Role of free DNA ends and protospacer adjacent motifs for CRISPR DNA uptake in Pyrococcus furiosus

Abstract

To acquire CRISPR–Cas immunity against invasive mobile genetic elements, prokaryotes must first integrate fragments of foreign DNA into their genomic CRISPR arrays for use in future invader silencing. Here, we found that the hyperthermophilic archaeaon, Pyrococcus furiosus, actively incorporates DNA fragments (spacers) from both plasmid (foreign) and host genome (self) sequences into its seven CRISPR loci. The majority of new spacers were derived from DNA immediately downstream from a 5′-CCN-3′ protospacer adjacent motif (PAM) that is critical for invader targeting. Interestingly, spacers were preferentially acquired from genome or plasmid regions corresponding to active transposons, CRISPR loci, ribosomal RNA genes, rolling circle origins of replication, and areas where plasmids recombined with the host chromosome. A common feature of the highly sampled spacers is that they arise from DNA regions expected to undergo DNA nicking and/or double-strand breaks. Taken together with recent results from bacterial systems, our findings indicate that free DNA termini and PAMs are conserved features important for CRISPR spacer uptake in diverse prokaryotes and CRISPR–Cas systems. Moreover, lethal self-targeting by CRISPR systems may contribute to host genome stability by eliminating cells undergoing active transposon mobility or chromosomal uptake of autonomously replicating foreign mobile genetic elements.

CRISPR–Cas adaptation in Escherichia coli requires RecBCD helicase but not nuclease activity, is independent of homologous recombination, and is antagonized by 5′ ssDNA exonucleases

“…The previous study used BL-21AI strain ( E. coli B) while we used E. coli K-12, which could be the reason for the observed difference. Another study ( 23 ) reported that P. furiosus cells acquired 96–99% of the unique spacers from the chromosome compared to 1–4% of new spacers derived from a plasmid expressing Cas proteins.…”

Detection of CRISPR adaptation

“…Examples of such selections include obtaining colonies of BIMs (bacteriophage insensitive mutants) ( Figure 1A) [4, 21,22,31,32] or PIMs ( plasmid interfering mutants) ( Figure 1B) [30,31,33]. These methods are cheap and do not require genetic manipulation of cells under study but they are biased towards interference-proficient spacers acquired from MGEs and thus cannot be used to detect spacers that do not lead to interference against MGEs or lead to self-interference due to acquisition of a spacer from cell's own genome (depending on the CRISPR-Cas subtype, when interference is inactivated, such spacers can constitute from 2 to 99% of acquired spacers [23,26,34,35]).…”

“…To analyze millions of CRISPR arrays in a single experiment, high-throughput sequencing (HTS) is usually used [38][39][40]. This allows one to study biases in spacer length, the distribution of corresponding protospacers along different DNA sources and their nucleotide composition [34,35,[38][39][40][41][42][43][44][45][46][47][48][49][50][51][52]. In principle, with sufficient sequencing depth, HTS of total genomic DNA purified from a culture should reveal reads corresponding to expanded arrays [53].…”

Archaeal DNA Repair Mechanisms

“…DSBs can be generated "accidentally" by missteps of information processing machineries, i.e., by mistiming of replication, replication-transcription complex conflicts, and replication or transcription through existing DNA damage/secondary structures [13][14][15][16]. DSBs are also purposefully generated as essential intermediates of many nucleic acid metabolism pathways [17][18][19][20] and if such pathways are aborted prematurely, intermediate complexes may be released inappropriately. Unchecked DSBs can be extremely detrimental to cellular health, causing arrests of replication and transcription which may lead to apoptosis.…”