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Structures of apo Cas12a and its complex with crRNA and DNA reveal the dynamics of ternary complex formation and target DNA cleavage [1]

['Li Jianwei', 'Department Of Biological Sciences', 'National University Of Singapore', 'Singapore', 'Chacko Jobichen', 'Satoru Machida', 'Sun Meng', 'Randy J. Read', 'Department Of Haematology', 'University Of Cambridge']

Date: 2023-03

Abstract Cas12a is a programmable nuclease for adaptive immunity against invading nucleic acids in CRISPR–Cas systems. Here, we report the crystal structures of apo Cas12a from Lachnospiraceae bacterium MA2020 (Lb2) and the Lb2Cas12a+crRNA complex, as well as the cryo-EM structure and functional studies of the Lb2Cas12a+crRNA+DNA complex. We demonstrate that apo Lb2Cas12a assumes a unique, elongated conformation, whereas the Lb2Cas12a+crRNA binary complex exhibits a compact conformation that subsequently rearranges to a semi-open conformation in the Lb2Cas12a+crRNA+DNA ternary complex. Notably, in solution, apo Lb2Cas12a is dynamic and can exist in both elongated and compact forms. Residues from Met493 to Leu523 of the WED domain undergo major conformational changes to facilitate the required structural rearrangements. The REC lobe of Lb2Cas12a rotates 103° concomitant with rearrangement of the hinge region close to the WED and RuvC II domains to position the RNA–DNA duplex near the catalytic site. Our findings provide insight into crRNA recognition and the mechanism of target DNA cleavage.

Citation: Jianwei L, Jobichen C, Machida S, Meng S, Read RJ, Hongying C, et al. (2023) Structures of apo Cas12a and its complex with crRNA and DNA reveal the dynamics of ternary complex formation and target DNA cleavage. PLoS Biol 21(3): e3002023. https://doi.org/10.1371/journal.pbio.3002023 Academic Editor: Jacob E. Corn, ETH Zurich, SWITZERLAND Received: May 5, 2022; Accepted: February 6, 2023; Published: March 14, 2023 Copyright: © 2023 Jianwei et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: Structural data is deposited at the PDB, including the X-ray crystal structure of Lb2Cas12a (PDB: 8H9D) and cryo-EM structure of the Lb2Cas12a+crRNA+DNA ternary complex (PDB: 8I54). Funding: J.S. acknowledges support from Ministry of Education Singapore (https://www.moe.gov.sg/) grants R-154-000-C07-114 (Tier 1) and R154-000-A39-112 (Tier 2). RJR acknowledges the support of the Wellcome Trust (grant 209407/Z/17/Z). L J is a graduate scholar in receipt of a research scholarship from the National University of Singapore. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist. Abbreviations: BH, Bridge Helix; DETECTR, DNA Endonuclease Targeted CRISPR Trans Reporter; DLS, dynamic light scattering; dsDNA, double-stranded DNA; FRET, fluorescence resonance energy transfer; NHEJ, nonhomologous end-joining; PAM, protospacer adjacent motif; PISA, Protein Interfaces, Surfaces and Assemblies

Introduction The CRISPR–Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) system is a bacterial adaptive immune system against invading nucleic acids [1,2]. CRISPR–Cas systems have been extensively studied over the past decade [3–10], and knowledge of their unique activity has paved the way for the development of next-generation, high-throughput genome editing tools [5,11,12]. Among the various Cas enzymes, the type V-A effector protein, Cas12a, previously termed Cpf1 [3,13,14], is known to process precursor-crRNA into mature crRNA without the requirement of transactivating RNA, a small trans-encoded RNA, which mediates the maturation of crRNA in CRISPR–Cas9. Instead, Cas12a targets the invading DNA through RNA–DNA base-pairing [14,15]. Cas12a recognizes a 5′ TTTN- protospacer adjacent motif (PAM) [3,16,17] and produces staggered ends at a position 18 to 20 bp downstream of PAM [3]; this differs significantly from the well-characterized S. pyogenes Cas9, which recognizes a 3′ NGG- PAM [3,16–18] in target double-stranded DNA (dsDNA) and produces blunt ends at a position 3 bp upstream of the PAM. The double-strand breaks in dsDNA are joined by nonhomologous end-joining (NHEJ) repair. In CRISPR–Cas12a, a PAM that is 18 nt from the double-strand cleavage site provides the possibility for secondary cleavage after NHEJ repair. After cleaving the target dsDNA, Cas12a acquires indiscriminate ssDNase activity, referred to as trans-cleavage activity [17,19,20]. This property allows Cas12a to be used to detect trace amounts of DNA, for example, trace amounts