In vitro assembly and activity of an archaeal CRISPR-Cas type I-A Cascade interference complex

André Plagens,Henning Urlaub,Kundan Sharma,Elena Conti,Michael Daume,Vanessa Tripp,Ajla Hrle,Andreas Klingl,Lennart Randau

doi:10.1093/nar/gku120

Abstract

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas) systems of type I use a Cas ribonucleoprotein complex for antiviral defense (Cascade) to mediate the targeting and degradation of foreign DNA. To address molecular features of the archaeal type I-A Cascade interference mechanism, we established the in vitro assembly of the Thermoproteus tenax Cascade from six recombinant Cas proteins, synthetic CRISPR RNAs (crRNAs) and target DNA fragments. RNA-Seq analyses revealed the processing pattern of crRNAs from seven T. tenax CRISPR arrays. Synthetic crRNA transcripts were matured by hammerhead ribozyme cleavage. The assembly of type I-A Cascade indicates that Cas3′ and Cas3′′ are an integral part of the complex, and the interference activity was shown to be dependent on the crRNA and the matching target DNA. The reconstituted Cascade was used to identify sequence motifs that are required for efficient DNA degradation and to investigate the role of the subunits Cas7 and Cas3′′ in the interplay with other Cascade subunits.

Highlights

The coevolution of viruses with their prokaryotic hosts led to the development of specific and highly divergent antiviral prokaryotic immune systems
Seven Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) loci with a total of 142 spacer sequences were identified, and CRISPR RNAs (crRNAs) transcripts were verified for five clusters (TTX_1, 4, 5–7) via northern blot detection, whereas two clusters (TTX_2–3) showed no distinct processing pattern [41]
Type I CRISPR-encoded interference is mediated by a crRNA-guided Cas ribonucleoprotein complex for antiviral defense (Cascade) complexed with Cas3 to degrade the foreign target DNA

Summary

Introduction

The coevolution of viruses with their prokaryotic hosts led to the development of specific and highly divergent antiviral prokaryotic immune systems. One complex group of adaptive immune systems that is widespread in bacterial and archaeal genomes is termed Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-CRISPR-associated (Cas). Cells that harbor these systems can be immunized against the attack of viruses by the integration of a virus-derived genome fragment into the host genome [1]. The adaptation, the injected viral DNA is recognized and a fragment is inserted into the host CRISPR array [7,8,9] This activity is often dependent on a short conserved sequence (2–5 bp) defined as the protospacer adjacent motif (PAM) that flanks the original spacer sequence (termed protospacer) in the viral genome [10,11]. During a repeated viral attack, the mature crRNAs can be incorporated into a large Cas ribonucleoprotein interference complex to target the viral DNA for degradation [19,20,21]

Methods

Results

Conclusion