In two recent studies in Molecular Cell, Wright et al. (2019) report complete spacer integration by a Cas1 mini-integrase and Edraki et al. (2019) describe accurate genome editing by a small Cas9 ortholog with less stringent PAM requirements.

Integrating short DNA fragments at the correct leader-repeat junction is key to successful CRISPR-Cas memory formation. The Cas1-2 proteins are responsible to carry out this process. However, the CRISPR adaptation process additionally requires a DNA element adjacent to the CRISPR array, called leader, to facilitate efficient localization of the correct integration site. In this work, we introduced the core CRISPR adaptation genes cas1 and cas2 from the Type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and assessed spacer integration efficiency. Truncation of the leader resulted in a significant reduction of spacer acquisition levels and revealed the importance of different conserved regions for CRISPR adaptation rates. We found three conserved sequence motifs in the leader of I-D CRISPR arrays that each affected spacer acquisition rates, including an integrase anchoring site. Our findings support the model in which the leader sequence is an integral part of type I-D adaptation in Synechocystis sp. acting as a localization signal for the adaptation complex to drive CRISPR adaptation at the first repeat of the CRISPR array.

CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which the role is unknown. Here, we introduced the CRISPR adaptation genes cas1, cas2, and cas4 from the type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and observed that cas4 is strictly required for the selection of targets with protospacer adjacent motifs (PAMs) conferring I-D CRISPR interference in the native host Synechocystis. We propose a model in which Cas4 assists the CRISPR adaptation complex Cas1-2 by providing DNA substrates tailored for the correct PAM. Introducing functional spacers that target DNA sequences with the correct PAM is key to successful CRISPR interference, providing a better chance of surviving infection by mobile genetic elements. Kieper et al. demonstrate that the ubiquitous protein Cas4 assists Cas1 and Cas2 in the selection of new CRISPR spacers with a PAM licensing efficient CRISPR interference.

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.

Prokaryotes use a mechanism called priming to update their CRISPR immunological memory to rapidly counter revisiting, mutated viruses, and plasmids. Here we have determined how new spacers are produced and selected for integration into the CRISPR array during priming. We show that Cas3 couples CRISPR interference to adaptation by producing DNA breakdown products that fuel the spacer integration process in a two-step, PAM-associated manner. The helicase-nuclease Cas3 pre-processes target DNA into fragments of about 30–100 nt enriched for thymine-stretches in their 3′ ends. The Cas1-2 complex further processes these fragments and integrates them sequence-specifically into CRISPR repeats by coupling of a 3′ cytosine of the fragment. Our results highlight that the selection of PAM-compliant spacers during priming is enhanced by the combined sequence specificities of Cas3 and the Cas1-2 complex, leading to an increased propensity of integrating functional CTT-containing spacers.