A complex of Cas proteins 5, 6, and 7 is required for the biogenesis and stability of crRNAs in Haloferax volcanii [Microbiology]

January 23rd, 2014 by Brendel, J., Stoll, B., Lange, S. J., Sharma, K., Lenz, C., Stachler, A.-E., Maier, L.-K., Richter, H., Nickel, L., Schmitz, R. A., Randau, L., Allers, T., Urlaub, H., Backofen, R., Marchfelder, A.

The CRISPR-Cas (clustered regularly interspaced short palindromic repeats/ CRISPR associated) system is a prokaryotic defence 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 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 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 loss of mature crRNAs and interference. However, cells that have the complete cas gene cluster (cas1-8) 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 (His41, Gly256 and Gly258) that are essential for pre-crRNA cleavage, while the mutation of two amino acids (Ser115 and Ser224) 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.