While restriction enzymes (REs) remain the gold-standard for manipulating DNA _in vitro,_ they have notable drawbacks including a dependence on short binding motifs that constrain their ability to cleave DNA substrates. Here we overcome limitations of REs by developing an optimized molecular workflow that leverages the PAMless nature of a CRISPR-Cas enzyme named SpRY to cleave DNA at practically any sequence. Using SpRY for DNA digests (SpRYgests), we establish a method that permits the efficient cleavage of DNA substrates at any base pair. We demonstrate the effectiveness of SpRYgests using more than 130 gRNAs, illustrating the versatility of this approach to improve the precision of and simplify several cloning workflows, including those not possible with REs. We also optimize a rapid and simple one-pot gRNA synthesis protocol, which reduces cost and makes the overall SpRYgest workflow comparable to that of RE digests. Together, SpRYgests are straightforward to implement and can be utilized to improve a variety of DNA engineering applications.

**Competing Interest Statement**

K.A.C. and B.P.K are inventors on patents and/or patent applications filed by Mass General Brigham that describe genome engineering technologies. B.P.K. is a consultant for Avectas Inc., EcoR1 capital, and ElevateBio, and is an advisor to Acrigen Biosciences and Life Edit Therapeutics.

### **Plasmids and oligonucleotides**

Descriptions and Addgene IDs for all plasmids used in this study are available in **Sup. Table 1**; new plasmids have been deposited with Addgene (https://www.addgene.org/Benjamin_Kleinstiver/). A list of all SpRYgest target sites is provided in **Sup. Table 2** that includes spacer sequences, PAMs, and gRNA generation methods. Oligonucleotide sequences and descriptions are available in **Sup. Table 3**. Target sites for TtAgo are listed in **Sup. Table 4**. Additional details for plasmids and oligonucleotides (oligos) are provided below in the respective sections. The SpOT-check computed off-target profiles for all gRNAs used in this study are available in **Sup. Table 5**.

### **Human cell culture**

Human HEK 293T cells (ATCC) were cultured in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% heat-inactivated FBS (HI-FBS) and 1% penicillin/streptomycin. The supernatant media from cell cultures was analyzed monthly for the presence of mycoplasma using MycoAlert PLUS (Lonza).

### **Expression of and normalization of SpCas9-containing human cell lysates**

Expression plasmids encoding WT SpCas9, SpG, and SpRY each with a -P2A-EGFP signal (RTW3027, RTW4177 and RTW4830, respectively) were used to generate human cell lysates containing SpCas9 proteins. Approximately 20-24 hours prior to transfection, 1.5×10**5** HEK 293T cells were seeded in 24-well plates. Transfections containing 500 ng of human codon optimized nuclease expression plasmid and 1.5 μL TransIT-X2 were mixed in a total volume of 50 μL of Opti-MEM, incubated at room temperature for 15 minutes, and added to the cells. The lysate was harvested after 48 hours by discarding the media and resuspending the cells in 100 μL of gentle lysis buffer (containing 1X SIGMAFAST Protease Inhibitor Cocktail, EDTA-Free (Sigma), 20 mM Hepes pH 7.5, 100 mM KCl, 5 mM MgCl2, 5% glycerol, 1 mM DTT, and 0.1% Triton X-100). The amount of SpCas9 protein was approximated from the whole-cell lysate based on EGFP fluorescence. SpCas9 lysates were normalized to 180 nM fluorescien (Sigma) based on a standard curve. Fluorescence was measured in 384-well plates on a DTX 880 Multimode Plate Reader (Beckman Coulter) with λex = 485 nm and λem= 535 nm.

