Allosteric control of type I-A CRISPR-Cas3 complexes and establishment as effective nucleic acid detection and human genome editing tools.

Detailed methods are provided in the online version of this paper and include the following: RESOURCE AVAILABILITY Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Dr. Ailong Ke ( ailong.ke@cornell.edu ).

Materials availability

All unique/stable reagents generated in this study are available from the lead contact with a completed Materials Transfer Agreement.

Data and code availability

The models and maps for the structures presented have been deposited in the PDB and EMDB. The accession numbers are listed in the key resources table . Uncropped original gel and microscopy images have been deposited on Mendeley Data: https://doi: 10.17632/ry9tf59h3k.1 . These are all publicly available as of the date of publication. This paper does not report original code. Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell lines and culture

HAP1 cell culture This GFP-tagged diploid HAP1 cell line was a gift from Yan Zhang’s lab ( Tan et al., 2022 ). Cells were cultured in DMDM (Gibco) supplemented with 10% FBS (Gibco) at 37 °C and 5% CO2 in a humidified incubator. Cells were suspended using Trypsin-EDTA solution (GIBCO) and split every 2 to 3 days.

HEK293 cell culture

HEK293 cells were cultured in DMDM (Gibco) supplemented with 10% FBS (Gibco) at 37 °C and 5% CO2 in a humidified incubator. Cells were suspended using Trypsin-EDTA solution (GIBCO) and split every 2 to 3 days. Bacterial strain Escherichia coli BL21 (DE3) E. coli BL21 (DE3) cells were used for protein production. Cells were grown in Lysogeny Broth (LB) supplemented with appropriate antibiotics. Escherichia coli BL21 AI E. coli BL21 AI cells were used for assaying for CRISPR interference by the Pfu I-A system. Cells were grown in Lysogeny Broth (LB) supplemented with appropriate antibiotics. Escherichia coli DH5 alpha E. coli DH5α was used for cloning. Cells were grown at 37 C in LB supplemented with appropriate antibiotics.

