key: cord-0315276-2xyzgsaa
authors: Yoshimi, Kazuto; Takeshita, Kohei; Kodera, Noriyuki; Shibumura, Satomi; Yamauchi, Yuko; Omatsu, Mine; Kunihiro, Yayoi; Yamamoto, Masaki; Mashimo, Tomoji
title: Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3
date: 2021-07-18
journal: bioRxiv
DOI: 10.1101/2021.07.18.452824
sha: ffd77c5eace7e4e457cf3824d10a42e589b374a7
doc_id: 315276
cord_uid: 2xyzgsaa

Type I CRISPR-Cas3 uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. Despite many studies using cryoEM and smFRET, the precise mechanism of Cas3-mediated cleavage and degradation of target DNA remains elusive. Here we reconstitute the CRISPR-Cas3 system in vitro to show how the Escherichia coli Cas3 (EcoCas3) with EcoCascade exhibits collateral non-specific ssDNA cleavage and target specific DNA degradation. Partial binding of EcoCascade to target DNA with tolerated mismatches within the spacer sequence, but not the PAM, elicits collateral ssDNA cleavage activity of recruited EcoCas3. Conversely, stable binding with complete R-loop formation drives EcoCas3 to nick the non-target strand (NTS) in the bound DNA. Helicase-dependent unwinding then combines with trans ssDNA cleavage of the target strand and repetitive cis cleavage of the NTS to degrade the target dsDNA substrate. High-speed atomic force microscopy demonstrates that EcoCas3 bound to EcoCascade repeatedly reels and releases the target DNA, followed by target fragmentation. Together, these results provide a revised model for collateral ssDNA cleavage and target dsDNA degradation by CRISPR-Cas3, furthering understanding of type I CRISPR priming and interference and informing future genome editing tools.

TS flanked by a PAM (AAAC) or a nonPAM (TTTG) (Fig. 2d) , as previously reported 40, 47 . 185

In contrast, EcoCas3 was partially activated by a TS with a PAM (TTC) but a TS with a 186 nonPAM (GGT) prevented any activity (Fig. 2c) . We then tested TS PAM specificity for all 187 64 possible target sites (Fig. 2e) . The PAM specificities for ssDNA-activated collateral 188 cleavage were similar to those of dsDNA-activated collateral cleavage (Fig. 2a) , although the 189 activity was mostly lower for ssDNA-activated cleavages, except for when the third 190 nucleotide of the PAM was C, such as TAC, AGC, GTC, GAC, and GGC, when the relative 191 fluorescence was increased (Fig. 2e) . 192

Base-pairing between the TS and NTS leads to correct Cascade binding of the NTS, 193 accessibility of the EcoCas3 cleavage site, and degradation of the target DNA 23, 49 . We 194 observed that dsDNA containing an unpaired PAM between NTS-nonPAM (CCA) and PAM (TTC) partially activated EcoCas3 for collateral cleavage (Fig. 2f) . This is not 196 consistent with a previous report that showed dsDNA with an unpaired PAM did not activate 197 Complete R-loop formation by the Cascade/crRNA complex recruits the Cas3 209 helicase/nuclease, which repeatedly cleaves the NTS via the HD domain's single catalytic site 210 31, 32 . It remains unknown how EcoCas3 cleaves the TS and progressively degrades the 211 dsDNA substrate (Fig. 1a) . Considering the collateral non-specific ssDNA cleavage in trans, 212

we hypothesized that the TS can be cleaved in trans, following cis cleavage of the NTS after 213 target dsDNA unwinding by the helicase properties of Cas3. To test this, we designed 214 fluorescently-labeled target dsDNA substrates, 5′-NTS-FAM, and 5′-TS-TAMRA, to 215 visualize dsDNA cleavage by EcoCas3 (Extended data Fig. 9a ). In control experiments, 216

SpCas9 cleaved both NTS and TS at 3-4 nucleotides upstream of the PAM site, as expected 217 (Fig. 3a) . In contrast, the highest peak of EcoCas3 cleavage was 10-11 nucleotides 218 downstream of the PAM site on the NTS, while several peaks upstream of the PAM site 219 demonstrated repetitive cleavage of the NTS. We also observed repetitive cleavage of dozens 220 of nucleotides upstream of the TS PAM, which was likely reeled by EcoCas3 helicase 221 activity and cleaved by its trans cleavage activity (Fig. 3a) . 222

