Structure reveals mechanism of CRISPR RNA-guided nuclease recruitment and anti-CRISPR viral mimicry

Rollins, MaryClare F.; Chowdhury, Saikat; Golden, Sarah; Miettinen, Heini M.; Santiago-Frangos, Andrew; Faith, Dominick; Lawrence, C. Martin; Lander, Gabriel C.; Wiedenheft, Blake

doi:10.1101/453720

Cited by 23 publications

(65 citation statements)

References 76 publications

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“…S1-S4). Similarly to other Csy-Acr complexes (11)(12)(13)(14), the maps exhibit an asymmetric spiral, with one Cas6f subunit in the head, one Cas5f and one Cas8f in the tail, and six Cas7f comprising the spiral backbone (Fig. 1).…”

Section: Resultsmentioning

confidence: 76%

“…S11B). The Cas3 protein (P. aeruginosa) was proposed to interact with the C-terminal helical bundle of Cas8f (11). Further biochemical and structure experiments are needed to elucidate whether AcrF9 also inhibits Cas2/ 3-mediated DNA degradation by competing for the same binding domains on Cas8f.…”

Section: Resultsmentioning

confidence: 99%

“…4 B and C). Residue K247 of Cas8f has been shown to promote foreign DNA duplex splitting by wedging into the strands, which is a prerequisite for DNA binding to Csy (11). Therefore, AcrF6 may interfere with DNA duplex splitting and subsequent DNA-crRNA hybridization to inhibit its degradation.…”

Section: Resultsmentioning

confidence: 99%

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Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR–Cas complex revealed by cryo-EM

Zhang

Wang

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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Prokaryotes and viruses have fought a long battle against each other. Prokaryotes use CRISPR–Cas-mediated adaptive immunity, while conversely, viruses evolve multiple anti-CRISPR (Acr) proteins to defeat these CRISPR–Cas systems. The type I-F CRISPR–Cas system in Pseudomonas aeruginosa requires the crRNA-guided surveillance complex (Csy complex) to recognize the invading DNA. Although some Acr proteins against the Csy complex have been reported, other relevant Acr proteins still need studies to understand their mechanisms. Here, we obtain three structures of previously unresolved Acr proteins (AcrF9, AcrF8, and AcrF6) bound to the Csy complex using electron cryo-microscopy (cryo-EM), with resolution at 2.57 Å, 3.42 Å, and 3.15 Å, respectively. The 2.57-Å structure reveals fine details for each molecular component within the Csy complex as well as the direct and water-mediated interactions between proteins and CRISPR RNA (crRNA). Our structures also show unambiguously how these Acr proteins bind differently to the Csy complex. AcrF9 binds to key DNA-binding sites on the Csy spiral backbone. AcrF6 binds at the junction between Cas7.6f and Cas8f, which is critical for DNA duplex splitting. AcrF8 binds to a distinct position on the Csy spiral backbone and forms interactions with crRNA, which has not been seen in other Acr proteins against the Csy complex. Our structure-guided mutagenesis and biochemistry experiments further support the anti-CRISPR mechanisms of these Acr proteins. Our findings support the convergent consequence of inhibiting degradation of invading DNA by these Acr proteins, albeit with different modes of interactions with the type I-F CRISPR–Cas system.

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Section: Resultsmentioning

confidence: 76%

Section: Resultsmentioning

confidence: 99%

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Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR–Cas complex revealed by cryo-EM

Zhang

Wang

et al. 2020

Proc. Natl. Acad. Sci. U.S.A.