Plasmids and cloning

Plasmids, primers, and RNA guides used in this work are listed in Supplementary Tables 1 , 2 , and 3 , respectively. Cloning was performed in E. coli DH5α. The Type I-A cas operon from Pyrococcus furiosus DSM 3638 was PCR-amplified using the iproof Polymerase (BioRad) and cloned into the pCDFDuet vector, giving rise to Plasmid pCascade/Cas3. For plasmid pCRISPR (or pcrRNA), a series of synthetic constructs composed of T7 promotor, CRISPR array (repeat-spacer-repeat with Cas6 or ribozyme derived 5’ handle-spacer) were introduced into the high copy number vector pRSFDuet. For purification, cas8a was cloned into pCDFduet with a N-terminal Twin-strep tag. cas11-cas7-cas5 operon was inserted into pETDuet with a C-terminal His tag on Cas5a. Cas3 HD and Cas3 HEL were expressed individually from the pRSFDuet vector with a N-terminal His tag. All plasmids were verified by Sanger sequencing. Bacterial transformations were carried out using chemically competent cells, and transformants were selected on LB agar plate supplemented with the appropriate antibiotics. METHOD DETAILS Protein expression and purification pCDFDuet-Twin-Strep-Cas8a ( Strep R ), pETDuet- Cas11-Cas7-Cas5 ( Amp R )and pcrRNA-Cas6 ( Kan R ) were co-transformed into E. coli BL21 (DE3) cell under the appropriate antibiotic selection. A 4 liter cell culture was grown in LB medium at 37 °C until an optical density of 0.6 at 600 nm. The culture temperature was then reduced to 20°C and incubated for an additional 1 hour. Expression was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 20°C overnight. Cells were harvested by centrifugation and resuspended in 100 mL buffer A containing 50 mM HEPES pH 7.5, and 300 mM NaCl, 10% glycerol, and 2 mM β-ME. Cells were lysed by sonication, and the debris was cleared using centrifugation at 12,000 rpm for 50 min at 4 °C. The supernatant was applied onto a pre-equilibrated 4 mL streptavidin column (Twin-strep purification). After washing with 50 ml of buffer A, the protein was eluted with 20 ml buffer B (50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 2.5 mM desthiobiotin). The sample was then concentrated to 1 ml and loaded onto a Superdex 200 16/60 size-exclusion column (GE Healthcare) equilibrated with buffer C (10 mM HEPES pH 7.5, 300 mM NaCl), the peak fractions of Cascade complex were pooled and snap-frozen in liquid nitrogen for later usage. For Cas3 HD and Cas3 HEL purification, pRSFDuet-Cas3HD or Cas3HEL (Kan R ) was individually transformed into E. coli BL21 (DE3), expressed using the same procedure as described above. Cells were harvested by centrifugation and lysed by sonication in 80 ml of buffer A containing 50 mM HEPES pH 7.5, 500 mM NaCl and 20 mM imidazole, 10% glycerol, and 2 mM β-ME. The lysate was centrifuged at 12,000 rpm for 50 min at 4 °C, and the supernatant was applied to a pre-equilibrated 4 mL Ni-NTA column. After washing with 100 ml of buffer A, the protein was eluted with 20 ml buffer B (50 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 300 mM imidazole, and 2 mM β-ME), concentrated to 1.5 mL and further purified on Superdex 200 16/60 equilibrated with buffer C (10 mM HEPES pH 7.5, 300 mM NaCl), the peak fractions were pooled and snap-frozen in liquid nitrogen for later usage. RNA extraction and ureal gel running 20 μL of Cascade sample at 2 μM and 20 μL phenol-chloroform solution was mixed together and vortexed vigorously for 2 min at room temperature. The aqueous and organic phases were separated by 13,000 rpm centrifuge for 15 min at room temperature. 10 μL sample was taken from the aqueous phase (top layer), mixed with 15 μL of formamide loading dye, heat-denatured at 95 °C for 10 min, and immediately loaded to a 12% ureal-polyacrylamide (PAGE) gel. After 50 minutes of electrophoresis at 25 watts, the gel was stained with EtBr to for 10 min, destained in water for 10 minutes, and scanned with the ChemiDoc imaging system (Bio-Rad) at appropriate wavelength. In vivo assay for Cascade and Cas3–mediated interference pCDFduet-Cascade/Cas3 (StrepR), pRSFDuet-crRNA-Cas6 (KanaR)and pETDuet-Targets (AmpR)with different PAM sequence were co-transformed into the E. coli BL21AI cell line and grown on LB agarose plates containing kanamycin (50 μg/ml), ampicillin (100 μg/ml), streptomycin (30 μg/ml). After transformation, a single colony were cultured at 37°C in nonselective LB medium (Strep + Kana) to O.D.600 of 0.3, at which point the expression of Pfu Cascade, Cas3, and pre-crRNA was induced for 12 h by the addition of 0.5% L-arabinose and 1 mM IPTG. Each cell culture was then divided into two equal volumes and plated onto Strep + Kana + Amp LB plates (selective for pETDuet-target) and Strep + Kan plates (non-selective for the target plasmid) in a series of dilutions. The number of colonies on each plate was counted after overnight incubation at 37°C. The CRISPR interference efficiency was reflected in the ratio of colony-forming units on the nonselective over selective plates. Each experiment was repeated three times to calculate the s.d. The experimental procedure is illustrated in Supplementary Figure 1K and Table 1 .

