The Cas4-Cas1-Cas2 complex mediates precise prespacer processing during CRISPR adaptation.

Hayun Lee,Yukti Dhingra,Dipali G Sashital

doi:10.7554/elife.44248

Abstract

CRISPR adaptation immunizes bacteria and archaea against viruses. During adaptation, the Cas1-Cas2 complex integrates fragments of invader DNA as spacers in the CRISPR array. Recently, an additional protein Cas4 has been implicated in selection and processing of prespacer substrates for Cas1-Cas2, although this mechanism remains unclear. We show that Cas4 interacts directly with Cas1-Cas2 forming a Cas4-Cas1-Cas2 complex that captures and processes prespacers prior to integration. Structural analysis of the Cas4-Cas1-Cas2 complex reveals two copies of Cas4 that closely interact with the two integrase active sites of Cas1, suggesting a mechanism for substrate handoff following processing. We also find that the Cas4-Cas1-Cas2 complex processes single-stranded DNA provided in cis or in trans with a double-stranded DNA duplex. Cas4 cleaves precisely upstream of PAM sequences, ensuring the acquisition of functional spacers. Our results explain how Cas4 cleavage coordinates with Cas1-Cas2 integration and defines the exact cleavage sites and specificity of Cas4.

Highlights

Bacteria and archaea use an adaptive immune system composed of clustered regularly interspaced short palindromic repeats (CRISPR) arrays and CRISPR-associated (Cas) proteins to defend against infection (Barrangou et al, 2007; Brouns et al, 2008; Marraffini and Sontheimer, 2008)
The locus is transcribed and processed into short CRISPR RNAs, which assemble with Cas proteins to form a RNA-guided surveillance complex
We previously showed that B. halodurans type I-C Cas4 associates tightly with Cas1 but were unable to obtain the Cas4-Cas1-Cas2 complex due to instability of the Cas1-Cas2 complex in this system (Lee et al, 2018)

Summary

Introduction

Bacteria and archaea use an adaptive immune system composed of clustered regularly interspaced short palindromic repeats (CRISPR) arrays and CRISPR-associated (Cas) proteins to defend against infection (Barrangou et al, 2007; Brouns et al, 2008; Marraffini and Sontheimer, 2008). Some systems have two cas genes that work together to define the PAM, length and orientation of spacers, suggesting that the two Cas proteins are involved in processing each end of the prespacer and that they may be present during integration (Shiimori et al, 2018). The Cas4-Cas1-Cas complex processes single-strand DNA when an activator duplex DNA is provided in trans Using this ssDNA cleavage assay, we show that the Cas4-Cas1Cas complex is highly specific for PAM sequences and cleaves precisely upstream of the PAM. The PAM must be positioned within a single-strand region for optimal cleavage, but Cas can cleave at various locations within this single-stranded overhang These findings provide the first structural information of the Cas4-Cas1-Cas adaptation complex and reveal the precision and specificity of prespacer processing prior to integration

Results

Discussion

Materials and methods

PAM sites interspersed with 4-nt

Funding Funder