SEE CRISPR-Cas Part I

CRISPR adaptation (continuation CRISPR adaptation from CRISPR-Cas Part I)

Primed spacer adaptation so far has been demonstrated only in type I systems (50, 65, 66). This priming mechanism constitutes a positive feedback loop that facilitates the acquisition of new spacers from formerly encountered genetic elements (67). Priming can occur even with spacers that contain several mismatches, making them incompetent as guides for targeting the cognate foreign DNA (67). Based on PAM selection, functional spacers are preferentially acquired during naïve adaptation. This initial acquisition event triggers a rapid priming response after subsequent infections. Priming appears to be a major pathway of CRISPR adaptation, at least for some type I systems (65). Primed adaptation strongly depends on the spacer sequence (68), and the acquisition efficiency is highest in close proximity to the priming site. In addition, the orientation of newly inserted spacers indicates a strand bias, which is consistentwith the involvement of singlestranded adaption intermediates (69). According to one proposed model (70), replication forks in the invader’s DNA are blocked by the Cascade complex bound to the priming crRNA, enabling the RecG helicase and the Cas3 helicase/nuclease proteins to attack the DNA. The ends at the collapsed forks then could be targeted by RecBCD, which provides DNA fragments for new spacer generation (70). Given that the use of crRNA for priming has much less strict sequence requirements than direct targeting of the invading DNA, priming is a powerful strategy that might have evolved in the course of the host-parasite arms race to reduce the escape by viral mutants, to provide robust resistance against invading DNA, and to enhance self/nonself discrimination. Naïve as well as primed adaptation in the subtype I-F system of Pseudomonas aeruginosa CRISPR-Cas require both the adaptation and the effector module (69).

In the type II-A system, the Cas9-tracrRNA complex and Csn2 are involved in spacer acquisition along with the Cas1-Cas2 complex (53, 71); the involvement of Cas9 in adaptation is likely to be a general feature of type II systems. Although the key residues of Cas9 involved in PAM recognition are dispensable for spacer acquisition, they are essential for the incorporation of new spacers with the correct PAM sequence (71).

The involvement of Cas9 in PAM recognition and protospacer selection (71) suggests that in type II systems Cas1 may have lost this role. Similarly, Cas4 that is present in subtypes IA-D and II-B has been proposed to be involved in the CRISPR adaptation process, and this prediction has been validated experimentally for type I-B (65). Cas4 is absent in the subtype II-C system of Campylobacter jejuni. Nonetheless, a conserved Cas4-like protein found in Campylobacter bacteriophages can activate spacer acquisition to use host DNA as an effective decoy to bacteriophage DNA. Bacteria that acquire self-spacers and escape phage infection must either overcome CRISPR-mediated autoimmunity by loss of the interference functions, leaving them susceptible to foreign DNA invasions, or tolerate changes in gene regulation (72). Furthermore, in subtypes I-U and V-B, Cas4 is fused to Cas1, which implies cooperation between these proteins during adaptation. In type I-F systems, Cas2 is fused to Cas3 (19), which suggests a dual role for Cas3 (20): involvement in adaptation as well as in interference. These findings support the coupling between the adaptation and interference stages of CRISPR-Cas defense during priming.

Summary of the most extensively characterized CRISPR endoribonucleases^[1][2]

Representatives of class 1 and class 2

Class 1 CRISPR-Cas systems are considered to be the evolutionary ancestral systems. The class 2 systems have evolved from class 1 systems via the insertion of transposable elements encoding various nucleases, and are now being used as tools for genome editing.^[2]

• Class 1:

(4qyz).

(3x1l).

• Class 2:

(5f9r).

5b43.

Class 1

CRISPR subtype I-A (Cascade) - SEE CRISPR subtype I-A

CRISPR subtype I-B (Cascade) - SEE CRISPR subtype I-B

CRISPR subtype I-C (Cascade) - SEE CRISPR subtype I-C

CRISPR subtype I-E (Cascade) - SEE CRISPR subtype I-E

CRISPR subtype I-F (Cascade) - SEE CRISPR subtype I-F

CRISPR subtype III-A (Csm complex) - SEE CRISPR subtype III-A (Csm complex)

CRISPR subtype III-B (Cmr complex)

CRISPR subtype Orphan

• TthCas6A (TTHB78) from T. thermophilus. (4c8z). Other representatives: 4c8y, 4c97.

CRISPR type IV (Csf1)

Class 2

CRISPR type II - SEE CRISPR-Cas9

CRISPR type V (Cpf1) - SEE CRISPR type V (Cpf1)

CRISPR type VI (C2c2)

See aslo

• Cas9
• Endonuclease