Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements. Since its discovery it has been developed into numerous applications like genome editing and regulation of transcription in eukaryotes and bacteria. For archaea currently no tools for transcriptional repression exist. Because molecular biology analyses in archaea become more and more widespread such a tool is vital for investigating the biological function of essential genes in archaea. Here we use the model archaeon Haloferax volcanii to demonstrate that its endogenous CRISPR-Cas system I-B can be harnessed to repress gene expression in archaea. Deletion of cas3 and cas6b genes results in efficient repression of transcription. crRNAs targeting the promoter region reduced transcript levels down to 8%. crRNAs targeting the reading frame have only slight impact on transcription. crRNAs that target the coding strand repress expression only down to 88%, whereas crRNAs targeting the template strand repress expression down to 8%. Repression of an essential gene results in reduction of transcription levels down to 22%. Targeting efficiencies can be enhanced by expressing a catalytically inactive Cas3 mutant. Genes can be targeted on plasmids or on the chromosome, they can be monocistronic or part of a polycistronic operon.
Highlights
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system is used by bacteria and archaea to fend off foreign genetic elements
CRISPR-Cas2 (Clustered Regularly Interspaced Short Palindromic Repeats)-CRISPR associated) is a defense mechanism found in bacteria and archaea to fend off foreign genetic elements
Many different versions of the CRISPR-Cas system have been discovered and they have been grouped into two classes and six major types that are subdivided into several subtypes (I-A-F, I-U, IIA-C, III-A-D, and V-A-B) [6, 7]
Summary
It consists of eight Cas proteins (Cas, Cas6b, Cas, and Cas8b) and three CRISPR loci that are constitutively expressed [30]. Short motif in the invader sequence: the protospacer adjacent motif (PAM), it is located directly adjacent to the sequence targeted in the DNA The fact that the Haloferax type I-B system recognizes six PAM sequences makes the selection of target sequences in the gene to be down-regulated much easier. For Haloferax a Cas6b independent way of crRNA production was developed making crRNA production easier and allowing interference reactions in a ⌬cas6b strain [34]. Deletion of the genes for the Cas and Cas6b proteins and expression of specific crRNAs result in repression of the reporter gene. Repression efficiencies can be enhanced by addition of a Cas mutant
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