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.
Type I CRISPR systems feature a sequential dsDNA target searching and degradation process, by crRNA-displaying Cascade and nuclease-helicase fusion enzyme Cas3, respectively. Here we present two ...cryo-EM snapshots of the Thermobifida fusca type I-E Cascade: (1) unwinding 11 bp of dsDNA at the seed-sequence region to scout for sequence complementarity, and (2) further unwinding of the entire protospacer to form a full R-loop. These structures provide the much-needed temporal and spatial resolution to resolve key mechanistic steps leading to Cas3 recruitment. In the early steps, PAM recognition causes severe DNA bending, leading to spontaneous DNA unwinding to form a seed-bubble. The full R-loop formation triggers conformational changes in Cascade, licensing Cas3 to bind. The same process also generates a bulge in the non-target DNA strand, enabling its handover to Cas3 for cleavage. The combination of both negative and positive checkpoints ensures stringent yet efficient target degradation in type I CRISPR-Cas systems.
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•Snapshot of a seed crRNA-DNA duplex formation—an important R-loop intermediate•PAM recognition bends dsDNA, leading to DNA melting and seed-bubble formation•Full R-loop formation by Cascade triggers conformational changes, licensing Cas3 binding•Full R-loop formation triggers non-target strand bulge, enabling Cas3 nicking
Cryo-EM structures of type I CRISPR-Cas system resolve the mechanisms governing the PAM-dependent R-loop formation, Cas3 recruitment, and substrate handover processes.
CRISPR-Cas systems enable microbial adaptive immunity and provide eukaryotic genome editing tools. These tools employ a single effector enzyme of type II or V CRISPR to generate RNA-guided, precise ...genome breaks. Here we demonstrate the feasibility of using type I CRISPR-Cas to effectively introduce a spectrum of long-range chromosomal deletions with a single RNA guide in human embryonic stem cells and HAP1 cells. Type I CRISPR systems rely on the multi-subunit ribonucleoprotein (RNP) complex Cascade to identify DNA targets and on the helicase-nuclease enzyme Cas3 to degrade DNA processively. With RNP delivery of T. fusca Cascade and Cas3, we obtained 13%–60% editing efficiency. Long-range PCR-based and high-throughput-sequencing-based lesion analyses reveal that a variety of deletions, ranging from a few hundred base pairs to 100 kilobases, are created upstream of the target site. These results highlight the potential utility of type I CRISPR-Cas for long-range genome manipulations and deletion screens in eukaryotes.
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•T. fusca type I CRISPR-Cas enables long-range genome deletions in human cells•The target-binding complex Cascade and helicase-nuclease Cas3 are both required•Genome engineering by type I CRISPR is crRNA guided and programmable•Heterogeneous deletions up to 100 kb are induced upstream of a genomic target site
Dolan et al. demonstrate that T. fusca type I CRISPR-Cas can generate a spectrum of large genome deletions in human cells. Cascade and Cas3 together induce heterogeneous DNA lesions upstream of a single CRISPR-targeted site, highlighting their potential utilities for long-range genome manipulation and deletion screen.
Clustered regularly interspaced short palindromic repeats (CRISPRs) and the cas (CRISPR-associated) operon form an RNA-based adaptive immune system against foreign genetic elements in prokaryotes. ...Type I accounts for 95% of CRISPR systems, and has been used to control gene expression and cell fate. During CRISPR RNA (crRNA)-guided interference, Cascade (CRISPR-associated complex for antiviral defence) facilitates the crRNA-guided invasion of double-stranded DNA for complementary base-pairing with the target DNA strand while displacing the non-target strand, forming an R-loop. Cas3, which has nuclease and helicase activities, is subsequently recruited to degrade two DNA strands. A protospacer adjacent motif (PAM) sequence flanking target DNA is crucial for self versus foreign discrimination. Here we present the 2.45 Å crystal structure of Escherichia coli Cascade bound to a foreign double-stranded DNA target. The 5'-ATG PAM is recognized in duplex form, from the minor groove side, by three structural features in the Cascade Cse1 subunit. The promiscuity inherent to minor groove DNA recognition rationalizes the observation that a single Cascade complex can respond to several distinct PAM sequences. Optimal PAM recognition coincides with wedge insertion, initiating directional target DNA strand unwinding to allow segmented base-pairing with crRNA. The non-target strand is guided along a parallel path 25 Å apart, and the R-loop structure is further stabilized by locking this strand behind the Cse2 dimer. These observations provide the structural basis for understanding the PAM-dependent directional R-loop formation process.