of viral genome in DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR) assays [17,19–21]. Thus, CRISPR–Cas12a is an attractive alternative strategy for next-generation genome editing and diagnostics. Several Cas12a orthologs have been utilized for genome editing to date [14,22,23]. These orthologs share various structural features, as identified through X-ray crystallography and Cryo-EM structural analysis of crRNA-bound and crRNA-DNA complexes of the Cas enzyme bound to crRNA and DNA fragments. Typically, Cas12a exhibits a bilobed architecture: a recognition lobe (the REC1 and REC2 domains), and a nuclease (NUC) lobe (the WED, PI, RuvC, BH, and Nuc domains) for cleaving nucleic acids [24–26]. The crRNA pseudoknot structure is anchored in the WED domain and extensively interacts with the RuvC and REC2 domains [24,25]. The 3′ tail of the crRNA hybridizes to the target DNA strand, forming an R-loop [26], which is accompanied by interactions with the REC and NUC lobes. A molecular dynamics study of Francisella novicida Cas12a indicated flexibility of the PI domain when in complex with crRNA and rigidity when the PI domain is engaged as part of the Cas12a+crRNA+DNA ternary complex [27]. Single-molecule fluorescence resonance energy transfer (FRET) studies of FnCas12a and Lachnospiraceae bacterium ND2006 (Lb) Cas12a further demonstrate that Cas12a adopts a compact shape in the crRNA-bound conformation [28,29], with the lobes undergoing slight opening upon DNA binding. Curiously, FRET studies indicate that apo Cas12a has an additional elongated conformation distinct from the semiclosed conformation of Cas12a+crRNA+DNA [28,29]. These studies collectively suggest the presence of multiple conformations in equilibrium, with respective domain flexibility differing in each conformation [28,29]. Indeed, conformational equilibrium is necessary for the activity of Cas12a, as suggested by the activity of the AcrVA4 inhibitor, which suppresses the activity of LbCas12a by making the RNA-bound compact structure rigid [30]. High structural plasticity is implicated in the mechanism of crRNA capture and substrate binding, and yet, despite the wealth of knowledge available, the structural transitions from apo Cas12a to the RNA/DNA-bound forms are not fully understood. Among the Cas12a orthologs, Lachnospiraceae bacterium MA2020 (Lb2) Cas12a is the smallest in molecular weight and recognizes a short spacer (14 nt) for cleavage and the creation of indels with high fidelity [23]. Here, we report the crystal structures of apo Lb2Cas12a and the Lb2Cas12a+crRNA binary complex as well as the cryo-EM structure of the Lb2Cas12a+crRNA+DNA ternary complex. These structures reveal distinct conformations of Lb2Cas12a at each interaction stage. Furthermore, through functional studies, we identified the mechanism of crRNA binding and targeted DNA cleavage.

Discussion In this study, we report the crystal structures of apo Lb2Cas12a and the Lb2Cas12a+crRNA binary complex, along with the cryo-EM structure of the Lb2Cas12a+crRNA+DNA ternary complex. Previous negative-staining EM [25] and SAXS (small-angle X-ray scattering) data [39] suggest that apoCas12a orthologs adopt an elongated shape. Our crystal structure reveals that Apo Lb2Cas12a exhibits a unique, elongated structure, which is supported by DLS and gel filtration chromatography profiles. Furthermore, we demonstrate that the Lb2Cas12a+crRNA binary complex adopts a compact triangular structure, which is consistent with previous findings in orthologs [25,35]. We demonstrate that structural rearrangement of the hinge loop into an α-helix (Leu477–Lys493) leads to the formation of a hydrophobic cluster among Trp871, Tyr484, Leu491, and Thr492. Meanwhile, several hydrogen bonds are formed between Asp39, Tyr149, and Asn504 and between Glu875 and Thr492, with this compact conformation further stabilized by hydrophobic interactions between the newly formed α-helix and the conserved Trp871 located between the BH and RuvC II domains. Thus, our findings indicate that the formation of the α-helix (Leu477–Lysr493)—induced by crRNA—is a prerequisite for the formation of the PAM-binding channel. The 2D class averages of negative staining images from our study suggested that apo Lb2Cas12a adopts a variety of conformations, including both elongated-open and compact-closed conformations, whereas Lb2Cas12a-crRNA adopts a uniform compact conformation. These findings indicate that the 2 lobes in apoLb2Cas12a are dynamic. We propose that