Fluorescently labeled prespacer substrate preparation Fluorescent

DNA oligos ( Supplementary Table 2 ) for biochemistry were synthesized (Integrated DNA Technologies) with either a 5AmMC6 or 3AmMO label, fluorescently labeled in-house by Cy3 or Cy5-NHS dye (Lumiprobe), and annealed at equimolar amount, and native PAGE purified to remove unannealed ssDNA. Electrophoretic mobility shift assay 5 nM final concentration of fluorescently labeled target DNA was incubated with titrations of Pfu Cascade or Cas3/Cascade complex in a 20 μL total reaction volume containing 50 mM Tris pH 8.0, 150 mM NaCl, and 10% glycerol. After a 15-minute incubation at 42°C or specified condition in the figure legends,10 μL of each sample was loaded onto 1% agarose gel equilibrated in 0.5 × TBE buffer. Electrophoresis was performed at 60 V for 40 min in cold room. The fluorescent signals from the gel were recorded using a ChemiDoc imaging system (Bio-Rad). For the fluorescence labeled protein EMSA assay in Fig. 1C ., the proteins were labeled using corresponding fluorophore (Sulfo-Cy3 or Cy5, Lumiprobe) following our previously published protocol ( Xiao et al., 2017 ). The 20 μL binding experiments used 300 nM Cy3-Cas3, 100 nM Cy5-Cascade, and 20 nM FAM-DNA in single tube or combined two or three components in one tube. Incubation and electrophoresis were carried out as above.

Plasmid transformation assay in P. furiosus

P. furiosus strains were generated via the previously described pop-in/pop-out marker technique ( Elmore et al. 2016 ). Cultures and media were prepared as described previously (Lipscomb et al. 2011). Incubations were performed under anaerobic conditions at 95°C in defined P. furiosus media. Liquid cultures were grown to mid to late log phase, and 200 μL of culture was combined with 100 ng of plasmid DNA (in 4.0 μL). The mixtures were plated on solid defined media and incubated for ~62 h. Following incubation, colonies on each plate were enumerated, and transformation efficiency (Colony Formation Units/μg plasmid DNA) was calculated and plotted logarithmically. This assay was carried out with a minimum of three replicates. Affinity pull-down assay 15 μg of strep-tagged Pfu Cascade and 15 μg of untagged Pfu Cas3 complex were mixed and incubated with 10 μL of strep resin at 4°C for 30 min in a binding buffer (50 mM HEPES pH7.5, 10% glycerol, 300 mM NaCl), in a total assay volume of 50 μL. The strep resin was pelleted by centrifugation at ~100 g for 30 seconds, washed 3 times with 200 μL of the corresponding binding buffer, then eluted with 70 μL of elution buffer (50 mM HEPES pH7.5, 300 mM NaCl, 2.5 mM desthiobiotin and 10% glycerol). Eluted proteins were separated on 12% SDS-PAGE and stained by Coomassie blue. Cascade-mediated Cas3 DNA cleavage assay and collateral activity assay The 127 bp dsDNA substrate was produced from PCR using 5′-fluorescently labeled primers (Cy3 at NTS and Cy5 at TS). The reaction mixture was prepared from 100 nM final concentration of Pfu Cascade, 100 nM Pfu Cas3 (HEL+HD) and 10 nM substrate in a cleavage buffer containing 10 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl 2 and 100 μM CoCl 2 . The reaction was incubated at 58°C (or indicated temperatures in the figures) for 30 min. The collateral cleavage activity of pfu Cas3 used 10 nM FAM-ssDNA reporter instead. After incubation, the nucleic acids were phenol-chloroform extracted and separated on a 12% denaturing polyacrylamide gel. Fluorescent signals were recorded using a Typhoon™ scanner (Amersham). HASTE detection assay 22 samples with the corresponding concentration of target plasmid were mixed with the PCR system by introducing primers and iProof polymerase in 50 μl total reaction volume. After 25 cycles (this step is taken for 15 min), 1 μl of each sample was combined with 20 μl HASTE tool (100 nM final concentration of Pfu Cascade, 100 nM Pfu Cas3 and 100 nM ssDNA-FQ reporter) respectively, and incubated at 60 °C for 15 min. Finally, all reaction tubes were scanned using a BioRad ChemiDoc imager. For the comparison, all samples were also resolved after electrophoresis on 1% agarose gels. Lateral flow assay for Pfu Cascade-Cas3 based nucleic acid detection 20 μL cleavage reactions were prepared and incubated at 58°C for 30 min with 1 μM final concentration of FAM-ssDNA-biotin reporter. Afterwards, 10 μL 30% PEG 6k and 30 μL of HybriDetect 1 assay buffer (Milenia) was added to the reaction and allowed to diffuse on a HybriDetect 1 lateral flow strip (Milenia) for 5 min. RNP electroporation of GFP-HAP1 and GFP-HEK293 cells The GFP-tagged diploid HAP1 cell line was a gift from Yan Zhang’s lab ( Tan et al., 2022 ). The GFP-tagged HEK293 cell line was purchased from GenTarget. The two cell lines were maintained in similar fashion, in DMDM (Gibco) supplemented with 10% FBS (Gibco) at 37 °C and 5% CO2 in a humidified incubator. The cells were electroporated using the Neon Transfection system (ThermoFisher) according to the manufacturer’s instructions. Briefly, HAP1 cells were individualized with 0.05% Trypsin-EDTA solution (GIBCO), washed once with DMDM (*give description), 10% FBS and resuspended in Neon buffer R to a concentration of 5×10 6 cells/mL. 20–40 pmol of NLS- Pfu Cascade/NLS- Pfu Cas3 complex were mixed with approximately 5×10 4 cells in buffer R (Neon Transfection system) in a total volume of 14 μL. Each mixture was electroporated using a 10 μL Neon tip (1450 V, 13 ms, 4 pulses) and plated in 6-well tissue culture plates containing 2 mL IMDM, 10% FBS. Flow cytometry analysis, FACS sorting and single cell isolation Cells were individualized with 0.05% Trypsin-EDTA solution (GIBCO) 5 days after electroporation and resuspended in 1× PBS before experiments. For analysis, individualized cells were analyzed on a BD Biosciences FACSAria Fusion using the 488nm laser for EGFP. Data analysis was performed using FlowJo® v10.4.1 in Flow Cytometry Facility of Cornell university. Sorted cells were then cultured in tissue culture incubator with 5% CO2 at 37°C for other usage.

