CRISPR-Cas9 enrichment, a new strategy in microbial metagenomics to investigate complex genomic regions: the case of an environmental integron.

Environmental integrons are ubiquitous in natural microbial communities, but they are mostly uncharacterized and their role remains elusive. Thus far, research has been hindered by methodological limitations. Here, we successfully used an innovative approach combining CRISPR-Cas9 enrichment with long-read nanopore sequencing to target, in a complex microbial community, a putative adaptive environmental integron, InOPS, and to unravel its complete structure and genetic context. A contig of 20 kb was recovered containing the complete integron from the microbial metagenome of oil-contaminated coastal sediments. InOPS exhibited typical integron features. The integrase, closely related to integrases of marine Desulfobacterota, possessed all the elements of a functional integron integrase. The gene cassettes harboured mostly unknown functions hampering inferences about their ecological importance. Moreover, the putative InOPS host, likely a hydrocarbonoclastic marine bacteria, raises questions as to the adaptive potential of InOPS in response to oil contamination. Finally, several mobile genetic elements were intertwined with InOPS highlighting likely genomic plasticity, and providing a source of genetic novelty. This case study showed the power of CRISPR-Cas9 enrichment to elucidate the structure and context of specific DNA regions for which only a short sequence is known. This method is a new tool for environmental microbiologists working with complex microbial communities to target low abundant, large or repetitive genetic structures hardly recover by classical metagenomics. More precisely, here, it offers new perspectives to comprehensively assess the eco-evolutionary significance of environmental integrons.