### **Production of gRNAs**

The DNA substrates required to transcribe gRNAs were generated via two methods. First, plasmids for IVT of SpCas9 gRNAs were generated by annealing and ligating duplexed oligos (see **Sup. Table 3**) corresponding to spacer sequences into BsaI-digested MSP3485 for T7 promoter-driven transcription of gRNAs. The derivative pT7-spacer-gRNA plasmids were digested with HindIII (NEB) to permit run-off transcription near the 3’ end of the SpCas9 gRNA. Secondly, oligo-derived DNA templates for IVT were generated by combining a target specific oligo (encoding a T7 promoter, spacer sequence, and partial sequence of the SpCas9 crRNA) and a common SpCas9 gRNA scaffold oligo (oKAC682), and then incubating with either Klenow Fragment (3’→5’ exo-) (New England Biolabs (NEB), M0212L) in 1x NEBuffer 2 at 37 °C for 30 minutes, or Q5 polymerase (NEB) using the following program: 2 minutes 98 °C; 5 cycles of (10 seconds 98 °C, 10 seconds 65 °C, 30 seconds 72 °C); 5 minutes 72 °C. Plasmid or oligo-derived transcription templates were cleaned up using a MinElute PCR Purification Kit (Qiagen). SpCas9 gRNAs were transcribed at 37 °C for 16 hours using the T7 RiboMAX Express Large Scale RNA Production Kit (Promega). For gRNAs utilized in _in vitro_ cleavage reactions containing SpRY from human cell lysates, the 37 °C incubation was followed by the addition of 1 μL RQ1 DNase at 37 °C for 15 minutes to degrade the DNA template. The DNase treatment step was omitted when preparing most gRNAs utilized for scaled-up SpRYgest reactions with purified SpRY protein. Following transcription and optional DNase treatment, gRNAs were purified using paramagnetic beads (prepared as previously described**28**; GE Healthcare Sera-Mag SpeedBeads from Fisher Scientific, washed in 0.1X TE and suspended in 20% PEG-8000 (w/v), 1.5 M NaCl, 10 mM Tris-HCl pH 8, 1 mM EDTA pH 8 and 0.05% Tween20) and refolded by heating to 90 °C for 5 minutes and then cooling to room temperature at 1 °C every 2 seconds. Synthetic gRNAs were purchased from Synthego.

For one-pot gRNA IVT reactions, we utilized two general methods. First, gRNAs were generated using the EnGen sgRNA Synthesis Kit (NEB, E3322S) according to the manufacturer recommended protocol, or the EnGen sgRNA Synthesis Kit with increased oligo concentrations (final concentrations of 0.75 μM target-specific oligo and 0.75 μM common SpCas9 gRNA scaffold oligo (oKAC682)). The DNase step was omitted. Second, we also optimized a separate one-pot gRNA synthesis method using other commercial reagents. In this second method, 20 μL one-pot reactions were assembled containing final amounts or concentrations of 2.5 U Klenow Fragment (3’→5’ exo-), target-specific oligo at 0.5 or 1.5 μM (for standard or scaled-up reactions, respectively), common SpCas9 gRNA scaffold oligonucleotide (oKAC682) at 0.25 or 0.75 μM (for standard or scaled-up reactions, respectively), 125 μM dNTPs, 1x RiboMAX Express T7 Buffer (Promega, P1320), and 2 μL T7 Express Enzyme Mix (Promega, P1320) and incubated at 37 °C for 4 hours unless otherwise indicated. Appropriately scaled 5 μL reactions were assembled for smaller-scale one-pot reactions. For one-pot gRNAs used in SpRYgest reactions, the Promega recommended RQ1 DNase treatment was omitted and no clean-up of the gRNA was performed. To quantify gRNA yield, separate IVT reactions were performed that included the RQ1 DNase step and were purified using paramagnetic beads. Note that gRNA yield will vary based on incubation time.