To confirm the NTS and TS cleavages mediated by nuclease/helicase activities of 223 EcoCas3, we tested a dnCas3 HD domain mutant and a dhCas3 SF2 domain mutant in the 224 dsDNA cleavage assay (Fig. 3b) . The dnCas3 mutant cleaved neither NTS nor TS, indicating 225 that the single catalytic domain of EcoCas3 plays a role in generating double-strand breaks 226 (DSBs). Notably, the dhCas3 mutant cleaved the NTS in cis, but not the TS in trans (Fig. 3b) , 227 which was not consistent with the assay's collateral cleavage results where the dhCas3 mutant 228 cleaved non-specific ssDNA in trans (Fig. 1f) . The dsDNA cleavage assay for wild-type 229

EcoCas3 and dhCas3 mutant in ATP-free reaction buffer resulted in cis cleavage of the NTS, 230 but no trans cleavage of the TS ( Fig. 3c and Extended data Fig. 9b ). Together, these results 231 indicate that the dhCas3 mutant (S483A/T485A) works as an EcoCas3 Nickase and that the helicase activity of EcoCas3 is indispensable not only for repetitive cis cleavage of the NTS, 233 but also for trans cleavage of the reeled TS. 234

To further characterize cis and trans cleavage by EcoCas3, we compared 30 sec 235 (short) and 5 min (long) incubation times for the dsDNA cleavage assay. More prolonged 236 incubation increased repetitive cleavage of the NTS in cis and the TS in trans (Extended data 237 Fig. 10a ). We also observed that progressive cis and trans cleavages showed similar patterns 238 in the repetitive experiments and the short and long incubation experiments, depending on the 239 target DNA sequence ( Fig. 3 and Extended data Fig. 10a ). The sizes of many cleaved 240 fragments were between 30-60 bps, which may be used for CRISPR adaptations as We previously reported that a single mismatch within the seed region markedly affected 246 target DNA degradation in the EcoCascade/Cas3 system 11 . We therefore investigated the 247 effect of mismatch for each nucleotide in the 32-nt spacer on collateral ssDNA cleavage 248 activity. A single mismatch in the spacer region, even within the seed region (positions 1-8), 249 resulted in little or no effect on collateral cleavage activity (Extended data Fig. 11a,b) . In the 250

LbaCas12a system, 1-3 mismatches in the seed region also did not affect collateral cleavage 251 activity (Extended data Fig. 11c) , consistent with previous reports 48, 51 . Previous in vitro 252 analysis revealed the effect of single mismatches in the target sequence, which slow the rate 253 of R-loop formation and target-strand cleavage by Cas12a 52, 53 . To investigate whether 254

Cascade-binding and R-loop-formation are linked with collateral cleavage and target DNA 255 degradation, we sought to characterize Cascade-target DNA binding kinetics using a Bio-layer interferometry (BLI) biosensor 54 . Corresponding to the collateral cleavage assay results 257 ( Fig. 2c) , crRNA-complementary TS-ssDNA showed associations with EcoCascade but not 258 with non-complementary NTS-ssDNA (Extended data Fig. 12a and Supplementary Table 1) . 259

Notably, the crRNA-complementary TS-PAM (TTC) showed higher association than that of 260 TS-nonPAM (GGT) or -PAMless (Extended data Fig. 12a ). Moreover, dsDNAs containing a 261 paired PAM (AAG-TTC) showed the maximum EcoCascade-target DNA binding (Extended 262 data Fig. 12b and Supplementary Table 1) , which corresponds to the results of the collateral 263 cleavage assay (Fig. 2f) . Unpaired PAM between TS-PAM (TTC) and NTS-nonPAM (CCA) 264 indicated a lower association, and unpaired PAM between NTS-PAM (AAG) and TS-265 nonPAM (GGT) showed little association (Extended data Fig. 12b ). Taken together, BLI can 266 provide solid information on the affinity and stability of interactions as previously reported 54 . 267