the dynamic conformation is physiologically relevant for 2 reasons. First, it exposes the peptide bonds of the flexible linkers for hydrolysis. Indeed, apo Lb2Cas12a was more susceptible to degradation by trypsin than Lb2Cas12a+crRNA. Hence, we speculate that the elongated conformation of apo Lb2Cas12a improves its rate of elimination from the cell without mounting an unnecessary immune response. Second, the dynamic conformation favors the binding of crRNA, with basic residues exposed to the solvent. Notably, the DLS and gel-filtration chromatography results both suggest that the diameter of apo Lb2Cas12a varies with respect to the buffer pH. We propose that the altered surface charge that occurs in response to this varied environment triggers the opening and closing of the lobes (Fig 5). In the process of crRNA binding, the negatively charged crRNA backbone is anchored into the RNA-binding pocket through electrostatic interactions. The positively charged cavity formed by the REC lobe and the WED domain is attracted to the crRNA, and these regions are then drawn closer to the NUC lobe. The N-terminal flexible loop of the WED domain allows for rotation and translation of the REC lobe to occur commensurate with the electrostatic forces. The basic residues located in the BH region between Arg864 and Lys870 then interact with the REC2 domain to stabilize this new RNA-bound conformation. PPT PowerPoint slide

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TIFF original image Download: Fig 5. Model of crRNA binding and DNA cleavage triggering conformational changes in Lb2Cas12a. Apo Lb2Cas12a adopts dynamic conformation. The negatively charged crRNA is captured by the positively charged RNA-binding pocket and triggers the REC lobe to rotate to the NUC lobe side to complete the first conformational rearrangement. The PAM sequence on dsDNA is recognized by PI domain and forms a heteroduplex with crRNA to trigger the second conformational rearrangement of REC lobe. The catalytic sites in RuvC and Nuc domain are marked by a triangular gap, and the positively charged RNA-binding sites are indicated by a polygonal split. Different domains are distinguished by color. https://doi.org/10.1371/journal.pbio.3002023.g005 In the transition to a ternary complex, mutation of the conserved residues responsible for DNA binding of the PI domain (Pro567Ala, Lys580Ala, and Lys581Ala) was not sufficient to inactivate the complex, whereas deletion of the entire PI domain completely blocked dsDNA cleavage activity (S10C Fig). Consistently, the entire PI domain, which is connected by 2 flexible loops, rotates 38° to recognize the PAM sequence. Subsequently, the DNA unwinds and forms an R-loop structure with crRNA, which results in expansion of the channel formed by the REC and NUC lobes. The cleaved target DNA is insufficiently long to stabilize the DNA-REC lobe contact through the finger helix (234Lys–244Gln), which leads to further expansion of the channel and its subsequent preparation for trans-cleavage. PAM is recognized by the conserved PI domain through Lys575 and Lys518. These residues are conserved in the Cas12a family. Modification of these residues may allow Lb2Cas12a to recognize noncanonical PAM sequences [32]. The unique feature of Lb2Cas12a in recognizing TTNN PAM sequences is an advantage, as it provides the opportunity to target more genes using a crRNA array. In summary, in reporting the crystal structure of apo Lb2Cas12a and the Lb2Cas12a+crRNA binary complex, we have identified the conformational rearrangements and mode of crRNA binding. We demonstrate that the crRNA-bound, compact form of Lb2Cas12a is achieved through a unique 103° rotation of the REC lobe and through further interaction between the REC and NUC lobes to maintain and stabilize this compact conformation. This evidence demonstrates that crRNA binding and rearrangement of the hinge loop precedes the formation of a PAM-binding channel. Finally, the Lb2Cas12a+crRNA+DNA ternary complex structure reveals bilobed movement and rearrangement of the hinge region, including the PI and WED domains and the connective loops, demonstrating that PAM is recognized by the conserved Lys575 and Lys518 residues and that the RNA–DNA duplex is formed with a minimum of 14 bp. Overall, this study offers snapshots of the catalytic activation process, beginning with the RNA-free elongated conformation and crRNA-bound compact-closed conformation resulting in the DNA-bound, post-cleavage conformation.

