Research Article

Structure basis for RNA-guided DNA degradation by Cascade and Cas3

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Science  06 Jul 2018:
Vol. 361, Issue 6397, eaat0839
DOI: 10.1126/science.aat0839

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Target degradation of Type I CRISPR

The CRISPR adaptive immune systems defend bacteria against invaders. Type I CRISPR-Cas systems, the most prevalent type, use a Cascade complex to search the target DNA that is then degraded by Cas3 protein. Xiao et al. report cryo–electron microscopy structures of the Type I-E Cascade/Cas3 complex in the pre– and post–DNA-nicking states. These structures reveal how Cas3 captures Cascade only in its correct conformation to reduce off-targeting and how Cas3 switches from the initial DNA-nicking mode to the processive DNA degradation mode.

Science, this issue p. eaat0839

Structured Abstract

INTRODUCTION

Type I CRISPR-Cas, the most prevalent CRISPR system, features a sequential target-searching and -degradation process. First, the multisubunit surveillance complex Cascade (CRISPR associated complex for antiviral defense) recognizes the matching double-stranded DNA target flanked by an optimal protospacer-adjacent motif (PAM), promotes the heteroduplex formation between CRISPR RNA (crRNA) and the target strand (TS) DNA, and displaces the nontarget strand (NTS) DNA, resulting in R-loop formation at the target site. The helicase-nuclease fusion enzyme Cas3 is then specifically recruited to Cascade/R-loop and nicks and processively degrades the DNA target. High-resolution structures of Type I-E Cascade/R-loop and Cas3/single-stranded DNA (ssDNA) complexes from Thermobifida fusca elucidate the PAM recognition and R-loop formation mechanism. However, the Cas3 recruitment and the DNA-nicking and -degradation mechanisms remain elusive.

RATIONALE

We reconstituted the TfuCascade/R-loop/Cas3 ternary complex and captured structures of the pre– and post–R-loop–nicking states using single-particle cryo–electron microscopy (cryo-EM). Together, these results provide the structural basis to understand crRNA-guided DNA degradation in Type I CRISPR-Cas systems.

RESULTS

We determined the TfuCascade/R-loop/Cas3 cryo-EM structure in the pre–NTS-nicking state at 3.7-Å resolution. Binding of Cas3 does not introduce further conformational changes to the R-loop–forming Cascade, suggesting that Cascade-Cas3 interaction for the most part features a conformation-capture rather than an induced-fit mechanism. The Cas3-Cascade interaction is exclusively mediated by the Cse1 subunit in Cascade. The recognitions are complementary in charge and surface contour to Cascade/R-loop but not to the apolipoprotein and seed-bubble states of Cascade. This is because before full R-loop formation, the C-terminal domain of Cse1 is in an alternative orientation. By making extensive contacts to both domains of Cse1, Cas3 is able to sense the altered surface landscape of Cse1 and reject the Cascade in such functional states. The conditional recruitment of Cas3 to Cascade serves as a mechanism to avoid mis-targeting a DNA with only partial complementarity [see the figure, (A)].

Moreover, we provided direct evidence that a substrate hand-over mechanism is essential for Type I-E CRISPR interference. The HD nuclease of Cas3 directly captures the NTS for strand-nicking, and this action bypasses the helicase moiety completely. Substrate capture relies on the presence of a flexible bulge in the NTS, and the nicking site preference is predetermined by the path of the recruitment pathway.

We further determined the post–NTS-nicking structure at 4.7-Å resolution, which allowed us to identify structural changes accompanying the strand-nicking reaction. The structure reveals that the entire NTS strand in the R-loop region disappears from its original path because of increased flexibility. Upon adenosine 5′-triphosphate (ATP) hydrolysis, the PAM-proximal half of NTS spontaneously relocates to the opening of the Cas3 helicase. It follows that upon ATP hydrolysis, the Cas3 helicase would feed the ssDNA through itself and further into its HD nuclease, entering into a processive DNA degradation mode [see the figure, (B)].

CONCLUSION

We completed the structure-function characterization of the molecular events that lead to Type I-E CRISPR interference. The onset of CRISPR interference is tightly controlled at the Cas3 recruitment step as a mechanism to reduce off-targeting. Upon NTS-nicking, however, Type I systems excel at target destruction because Cas3 degrades DNA processively rather than stopping at generating a double-strand break. Such characteristics may explain why Type I evolved to be the most prevalent CRISPR-Cas system found in nature. It would be interesting to see whether the Type I system may be repurposed into a genome-editing tool with distinct utilities from that of Cas9.

Type I-E CRISPR interference by Cascade and Cas3 zooming into focus.

(A) Mechanism for R-loop–dependent Cas3 recruitment explained. This prevents mistargeting partial-matching DNA sequences. (B) Structural rearrangements after R-loop–nicking by Cas3. The NTS DNA spontaneously relocates to the opening of Cas3 helicase, ready to be threaded for processive degradation.

Abstract

Type I CRISPR-Cas system features a sequential target-searching and degradation process on double-stranded DNA by the RNA-guided Cascade (CRISPR associated complex for antiviral defense) complex and the nuclease-helicase fusion enzyme Cas3, respectively. Here, we present a 3.7-angstrom-resolution cryo–electron microscopy (cryo-EM) structure of the Type I-E Cascade/R-loop/Cas3 complex, poised to initiate DNA degradation. Cas3 distinguishes Cascade conformations and only captures the R-loop–forming Cascade, to avoid cleaving partially complementary targets. Its nuclease domain recruits the nontarget strand (NTS) DNA at a bulged region for the nicking of single-stranded DNA. An additional 4.7-angstrom-resolution cryo-EM structure captures the postnicking state, in which the severed NTS retracts to the helicase entrance, to be threaded for adenosine 5′-triphosphate–dependent processive degradation. These snapshots form the basis for understanding RNA-guided DNA degradation in Type I-E CRISPR-Cas systems.

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