Unexpected diversity of CRISPR unveils some evolutionary patterns of repeated sequences in Mycobacterium tuberculosis

Abstract

Background Diversity of the CRISPR locus of Mycobacterium tuberculosis complex has been studied since 1997 for molecular epidemiology purposes. By targeting solely the 43 spacers present in the two first sequenced genomes (H37Rv and BCG), it gave a biased idea of CRISPR diversity and ignored diversity in the neighbouring cas-genes. Results We set up tailored pipelines to explore the diversity of CRISPR-cas locus in Short Reads. We analyzed data from a representative set of 198 clinical isolates as evidenced by well-characterized SNPs. We found a relatively low diversity in terms of spacers: we recovered only the 68 spacers that had been described in 2000. We found no partial or global inversions in the sequences, letting always the Direct Variant Repeats (DVR) in the same order. In contrast, we found an unexpected diversity in the form of: SNPs in spacers and in Direct Repeats, duplications of various length, and insertions at various locations of the IS6110 insertion sequence, as well as blocks of DVR deletions. The diversity was in part specific to lineages. When reconstructing evolutionary steps of the locus, we found no evidence for SNP reversal. DVR deletions were linked to recombination between IS6110 insertions or between Direct Repeats. Conclusion This work definitively shows that CRISPR locus of M. tuberculosis did not evolve by classical CRISPR adaptation (incorporation of new spacers) since the last most recent common ancestor of virulent lineages. The evolutionary mechanisms that we discovered could be involved in bacterial adaptation but in a way that remains to be identified.

CRISPRbuilder-TB: “CRISPR-builder for tuberculosis”. Exhaustive reconstruction of the CRISPR locus in mycobacterium tuberculosis complex using SRA

“…We reconstructed the CRISPR-Cas locus of 434 strains representing the diversity of the MTC lineages and showing interesting features ( Fig 2 , sample #3). As explained previously, these results and lessons is the subject of the companion article [ 1 ]. Yet we want here to highlight some of the patterns we observed that may be observed in the CRISPRs of other species.…”

“…In contrast, CRISPR-builder TB could retrieve IS 6110 sequences insertion positions as shown on strains for which reliable complete genome sequences were available (for instance H37Ra, L4.9, S3 Table , S2 Text ). On other SRR, CRISPRbuilder-TB retrieved between 0 and 2 copies of IS 6110 in the CRISPR locus, in either orientation ( S7 Table , sheet “IS”, and see also companion article Refrégier et al [ 1 ]). The tendency of Crass tool to miss duplications and/or insertions and the neighbouring spacers was further confirmed when applying it to MTC SRR (for instance, on ERR025424, lineage 4.1.2.1, coverage = 555.97, reads length = 116, 8/38 spacers were detected, moreover with errors in their sequences; ERR751335 from L5, S2 Text ).…”

“…The CRISPR locus of Mycobacterium tuberculosis complex (MTC) the agent of tuberculosis (TB) was first described in 1993 under the “Direct Repeat” locus designation [ 1 , 2 ]. It is made of 36 nucleotides-repeats interspaced by unique spacers of a mean of 37nt (interval: 25-45nt).…”

“…Applied on TB SRA without any genome assembly step, CRISPRbuilder-TB proved reliable and robust with reads of more than 75bp. The remarkable elements that were found in this locus by MTC lineages is the subject of a separate publication [ 1 ]. We emphasize its usefulness by showing that close to 50% of MTC complete genomes available in public NCBI database exhibit unreliable CRISPRs, and by detailing the different kind of evolutionary events that can affect CRISPRs, at least in MTC genomes.…”

Comparative Genomic Analysis of Mycobacteriaceae Reveals Horizontal Gene Transfer-Mediated Evolution of the CRISPR-Cas System in the Mycobacterium tuberculosis Complex

“…Therefore, lack of any shared spacer sequences may be due to the phylogenetic distance of M. canettii from MTBC members, which are mainly variants of M. tuberculosis species and so phylogenetically more related to each other ( 7 , 21 ). Another possibility could be the inability of M. tuberculosis to incorporate new spacers, unlike M. canettii ( 2 , 24 ).…”