Materials and methods Lb2Cas12a expression and purification Lb2Cas12a gene was purchased from Addgene and reconstituted in the pET28b vector. Lb2Cas12a or mutants with pET 28b-N-6×His tag was heterologously expressed in BL21(DE3) pLysS E. coli bacteria. The engineered bacteria in LB medium were placed in a shaker incubator at 37°C, 220 rpm until it reached an OD600 = 0.6, then added IPTG to a final concentration of 0.4 mM and cultured for 16 h at 16°C, 220 rpm. The harvested bacteria were resuspended in lysis buffer (1 mM DTT, 25 mM KH 2 PO 4 (pH7.0), 10% glycerol, 500 mM NaCl, 25 mM Tris (pH 7.4), 1 mM EDTA). The high-pressure homogenizer (Avestin) with a cooling system was used for the lysis. The lysed bacteria were centrifuged at 40,000 rpm, 4°C for 1 h by Beckman Ultracentrifuge Type 45Ti. The supernatant was incubated with RNase A at 16°C for 15 min to remove endogenous RNA. Subsequently, the harvested supernatant was purified using Ni2+ affinity chromatography column (GE Healthcare) combined with ÄKTA purification system (GE Healthcare). Further purification was carried out by size-exclusion chromatography using the HiLoad Superdex 200 26/60 prep column (GE Healthcare). The purified protein was dialyzed against 20 mM Tris-HCl, 100 mM NaCl (pH 7.4) solution until use. Crystallization and structure determination For crystallization, the purified Lb2Cas12a (10 mg/ml) and crRNA were mixed in a molar ratio of 1:2 and incubated at 4°C for 20 min. The Lb2Cas12a-crRNA complex and screening solution were mixed in 1:1 ratio and crystallized at 20°C by hanging drop vapor diffusion method. Lb2Cas12a-crRNA crystals were grown in 10 mM MgCl 2 , 0.1 M sodium cacodylate (pH 6.8), 17% PEG1000, and 1 mM DTT. The complex crystals were briefly soaked in cryoprotectant solution containing 25% w/v D- (+)-Glucose monohydrate and flash cooled at 100 K. Diffraction data were collected at the NSRRC, Taiwan TPS05A beamline at 0.99 Å wavelength. HKL2000 program [40] was used for data processing. The Matthews coefficient was 3.2 Å3/Da [41] with 61.55% solvent content and 2 molecules in the asymmetric unit. The complex structure of Lb2Cas12a-crRNA was determined by molecular replacement using Phenix-Phaser program [42] and the individual REC and NUC coordinates of Lachnospiraceae bacterium ND2006 LbCas12a (PDB: 5ID6) were used as the search models. Several rounds of model building were done using COOT program [43] followed by refinement using Phenix-Refine [44]. The final model had good stereochemistry, with 99.5% residues falling within the allowed regions of the Ramachandran plot. Although the average B factor is around 90Å2, the model has good electron density map well covering the model (S15 Fig). Molecular graphic images were prepared using CueMol2 and pymol program. Cryo-EM sample preparation and data collection Purified Lb2Cas12a, crRNA with 20 nt guide sequences, and DNA were mixed in a ratio of 1:1.5:2 and remove excess nucleic acid by gel filtration chromatography. The fresh sample was purified at 0.5 mg/ml in buffer containing 20 mM HEPES-Na (pH 7), 150 mM NaCl, 10 mM MgCl 2 , 5 mM DTT. The cryo-EM data were collected at CBIS CryoEM Facility, National University of Singapore. Four microliters of sample were applied on glow-discharged UltrAufoil R1.2/1.3 (Quantifoil) and blotted for 1 s in 22°C with 100% humidity, a wait time of 15 s, a drain time of 0 s, and a force of −5 using FEI Vitrobot Marc IV. The grid was plunge-frozen in liquid ethane cooled by liquid nitrogen. The frozen-hydrated grid was loaded into Titan Krios cryo-electron microscope equipped with Gatan K3 direct-electron counting camera and operated at 300 keV, and 35-frame movies were collected at 81,000× magnification in counting mode with a physical pixel size of 1.105 Å/pixel. The images were recorded at defocus range of 0.5 to 2.5 μm. The exposure time was 3.49 s. The dose was 45 e/Å per movie stack. The 2,560 stacks of 35-frame movies were collected, using SerialEM program (FEI; Thermo Fisher Scientific). Image processing The micrographs were pre-processed by Relion-3.1.1, and 2D- and 3D-classifications were done in cryoSPARC-3.2.0 [45]. The movie frames were aligned by MotionCor2, using Relion’s own