To further investigate the relationship between the R-loop-formation and EcoCas3-268 mediated collateral ssDNA cleavage and dsDNA degradation, we assayed different length R-269 loop formations, from 0-32 nucleotides (n0, n6, n12, n18, n24, n30, and n32) (Fig. 3d) . BLI 270 revealed that crRNA-DNA hybridization with 0-12 base-pairs (n0, n6, n12) including seed 271 sequences did not show any association, while 18-30 base-pairs (n18, n24, n30) produced a 272 degree of association. Furthermore, a complete match for 32 base-pairs (n32) resulted in 273 stable and emphatic Cascade binding, similar to locked R-loop formation reported previously 274 15, 19, 28 (Fig. 3e and Supplementary Table 1 ). In the collateral cleavage assay, n0, n6, and n12 275 did not show any cleavage activity, while n18-n32 R-loop formations increasingly promoted 276 trans cleavage of ssDNA (Fig. 3f ). In the dsDNA cleavage assay, n0-n24 did not show any 277 cleavage of either NTS or TS DNA (Fig. 3g ). This means that collateral cleavage does not 278 need the nicking activity on the NTS (n18 and n24). Only the n30 and n32 sequences 279 underwent repetitive cleavage on both the NTS and TS, and progressive cleavage of target 280 dsDNA substrates (Fig. 3g) . Taken together, these results show two Cascade binding modes. EcoCascade RNPs onto a 3-aminopropyl-trietoxy silane-mica surface (APTES-mica) 56 . We 294 observed that the EcoCascade RNP ran from one end to the other through the target DNA, 295

presumably searching for the right PAM site and spacer sequences ( Fig. 4b and video 1 and 296 2). We also found that many EcoCascade RNPs formed a stable multibody and stuck to the 297 expected target site. Notably, we observed a typical DNA bend at the EcoCascade-RNP-298 binding site for stable R-loop formation, as previously indicated by cryo-EM 15, 18 and 299 smFRET studies 16, 57 . During the observation periods, the EcoCascade RNPs bound tightly to 300 the target DNAs without dissociating, consistent with previous smFRET analyses 16, 20, 57 . By 301 applying excessive force, the EcoCascade RNP body was broken and separated into multiple 302

Cas effector components (Extended data Fig. 13a and video 3) . 303

Next, we injected EcoCas3 proteins after fixing the EcoCascade RNPs with the 645-304 bp target DNA in ATP-free reaction buffer to reproduce EcoCas3-mediated nicking at the target site. EcoCas3 did not make any single-strand breaks (SSBs) per se but together with 306 the Cascade RNPs, several SSB-like DNA bends at the target site were observed (Fig. 4c) . 307

Notably, the shape of DNA bending was similar to that of artificially nicked dsDNA using 308

Nb.BsrDI nicking endonucleases (Extended data Fig. 13b ). Furthermore, we observed 309 dynamic movement of the EcoCas3-Cascade RNP along the target DNA, which suddenly 310 bound to the target site and disconnected from the DNA, with the bent DNA appearing as an 311 SSB shape ( Fig. 4c and video 4) . In contrast, in ATP-containing reaction buffer, we detected 

Up until now it has been unclear how a single HD nuclease domain in Cas3 can cause DSBs 321 at target sites and long-range unidirectional deletions upstream of target sites 10, 11, 13, 15, 25, 29, 322 30 . We believe that this is the first report to use hs-AFM to capture the dynamic movements of 323 CRISPR-Cas3 interference at the single molecule level. The hs-AFM results clarify that the 324 EcoCascade/crRNA complex searches for and binds to target DNA, and recruited EcoCas3 325 bound to EcoCascade then reels and loops the target dsDNA, and subsequently cleaves it (Fig.  326   5 ). This is consistent with a reeling model in which Cas3 remains associated with Cascade to 327 cleave ssDNA by a reeling mechanism 12 . However, it remained unknown how EcoCas3 328 cleaves the reeled TS and progressively degrades the dsDNA substrate (Fig. 1a) . Our results 329 from collateral ssDNA cleavage assays and dsDNA cleavage assays revealed that Cas3 330 repeatedly cleaves the NTS by helicase activity in cis. Simultaneously, the TS reeled by the 331 helicase property of Cas3 can be cleaved by non-specific ssDNA cleavage activity in trans. 332

