CasA mediates Cas3-catalyzed target degradation during CRISPR RNA-guided interference

Abstract

In bacteria, the clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) DNA-targeting complex Cascade (CRISPR-associated complex for antiviral defense) uses CRISPR RNA (crRNA) guides to bind complementary DNA targets at sites adjacent to a trinucleotide signature sequence called the protospacer adjacent motif (PAM). The Cascade complex then recruits Cas3, a nuclease-helicase that catalyzes unwinding and cleavage of foreign double-stranded DNA (dsDNA) bearing a sequence matching that of the crRNA. Cascade comprises the CasA-E proteins and one crRNA, forming a structure that binds and unwinds dsDNA to form an R loop in which the target strand of the DNA base pairs with the 32-nt RNA guide sequence. Single-particle electron microscopy reconstructions of dsDNA-bound Cascade with and without Cas3 reveal that Cascade positions the PAM-proximal end of the DNA duplex at the CasA subunit and near the site of Cas3 association. The finding that the DNA target and Cas3 colocalize with CasA implicates this subunit in a key target-validation step during DNA interference. We show biochemically that base pairing of the PAM region is unnecessary for target binding but critical for Cas3-mediated degradation. In addition, the L1 loop of CasA, previously implicated in PAM recognition, is essential for Cas3 activation following target binding by Cascade. Together, these data show that the CasA subunit of Cascade functions as an essential partner of Cas3 by recognizing DNA target sites and positioning Cas3 adjacent to the PAM to ensure cleavage.

Fig. 1.

Cascade binding to dsDNA positions the PAM near CasA. (A) Subunit organization of Cascade and schematic of crRNA (green) and target DNA (sky blue). The PAM and protospacer of the target are depicted in orange and dark blue, respectively. The subunits of Cascade are colored as follows: purple, CasA (Cse1); yellow, CasB (Cse2); light blue and gray, CasC (Cas7); orange, CasD (Cas5e); and red, CasE (Cas6e). We use the Cas protein nomenclature from Brouns et al. (3), but the more recent protein names are included in parentheses (36). (B) CryoEM reconstruction of dsDNA target-bound Cascade at 9-Å resolution (0.5 FSC criterion) with subunits and target labeled and colored as in A. (Right) A larger distance between CasC5 (C5) and CasC6 (C6) relative to that between C4 and C5 allows the accommodation of the target DNA duplex. (C) Docking of the CasA crystal structure (24) (purple) and a modeled 17-bp B-form dsDNA (sky blue) into the EM density. The L1 loop of CasA is labeled.

Fig. 2.

Cas3 interacts with CasA. (A) (Left) Negative-stain reconstruction of TEV-cleaved Cas3-bound dsDNA–Cascade at 20-Å resolution (0.5 FSC criterion) with subunits and Cas3 labeled. (Right) Reference-free 2D class average of the dsDNA–Cascade complex, the corresponding reference-free 2D class average of Cas3–dsDNA–Cascade, and the difference map between these structures. The width of the boxes is ∼288 Å. (B) Cas3 density (outlined semitransparent blue surface) from the negative-stain reconstruction (A) mapped onto the cryoEM reconstruction of dsDNA-bound Cascade.

Fig. 3.

Target cleavage by Cas3 requires base pairing within the PAM sequence and integrity of the PAM-binding loop in CasA. (A) Native gel electrophoretic mobility-shift assay showing that all dsDNA substrates tested for Cas3-mediated degradation are fully bound by Cascade under the conditions used in the cleavage assays. Red text indicates a nucleotide mutation that changes the PAM to a sequence other than one of the four functional motifs. Cascade (1 µM) was incubated with 0.1–0.5 nM [³²P]dsDNA at 37 °C for 30 min before gel-shift analysis by 10% native PAGE. NTS, non-target strand; TS, target strand. (B) All four PAM sequences that function in vivo also permit target cleavage in vitro. DNA cleavage reactions were performed using target dsDNAs with different PAM sequences at a final concentration of 1 nM in 1× reaction buffer. In the presence of 2 mM ATP, 1 μM targeting Cascade, and 500 nM MBP-tagged Cas3, all four targets are cleaved after incubation at 37 °C for 30 min. (C) PAM base-pair mismatches prevent efficient target cleavage by Cas3. Targeting Cascade was prebound to each dsDNA before addition of ATP and MBP-Cas3. (D) Quantified cleavage percentages for each condition in the presence of Cas3 and ATP are the average of three (E) or four (C) independent replicates; error bars represent ±1 SD. All values have been normalized to the WT cleavage efficiency. (E) Denaturing polyacrylamide gel depicting the cleavage defect caused by mutation of Asn131 in the L1 loop of CasA. Wild-type or N131A CasA (1 μM) was added back to 1 μM Strep-tag II–tagged CasB–E to reconstitute the Cascade complex and prebound to a 1 nM dsDNA target with a functional PAM before addition of ATP and MBP-Cas3.

Fig. 4.

Model for target recognition and Cas3 recruitment by Cascade. (A) Cascade searches dsDNA for PAM sites. (B) CasA recognizes the PAM sequence via the L1 loop. (C) Once CasA has located a bona fide target, a rearrangement of Cascade facilitates crRNA–protospacer DNA hybridization. (D) CasA positions the displaced, non-target strand for recruitment of the transacting Cas3 nuclease. (E) Following initial nicking of the displaced strand, Cas3 loads onto the newly formed ssDNA end and translocates along the substrate during processive degradation of the target dsDNA.