92 Weijin Road, 300072 Tianjin, China.

出版信息

Nucleic Acids Res. 2023 Feb 22;51(3):1473-1487. doi: 10.1093/nar/gkac1270.

DOI:10.1093/nar/gkac1270
PMID:36651298
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC9943656/
Abstract

Type V-A CRISPR-(d)Cas system has been used in multiplex genome editing and transcription regulation in both eukaryotes and prokaryotes. However, mRNA degradation through the endonuclease activity of Cas12a has never been studied. In this work, we present an efficient and powerful tool to induce mRNA degradation in the yeast Saccharomyces cerevisiae via the catalytic activity of (d)Cas12a on pre-crRNA structure. Our results point out that dFnCas12a, (d)LbCas12a, denAsCas12a and two variants (which carry either NLSs or NESs) perform significant mRNA degradation upon insertion of pre-crRNA fragments into the 5'- or 3' UTR of the target mRNA. The tool worked well with two more Cas12 proteins-(d)MbCas12a and Casϕ2-whereas failed by using type VI LwaCas13a, which further highlights the great potential of type V-A Cas proteins in yeast. We applied our tool to the construction of Boolean NOT, NAND, and IMPLY gates, whose logic operations are fully based on the control of the degradation of the mRNA encoding for a reporter protein. Compared to other methods for the regulation of mRNA stability in yeast synthetic gene circuits (such as RNAi and riboswitches/ribozymes), our system is far easier to engineer and ensure very high performance.

摘要

V-A 型 CRISPR-(d)Cas 系统已被用于真核生物和原核生物中的多重基因组编辑和转录调控。然而，Cas12a 的内切酶活性导致的 mRNA 降解从未被研究过。在这项工作中，我们通过 Cas12a 在 pre-crRNA 结构上的催化活性，在酵母酿酒酵母中提供了一种有效且强大的诱导 mRNA 降解的工具。我们的结果表明，dFnCas12a、(d)LbCas12a、denAsCas12a 和两种变体（携带 NLS 或 NES）在将 pre-crRNA 片段插入靶 mRNA 的 5'或 3'UTR 时，能显著降解 mRNA。该工具在使用更多两种 Cas12 蛋白-(d)MbCas12a 和 Casϕ2 时效果良好，而使用 VI 型 LwaCas13a 时则失败，这进一步突出了 V-A 型 Cas 蛋白在酵母中的巨大潜力。我们将我们的工具应用于布尔非、与非和蕴涵门的构建，其逻辑操作完全基于对报告蛋白编码 mRNA 降解的控制。与酵母合成基因电路中用于调节 mRNA 稳定性的其他方法（如 RNAi 和核糖开关/核酶）相比，我们的系统更容易设计，并能确保非常高的性能。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec7f/9943656/70f6b15dd0bc/gkac1270fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec7f/9943656/6fd75a7acff2/gkac1270fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec7f/9943656/e9826ac66883/gkac1270fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec7f/9943656/0acde94a45a2/gkac1270fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec7f/9943656/df860e200e50/gkac1270fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec7f/9943656/5c79612381cb/gkac1270fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec7f/9943656/70f6b15dd0bc/gkac1270fig6.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec7f/9943656/6fd75a7acff2/gkac1270fig1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec7f/9943656/e9826ac66883/gkac1270fig2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec7f/9943656/0acde94a45a2/gkac1270fig3.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec7f/9943656/df860e200e50/gkac1270fig4.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec7f/9943656/5c79612381cb/gkac1270fig5.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/ec7f/9943656/70f6b15dd0bc/gkac1270fig6.jpg

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