The hs-AFM analysis also revealed that Cascade-bound Cas3 repeatedly reels and releases 333 the target DNA upstream of the PAM site, followed by target degradation. (Extended data Fig.  334 14). Although these results are inconsistent with a translocation model (Fig. 1a) We also find that this partial Cascade binding can recruit Cas3 to mediate non-specific 353 ssDNA cleavage in trans, but can also interrupt dsDNA cleavage in cis (Fig. 3f,g) . Moreover, this collateral ssDNA cleavage tolerates mismatches within the spacer sequences (Extended 355 data Fig. 11a,b) , in contrast to our previous findings with target dsDNA cleavage 11 . Previous 356 in vivo studies have revealed that the CRISPR-Cas system acquires new spacer sequences 357 from escape mutants that carry mutations in PAM and protospacer sequences, known as 358 primed CRISPR adaptation or priming 19, 20, 57, 58 . These findings suggest that the type I 359 CRISPR system uses the collateral ssDNA cleavage for the priming process (Fig. 5 ). This is The CRISPR-Cas3 system potently degrades phage and viral DNA. It is probably 389 more powerful than Cas9 and Cas12, which carry small mutations 2-4 . However, if Cas3 is too 390 powerful, it may have the potential for self-attack, from which Cas3 must escape. EcoCas3 391 has a longer spacer sequence of 27 nucleotides compared with the 20 nucleotides of Cas9 or 392 the 24 nucleotides of Cas12, which may increase the specificity for target recognition. 393

EcoCas3 has maximal cleavage activity at 37°C, although EcoCas3 protein is sensitive to 394 temperature-dependent aggregation at 37°C (Extended data Fig. 3 ), which may also decrease 395 self-attack. The specific PAM recognition by EcoCascade can also enable escape from self-396 attack (Fig. 2) (Fig. 2) . In the CRISPR-Cas3 system, the PAM plays an important role in self-and 406 non-self-discrimination, and PAM recognition by Cas effectors is the initial step following 407 the formation of an R-loop structure with the crRNA 18, 27 . 408

In conclusion, we found that the partial binding of EcoCascade to target DNA can 

We employed a method to express recombinant EcoCas3 at a low temperature using a 516 baculovirus expression system. Briefly, we cloned an EcoCas3 cDNA with a octa-histidine 517 tag and a six asparagine-histidine repeat tag into a pFastbac-1 plasmid (Thermo Fisher 518

Scientific, Waltham, Massachusetts, USA) according to the manufacturer's instructions 519 (Extended data Fig. 2a) . The TEV protease recognition site was also inserted between the 520 tags and EcoCas3 to enable tag removal. Self-ligation of the PCR product generated the 521 were then collected and stored at -80°C until use. The expressed EcoCas3-tag fusion proteins 528 were purified using nickel affinity resin (Ni-NTA, Qiagen, Hilden, Düsseldorf, Germany). To 529 remove tags, purified protein was digested with TEV protease and then further purified by 530 size-exclusion chromatography using Superdex 200 Increase 10/300 GL (Thermo Fisher 531 Scientific) in 0.2 M NaCl, 10% glycerol, 1 mM DTT, and 20 mM HEPES-Na (pH 7.0). 532

Cascade from E. coli and CRISPR RNA complex (EcoCascade/crRNA) was 533 produced as described previously 23, 38 . Briefly, we cloned EcoCas11 with a hexahistidine tag 534 and HRV3C protease recognition site, EcoCascade operon, and pre-crRNA into pCDFDuet-1, 535 pRSFDuet-1, and pACYCDuet-1 plasmids, respectively (Extended data Fig. 2c ). Sequences 536 cloned in these plasmids are also listed in Supplementary Table 2 . Then, we transformed 537 JM109(DE3) with three plasmids to express EcoCascade/crRNA recombinant protein 538 complex. Expressed recombinant EcoCascade-crRNA was purified using Ni-NTA resin. 539

After removal of the hexahistidine tag by HRV3C protease, EcoCascade-crRNA was further 540 purified by size-exclusion chromatography in 350 mM NaCl, 1 mM DTT, and 20 mM 541 HEPES-Na (pH 7.0). 542 543 Thermal stability assay of EcoCas3 544 Thermal stability was evaluated by nanoDSF using the Tycho NT.6 system (NanoTemper 545

Technologies GmbH, München, Germany) 68 . Also, Thermal stability at a constant 37°C was 546 measured by a thermal shift assay using a Mx3000p real-time PCR instrument (Agilent 547 technologies, Santa Clara, California, USA) and SYPRO orange (Thermo Fisher Scientific) 69 . targeted sequence variants were also designed to examine collateral ssDNA cleavage activity. 557

