A Complex of Cas Proteins 5, 6, and 7 Is Required for the Biogenesis and Stability of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-derived RNAs (crRNAs) in Haloferax volcanii

Jutta Brendel,Britta Stoll,Sita J Lange,Kundan Sharma,Christof Lenz,Aris-Edda Stachler,Lisa-Katharina Maier,Hagen Richter,Lisa Nickel,Ruth A Schmitz,Lennart Randau,Thorsten Allers,Henning Urlaub,Rolf Backofen,Anita Marchfelder

doi:10.1074/jbc.m113.508184

Abstract

The clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR-Cas) system is a prokaryotic defense mechanism against foreign genetic elements. A plethora of CRISPR-Cas versions exist, with more than 40 different Cas protein families and several different molecular approaches to fight the invading DNA. One of the key players in the system is the CRISPR-derived RNA (crRNA), which directs the invader-degrading Cas protein complex to the invader. The CRISPR-Cas types I and III use the Cas6 protein to generate mature crRNAs. Here, we show that the Cas6 protein is necessary for crRNA production but that additional Cas proteins that form a CRISPR-associated complex for antiviral defense (Cascade)-like complex are needed for crRNA stability in the CRISPR-Cas type I-B system in Haloferax volcanii in vivo. Deletion of the cas6 gene results in the loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1-8b) removed and are transformed with the cas6 gene are not able to produce and stably maintain mature crRNAs. crRNA production and stability is rescued only if cas5, -6, and -7 are present. Mutational analysis of the cas6 gene reveals three amino acids (His-41, Gly-256, and Gly-258) that are essential for pre-crRNA cleavage, whereas the mutation of two amino acids (Ser-115 and Ser-224) leads to an increase of crRNA amounts. This is the first systematic in vivo analysis of Cas6 protein variants. In addition, we show that the H. volcanii I-B system contains a Cascade-like complex with a Cas7, Cas5, and Cas6 core that protects the crRNA.

Highlights

The Cas6 protein is required for generating CRISPR-derived RNA (crRNA) in CRISPR-Cas I and III systems
The Cas6 protein is necessary for crRNA production but not sufficient for crRNA maintenance in Haloferax
A CRISPR-associated complex for antiviral defense (Cascade)-like complex is required in the type I-B system for a stable crRNA population

Summary

Conclusion

A Cascade-like complex is required in the type I-B system for a stable crRNA population. During the second stage (the expression stage), the CRISPR RNA (pre-crRNA) is transcribed and subsequently processed into the mature crRNAs. In the third stage (the interference stage), Cas proteins, together with crRNAs, identify and degrade the invader. The crRNAs generated by the Cas proteins usually contain a 2Ј-3Ј phosphate group [14]; only processing by the type I-F Cas (Cas6f) results in a non-cyclic phosphate group [24]. Whereas in system III, the Cas protein acts alone, in type I-A, I-E, and I-F systems, the respective Cas protein is part of the CRISPR-associated complex for antiviral defense (Cascade) [9]. We show that the H. volcanii I-B system contains a Cascade-like complex consisting of at least Cas, Cas, and Cas proteins as well as crRNA

EXPERIMENTAL PROCEDURES

RESULTS

DISCUSSION