Dynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3

Expression and purification of EcoCas3 and EcoCascade/crRNA

We employed a method to express recombinant EcoCas3 at a low temperature using a baculovirus expression system. Briefly, we cloned an EcoCas3 cDNA with a octa-histidine tag and a six asparagine-histidine repeat tag into a pFastbac-1 plasmid (Thermo Fisher Scientific, Waltham, Massachusetts, USA) according to the manufacturer’s instructions (Extended data Fig. 2a). The TEV protease recognition site was also inserted between the tags and EcoCas3 to enable tag removal. Self-ligation of the PCR product generated the mutant Cas3, such as H74A (dead nickase; dn) and S483A and T485A double mutant (dead helicase; dh) with PrimeSTAR MAX (Takara Bio, Kyoto, Japan). Coding sequences cloned in the plasmids are listed in Supplementary Table 2.

Expression of EcoCas3-tag fusion proteins in Sf9 cells. We infected Sf9 cells with baculovirus at a multiplicity of infection (MOI) of two at 28°C for 24 h. Then, we changed the culture temperature to 20°C four days after infection for protein expression. Sf9 cells were then collected and stored at -80°C until use. The expressed EcoCas3-tag fusion proteins were purified using nickel affinity resin (Ni-NTA, Qiagen, Hilden, Düsseldorf, Germany). To remove tags, purified protein was digested with TEV protease and then further purified by size-exclusion chromatography using Superdex 200 Increase 10/300 GL (Thermo Fisher Scientific) in 0.2 M NaCl, 10% glycerol, 1 mM DTT, and 20 mM HEPES-Na (pH 7.0).

Cascade from E. coli and CRISPR RNA complex (EcoCascade/crRNA) was produced as described previously 23, 38. Briefly, we cloned EcoCas11 with a hexahistidine tag and HRV3C protease recognition site, EcoCascade operon, and pre-crRNA into pCDFDuet-1, pRSFDuet-1, and pACYCDuet-1 plasmids, respectively (Extended data Fig. 2c). Sequences cloned in these plasmids are also listed in Supplementary Table 2. Then, we transformed JM109(DE3) with three plasmids to express EcoCascade/crRNA recombinant protein complex. Expressed recombinant EcoCascade-crRNA was purified using Ni-NTA resin. After removal of the hexahistidine tag by HRV3C protease, EcoCascade-crRNA was further purified by size-exclusion chromatography in 350 mM NaCl, 1 mM DTT, and 20 mM HEPES-Na (pH 7.0).

Thermal stability assay of EcoCas3

Thermal stability was evaluated by nanoDSF using the Tycho NT.6 system (NanoTemper Technologies GmbH, München, Germany) 68. Also, Thermal stability at a constant 37°C was measured by a thermal shift assay using a Mx3000p real-time PCR instrument (Agilent technologies, Santa Clara, California, USA) and SYPRO orange (Thermo Fisher Scientific) 69.

Single and double-stranded DNA preparation

To detect in vitro DNA cleavage activity of CRISPR-Cas3 proteins, targeted sequences of EMX1 with PAM variants (AAG or CCA) were cloned into a pCR4Blunt-TOPO plasmid vector (Thermo Fisher Scientific) according to the manufacturer’s protocol. For collateral DNA cleavage assays, 60 bp activator fragments of hEMX1 and mTyr (which included a target site) were designed and purchased. Targeted sequences for CRISPR-Cas3, CRISPR-Cas12a and CRISPR-Cas9 are listed in Supplementary Table 3. PAM sequence variants and targeted sequence variants were also designed to examine collateral ssDNA cleavage activity. Biotin-labeled fragments were also purchased for protein-DNA interaction analysis. For fragment analysis, fluorescence-labeled primers were designed and the DNA fragment amplified from genomic DNA of HEK293T cells using Gflex DNA polymerase (Takara-bio). Amplicons were purified using NucleoSpin Gel and a PCR Clean-up kit (Takara-bio) according to the manufacturer’s protocols. A DNA fragment for hs-AFM was also amplified with non-labeled primers. All sequences of primers and donor DNAs are listed in Supplementary Table 4 and 5, respectively.

In vitro DNA cleavage activity

To analyze DNA cleavage activity, 1.6 nM of plasmid with or without targeted sequences were added to 115 nM EcoCascade-crRNA complex, 250 nM EcoCas3, and 2.5 mM ATP in CRISPR-Cas3 working buffer (60 mM KCl, 10 mM MgCl2, 10 µM CoCl2, 5 mM HEPES-KOH, pH 7.5), as previously described 40, 41, 45. After incubation at 37°C, samples were detected by either electrophoresis or with the MultiNa microchip electrophoresis system and the DNA-12,000 kit (Shimadzu, Kyoto, Japan).

