CRISPR (clustered regularly interspaced short palindromic repeat) arrays and cas (CRISPR-associated) genes of prokaryotes form a unique immune system that confers adaptive immunity against invading mobile genetic elements (MGEs), such as viruses [1] and plasmids [2]. CRISPR-Cas systems contain short sequences, the so-called spacer sequences, that are incorporated into the CRISPR array following an invasion of a foreign nucleic acid [1]. Spacer sequences serve as a memory of previous infections, thereby allowing for sequence-specific targeting of the MGE [3]. In addition, recent studies showed that CRISPR-Cas systems have potential regulatory functions [4] and mediate immunity of certain archaea against their archaeal episymbionts [5]. Despite all the mechanisms of spacer acquisition described so far, a matching protospacer can only be detected for a small proportion of all spacers in genomic databases [6]. Therefore, we tested the hypothesis that spacers originate from chromosomal DNA of prokaryotic genomes from the same environment. Using metagenomics, we analyzed the CRISPR-Cas systems of metagenome-assembled genomes from two subsurface environments. Spacer-to-protospacer matching revealed that spacers were frequently acquired from chromosomal DNA of different prokaryotes inhabiting the same environment. Further, our results suggest that the spacer acquisition from prokaryotic DNA occurs in almost all types of CRISPR-Cas systems. Analyses of the host genomes revealed a frequent presence of genes encoding proteins potentially involved in the uptake or breakdown of environmental DNA. Based on these findings, we propose that DNA uptake by the cell facilitates the acquisition of spacers from prokaryotic DNA, contributing to the diversity of spacers found in CRISPR arrays from natural environments. Taken together, our results provide a possible explanation for the origin of some of the many unmatched spacers in public databases.
[1] Barrangou et al. (2007), Science, 315(5819), 1709-1712.
[2] Marraffini and Sontheimer (2008), Science, 322(5909), 1843-1845.
[3] Brouns et al. (2008), Science, 321(5891), 960-964.
[4] Stachler et al. (2017), Nucleic Acids Research, 45(9), 5208-5216.
[5] Esser et al. (2023), Nature Microbiology, 8(9), 1619-1633.
[6] Shmakov et al. (2017), mBio, 8(5), e01397-17.