Efficient CRISPR-Mediated Post-transcriptional Gene Silencing in a Hyperthermophilic Archaeon Using Multiplexed crRNA Expression

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) -mediated RNA degradation is catalyzed by a type III system in the hyperthermophilic archaeon Sulfolobus solfataricus. Earlier work demonstrated that the system can be engineered to target specifically mRNA of an endogenous host reporter gene, namely the β-galactosidase in S. solfataricus. Here we investigated the effect of single and multiple spacers targeting the mRNA of a second reporter gene, α-amylase, at the same and at different locations respectively, by using a minimal CRISPR (miniCR) locus supplied on a viral shuttle vector. The use of increasing numbers of spacers reduced mRNA levels at progressively higher levels, with three crRNAs (CRISPR RNAs) leading to approximately 70 to 80% reduction and five spacers resulting in an α-amylase gene knockdown of > 90% measured on both mRNA and protein activity levels. Our results indicate that this technology can be used to increase or modulate gene knockdown for efficient post-transcriptional gene silencing in hyperthermophilic archaea and potentially also in other organisms.

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