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

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.