ABSTRACT
The immunization of bacteria and archaea against invading viruses via CRISPR adaptation is critically reliant on the efficient capture, accurate processing, and integration of CRISPR spacers into the host genome. The adaptation proteins Cas1 and Cas2 are sufficient for successful spacer acquisition in some CRISPR-Cas systems. However, many CRISPR-Cas systems additionally require the Cas4 protein for efficient adaptation. Cas4 has been implied in the selection and processing of spacer precursors, but the detailed mechanistic understanding of how Cas4 contributes to CRISPR adaptation is lacking. Here, we biochemically reconstitute the CRISPR-Cas type I-D adaptation system and show two functionally distinct adaptation complexes: Cas4-Cas1 and Cas1-Cas2. The Cas4-Cas1 complex recognizes and cleaves protospacer adjacent motif (PAM) sequences in 3' overhangs in a sequence-specific manner, while the Cas1-Cas2 complex defines the cleavage of non-PAM sites via host-factor nucleases. Both sub-complexes are capable of mediating half-site integration, facilitating the integration of processed spacers in the correct interference-proficient orientation. We provide a model in which an asymmetric adaptation complex differentially acts on PAM- and non-PAM-containing overhangs, providing cues for the correct orientation of spacer integration.
Subject(s)
Binding Sites , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems , Gene Editing , Nucleotide Motifs , CRISPR-Associated Proteins/chemistry , CRISPR-Associated Proteins/genetics , DNA, Bacterial , Gene Editing/methods , Gene Order , Multiprotein Complexes , Plasmids/chemistry , Plasmids/genetics , Protein Binding , Protein MultimerizationABSTRACT
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.
Subject(s)
CRISPR-Cas Systems/genetics , Clustered Regularly Interspaced Short Palindromic Repeats/genetics , Escherichia coli/genetics , Escherichia coli/metabolism , Protein Sorting Signals/genetics , Protein Sorting Signals/physiology , Synechocystis/genetics , Synechocystis/metabolismABSTRACT
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.
Subject(s)
CRISPR-Cas Systems , Gene EditingABSTRACT
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.
Subject(s)
CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems/genetics , Humans , Synechocystis/geneticsABSTRACT
CRISPR-Cas adaptive immune systems capture DNA fragments from invading bacteriophages and plasmids and integrate them as spacers into bacterial CRISPR arrays. In type I-E and II-A CRISPR-Cas systems, this adaptation process is driven by Cas1-Cas2 complexes. Type I-F systems, however, contain a unique fusion of Cas2, with the type I effector helicase and nuclease for invader destruction, Cas3. By using biochemical, structural, and biophysical methods, we present a structural model of the 400-kDa Cas14-Cas2-32 complex from Pectobacterium atrosepticum with bound protospacer substrate DNA. Two Cas1 dimers assemble on a Cas2 domain dimeric core, which is flanked by two Cas3 domains forming a groove where the protospacer binds to Cas1-Cas2. We developed a sensitive in vitro assay and demonstrated that Cas1-Cas2-3 catalyzed spacer integration into CRISPR arrays. The integrase domain of Cas1 was necessary, whereas integration was independent of the helicase or nuclease activities of Cas3. Integration required at least partially duplex protospacers with free 3'-OH groups, and leader-proximal integration was stimulated by integration host factor. In a coupled capture and integration assay, Cas1-Cas2-3 processed and integrated protospacers independent of Cas3 activity. These results provide insight into the structure of protospacer-bound type I Cas1-Cas2-3 adaptation complexes and their integration mechanism.
Subject(s)
Bacterial Proteins/metabolism , CRISPR-Cas Systems/physiology , Endonucleases/metabolism , Multienzyme Complexes/metabolism , Pectobacterium/enzymology , Bacterial Proteins/genetics , Endonucleases/genetics , Multienzyme Complexes/genetics , Pectobacterium/geneticsABSTRACT
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.
Subject(s)
Adaptation, Physiological , Archaea/immunology , Bacteria/immunology , CRISPR-Associated Proteins/metabolism , CRISPR-Cas Systems/physiology , Archaea/virology , Bacteria/virology , CRISPR-Associated Proteins/chemistry , CRISPR-Associated Proteins/classification , CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems/genetics , DNA/metabolism , RNA/metabolismABSTRACT
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.
Subject(s)
CRISPR-Associated Proteins/genetics , CRISPR-Cas Systems , Clustered Regularly Interspaced Short Palindromic Repeats , DNA Helicases/genetics , DNA/genetics , Escherichia coli Proteins/genetics , Plasmids/metabolism , RNA, Bacterial/genetics , Binding Sites , CRISPR-Associated Proteins/chemistry , CRISPR-Associated Proteins/metabolism , Cloning, Molecular , DNA/chemistry , DNA/metabolism , DNA Cleavage , DNA Helicases/chemistry , DNA Helicases/metabolism , Escherichia coli/genetics , Escherichia coli/metabolism , Escherichia coli Proteins/chemistry , Escherichia coli Proteins/metabolism , Gene Expression , Kinetics , Models, Molecular , Nucleic Acid Conformation , Nucleotide Motifs , Plasmids/chemistry , Protein Binding , Protein Interaction Domains and Motifs , RNA, Bacterial/chemistry , RNA, Bacterial/metabolism , Recombinant Proteins/chemistry , Recombinant Proteins/genetics , Recombinant Proteins/metabolism , Substrate SpecificityABSTRACT
CRISPR-Cas systems are adaptive immune systems in prokaryotes that provide protection against viruses and other foreign DNA. In the adaptation stage, foreign DNA is integrated into CRISPR (clustered regularly interspaced short palindromic repeat) arrays as new spacers. These spacers are used in the interference stage to guide effector CRISPR associated (Cas) protein(s) to target complementary foreign invading DNA. Cas1 is the integrase enzyme that is central to the catalysis of spacer integration. There are many diverse types of CRISPR-Cas systems, including type I-F systems, which are typified by a unique Cas1-Cas2-3 adaptation complex. In the present study we characterize the Cas1 protein of the potato phytopathogen Pectobacterium atrosepticum, an important model organism for understanding spacer acquisition in type I-F CRISPR-Cas systems. We demonstrate by mutagenesis that Cas1 is essential for adaptation in vivo and requires a conserved aspartic acid residue. By X-ray crystallography, we show that although P. atrosepticum Cas1 adopts a fold conserved among other Cas1 proteins, it possesses remarkable asymmetry as a result of structural plasticity. In particular, we resolve for the first time a flexible, asymmetric loop that may be unique to type I-F Cas1 proteins, and we discuss the implications of these structural features for DNA binding and enzymatic activity.