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I understand CRISPR-mediated bacterial immunity to occur in the following simplified steps:

  1. A CRISPR array is transcribed from promoters in the leader sequence to yield a precursor CRISPR RNA (pre-crRNA).
  2. The pre-crRNA is processed by ribonucleases (e.g. Cas6 in Type III systems) to create mature crRNAs.1
  3. 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

References

  1. 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.
  2. 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.
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  • $\begingroup$ What difference will it make in the systems you are considering if there is co-transcriptional processing? $\endgroup$
    – jakebeal
    Sep 19 at 19:43
  • $\begingroup$ It may not make a difference. This question pertains to my research interests and has implications for the interpretation of my data. $\endgroup$
    – acvill
    Sep 20 at 0:23
  • $\begingroup$ I don't know the answer myself, but if it would make a difference in the interpretation of your data, it seems like that might be a good hook to build off of. $\endgroup$
    – jakebeal
    Sep 20 at 0:30
  • $\begingroup$ Thanks, @jakebeal. I'll take that into consideration if I don't get any answers in the next few days. $\endgroup$
    – acvill
    Sep 20 at 0:51

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