DNA-guided CRISPR–Cas12a effectors for programmable RNA recognition and cleavage – Nature Biotechnology

Materials

Acidaminococcus sp. Cas12a (AsCas12a) and Lachnospiraceae bacterium Cas12a (LbCas12a) were from Tolo Biotech. AsCas12a Ultra was from Integrated DNA Technologies, Inc. EnGen LbaCas12a and NEbuffer r2.1 were from New England Biolabs. The RNA sequences and ssDNA sequences (Supplementary Table 1) were from GENEWIZ. HEPES buffer, magnesium chloride, Lipofectamine 3000 and UltraPure diethyl pyrocarbonate (DEPC)-treated H₂O were from Thermo Fisher Scientific. TwistAmp basic kit was from TwistDx. DNA amplification powder kit and RNA-amplification powder kit were from Amp-future. Ribonucleotide solution mix, deoxynucleotide solution mix, RNase inhibitor and T7 RNA polymerase were from New England Biolabs. TIANamp Virus DNA/RNA Kit was from Tiangen. PCR amplification premix kit was from Takara, MolPure Flash Cell/Tissue Total RNA Kit and RT–qPCR mix Hifair Advanced One Step RT–qPCR SYBR Green Kit were from Yeasen Biotechnology. RNA sequencing was performed by GENEWIZ. BeyoBlue Plus Coomassie Blue Super-Fast Staining Solution was from Beyotime. HEK293T (IM-H222) cells were from Xiamen Immocell Biotechnology Co., Ltd.

RNA cleavage assays

Unless otherwise stated, cleavage reactions contained 5′-FAM-labeled target RNA or label-free target RNA (100 nM), 200 nM Cas12a and 400 nM crDNA. The Cas12a–crDNA complex was first prepared, incubated for 5 min at room temperature before being mixed with target RNA, incubated at 37 °C for 60 min then resolved by 5% native PAGE (37.5: 1) at 4 °C (0.25× TBE buffer), 250 V for 12 min. RNA was visualized using a Bio-Rad gel imaging system.

RNA cleavage site mapping

A hydrolysis ladder (OH⁻) was obtained by incubating 0.5 μl 100 μM 5′-FAM labeled target ssRNA in hydrolysis buffer at 95 °C for 30 min, before quenching on ice. The control ladder was a synthetic 5′-FAM-labeled 24-nt RNA fragment derived from target ssRNA.

The reaction was quenched by Inactivation/Precipitation Buffer (Thermo Fisher Scientific) and precipitation by microcentrifuge at 38,200g for 15 min at 4 °C before adding RNA gel-loading buffer (90% glycerol and 10% formamide). All products were resolved by 15% denaturing PAGE.

Electrophoretic mobility shift assays

Unless otherwise stated, to avoid dissociation of the Cas12a–crDNA or Cas12a–crRNA, complex at low concentrations during target ssRNA binding experiments, binding reactions contained a constant excess ratio of Cas protein and crDNA/crRNA/gRNA (2:1), and 50 nM of 5′-FAM-labeled target ssRNA. Reactions were incubated at 37 °C for 60 min before being resolved by 6% native PAGE (37.5:1) at 4 °C (0.25× TBE buffer), 120 V for 60 min. RNA and DNA were visualized by Bio-Rad gel imaging system.

For ternary complex analysis, binding reactions contained constant 5′-FAM-labeled target RNA (50 nM), then preparation of 200 nM Cas12a–crDNA, or Cas12a–crRNA complex (at a ratio of 2:1) and incubating for 30 min at room temperature, then gradient dilution to target concentration.

For binary complex analysis, binding reactions contained constant crDNA (50 nM), and increasing concentrations of Cas12a.

