Spontaneous CRISPR loci generation in vivo by non-canonical spacer integration

Abstract

The adaptation phase of CRISPR-Cas immunity depends on the precise integration of short segments of foreign DNA (spacers) into a specific genomic location within the CRISPR locus by the Cas1-Cas2 integration complex. Although off-target spacer integration outside of canonical CRISPR arrays has been described in vitro, no evidence of non-specific integration activity has been found in vivo. Here, we show that non-canonical off-target integrations can occur within bacterial chromosomes at locations that resemble the native CRISPR locus by characterizing hundreds of off-target integration locations within Escherichia coli. Considering whether such promiscuous Cas1-Cas2 activity could have an evolutionary role through the genesis of neo-CRISPR loci, we combed existing CRISPR databases and available genomes for evidence of off-target integration activity. This search uncovered several putative instances of naturally occurring off-target spacer integration events within the genomes of Yersinia pestis and Sulfolobus islandicus. These results are important in understanding alternative routes to CRISPR array genesis and evolution, as well as in the use of spacer acquisition in technological applications.

Comparative genomics and evolution of trans-activating RNAs in Class 2 CRISPR-Cas systems

“…Ultimately, the problem of the tracrRNA origin is tightly linked with the origin of the repeats themselves in different CRISPR-Cas variants. There is currently no conclusive evidence of either divergent or convergent origin of the repeats although the lack of sequence similarity between and sometimes even within subtypes and the recently demonstrated ability of the Cas1-Cas2 protein complex to generate CRISPR arrays de novo [50] appear to favor convergence.…”

Detection of CRISPR adaptation

“…In principle, with sufficient sequencing depth, HTS of total genomic DNA purified from a culture should reveal reads corresponding to expanded arrays [53]. In a model system of E. coli cultures overproducing the Cas1-Cas2 adaptation protein complex and transformed with spacer-sized oligonucleotides, ∼350× genomic coverage allowed to confidently detect CRISPR array expansion that occurred in ∼10% of cells [54]. Moreover, rarer off-target integration events elsewhere in the genome were also detected [54].…”

“…While clearly powerful and unbiased, the shotgun sequencing approach requires high sequencing coverage and provides very low (dozens) numbers of reads corresponding to expanded arrays making it unsuitable for studies aimed at qualitative understanding of spacer selection preferences [54]. Therefore, the common strategy is to prepare PCR amplicons of arrays from cultures undergoing CRISPR adaptation and then subject them for HTS [38][39][40].…”

“…The obvious drawback of this method is that it requires prior knowledge about acquired spacer(s) and thus cannot be applied to study spacer acquisition in systems with unknown adaptation preferences. However, it is very powerful when studying acquisition from spacer-sized oligonucleotides transformed into cells [51,54,57]. Another modification uses a leader-specific primer and 'degenerate' primers matching the repeat sequence and containing one extra 3 0 -end nucleotide ( Figure 2B) [42].…”

A Functional Mini-Integrase in a Two-Protein Type V-C CRISPR System

“…Integration is specific to the CRISPR locus, with the preferred site being the first repeat adjacent to the leader, an upstream sequence that also contains the promoter for the CRISPR array (Yosef et al, 2012;Nuñ ez et al, 2014Nuñ ez et al, , 2015bWei et al, 2015). Cas1-Cas2 integrases have intrinsic target specificity, although host factors have been shown to improve integration specificity in some hosts (Nuñ ez et al, 2016;Yoganand et al, 2017;Nivala et al, 2018;Fagerlund et al, 2017;Rollie et al, 2018).…”