Creating memories: molecular mechanisms of CRISPR adaptation

Abstract

Prokaryotes use clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) proteins as an adaptive immune system. CRISPR-Cas systems preserve molecular memories of infections by integrating short fragments of foreign nucleic acids as spacers into the host CRISPR array in a process termed 'adaptation'. Functional spacers ensure a robust immune response by Cas effectors, which neutralizes subsequent infection through RNA-guided interference pathways. In this review, we summarize recent discoveries that have advanced our understanding of adaptation, with a focus on how functional spacers are generated and incorporated through many widespread, but type-specific, mechanisms. Finally, we highlight future directions and outstanding questions for a more thorough understanding of CRISPR adaptation.

Keywords: CRISPR-Cas; Cas1-Cas2; Cas4; Cas9; PAM; spacers.

Figure 1.. Overview of CRISPR-Cas systems.

CRISPR-Cas operons contain a CRISPR, made up of repeats (black diamonds) and spacers (colored boxes) and cas genes expressing Cas proteins involved in adaptation (yellow) or interference (blue). The adaptation Cas proteins capture and integrate short fragments from foreign genomes into the CRISPR array as new spacers. The array is transcribed into a pre-crRNA and processed into crRNAs, allowing formation of an effector complex with multiple Cas proteins (class 1) or a single Cas protein (class 2). Class 1 type I systems use CRISPR-associated complex for antiviral defense (Cascade) complexes and type III systems use Csm-(III-A)/Cmr-(III-B) effector complexes. Cascade recognizes DNA targets and recruits Cas3 nuclease for degradation. In type III systems, the effector complex provides immunity in transcription-dependent manner, exhibiting both target RNA and ssDNA cleavage activities. Class 2 systems encompass a single effector protein, Cas9 in type II, Cas12 in type V, and Cas13 in type VI. The crRNA-guided effector complexes target the foreign nucleic acids that are complementary to the crRNA sequences. Type II Cas9 and type V Cas12 cleaves target DNA, resulting in dsDNA breaks. Type VI uses Cas13 for degradation of target RNAs.

Figure 2.. Model of CRISPR adaptation mechanisms.

(A-B) Prespacers may be generated through the activity of RecBCD or AddAB, in Gram negative and positive bacteria, respectively, or through interference activity by type I (A) or type II (B) effector complexes. (C-E) Models for prespacer selection and processing in (C) E. coli type I-E, (D) G. sulfurreducens type I-G, and (E) S. thermophilus type II-A systems. Type I systems acquire spacers from RecBCD or Cascade-Cas3 activities. RecBCD or Cascade-Cas3 activities provide ssDNA substrates for naïve or primed adaptation. In type I systems, Cas1-Cas2 (C) or Cas4-Cas1-Cas2 (D) complex captures ssDNA with PAM sequences and facilitates annealing of complimentary ssDNAs. Non-PAM overhangs are cleaved by DnaQ exonuclease while PAM end overhangs are protected by Cas1 (C) or Cas4 (D). IHF may induce bending of the leader (green), allowing recognition of upstream leader motifs by Cas1-Cas2. The adaptation complex integrates the processed non-PAM end first at the junction of the leader (green) and the first repeat (black) (L-site integration) followed by the cleavage of PAM-end by DnaQ in Cas1-Cas2 (C) or Cas4 in Cas4-Cas1-Cas2 (D) complex. DnaQ or Cas4 dissociates from the complex after the cleavage and the processed PAM-ends are integrated at the junction of the repeat and spacer (blue) (S-site integration). (E) Type II-A systems acquire spacers from AddAB and Cas9 activities. Cas9 is required for both naïve and primed adaptation for selecting prespacers flanking PAM sequences. Csn2-Cas1-Cas2 multisubunit complex binds dsDNA substrates. The exact roles of Cas9 and Csn2 in this process remain unknown. Cas1-Cas2 is sufficient for integration at L-site and S-sites. After integration, gaps are filled through transcription-coupled repair mechanism.

Figure 3:. PAM-recognition in type I adaptation complexes.

(A) Type I-E adaptation complexes are composed of two Cas1 dimers (yellow/beige) sandwiching a Cas2 dimer (salmon/pink) and lacks a Cas4 subunit. The prespacer (white/gray) spans the length of the Cas2 subunits, allowing threading of 3’ overhangs into the Cas1 active sites. (B) The PAM in one overhang is recognized by Cas1 and protected from degradation by exonucleases by a loop in the C-terminal tail of Cas1. The CTT PAM-complementary sequence is recognized through a combination of specific hydrogen-bonding interactions and stacking interactions with the two Cas1 subunits. (C) Other type I systems (excluding type I-E) contain Cas4 subunits that assemble with the core Cas1-Cas2 complex. The type I-G system contains a Cas4-1 fusion protein, in which one Cas4 domain (blue) recognizes the PAM-end of the prespacer. (D) Specific recognition of the GAA PAM-complementary sequence is recognized by several residues adjacent to an Fe₄S₄ cluster, mainly through stacking and van der Waals interactions that recognize the shape of the sequence and a few specific hydrogen-bonding interactions. (A-B) PDB ID: 5DQZ; (C-D) PDB ID: 7MI5).

Figure 4:. Integration of prespacers at first repeat of CRISPR array.

(A) Integration may be directed to the first repeat in the CRISPR array through interactions between Cas1 and upstream regions of the leader (green). IHF (tan) induces DNA bending in the leader, enabling this interaction. (B) Integration at the leader-repeat junction (L-site integration) occurs within the Cas1 active site. Proteins are colored as in Figure 3, DNA as in Figure 2. (C-E) Structures capturing half-site (C, E) and full-site (D, F) integration products for type I-G (C, D) and II-A (E, F). (C) L-site integration triggers cleavage of the PAM (red) by Cas4, likely through activation based on interactions between the repeat and the Cas1 domain. (D) PAM cleavage leads to dissociation of the Cas4 domain and integration of the PAM end at the repeat-spacer junction (S-site integration). (E-F) Type II-A Cas1-Cas2 performs half-site integration first at the L-site €, followed by S-site integration (F). (A-B) PDB ID: 5WFE; (C) PDB ID: 7MIB; (D) PDB ID: 7MI9; (E) PDB ID: 5XVO; (F) PDB ID: 5XVP.