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“…Large conformation changes in the Csy complex occurring upon target DNA-binding lead to 69 exposure of a Cas8f domain that recruits Cas3, the helicase-nuclease that mediates processive 70 degradation of the targeted DNA (Rollins et al, 2019). 71…”

Section: Introduction 45mentioning

confidence: 99%

“…255 After Cas8f recognizes the PAM, this subunit destabilizes the DNA duplex, leading to base 256 pairing of one DNA strand to the crRNA and to the formation a single-stranded loop, referred to 257 as the R-loop, by the other strand. During this process of PAM recognition, Cas8f undergoes a 258helical rotation to expose a positively charged channel that binds and stabilizes the R-loop 259(Rollins et al, 2019). The R-loop binding channel is not sequence specific, as it must bind to a 260 DNA sequence that matches any spacer.…”

mentioning

confidence: 99%

An anti-CRISPR protein induces strong non-specific DNA binding activity in a CRISPR-Cas complex

Trost

Müller-Esparza

et al. 2020

Preprint

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Phages and other mobile genetic elements express anti-CRISPR proteins (Acrs) to protect their genomes from destruction by CRISPR-Cas systems. Acrs usually block the ability of CRISPR-Cas systems to bind or cleave their nucleic acid substrates. Here, we investigate an unusual Acr, AcrIF9, that induces a gain-of-function to a type I-F CRISPR-Cas (Csy) complex, causing it to bind strongly to DNA that lacks both a PAM sequence and sequence complementarity. We show that specific and non-specific dsDNA compete for the same site on the Csy:AcrIF9 complex with rapid exchange, but specific ssDNA appears to still bind through complemetarity to the CRISPR RNA. We also demonstrate that induction of non-specific DNA-binding is a conserved property of diverse AcrIF9 homologues, implying that this activity contributes the biologically relevant function of this Acr family. AcrIF9 provides another example of the surprising variety of mechanisms by which Acrs inhibit CRISPR-Cas systems.

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Allosteric regulation in CRISPR/Cas1-Cas2 protospacer acquisition mediated by DNA and Cas2

Long

Dai

Chao

et al. 2021

Biophysical Journal

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Cas1 and Cas2 are highly conserved proteins across clustered-regularly-interspaced-short-palindromic-repeat-Cas systems and play a significant role in protospacer acquisition. Based on crystal structure of twofold symmetric Cas1-Cas2 in complex with dual-forked protospacer DNA (psDNA), we conducted all-atom molecular dynamics simulations to study the psDNA binding, recognition, and response to cleavage on the protospacer-adjacent-motif complementary sequence, or PAMc, of Cas1-Cas2. In the simulation, we noticed that two active sites of Cas1 and Cas1' bind asymmetrically to two identical PAMc on the psDNA captured from the crystal structure. For the modified psDNA containing only one PAMc, as that to be recognized by Cas1-Cas2 in general, our simulations show that the non-PAMc association site of Cas1-Cas2 remains destabilized until after the stably bound PAMc being cleaved at the corresponding association site. Thus, long-range correlation appears to exist upon the PAMc cleavage between the two active sites ($10 nm apart) on Cas1-Cas2, which can be allosterically mediated by psDNA and Cas2 and Cas2' in bridging. To substantiate such findings, we conducted repeated runs and further simulated Cas1-Cas2 in complex with synthesized psDNA sequences psL and psH, which have been measured with low and high frequency in acquisition, respectively. Notably, such intersite correlation becomes even more pronounced for the Cas1-Cas2 in complex with psH but remains low for the Cas1-Cas2 in complex with psL. Hence, our studies demonstrate that PAMc recognition and cleavage at one active site of Cas1-Cas2 may allosterically regulate non-PAMc association or even cleavage at the other site, and such regulation can be mediated by noncatalytic Cas2 and DNA protospacer to possibly support the ensued psDNA acquisition.

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Structure reveals mechanism of CRISPR RNA-guided nuclease recruitment and anti-CRISPR viral mimicry

Abstract: Bacteria and archaea have evolved sophisticated adaptive immune systems that rely on CRISPR RNA (crRNA)guided detection and nuclease-mediated elimination of invading nucleic acids. Here we present the cryo-EM

Cited by 23 publications

References 76 publications

Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR–Cas complex revealed by cryo-EM

Inhibition mechanisms of AcrF9, AcrF8, and AcrF6 against type I-F CRISPR–Cas complex revealed by cryo-EM

An anti-CRISPR protein induces strong non-specific DNA binding activity in a CRISPR-Cas complex

Allosteric regulation in CRISPR/Cas1-Cas2 protospacer acquisition mediated by DNA and Cas2

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