implementation. Contrast transfer function was estimated by CTFFIND-4.1. The particles were first LoG-picked and then template-picked on the same set of micrographs. The duplicate picks were removed from the combined particle sets. The particles were extracted with box size 642 pix2 by 4-fold binning (4.420 Å/pix) in Relion-3.1.1 and imported to cryoSPARC-3.2.0. Suboptimal particle images were removed by multiple rounds of 2D classification and class selection. The selected particles were exported to Relion-3.1.1 using pyem command csparc2star.py–copy-micrograph-coordinates and re-extracted with box size 1282 pix2 by 2-fold binning (2.210 Å/pix). The particle duplicates were removed by inter-particle 30 Å cutoff. The particles were again imported to cryoSPARC-3.2.0 and subjected to initial 3D modeling by Ab-initio Reconstruction with the number of models 3 and class similarity 0, followed by Heterogeneous Refinement. The particle set belonging to the least represented class was discarded. The particle sets belonging to remaining two 3D-classes were retained and subjected to another round of Heterogeneous Refinement using the previous three 3D-classes as the volume input. After 3 times Heterogeneous Refinement, the best 3D-class reaches 5.0 Å resolution (S11 and S12 Figs). The particles were exported again using csparc2star.py to Relion-3.1.1 and re-extracted with un-binned box size 2562 pix2 (1.105 Å/pix). The volume output belonging to the best 3D-class of Heterogeneous Refinement was rescaled to angpix 1.105 and re-sized to box size 2563 pix3 using relion_image_handler and imported to Relion-3.1.1 as 3D-reference. The re-extracted particles and the imported volume were subjected to 3D auto-refinement without masking. Following mask creation and post-processing, the images were Ctf-refined 3 times in order: (1) beam tilt; (2) anisotropic magnification; and (3) defocus per particle and astigmatism per micrograph. The images were Bayesian polished with the trained optimal parameters on the original output of MotionCorr2 in Relion-3.1.1. The polished particles were used for the second 3D-auto refinement without masking. The volume output from the first 3D-auto refinement was used as reference map with initial low-pass filter 40 Å. The volume from the second 3D auto-refinement was used to create a mask with low-pass filter 20 Å, initial binarization threshold 0.004 and extension of binary map threshold by 7 pixels, adding a soft edge of 8 pixels. The new mask and the output from the second 3D auto-refinement were subjected to post-processing with automatic sharpening, resulting in 4.28 Å map. The post-processing output was used for the second round of Ctf-refinement and Bayesian polishing as described above. The polished particles, the mask, and output volume from the second 3D auto-refinement before sharpening were subjected to the third 3D auto-refinement, which resulted in map resolution 4.49 Å before sharpening and 4.09 Å after sharpening. The second polished particles were imported to cryoSPARC-3.2.0 and performed Ab initio Reconstruction and Non-uniform Refinement without further particle sorting. The map quality was improved to 3.95 Å. Lb2Cas12a/crRNA/DNA model building The initial model was prepared by using SWISS-MODEL online suite based on the structure 5XUS as a template. Phenix dock-in-map [46] was used for initial model building followed by manual model building in COOT [47] using the cryo-EM map. After initial model building, the model was refined against the EM-derived maps using the phenix.real-space-refinement tool from the PHENIX software package [46], employing rigid body, local grid, NCS, and gradient minimization. The model was used to sharpen the map in CCPEM-1.6.0, and further rebuilt by Flex-EM and ISOLDE-1.0.1. This model was then subjected to additional rounds of manual model-building and refinement which resulted in a final model-to-map cross-correlation coefficient of 0.72 for Lb2Cas12a/crRNA/DNA model. Stereo-chemical properties of the model were evaluated by Molprobity [48]. Molecular graphic images were prepared using CueMol2 (http://www.cuemol.org/en/) and Chimera programs [49]. RNA substrate in vitro transcription The primers were designed