Mycobacterium tuberculosis type III‐A CRISPR/Cas system crRNA and its maturation have atypical features

Abstract

Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems are prokaryotic adaptive immune systems against invading nucleic acids. CRISPR locus variability has been exploited in evolutionary and epidemiological studies of Mycobacterium tuberculosis, the causative agent of tuberculosis, for over 20 yr, yet the biological function of this type III-A system is largely unexplored. Here, using cell biology and biochemical, mutagenic, and RNA-seq approaches, we show it is active in invader defense and has features atypical of type III-A systems: mature CRISPR RNA (crRNA) in its crRNA-CRISPR/Cas protein complex are of uniform length (∼71 nt) and appear not to be subject to 3'-end processing after Cas6 cleavage of repeat RNA 8 nt from its 3' end. crRNAs generated resemble mature crRNA in type I systems, having both 5' (8 nt) and 3' (28 nt) repeat tags. Cas6 cleavage of repeat RNA is ion dependent, and accurate cleavage depends on the presence of a 3' hairpin in the repeat RNA and the sequence of its stem base nucleotides. This study unveils further diversity among CRISPR/Cas systems and provides insight into the crRNA recognition mechanism in M. tuberculosis, providing a foundation for investigating the potential of a type III-A-based genome editing system.-Wei, W., Zhang, S., Fleming, J., Chen, Y., Li, Z., Fan, S., Liu, Y., Wang, W., Wang, T., Liu, Y., Ren, B., Wang, M., Jiao, J., Chen, Y., Zhou, Yi., Zhou, Ya., Gu, S., Zhang, X., Wan, L., Chen, T., Zhou, L., Chen, Y., Zhang, X.-E., Li, C., Zhang, H., Bi, L. Mycobacterium tuberculosis type III-A CRISPR/Cas system crRNA and its maturation have atypical features.

Cyclic oligoadenylate signalling mediatesMycobacterium tuberculosisCRISPR defence

“…Cas6 was observed to cleave a CRISPR RNA substrate to generate the expected 8 nt 5'-handle ( Figure S1). This activity was stimulated by the addition of a divalent cation, as observed previously (23). However, the ability of Ca 2+ to support catalysis is suggestive of a role for the metal ion in RNA folding and stability rather than a direct role in the catalytic mechanism, and all other Cas6 enzymes studied are metal-independent enzymes (reviewed in (28)).…”

“…Little is known about the activity of the Mtb CRISPR system, although recent work suggests it can provide modest immunity against MGE (23). Here, we report the reconstitution and characterisation of the Mtb system both in vitro and in vivo, in E. coli.…”

Exhaustive reconstruction of the CRISPR locus inMycobacterium tuberculosiscomplex using short reads

“…To this collection of subpatterns of interest to be discovered in the CRISPR loci, we added: the beginning and end sequences of IS 6110 and its reverse complement (40 bp each time). CRISPR approximate borders: sequences corresponding to Rv2816c ( cas2 gene of the cas locus) and Rv2813c, reputed to border the CRISPR locus [10]. CRISPR exact-flanking sequences: the reads including the end of the cas2 gene have been extracted from a small collection of genomes from the database presenting spacer 1 in its “new spoligotype” to retrieve likely ancestral closest border to CRISPR locus.…”

“…CRISPR approximate borders: sequences corresponding to Rv2816c ( cas2 gene of the cas locus) and Rv2813c, reputed to border the CRISPR locus [10].…”

“…Their number and nature makes MTC CRISPR type fall into Type III-A group inside CRISPR-Cas taxonomy. It was recently shown to be active under its H37Rv form [9, 10]. Yet, part or the entire region is deleted in several MTC sublineages [11].…”