### **Expression and purification of SpRY and SpRY-HF1 proteins**

_E. coli_ codon optimized SpRY and SpRY-HF1 coding sequences including an N-terminal MKIEE tag and C-terminal SV40 NLS and 6x histidine tag were synthesized (GenScript, NJ, USA) and cloned into pET28 expression vectors. The SpRY and SpRY-HF1 expression constructs and were used to express and purify the proteins as described previously**29**. Briefly, _E. coli_ strain NiCo21(DE3) (C2529H from NEB) harbouring the recombinant construct was grown in 1-2 L of LB medium with 40 μg/mL Kanamycin at 30°C until mid-log phase. Overexpression of the target protein was induced by adding IPTG to a final concentration of 0.4mM with shaking overnight at 18°C. Cells were harvested and target protein expression was assessed by SDS PAGE prior to purification. Cells were disrupted by sonication in breakage buffer (50mM Tris-HCl (pH8.0), 300mM NaCl, 1mM EDTA, 1mM DTT, 2% (v/v) glycerol) supplemented with PMSF. The supernatant was passed through HiTrap DEAE Sepharose (Cytiva, MA, USA) in column buffer (20mM Tris(pH7.5) and 250mM NaCl) followed by subsequent purification on a HisTrap HP column (Cytiva). After 16x column volume wash in buffer containing 20mM T ris pH7.5, 250mM NaCl, 40mM imidazole, target proteins were eluted using a 40mM to 750mM imidazole gradient in the same buffer. Pooled fractions containing the proteins were further purified by loading onto HiTrap heparin HP columns (Cytiva), washed with 6 column volumes of a buffer containing 20mM Tris (pH8.0), 1mM EDTA, and 1mM DTT, and eluted using a 0.25 to 2M NaCl gradient in the same buffer. Pooled fractions were dialyzed in SEC column buffer (20mM HEPES (pH8.0), 250mM KCl, and 1mM DTT) and concentrated using an Amicon® Ultra-15 Centrifugal Filter Unit with 100 kDa molecular weight cut-off. Concentrated fractions were loaded on to a HiLoad 16/600 Superdex 200 pg column (Cytiva) using a 1 mL sample loop. Size exclusion chromatography was performed in SEC column buffer with a flow rate of 0.5mL/min. Eluted fractions were assessed by SDS-PAGE, pooled, dialyzed in storage buffer (20mM Tris (pH7.5), 300mM NaCl, 0.1mM EDTA, 1mM DTT and 50% (v/v) glycerol), and stored at −20°C. Protein concentration was determined by Bradford assay using BSA for standards.

### **_In vitro_** **cleavage reactions using SpCas9 from lysates**

Plasmid KAC833 linearized with HindIII (NEB) was used as the DNA substrate for most _in vitro_ cleavage reactions unless otherwise stated. SpCas9 ribonucleoprotein (RNP) complexes were formed by mixing 9 μL of SpCas9-containing normalized whole-cell lysate (normalized to 180 nM Fluorescein) with 11.25 pmol of transcribed or synthetic gRNA, and incubating for 5 minutes at 37 °C. Cleavage reactions were initiated by the addition of 34.82 fmol of linearized plasmid (digested with HindIII (NEB)) and buffer to bring the total reaction volume to 22.5 μL with a final composition of 10 mM HEPES pH 7.5, 150 mM NaCl, and 5 mM MgCl2. Reactions were performed at 37 °C and aliquots were terminated at timepoints of 1,6, 36 and 216 minutes by removing 5 μL aliquots, mixing with 5 μL of stop buffer (50 mM EDTA and 2 mg/ml Proteinase K (NEB)), and incubating at room temperature for 10-minutes. Cleavage fragments were purified using paramagnetic beads and quantified via QIAxcel capillary electrophoresis (Qiagen). The relative abundances of substrate and products were analyzed using QIAxcel ScreenGel Software (v1.5.0.16, Qiagen) and plotted using GraphPad Prism 9 (v9.2.0).