Trans-cleavage activity analysis

Unless otherwise stated, 5′-FAM- and 3′-BHQ1-labeled ssDNA probes were synthesized by GENEWIZ. For the general system, reactions were assembled with 50 nM Cas12a protein, 100 nM crDNA, 0.10 pM–20 nM activators and 500 nM probes in a total volume of 20 μl in PCR tube (Bio-Rad). The reaction was performed at 37 °C, with the buffer containing 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl₂ and 10 mg ml⁻¹ bovine serum albumin (BSA). The fluorescent signal (485 nm excitation and 535 nm emission) was monitored with a qPCR system (Bio-Rad) with an interval time of 1 min.

Michaelis–Menten modeling

To measure the trans-cleavage kinetics of DNA-guided and RNA-guided Cas12a enzymes, we first prepared 1 μM solutions of the Cas12a–crDNA or Cas12–crRNA complex. This was achieved by incubating a mixture of 1 μM synthetic crDNA or crRNA with at least twofold excess of the corresponding Cas12a at 4 °C for 5 min on an ice plate. Cas12a–crDNA or Cas12–crRNA complexes were then activated for trans-cleavage activity by mixing and incubating these complexes with synthetic RNA or dsDNA at 100 nM concentration at 4 °C for 5 min on an ice plate and then incubating on a heating plate at 37 °C for 30 min. The latter step yielded a solution with an activated Cas12a concentration of 100 nM. We performed the trans-cleavage kinetics assay using 1 nM activated Cas12a and varied ssDNA reporter concentrations of 1,000; 2,000; 4,000; 8,000; 16,000 and 32,000 nM. Two replicates were taken for each concentration. The reaction velocities versus reporter concentration data were fitted to the Michaelis–Menten equation using GraphPad Prism v.10 to obtain the k_cat.

Limited proteolysis

Limited trypsin proteolysis assays were performed using 2.5 µg of purified AsCas12a in the absence or presence of crDNA at a 1:1.2 molar ratio. Protein samples were incubated with trypsin at a protease-to-substrate mass ratio of 1:100. Proteolysis reactions were carried out at 37 °C and quenched after 10 min by the addition of SDS–PAGE loading buffer. Reaction products were resolved by SDS–PAGE on precast 5% polyacrylamide gels and visualized by Coomassie staining using BeyoBlue Plus Coomassie Blue Super-Fast Staining Solution.

ssRNA, ssDNA or dsDNA target binding K
_d
2 measurement using fluorescence polarization

Unless otherwise stated, FAM-labeled RNA or DNA oligonucleotides were procured from GENEWIZ. To prepare dsDNA substrates, the oligos were annealed at a concentration of 10 µM in an annealing buffer composed of 10 mM Tris-HCl (pH 7.5) and 100 mM NaCl. The solution was heated at 95 °C for 5 min and allowed to cool down gradually to room temperature. Once annealed, the dsDNA was diluted to a final concentration of 20 nM using a binding buffer containing 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, and 10 mg ml⁻¹ BSA.

To form the Cas DNP, or Cas RNP, Cas12a and its respective crDNA or crRNA were mixed at concentrations of 2 µM and 1 µM, respectively, in the binding buffer. This mixture was then incubated at room temperature (~25 °C) for 30 min. The DNP or RNP were subsequently serially diluted using the binding buffer. Equal volumes of the diluted DNP or RNP and target substrates were combined, resulting in a final concentration of 10 nM target. The reactions were incubated at room temperature for an additional 60 min. Fluorescence polarization reading of FAM fluorophore was then taken using the Molecular Devices FlexStation v.3 Multi-mode Microplate Reader (Thermo Fisher Scientific).

crDNA or crRNA binding K
_d
1 measurement using fluorescence polarization

Unless otherwise stated, FAM-labeled crDNA or crRNA oligonucleotides were procured from GENEWIZ. Cas12a was serially diluted using the binding buffer containing 10 mM Tris-HCl (pH 7.9), 50 mM NaCl, and 10 mg ml⁻¹ BSA. Equal volumes of the diluted Cas12a and crDNA or crRNA were combined, resulting in a final concentration of 10 nM guide nucleic acid. The reactions were allowed to incubate at room temperature for 60 min. Fluorescence polarization reading of FAM fluorophore was then taken using the Molecular Devices FlexStation 3 Multi-mode Microplate Reader (Thermo Fisher Scientific).