Prostate cancer (PCa) is one of the most prevalent cancers in men worldwide, and hormonal therapy plays a key role in the treatment of PCa. However, the drug resistance of hormonal therapy makes it ...urgent and necessary to identify novel targets for PCa treatment. Herein, dual-specificity tyrosine phosphorylation-regulated kinase 2 (DYRK2) is found and confirmed to be highly expressed in the PCa tissues and cells, and knock-down of DYRK2 remarkably reduces PCa burden in vitro and in vivo. On the base of DYRK2 acting as a promising target, we further discover a highly selective DYRK2 inhibitor YK-2-69, which specifically interacts with Lys-231 and Lys-234 in the co-crystal structure. Especially, YK-2-69 exhibits more potent anti-PCa efficacy than the first-line drug enzalutamide in vivo. Meanwhile, YK-2-69 displays favorable safety properties with a maximal tolerable dose of more than 10,000 mg/kg and pharmacokinetic profiles with 56% bioavailability. In summary, we identify DYRK2 as a potential drug target and verify its critical roles in PCa. Meanwhile, we discover a highly selective DYRK2 inhibitor with favorable druggability for the treatment of PCa.
Cas1 integrase associates with Cas2 to insert short DNA fragments into a CRISPR array, establishing nucleic acid memory in prokaryotes. Here we applied single-molecule FRET methods to the ...Enterococcus faecalis (Efa) Cas1-Cas2 system to establish a kinetic framework describing target-searching, integration, and post-synapsis events. EfaCas1-Cas2 on its own is not able to find the CRISPR repeat in the CRISPR array; it only does so after prespacer loading. The leader sequence adjacent to the repeat further stabilizes EfaCas1-Cas2 contacts, enabling leader-side integration and subsequent spacer-side integration. The resulting post-synaptic complex (PSC) has a surprisingly short mean lifetime. Remarkably, transcription effectively resolves the PSC, and we predict that this is a conserved mechanism that ensures efficient and directional spacer integration in many CRISPR systems. Overall, our study provides a complete model of spacer acquisition, which can be harnessed for DNA-based information storage and cell lineage tracing technologies.
Type I CRISPR-Cas systems employ multi-subunit effector Cascade and helicase-nuclease Cas3 to target and degrade foreign nucleic acids, representing the most abundant RNA-guided adaptive immune ...systems in prokaryotes. Their ability to cause long fragment deletions have led to increasing interests in eukaryotic genome editing. While the Cascade structures of all other six type I systems have been determined, the structure of the most evolutionarily conserved type I-B Cascade is still missing. Here, we present two cryo-EM structures of the Synechocystis sp. PCC 6714 (Syn) type I-B Cascade, revealing the molecular mechanisms that underlie RNA-directed Cascade assembly, target DNA recognition, and local conformational changes of the effector complex upon R-loop formation. Remarkably, a loop of Cas5 directly intercalated into the major groove of the PAM and facilitated PAM recognition. We further characterized the genome editing profiles of this I-B Cascade-Cas3 in human CD3
T cells using mRNA-mediated delivery, which led to unidirectional 4.5 kb deletion in TRAC locus and achieved an editing efficiency up to 41.2%. Our study provides the structural basis for understanding target DNA recognition by type I-B Cascade and lays foundation for harnessing this system for long range genome editing in human T cells.