Biotin-labeled fragments were also purchased for protein-DNA interaction analysis. For 558 fragment analysis, fluorescence-labeled primers were designed and the DNA fragment amplified from genomic DNA of HEK293T cells using Gflex DNA polymerase (Takara-bio). 560

Amplicons were purified using NucleoSpin Gel and a PCR Clean-up kit (Takara-bio) 561

according to the manufacturer's protocols. A DNA fragment for hs-AFM was also amplified 562 with non-labeled primers. All sequences of primers and donor DNAs are listed in 563

Supplementary Table 4 were both 300 sec to measure the interaction between ligands and analyte. These raw data 604 were analyzed using ForteBio analysis software. The binding sensorgram was locally fitted to a 1:1 Langmuir binding model with mass transport limitation. Sequences for the donor DNA 606 fragments were listed in Supplementary Table 5 . Table 4 . 635 To evaluate the temperature-dependent stability of purified EcoCas3, we employed a 654 modified nanoscale differential scanning fluorimetry method, nanoDSF, which determines 655 protein stability by measuring intrinsic tryptophan or tyrosine fluorescence using the Tycho 656 NT.6 system (NanoTemper Technologies GmbH) 68 the target DNA (Fig. 4b) . 737 Video 2. EcoCascade RNP sticks to the target site of the dsDNA to form R-loop architecture. 738 Video 3. Excessive forces disrupt EcoCascade RNP binding and separate the body into 739 multiple Cas effector components (Extended data Fig. 13a) . 740

Video 4. EcoCas3 combined with EcoCascade RNPs mediates nicking at the target site of the 741 600-bp DNA in ATP-free (-) reaction buffer (Fig. 4c) .

Video 5. EcoCas3-Cascade complex repeatedly reels and releases the longer side of the DNA, 743 then cleaves it with a DSB in ATP (+) reaction buffer (Extended data Fig. 14) . 744 Video 6. EcoCas3-Cascade complex reels the longer side of the DNA, then cleaves it with a 745 DSB in ATP (+) reaction buffer (Fig. 4d) . 

Evolutionary classification of CRISPR-Cas systems: a burst of 751 class 2 and derived variants

The next generation of CRISPR-Cas technologies 753 and applications

CRISPR-Cas guides the future of genetic engineering

Continues: Newly Discovered Systems Expand the CRISPR-Cas Toolkit

Programmed DNA destruction by miniature CRISPR-Cas14 760 enzymes

PAM recognition by miniature CRISPR-Cas12f nucleases triggers 762 programmable double-stranded DNA target cleavage

Repurposing endogenous type I 765

CRISPR-Cas systems for programmable gene repression

Genome editing using the endogenous type I CRISPR-Cas system in 769 <em>Lactobacillus crispatus</em>

Targeted transcriptional modulation with type I CRISPR-Cas 772 systems in human cells

Introducing a Spectrum of Long-Range Genomic Deletions in 774

Human Embryonic Stem Cells Using Type I CRISPR-Cas

CRISPR-Cas3 induces broad and unidirectional genome editing in 777 human cells

Repetitive DNA Reeling by the Cascade-Cas3

Complex in Nucleotide Unwinding Steps

Recruitment by the CRISPR RNA-Guided Cascade Complex

Cryo-EM Structures Reveal Mechanism and Inhibition of DNA 784

Targeting by a CRISPR-Cas Surveillance Complex

Structure Basis for Directional R-loop Formation and Substrate 786

Handover Mechanisms in Type I CRISPR-Cas System

Target Search by the CRISPR Surveillance Complex Cascade

Structure Reveals Mechanisms of Viral Suppressors that 791

Intercept a CRISPR RNA-Guided Surveillance Complex

Structural basis for promiscuous PAM recognition in type I-E 793

Directional R-Loop Formation by the CRISPR-Cas Surveillance 795

Complex Cascade Provides Efficient Off-Target Site Rejection

Surveillance and Processing of Foreign DNA by the Escherichia 798 coli CRISPR-Cas System

Crystal structure of the CRISPR RNA-guided 800 surveillance complex from Escherichia coli