Reporter assay for DNA and RNA cleavage

To characterize Cas3 collateral nucleic acid cleavage activities, 50 nM DNA activator templates were added to 100 nM EcoCascade-crRNA complex, 250 nM EcoCas3 and 2.5 mM ATP in CRISPR-Cas3 working buffer (60 mM KCl, 10 mM MgCl2, 10 µM CoCl2, 5 mM HEPES-KOH, pH 7.5). We used the DNase Alert kit (Integrated DNA Technologies, Coralville, IA USA) and the RNase Alert kit (Integrated DNA Technologies) for detecting DNase and RNase activity, respectively. To measure the ssDNA cleavage activity, we used the qPCR reporter probe for GAPDH (the sequence is listed in Supplementary Table 4) at 125 nM. Cleavage-related change in fluorescence signal of the probe was measured every 30 s for 60 min under incubation at 37°C using a Real-time PCR system (Bio-Rad Laboratories, Hercules, California, USA). Alternatively, M13mp18 single-stranded DNA (New England Biolabs, Ipswich, Massachusetts, USA) or pBluescript plasmid were added and incubated at 37°C. Samples were then electrophoresed on an agarose gel.

DNA fragment analysis

To analyze CRISPR DNA cleavage patterns in vitro, 16 nM DNA fragments amplified from HEK293 genomic DNA were added to 160 nM EcoCascade-crRNA complex, 400 nM EcoCas3 and 2 mM ATP in CRISPR-Cas3 working buffer (60 mM KCl, 10 mM MgCl2, 10 µM CoCl2, 5 mM HEPES-KOH pH 7.5). After incubation at 37°C, DNA samples were purified by ethanol precipitation. The length of DNA in samples was measured using GeneScan 600 LIZ dye Size Standard (Thermo Fisher Scientific) via a G5 dye set filter. All data were analyzed using PeakScanner software (Thermo Fisher Scientific).

Protein-DNA interaction assay

The evaluation of binding properties between EMX-EcoCascade (analyte) and target DNAs (ligands) was performed by bio-layer interferometry (BLI) using the Octet RED 96 system (ForteBio, Sartorius BioAnalytical Instruments, Fremont, California, USA). All ligands were biotinylated (20 µM) and immobilized on streptavidin biosensors. Kinetic titration series were performed in interaction buffer (PBS with 0.01% Tween 20, 0.02% BSA). Analyte concentration was 20 µM in the interaction buffer. The association and dissociation times were both 300 sec to measure the interaction between ligands and analyte. These raw data 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 fragments were listed in Supplementary Table 5.

High-speed atomic force microscopy (hs-AFM)

hs-AFM imaging was performed in solution using a laboratory-built hs-AFM setup as described previously 55. We used small cantilevers (BLAC10DS-A2, Olympus, Tokyo, Japan) with a nominal spring constant of 0.1 N/m, resonance frequency of ∼0.5 MHz, and a quality factor of ∼1.5 in the buffer. The cantilever’s free oscillation amplitude _A_0 and set-point amplitude were set at 1−2 nm and ∼0.9 × _A_0, respectively. To observe either the pre-mixed complex of EcoCascade/crRNA and dsDNA or the artificially nicked dsDNA at high spatial resolution, hs-AFM imaging was carried out in observation buffer (5 mM HEPES-KOH, pH 7.5, 30 mM KCl, 1 mM MgCl2, 2 μM CoCl2, 10% glycerol) at room temperature (∼25°C) using 3-aminopropyltriethoxysilane treated mica as described previously 55.

To observe dynamic behaviors of EcoCascade and EcoCas3 on dsDNA, we used a mica-supported lipid bilayer (mica-SLB) as a sample substrate. To observe EcoCascade binding to a target site, a lipid composition of 90:5:5 (w/w) DPPC:DPTAP:biotin-cap-DPPE was used. We deposited 5 nM dsDNA amplicon on the mica-SLB. Three minutes later the sample surface was rinsed with 20 μl observation buffer. We then immersed the sample stage in a liquid cell containing about 60 μl observation buffer, and hs-AFM imaging was performed in a room heated to ∼30°C with a heater. We added a drop (∼6 μl) of EcoCascade to the liquid cell during the hs-AFM observations, resulting in a final concentration of ∼20 nM. To observe DNA reeling and double-strand break generation by EcoCas3, the lipid composition used was 80:10:10 (w/w) DPPC:DPTAP:biotin-cap-DPPE. EcoCascade-DNA pre-assembled with 20 nM EcoCascade in observation buffer was placed on the mica-SLB together with 2 nM DNA at 37°C for 5 min. The sample surface was rinsed with 20 μl observation buffer and then imaged with hs-AFM, with a head temperature controlled at ∼37°C using a thermostatic cover. During the hs-AFM observations, a drop (∼6 μl) of EcoCas3 and ATP mixture was added to the liquid cell, at a final concentration of ∼100 nM and ∼2 mM, respectively. Primers for the donor DNA amplicons are listed in Supplementary Table 4.

Article TitleDynamic mechanisms of CRISPR interference by Escherichia coli CRISPR-Cas3


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.

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