### **_In vitro_** **cleavage reactions using purified SpRY**

Small-scale _in vitro_ cleavage reactions were performed as described above, except using 0.6-1 μM purified SpRY per reaction pool instead of 9 μL of SpCas9-containing normalized whole-cell lysate (0.6 μM in **Sup. Figs. 9a** and **9b** and 1 μM in **9c**). For scaled-up digests, 4 μg of supercoiled plasmid DNA was incubated at 37 °C for 3 hours with purified SpRY protein at a final concentration of 1 μM and IVT gRNA (prepared without DNase treatment) at a final concentration of 2 μM in Buffer 3.1 (NEB). Reactions were stopped by the addition of 1 μL of Proteinase K (NEB) and incubated at room temperature for 15 minutes. Cleavage fragments were resolved by 0.8% agarose gel electrophoresis with 1 μL of 1 kb Plus DNA Ladder (NEB) and visualized by ethidium bromide staining. Digestion products were purified using a QIAquick Gel Extraction Kit (Qiagen).

### **Molecular cloning reactions using purified SpRY**

The C-terminal P2A-EGFP sequence was added to SaABE8e or pCMV-PE2 (Addgene IDs 138500 and 132775, respectively), and the N-terminal BPNLS was added to an SpCas9 plasmid similar to pCMV-T7-SpCas9 (Addgene plasmid ID 139987) via isothermal assembly. Reactions contained approximately 5 μL of isothermal assembly mix (prepared similar to as previously described**12**) or NEBuilder HiFi (NEB), 0.01 pmol of plasmid linearized via SpRYgest, and 0.03 pmol of PCR product insert in a final volume of 10 μL, and incubated at 50°C for 60 minutes. Cloning reactions were transformed into chemically competent XL1-Blue _E. coli_ cells and grown at 37 °C for approximately 16 hours. Individual colonies were grown overnight at 37 °C, miniprepped (Qiagen), and fidelity of cloning was verified via Sanger sequencing. Saturation mutagenesis plasmid libraries for were constructed by incubating 0.02 pmol of BPK848 (linearized via SpRYgest) with 0.1 pmol of ~60bp ssDNA oligo (either oBK9102 or oBK9104) with 10 μL NEBuilder HiFi DNA Assembly Master Mix (NEB) in a final volume of 20 μL, and incubated at 50 °C for 15 minutes. NEBuilder reactions were cleaned up via MinElute (Qiagen) and eluted in 10 μL water, transformed into 100 μL of electrocompetent XL1-Blue _E. coli,_ and recovered in 3 mL of SOC for 1 h at 37 °C. Next, 2 μL of the transformation recovery media was plated on LB + chloramphenicol, where the number of colonies following overnight at 37 °C were used to estimate library complexity. The remaining recovery was grown overnight in 150 mL LB + chloramphenicol and plasmid DNA was isolated by MaxiPrep (Qiagen). The complexity of the SpCas9 catalytic and PAM domain libraries were estimated to be 77,400 and 292,600 respectively. Plasmid libraries were sequenced via Sanger sequencing and NGS. For NGS, PCR amplicons were generated from the plasmids using primer pairs oKAC1589/oKAC1590 (for the catalytic domain) or oKAC1591/oKAC1592 (for the PI domain) and sequenced on a MiSeq (Illumina) to a depth of 12,378 and 409,221 reads for the catalytic and PI domain libraries, respectively. The resulting data was analyzed using CRISPResso2**30** to generate allele tables.

### **_In vitro_** **cleavage reactions using TtAgo**

For TtAgo reactions, 5’-phosphorylated DNA guides were either purchased from Integrated DNA technologies or generated by incubating an unmodified oligonucleotide with T4 Polynucleotide Kinase (NEB) at 37 °C for 30 minutes, followed by heat-activation at 65°C for 20 min. Complexes of TtAgo programmed with ssDNA guides were prepared by combining final concentrations of 1 pmol TtAgo (NEB) and 2 pmol 5’-phosphorylated ssDNA guides and incubating at 70 °C for 20 minutes. Cleavage reactions were performed by combining TtAgo complexes with either 79.85 fmol of linearized KAC833 plasmid substrate (digested with PvuI, NEB) or 79.85 fmol supercoiled plasmid DNA (KAC1151 or MNW95) in ThermoPol buffer (NEB) with a final concentration of 10mM MgSO4. Reactions were performed at 80 °C for 60 minutes and terminated by the addition of 1 μL of Proteinase K (NEB). Cleavage fragments from pre-linearized substrates were purified using paramagnetic beads and quantified and analyzed as described above. Cleavage fragments from scaled-up plasmid DNA digests were resolved by 0.8% agarose gel electrophoresis and visualized by ethidium bromide staining.