to amplify DNA fragments with T7 promotor. In vitro transcription was based on standard procedures of RiboMAX Large Scale RNA Production System T7 (Promega). The reaction mixture was further purified by 7% polyacrylamide 8 M urea denaturation TBE gel and classical ethanol precipitation [50]. Dynamic light scattering (DLS) The purified Lb2Cas12a or mutant was incubated with RNA, DNA, or RNA/DNA-duplex. Subsequently, the apo or complex was dialyzed against the pH 6.2, pH 7.4, and pH 8.0 buffers (20 mM Tris-HCl, 100 mM NaCl). Further purification was performed by size exclusion chromatography using a HiLoad 16/600 Superdex 200 prep grade preparative column (GE Healthcare). Subsequently, 5 μl sample (1 mg/ml) was used for DLS (DynaPro NanoStar) experiments that was run at 4°C. Electrophoretic mobility shift assay Lb2Cas12a wild type or mutant (0–400 nM) was incubated with crRNA (100 nM) in the buffer that contained 20 mM HEPES 10 mM MgCl 2 , 100 mM NaCl, 1 mM DTT, 4% glycerol, pH 7.4 at 4°C for 15 min. Subsequently, the incubation mixture with native PAGE loading dye was used to perform 7% native polyacrylamide gel electrophoresis. The gel was stained with SYBR Safe at room temperature for 5 min and imaged via Imaging via ChemiDoc Imaging System (Bio-Rad Laboratories). dsDNA cleavage assay To generate Biotin-tagged substrates, DNA duplex was amplified by PCR reaction using forward or reverse primers labeled with 5′ Biosg (synthesized from IDT). The 20 μl cleavage reaction mixture containing 20 mM Tris-HCl, 50 mM NaCl, 1 mM DTT, 10 mM MgCl 2 (pH 7.4), 100 nM crRNA, 50 nM dsDNA, and 100 nM Lb2Cas12a WT or mutant was incubated at 37°C for 20 min. The reaction mixture was subjected to denaturation TBE-urea 7% PAGE and imaged using SYBR Safe stain and ChemiDoc MP Imaging System. The cleaved products were excised from the gel to perform sequencing analysis. The biotin-tagged cleavage products were detected by Chemiluminescent Nucleic Acid Detection Module (Thermo, 89880) and visualized by ChemiDoc Imaging System. The reaction was performed in independent triplicates. Oligonucleotides used in this assay are shown in S8 Table. ssDNA cleavage assay Single-stranded DNA activity was detected using M13mp18 ssDNA (New England Biolabs) as the substrate. The RNA-DNA duplex-triggered ssDNA cleavage reaction was performed according to the previous protocols [17]. The Mn2+-triggered ssDNA cleavage reaction was performed as follows: the 20 μl reaction mixture containing 30 nM M13mp18 ssDNA, 20 nM Lb2Cas12a, 20 mM Tris-HCl, 50 mM NaCl, 1 mM DTT (pH 7.4), 10 mM MgCl 2 or CaCl 2 or MnCl 2 or CuCl 2 was incubated at 37°C for 15 min. The reaction mixture was separated by 1.2% agarose gel. The gel was stained with SYBR Gold and the image was captured with ChemiDoc Imaging System. 2D Classification of negative staining apo Lb2Cas12a particles and Lb2Cas12a-crRNA The concentration of apo Lb2Cas12a and Lb2Cas12a-crRNA were diluted to 0.02 mg/ml to perform negative staining, 5 μl sample was loaded onto grid (carbon film 300 mesh copper, EMS) for 60 s and excess sample was removed with filter paper. Then, 5 μl stain solution (Uranyless EM Stain, EMS) was applied on the grid for 30 s and removed with filter paper. The grid was dried at room temperature for 10 min. The images were captured by Tecnai 12 (FEI 120kV, LaB6, magnification 52Kx). Trypsinization Approximately 5 μg of purified Lb2Cas12a was incubated with crRNA at a molar ratio of 1:1 for 15 min on ice. Subsequently, 5 μg apo Lb2Cas12a and Lb2Cas12a-crRNA complex were incubated with 0.1 μg trypsin at room temperature in the 10 μl reaction buffer containing 30 mM HEPES, 150 mM NaCl, 1 mM DTT (pH 7.4), 10 mM MgCl 2 , 0.02 μg trypsin, 5 μg apo Lb2Cas12a, or Lb2Cas12a-crRNA complex. Five replicates were set up for each apo Lb2Cas12a and Lb2Cas12a-crRNA complex reaction mixture. The reaction was terminated at 1 min, 3 min, 5 min, 10 min, and 15 min by adding SDS-PAGE loading buffer and heating at 100°C for 2 min. The reaction product was resolved by SDS-PAGE gel and imaged with ChemiDoc Imaging System.

Acknowledgments The authors acknowledge the synchrotron beamline TPS05A at NSRRC, Taiwan.

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