I understand CRISPR-mediated bacterial immunity to occur in the following simplified steps:
- A CRISPR array is transcribed from promoters in the leader sequence to yield a precursor CRISPR RNA (pre-crRNA).
- The pre-crRNA is processed by ribonucleases (e.g. Cas6 in Type III systems) to create mature crRNAs.1
- crRNAs are loaded into Cas proteins to create an active CRISPR-Cas complex.
My question pertains to the timing of steps 1 and 2. Is there any evidence for co-transcriptional processing of pre-crRNAs? Put another way, must the full pre-crRNA be transcribed prior to its cleavage by ribonucleases, or can single crRNAs be cleaved from the 5' end of the nascent pre-crRNA. My understanding is that cas6 recognizes and cleaves at the base of the repeat stem-loop,2 and that these stem loops form spontaneously after a repeat is transcribed, so my hunch is that co-transcriptional pre-crRNA cleavage probably can occur, though I'd like some evidence from the literature to support/refute my intuition. I realize mechanisms of CRISPR immunity are quite diverse, so I'd appreciate references pertaining to any CRISPR system.
Some search phrases I've tried in Google:
- co-transcriptional processing of crRNA
- co-transcriptional cleavage of crRNA
- crRNA processing concurrent with transcription
- ribonuclease processing of nascent pre-crRNA
- cas6 proximity to elongating rna polymerase
- Carte J, Wang R, Li H, Terns RM, Terns MP. Cas6 is an endoribonuclease that generates guide RNAs for invader defense in prokaryotes. Genes Dev. 2008 Dec 15;22(24):3489-96.
- Sokolowski RD, Graham S, White MF. Cas6 specificity and CRISPR RNA loading in a complex CRISPR-Cas system. Nucleic Acids Res. 2014 Jun;42(10):6532-41.