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


  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.
  • $\begingroup$ What difference will it make in the systems you are considering if there is co-transcriptional processing? $\endgroup$
    – jakebeal
    Sep 19, 2021 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, 2021 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, 2021 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, 2021 at 0:51

1 Answer 1


I originally posted this question in an attempt to understand a pattern I observed in my sequencing data. In the time since, I've formalized my interpretation as part of a manuscript, now posted on bioRxiv.

Some background: we performed precision run-on sequencing (PRO-seq) on bacterial cells derived from human stool samples. In short, PRO-seq involves supplementing permeabilized nuclei (or cell lysates, in the case of bacteria) with biotinylated NTPs, such that endogenous RNA polymerases incorporate biotin onto the 3' ends of nascent transcripts. Transcripts are then precipitated on streptavidin beads and sequenced, revealing (1) which genomic features are being actively transcribed and (2) the genome-wide and strand-specific positions of RNA polymerase with base-pair resolution.

Looking at the PRO-seq reads mapping to our metagenome-assembled genomes, we observe a wave-like pattern at loci annotated as CRISPR arrays. Figure 3 from our preprint gives one example:

PRO-seq signal across a Prevotella CRISPR array

(A) Coverage of PRO-seq and RNAseq reads across a CRISPR array in a US2 Prevotella sp. contig. Shaded boxes represent repeats. The large black arrow in each panel represents the leader sequence containing a putative promoter. Small black arrows in the “PRO-seq 5’ end” panel correspond to the predicted site of crRNA cleavage proximal at the base of the repeat stem loop.
(B) Predicted crRNA repeat secondary structure. The black arrow points to the phosphodiester bond that is likely cleaved by Cas5d during pre-crRNA processing, which marks the same position in the repeat as the small arrows in (A). The sequence logo shows perfect conservation of the repeat sequence for this array.
(C) PRO-seq captures nascent transcription of cas5d upstream of and contiguous with the CRISPR array.

Looking specifically at the position of the 5' ends of PRO-seq reads (bottom panel of A), we noticed a pile-up at the same position within each repeat corresponding to the putative crRNA cleavage site at the base of the repeat stem loop (arrow in B). Notably, transcription across this array coincides with transcription of a cas5d endoribonuclease gene upstream of the array (light blue rectangle in C).

Coming back to the title question...

Does ribonuclease processing of pre-crRNAs happen co-transcriptionally?

... I believe the answer is yes. Wave-like transcriptional profiles at CRISPR arrays are not a novel observation (see Figure 1 from Richter et al. 2012), but, importantly, our data include only nascent transcripts. This implies that crRNA cleavage by Cas5d is concurrent with pre-crRNA synthesis, at least in this human-associated Prevotella species.

  • 1
    $\begingroup$ Very nice answer as usual. Unfortunately the link for your manuscript doesn't seem to work (I'm being sent to a "DOI Not Found" page). $\endgroup$
    – tyersome
    Apr 27, 2022 at 3:19
  • 1
    $\begingroup$ @tyersome thanks for catching that. Fixed $\endgroup$
    – acvill
    Apr 27, 2022 at 10:18

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