基于内源III-B型CRISPR-Cas系统构建超嗜热火球菌Pyrococcus yayanosii A1的基因敲降系统

An endogenous type III-B CRISPR-Cas based gene knockdown system in Pyrococcus yayanosii A1

摘要:

[目的] 亚氏火球菌（Pyrococcus yayanosii） A1菌株的最适生长温度为98℃，最适生长压力为5.2×10⁴ Pa，是研究此类严格厌氧、超嗜热和兼性嗜压古菌的环境适应性机制的良好材料。本研究基于P.yayanosii A1基因组中Ⅲ-B型CRISPR-Cas系统，旨在建立可应用于此类古菌的基因表达敲降系统（gene knock-down system）。[方法] 人工构建的mini-CRISPR簇，由内源性CRISPR簇Group 1的重复序列（repeats）和待敲降的淀粉酶基因（PYCH_13690）中的原间隔序列（protospacer）组成。将该mini-CRISPR簇插入到具有辛伐他汀抗性的穿梭载体pLMOShhp中，使之转录与目的基因mRNA匹配的crRNA，引导Cmr复合物对其进行切割。[结果] 使用高静水压（HHP）诱导型启动子P_hhp诱导mini-CRISPR簇表达时，淀粉酶基因的mRNA数量在5.2×10⁴ Pa静水压时下调到原来的67.05%，在1.0×10² Pa静水压时下调为原来的49.69%；而使用组成型启动子P_hmtb诱导mini-CRISPR簇表达时，淀粉酶基因的mRNA数量在5.2×10⁴ Pa静水压时下调为原来的58.48%，在1.0×10² Pa静水压时下调为原来的23.97%。使用鲁戈氏碘液显色法对这两类基因表达敲降突变菌株的培养物中残留的淀粉含量进行分析，结果显示突变菌株的淀粉降解能力明显降低。[结论] 在P.yayanosii A1中建立了基于内源Ⅲ-B型CRISPR-Cas系统的基因敲降系统，可以用于抑制此类超嗜热嗜压古菌体内基因的表达。

Abstract:

[Objective] Pyrococcus yayanosii A1, with its optimum growth condition at 98℃ and 5.2×10⁴ Pa, was a laboratory mutant strain used in the studies of environmental adaptation mechanisms in a group of piezophilic hyperthermophilic archaea in Thermococcaceae. In this study, we established a new genetic tool for P. yayanosii A1, to knock down gene expression based on an endogenous active type Ⅲ-B CRISPR-Cas system.[Methods] The mini-CRISPR cluster was consisted the repeat sequences from endogenous CRISPR arrays Group I and a protospacer from a putative amylase gene (PYCH_13690). We inserted an artificial mini-CRISPR cluster into the shuttle vector pLMOShhp1, with the overexpression of the HMG-CoA (hydroxy methylglutaryl coenzyme A) reductase gene controlled by a constitutive promoter P_gdh, which conferred simvastatin resistance. And the mini-CRISPR cluster transformed crRNA, which guided the Cmr complex cut target mRNA. [Results] The ratio of mRNA cleavage of a high hydrostatic pressure inducible promoter P_hhp was 33% at 5.2×10⁴ Pa and 50% at 1.0×10² Pa, while the cleavage of constitutive promoter P_hmtb was 42% at 5.2×10⁴ Pa and 76% at 1.0×10² Pa. As a proof, the gene knockdown mutant degraded less starch than its parental strain A1 in vivo. [Conclusion] Thus, we successfully constructed a gene knockdown system based on type Ⅲ-B CRISPR-Cas system in P. yayanosii, which could be used in the genetic studies targeting essential genes in piezophilic hyperthermophiles.