Structural biology. Crystal structure of a 802

CRISPR RNA-guided surveillance complex bound to a ssDNA target

CasA mediates Cas3-catalyzed target degradation during 805

CRISPR RNA-guided interference

In vitro reconstitution of an Escherichia coli RNA-guided 807 immune system reveals unidirectional, ATP-dependent degradation of DNA target

Structures of the RNA-guided surveillance complex from a 813 bacterial immune system

PAM identification by CRISPR-Cas effector complexes: 815 diversified mechanisms and structures

Decision-Making in Cascade Complexes Harboring crRNAs of 817

Cas3 is a single-stranded DNA nuclease and ATP-dependent 819

helicase in the CRISPR/Cas immune system

Structure basis for RNA-guided 821 DNA degradation by Cascade and Cas3

Cas3 Protein-823 A Review of a Multi-Tasking Machine

Chemistry of Class 1 CRISPR-Cas effectors: binding, 825 editing, and regulation

Characterization and applications of Type I 827 CRISPR-Cas systems

Endogenous Type I CRISPR-Cas: From Foreign DNA Defense to 829

Structure and activity of the Cas3 HD nuclease MJ0384, an 831 effector enzyme of the CRISPR interference

High-temperature protein G 833 is essential for activity of the Escherichia coli clustered regularly interspaced short 834 palindromic repeats (CRISPR)/Cas system

Baculovirus expression systems for 837 recombinant protein production in insect cells

Structural basis for CRISPR RNA-guided DNA recognition by 839

Structural and biochemical analysis of nuclease domain of 841 clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 842 3 (Cas3)

CRISPR-Cas12a target binding unleashes indiscriminate single-844 stranded DNase activity

Nucleic acid detection with CRISPR-Cas13a/C2c2

ssODN-mediated knock-in with CRISPR-Cas for large genomic 848 regions in zygotes

CRISPR-Cas12-based detection of SARS-CoV-2

Clinical validation of a Cas13-based assay for the detection of 852 SARS-CoV-2 RNA

Rapid and accurate detection of novel coronavirus SARS-CoV-2 854 using CRISPR-Cas3. medRxiv

High-Throughput Characterization 856 of Cascade type I-E CRISPR Guide Efficacy Reveals Unexpected PAM Diversity

Mechanistic Insights into the cis-and trans-Acting DNase 859 Activities of Cas12a

CRISPR-Cas12a has 861 widespread off-target and dsDNA-nicking effects

Type I-E CRISPR-cas systems discriminate target from non-target 864

DNA through base pairing-independent PAM recognition

Detection of spacer precursors formed in vivo during primed 867 CRISPR adaptation

Target-dependent nickase activities of the CRISPR-Cas nucleases 869

Real-time observation of DNA target interrogation and product 871 release by the RNA-guided endonuclease CRISPR Cpf1 (Cas12a)

Basis for DNA Target Specificity of CRISPR-Cas12a

Bio-Layer Interferometry Analysis of the Target Binding Activity of CRISPR-Cas

Real-space and real-time dynamics of CRISPR-Cas9 visualized by 880 high-speed atomic force microscopy

Controlled immobilization of DNA 882 molecules using chemical modification of mica surfaces for atomic force microscopy: 883 characterization in air

Two distinct DNA binding modes guide dual roles of a CRISPR-885

Assembly and Translocation of a CRISPR-Cas Primed Acquisition 887

Conformational Control of Cascade 889

Interference and Priming Activities in CRISPR Immunity

Mechanism of foreign DNA selection 892 in a bacterial adaptive immune system

Massively Parallel Biophysical Analysis of CRISPR-Cas Complexes on 894

A 896 conformational checkpoint between DNA binding and cleavage by CRISPR-Cas9

Conformational control of 899 DNA target cleavage by CRISPR-Cas9

Structural Basis for Guide RNA Processing 901 and Seed-Dependent DNA Targeting by CRISPR-Cas12a

Mechanisms for target recognition and 904 cleavage by the Cas12i RNA-guided endonuclease

Structures of CRISPR Cas3 offer mechanistic insights into Cascade-907

DNA unwinding and degradation

Structural Basis for the Canonical and Non-canonical PAM 909 Recognition by CRISPR-Cpf1

Improving biosensor assay 911 development by determining sample quality with Tycho NT.6

Structure of the cross-beta spine of amyloid-like fibrils

Conformational Activation Promotes CRISPR-Cas12a Catalysis and 916