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 39-end processing after Cas6 cleavage of repeat RNA 8 nt from its 39 end. crRNAs generated resemble mature crRNA in type I systems, having both 59 (8 nt) and 39 (28 nt) repeat tags. Cas6 cleavage of repeat RNA is ion dependent, and accurate cleavage depends on the presence of a 39 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.
Original language | English (US) |
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Pages (from-to) | 1496-1509 |
Number of pages | 14 |
Journal | FASEB Journal |
Volume | 33 |
Issue number | 1 |
DOIs | |
State | Published - 2019 |
Keywords
- Cas6
- Hairpin
- Mature crRNA
- Metal ion dependence
ASJC Scopus subject areas
- Biotechnology
- Biochemistry
- Molecular Biology
- Genetics