Recombinase polymerase amplification

In the RPA reaction, a 50 μl RPA reaction mixture contained 29.5 μl primer-free rehydration buffer, 1.2 μl forward primer (20 μM), 0.75 μl reverse primer (20 μM), 5 μl sample, 2.5 μl Mg acetate (280 mM) and 11.05 μl DNase/RNase-free water. The reaction was then incubated in 37 °C for 20 min.

In the RT–RPA reaction, a 50 μl RT–RPA reaction mixture contained 29.5 μl primer-free rehydration buffer, 1.2 μl forward primer (20 μM), 0.75 μl reverse primer (20 μM), 5 μl sample, 2.5 μl Mg acetate (280 mM), 1 μl Script IV RT (200 U μl⁻¹) and 10.05 μl DNase/RNase-free water.

SLEUTH detection

An optimized 20-µl DNA-guided CRISPR–Cas12a reaction was formulated, consisting of 50 nM Cas12a, 500 nM crDNA, 500 nM fluorescence reporter, 1× NEB r2.1 buffer and 2 µl of amplification solution placed on the lid. The amplification reaction also included 0.125 U µl⁻¹ T7 RNA polymerase, 1× T7 RNA polymerase buffer, 0.25 mM rNTP, 0.48 µM primers and 1.6 µl of RPA or RT–RPA rehydration mixture (RPA kit from Amp-Future). The tube lid was incubated at 40 °C for 15 min to promote the production of RNA amplicons. Subsequently, 2 µl of the amplification solution was spun down and incubated at 37 °C for 30 min to allow the DNA-guided CRISPR–Cas12a reaction to proceed.

SARS-CoV-2 clinical sample collection

SARS-CoV-2 raw samples were obtained and inactivated in the Prince of Wales Hospital (Hong Kong). Clinical samples were pretreated for pure RNA collection using a TIANamp Virus DNA/RNA Kit (Tiangen; Beijing; China) and a fast column extraction kit within 20 mins.

SARS-CoV-2 detection with RT–qPCR

First, a 10 μl reaction mixture contained 0.1 μl Script IV reverse transcriptase enzyme, 0.5 μl reverse primer (10 μM), 1 μl dNTP mix (10 μM), 5 μl RNA template and 3.4 μl DEPC-treated H₂O was incubated for 10 mins at 50 °C. Notably, the RNA template-primer mix was preheated at 65 °C for 5 mins, and then incubated on ice for at least 1 min for annealing before reaction. Second, SARS-CoV-2 raw samples were validated with Takara Taq premix kit. In the qPCR reaction, a 25 μl reaction mixture contained 12.5 μl premix Taq, 0.5 μl forward primer (10 μM), 0.5 μl reverse primer (10 μM), 1 μl probe (10 μM), 2 μl cDNA template and 8.5 μl DEPC-treated H₂O. The qPCR program was 95 °C for 30 s, then 95 °C for 5 s, 60 °C for 20 s, 72 °C for 10 s for 40 cycles.

Analysis of SLEUTH fluorescence data

Each SLEUTH Cas12a cleavage reaction was added to a 100 μl tube and reacted in Bio-Rad PCR machine. Fluorescence measurements (FAM channel for SYBR Green based melting curve analysis or HEX channel for trans-cleavage signal) were then obtained every 60 s for a period of 1 h using the CFX Opus real-time PCR machine (Bio-Rad). The fluorescence signal datapoints were exported directly from the machine system that are background-subtracted signal treated signal. The s.e.m. (s.d.) of duplicates/triplicates are shown as error bars.

Plasmid construction

For mammalian expression, AsCas12a and mCherry were cloned into a pcDNA3.1 vector and linked by a P2A peptide to enable coexpression from a single transcript. EGFP was cloned separately into a pcDNA3.1 vector. Plasmid synthesis and assembly were carried out by GENEWIZ. Full construct sequences are provided in Supplementary Table 1.