### **Bacterial-based positive selection assay**

Target plasmids for the selection assays were generated by cloning duplexed oligonucleotides into XbaI and SphI-digested p11-lacY-wtx1 (Addgene ID 69056)**16** as previously described**7**, which contains an arabinose-inducible ccdB toxin gene. The derivative toxin-expressing plasmids contain target sites harboring either NGG or NGA PAMs (BPK740 and BPK754, respectively). To perform the selections, electrocompetent _E. coli_ BW25141(λDE3)**17** containing a toxin-expressing plasmid were transformed with BPK848-derived plasmids that express the SpCas9 variant libraries (encoding randomized codons in specified positions) in addition to a gRNA, both from separate T7 promoters. Following a 60-minute recovery in SOC media, transformations were spread on LB plates containing either chloramphenicol and 10 mM dextrose (non-selective) or chloramphenicol + 10 mM arabinose (selective). Transformation efficiency was assessed based on colony count from non-selective plates. The catalytic library selection resulted in approximately 9e4 colonies (from sampling approximately 87x library coverage). The PI domain library selections for NGG PAMs and NGA PAMs resulted in approximately 6e5 and 6.4e4 colonies (from sampling approximately 18x and 2x coverage of the libraries, respectively). Surviving colonies from selective plates were picked as single colonies for miniprep (Qiagen) followed by Sanger sequencing to verify the identities of the mutated amino acids.

Methods

The bacterial archetypal adaptive immune system, CRISPR-Cas, is thought to be non-functional in the best-studied bacterium, _Escherichia coli_ K-12. Instead, we demonstrate here that the _E. coli_ CRISPR-Cas system is active and inhibits its nine defective (i.e., cryptic) prophages. Specifically, deactivation of CRISPR-Cas via deletion of _cas2_, which encodes one of the two conserved CRISPR-Cas proteins, reduces growth by 40%, increases cell death by 700%, and prevents persister cell resuscitation; hence, CRISPR-Cas serves to inhibit the remaining deleterious effects of these cryptic prophages. Consistently, seven of the 13 _E. coli_ spacers contain matches to the cryptic prophages, and, after excision, CRISPR-Cas cleaves cryptic prophage CP4-57 and DLP-12 DNA. Moreover, we determine that the key genes in these cryptic prophages that CRISPR-Cas represses by cleaving the excised DNA include lysis protein YdfD of Qin and lysis protein RzoD of DLP-12. Therefore, we report the novel results that (i) CRISPR-Cas is active in _E. coli_ and (ii) CRISPR-Cas is used to tame cryptic prophages; i.e., unlike with active lysogens, CRISPR-Cas and cryptic prophages may stably exist.

CRISPR-Cas Controls Cryptic Prophages

### **Bacteria and growth conditions**

Bacteria (**Table S4**) were cultured in lysogeny broth**36** at 37°C. pCA24N-based plasmids**37** were retained in overnight cultures via chloramphenicol (30 μg/mL), and kanamycin (50 μg/mL) was used for deletion mutants, where applicable.

### **Spacer knockout**

To attempt to delete the CRISPR-Cas spacer region, the one-step inactivation method for single gene deletions**38** was utilized in which primers that included the sequences flanking the spacer region (**Table S5**) were used with plasmid pKD4 to allow for insertion of the kanamycin resistance cassette (1.5 kb) and FRT sites.

### **Persister cells**

Exponentially-growing cells (turbidity of 0.8 at 600 nm) were converted nearly completely to persister cells**18**,**20** by adding rifampicin (100 μg/mL) for 30 min to stop transcription, centrifuging, and adding LB with ampicillin (100 μg/mL) for 3 h to lyse non-persister cells. To remove ampicillin, cells were washed twice with 0.85% NaCl then re-suspended in 0.85% NaCl. Persister concentrations were enumerated via a drop assay**39**.

