Bacteria encode diverse anti-phage systems, such as CRISPR-Cas and restriction modification (RM), which limit infection by targeting phage DNA. We identified a DNA modification in phages, i.e., 5-arabinosyl-hydroxy-cytosine (5ara-hC), which adds arabinose to cytosines via a hydroxy linkage and protects phage from DNA targeting. The hydroxy linkage was common among arabinoslyated phages, with some arabinosylated phages encoding arabinose-5ara-hC transferases (Aat) that add a second or third arabinose to DNA. DNA arabinosylation enables evasion from DNA-targeting type I CRISPR-Cas and type II RM systems. However, arabinosylated phages remain sensitive to RNA-targeting CRISPR-Cas (type III and VI) and promiscuous type IV restriction endonucleases. 5ara-hC enables evasion of glycosylase defenses that target phages with glucosylated hydroxymethyl cytosines, and 5ara-ara-hC protects against some defenses capable of targeting 5ara-hC-modified phages. Collectively, this work identifies DNA modifications that enable phages to evade multiple defenses yet remain vulnerable to some systems that target RNA or modified nucleobases.

Adaptation of clustered regularly interspaced short palindromic repeats (CRISPR) arrays is a crucial process responsible for the unique, adaptive nature of CRISPR-Cas immune systems. The acquisition of new CRISPR spacers from mobile genetic elements has previously been studied for several types of CRISPR-Cas systems. In this study, we used a high-throughput sequencing approach to characterize CRISPR adaptation of the type V-A system from Francisella novicida and the type V-B system from Alicyclobacillus acidoterrestris. In contrast to other class 2 CRISPR-Cas systems, we found that for the type V-A and V-B systems, the Cas12 nucleases are dispensable for spacer acquisition, with only Cas1 and Cas2 (type V-A) or Cas4/1 and Cas2 (type V-B) being necessary and sufficient. Whereas the catalytic activity of Cas4 is not essential for adaptation, Cas4 activity is required for correct protospacer adjacent motif selection in both systems and for prespacer trimming in type V-A. In addition, we provide evidence for acquisition of RecBCD-produced DNA fragments by both systems, but with spacers derived from foreign DNA being incorporated preferentially over those derived from the host chromosome. Our work shows that several spacer acquisition mechanisms are conserved between diverse CRISPR-Cas systems, but also highlights unexpected nuances between similar systems that generally contribute to a bias of gaining immunity against invading genetic elements.

Bacteria and archaea are engaged in a constant arms race to defend against the ever-present threats of viruses and invasion by mobile genetic elements. The most flexible weapons in the prokaryotic defense arsenal are the CRISPR-Cas adaptive immune systems. These systems are capable of selective identification and neutralization of foreign DNA and/or RNA. CRISPR-Cas systems rely on stored genetic memories to facilitate target recognition. Thus, to keep pace with a changing pool of hostile invaders, the CRISPR memory banks must be regularly updated with new information through a process termed CRISPR adaptation. In this Review, we outline the recent advances in our understanding of the molecular mechanisms governing CRISPR adaptation. Specifically, the conserved protein machinery Cas1-Cas2 is the cornerstone of adaptive immunity in a range of diverse CRISPR-Cas systems.