RNA silencing reagent preparation

Complete Dulbecco’s Modified Eagle Medium (DMEM) was prepared by combining 445 ml of basal DMEM with 50 ml of fetal bovine serum and 5 ml of penicillin–streptomycin in a sterile mixing vessel. The mixture was inverted gently until homogeneous, then stored at 2–8 °C for up to 3 months. To prepare 1× phosphate-buffered saline (PBS), 5 ml of 10× PBS stock solution was diluted with 45 ml of ultrapure water in a sterile container, mixed by gentle inversion and stored at room temperature for up to 6 months.

Cell culture

HEK293T cells were maintained in complete DMEM and passaged every 2–3 days at a 1:3 split ratio. Confluent cultures in 6 cm dishes (approximately 5 × 10⁶ cells per dish) were monitored by phase-contrast microscopy for normal morphology and growth. For subculturing, approximately 30% of the total cell population (≈1.5 × 10⁶ cells) was harvested by aspirating medium, trypsinizing for 2 min at 37 °C, and neutralizing with twice the volume of complete DMEM. Cells were collected by centrifugation at 90g for 3 min, the supernatant was removed, and the pellet resuspended gently in 1 ml of complete DMEM. The cell suspension was then transferred into a fresh 6 cm dish containing 5 ml of prewarmed medium and incubated at 37 °C with 5% CO₂.

Cell transfection

For EGFP reporter system, 1 × 10⁵ cells per well were seeded in 48-well plates 24 h before transfection. Then, 200 ng of EGFP reporter plasmid, 400 ng of AsCas12a-mCherry or 0.3 µl of crDNA (100 µM) were mixed with 0.45 µl of Lipofectamine 3000, 0.6 µl P3000 (except crDNA) and 30 µl of Opti-MEM Reduced Serum Medium, respectively. Complexes were allowed to form for 15 min and then added dropwise to each well. Transfected cells were harvested 12 h post-transfection for flow cytometry, fluorescence microscopy observation and qPCR analysis.

For endogenous gene knockdown, 1 × 10⁵ cells per well were seeded in 48-well plates 24 h before transfection. Then, 400 ng of AsCas12a-mCherry or 0.3 µl of crDNA (100 µM) were mixed with 0.45 µl of Lipofectamine 3000, 0.6 µl P3000 (except crDNA) and 30 µl of Opti-MEM Reduced Serum Medium, respectively. Complexes were allowed to form for 15 min and then added dropwise to each well. Transfected cells were harvested 12 h post-transfection for qPCR and transcriptome sequencing analysis.

Flow cytometry for quantification of EGFP expression

At 12 h after transfection as described above, cells were trypsinized with 1× Trypsin-EDTA. Then the cells were resuspended in cytometry buffer (0.6% BSA, 2 mM EDTA, 10 mM HEPES, pH 7.4 in 1× DPBS). Each sample was passed through a 35-µm cell strainer. Cells were then analyzed in a BD FACSDiscover S8 Cell Sorter. FCS files were then analyzed in FloJo v.10.10 to obtain the MFI of EGFP fluorescence in mCherry–cy5-positive cells.

RT–qPCR for relative quantification of mRNA

Total RNA was extracted from samples using the MolPure Flash Cell/Tissue Total RNA Kit (Yeasen Biotech) according to the manufacturer’s instructions. Purified RNA was subsequently subjected to one-step RT–qPCR using the Hifair Advanced One Step RT–qPCR SYBR Green Kit. RT–qPCR was performed under the following cycling conditions: reverse transcription at 50 °C for 20 min, initial denaturation at 95 °C for 5 min, followed by 40 cycles of denaturation at 95 °C for 15 s and annealing/extension at 60 °C for 30 s. Fluorescence signals were acquired using a Bio-Rad real-time PCR system. Relative gene expression levels were quantified using the comparative Ct (2^−ΔΔCt) method, with the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) gene serving as the internal reference transcript.