### **Single-cell persister resuscitation**

Persister cells (5 μL) were added to 1.5% agarose gel pads containing M9 glucose (0.4 wt%) medium**40**, and single-cell resuscitation was visualized at 37°C via a light microscope (Zeiss Axio Scope.A1, bl_ph channel at 1000 ms exposure). For each condition, at least two independent cultures were used with 150 to 300 individual cells used per culture.

### **Membrane integrity assay**

To determine membrane integrity, the persister cells were analyzed with the LIVE/DEAD BacLight Bacterial Viability Kit (Molecular Probes, Inc., Eugene, OR, catalog number L7012). The fluorescence signal was analyzed via a Zeiss Axioscope.A1 using excitation at 485 nm and emission at 530 nm for green fluorescence and using excitation at 485 nm and emission at 630 nm for red fluorescence.

### **qRT-PCR**

To quantify transcription from the cryptic prophage lytic genes, RNA was isolated from persister cells that were resuscitated by adding M9 glucose (0.4%) medium for 10 min then washed with 0.85% NaCl and from exponential cells grown to a turbidity of 0.8; samples were cooled rapidly using ethanol/dry ice in the presence of RNA Later. RNA was isolated using the High Pure RNA Isolation Kit (Roche). The following qRT-PCR thermocycling protocol was used with the iTaq™ universal SYBR® Green One-Step kit (Bio-Rad): 95 °C for 5 min; 40 cycles of 95 °C for 15 s, 60 °C for 1 min for two replicate reactions for each sample/primer pair. The annealing temperature was 60°C for all primers (**Table S5**).

### **qPCR**

To quantify prophage excision and the levels of DNA flanking the CRISPR-Cas cleavage sites, total DNA (100 ng) was isolated from exponentially-growing and persister resuscitating cells using an UltraClean Microbial DNA Isolation Kit (Mo Bio Laboratories). Excised cryptic prophage was quantified using primers for each prophage excisionase (**Table S5**) that only yield a PCR product upon prophage excision, and the relative amount of each target gene was determined using reference gene _purM_. The level of cryptic prophage flanking the CRISPR-Cas cleave site was quantified using primers that flank each site (**Table S5**). The qPCR reaction performed using CFX96 Real Time System. The reaction and analysis was conducted using the StepOne Real-Time PCR System (Bio-Rad).

Materials & Methods

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.

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.

Gel extractions can be a low yield affair, but overnight incubation over a spin column can increase yields. This protocol details a modified QIAquick Gel Extraction protocol.

QIAquick Gel Extraction Kit 

1. Cut the desired band out of an agarose gel using a scalpel. 

2. Weigh the slice in a snap cap tube and add 3 volumes of QG buffer per volume of gel, assuming the gel density is roughly 1µl/mg. This can be whatever gel dissolving buffer (with chaotropic salts and guanidine thiocyanate) you typically use.

3. Add 1 volume of isopropanol to the tube and vortex.

4. Place a QIAquick spin column in a collection tube. Add the sample to the column and incubate over column overnight at room temp. 

5. Centrifuge spin column for 1 minute at 13,000 RPM. 

6. Discard flow through. Repeat step 5 if the sample volume was greater than 800 µl.

7. Add 750 µl of buffer PE to column and centrifuge for 1 minute at 13,000 RPM. Discard flow through.

8. Centrifuge for an additional 1 minute to remove all wash buffer.

9. Place column in collection tube and add 50 µl EB buffer or water directly to the silica portion of the column. Incubate up to 4 minutes then centrifuge the column for 1 minute to elute DNA.

Analyze DNA using a standard Nanodrop/general spectrophotometer protocol for 260/280 absorbance ratios. Store all samples in a -20 oC freezer.

Higher Yield Gel Extractions

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