CRISPR-Cas systems confer an adaptive immunity against viruses. Following viral injection, Cas1-Cas2 integrates segments of the viral genome (spacers) into the CRISPR locus. In type I CRISPR-Cas ...systems, efficient “primed” spacer acquisition and viral degradation (interference) require both the Cascade complex and the Cas3 helicase/nuclease. Here, we present single-molecule characterization of the Thermobifida fusca (Tfu) primed acquisition complex (PAC). We show that TfuCascade rapidly samples non-specific DNA via facilitated one-dimensional diffusion. Cas3 loads at target-bound Cascade and the Cascade/Cas3 complex translocates via a looped DNA intermediate. Cascade/Cas3 complexes stall at diverse protein roadblocks, resulting in a double strand break at the stall site. In contrast, Cas1-Cas2 samples DNA transiently via 3D collisions. Moreover, Cas1-Cas2 associates with Cascade and translocates with Cascade/Cas3, forming the PAC. PACs can displace different protein roadblocks, suggesting a mechanism for long-range spacer acquisition. This work provides a molecular basis for the coordinated steps in CRISPR-based adaptive immunity.
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•Cascade diffuses on DNA and transiently engages potential targets•Cascade/Cas3 stalling at protein roadblocks results in DNA double strand breaks•Cascade, Cas3, and Cas1-Cas2 form the primed acquisition complex (PAC)•The PAC displaces protein roadblocks in search of downstream protospacers
Single-molecule studies illuminate how the type I-E CRISPR-Cas interference and adaptation complexes interact and function to achieve primed spacer acquisition.
The COVID-19 pandemic is still affecting many people worldwide and causing a heavy burden to global health. To eliminate the disease, SARS-CoV-2, the virus responsible for the pandemic, can be ...targeted in several ways. One of them is to inhibit the 2'-
-methyltransferase (nsp16) enzyme that is crucial for effective translation of viral RNA and virus replication. For methylation of substrates, nsp16 utilizes
-adenosyl methionine (SAM). Binding of a small molecule in the protein site where SAM binds can disrupt the synthesis of viral proteins and, as a result, the replication of the virus. Here, we performed high-throughput docking into the SAM-binding site of nsp16 for almost 40 thousand structures, prepared for compounds from three libraries: Enamine Coronavirus Library, Enamine Nucleoside Mimetics Library, and Chemdiv Nucleoside Analogue Library. For the top scoring ligands, semi-empirical quantum-chemical calculations were performed, to better estimate protein-ligand binding enthalpy. Relying upon the calculated binding energies and predicted docking poses, we selected 21 compounds for experimental testing.
CRISPR-Cas nucleoproteins target foreign DNA via base pairing with a crRNA. However, a quantitative description of protein binding and nuclease activation at off-target DNA sequences remains elusive. ...Here, we describe a chip-hybridized association-mapping platform (CHAMP) that repurposes next-generation sequencing chips to simultaneously measure the interactions between proteins and ∼107 unique DNA sequences. Using CHAMP, we provide the first comprehensive survey of DNA recognition by a type I-E CRISPR-Cas (Cascade) complex and Cas3 nuclease. Analysis of mutated target sequences and human genomic DNA reveal that Cascade recognizes an extended protospacer adjacent motif (PAM). Cascade recognizes DNA with a surprising 3-nt periodicity. The identity of the PAM and the PAM-proximal nucleotides control Cas3 recruitment by releasing the Cse1 subunit. These findings are used to develop a model for the biophysical constraints governing off-target DNA binding. CHAMP provides a framework for high-throughput, quantitative analysis of protein-DNA interactions on synthetic and genomic DNA.
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•CHAMP enables massively parallel profiling of protein-nucleic acid interactions•CHAMP was used to measure off-target DNA binding by a CRISPR-Cas complex•Cascade decodes extended PAMs and binds DNA with a 3-nt periodicity•Cas3 binding is dependent on the PAM and PAM-proximal crRNA-DNA mismatches
Discarded next-gen sequencing chips provide a platform for analyzing protein-DNA interactions that reveals a novel proofreading mechanism used by the Cascade/Cas3 complex.
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