Structure prediction pipeline using AlphaFold3-guided molecular dynamic simulation

The initial crystal structures for Acidaminococcus sp. Cas12a (AsCas12a) were retrieved from the Protein Data Bank (PDB) under accession codes 4UN3 and 5B43, respectively.

To predict the 3D structure of our crDNA-guided RNA targeting tri-complex with cas12a, we used the following computational workflow: first, the primary sequences of the target DNA, guide RNA and the cas12a protein were submitted to the AlphaFold server for structure prediction. The resulting protein–nucleic acid complex model was then optimized and prepared for molecular dynamics simulation using the CHARMM-GUI webserver, generating a GROMACS-compatible topology. The system underwent energy minimization followed by equilibration under constant number of particles, volume and temperature (NVT ensemble) and constant number of particles, pressure and temperature (NPT ensemble). Finally, a 300-ns molecular dynamics simulation was performed to allow structural relaxation and to refine the binding interactions within the predicted complex. Similar workflow was adopted for PAM-related mutation with AsCas12a as a binary complex prediction.

Cryo grid preparation and data collection

AsCas12a Ultra (6.4 µM) was incubated with crDNA (final 13 µM) and RNA (final 13 µM) in 10 mM Tris-HCl pH 8.0 and 50 mM NaCl at 37 °C for 30 min to form the ternary complex. Then, 4 μl of 2.5-fold diluted ternary complex was then applied to holey carbon grids (C-flat R1.2/1.3 Au, 300 mesh) after glow discharge (15 mA, 45 s). After incubation for 10 s at 4 °C and 100% humidity, the grids were blotted for 4 s with blotting paper, and then plunged quickly into liquid ethane cooled by liquid nitrogen with a Mark IV Vitrobot (Thermo Fisher Scientific). The grids were loaded into a FEI Titan Krios G3i electron microscope (Thermo Fisher Scientific) equipped with a high-brightness field emission gun operated at 300 kV. Images were collected automatically with a K3 Summit direct electron detector (Gatan) using EPU software (Thermo Fisher Scientific) in counting mode and magnification of ×81,000, corresponding to a 1.05 Å physical pixel size. The slit width of the Gatan Imaging Filter (GIF) Bio Quantum was set to 20 eV. The defocus ranges, dose rate and other parameters during image acquisition of the dataset are listed in Supplementary Table 4.

Drift correction and CTF correction were performed in CryoSPARC v.4.7.1⁵⁵. Motion-corrected sums with dose-weighting were used for all subsequent image processing. Blob picking on 500 micrographs was first used to obtain the initial particle stack. After cleaning with two-dimensional classification and heterogenous refinement, the particles of classes with clear AsCas12a features were used as templates to pick on the full 2,030 micrographs. Two rounds of heterogenous refinements were then used to obtain a clean stack of 1,305,848 particles, which showed preferred orientation. Rebalance orientations in CryoSPARC v.4.7.1⁵⁶ was used twice to remove excessive side views, resulting in 277,781 particles as the final stack to reconstruct the consensuses map for subsequent 3D classifications in CryoSPARC v.4.7.1⁵⁵ and Relion v.5⁵⁶. The data processing flowchart is shown in Supplementary Fig. 9.

Model building

Models of PDB 8SFL were first docked into the final map using fit in map function of ChimeraX v.1.9⁵⁷. The models of bound crDNA and RNA were then fitted into the density, matching the distinct purine/pyrimidine patterns and their known sequences. Coot v.0.9.8.96⁵⁸ was used to adjust the model manually. The model was refined against the corresponding cryo-EM density map with phenix.real_space_refine module in PHENIX v.1.21.2-5419 package⁵⁹. The qualities of the final refined model were estimated with MolProbity⁶⁰. The figures on maps and refined models were generated with ChimeraX v.1.9. Refinement statistics are presented in Supplementary Table 4.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.