DNA lesion analysis by long-range PCR genotyping Genomic

DNA of HAP1 cells were isolated using Gentra Puregene Cell Kit (QIAGEN) per manufacturer protocol. Long-range PCRs were all done using iProof DNA Polymerase (Biorad). Products were resolved on 1% agarose gels, stained by EtBr dye and visualized with Chemidoc MP imager (Biorad). See Supplementary Table 2 for all primers used for long-range PCRs. To define lesion junctions shown in Figures 7 , lesion PCR reactions were purified using QIAquick PCR Purification Kit (QIAGEN), cloned into pJET vector (Thermo fisher), and transformed into DH5α cells. Plasmids from randomly picked single colonies were Sanger-sequenced to define the deletion boundaries. Cryo-EM data acquisition 4 μL of 0.6 mg/mL SEC-purified complexes were applied to a Quantifoil holey carbon grid (1.2/1.3, 400 mesh), which had been glow-discharged for 30 sec at 30 mA current. Grids were blotted at 8°C for 4 seconds with zero force setting, 100% humidity and plunge-frozen in liquid ethane using a Mark IV FEI/Thermo Fisher Vitrobot. Cryo-EM images were collected on a 200 kV Talos Arctica transmission microscope (Thermo Fisher) equipped with a K3 direct electron detector (Gatan). The total exposure time of each movie stack was ~ 3.5 s, leading to a total accumulated dose of 50 electrons per Å^2 which fractionated into 50 frames. Dose fractionated super-resolution movie stacks collected from the K3 direct electron detector were 2× binned to a pixel size of 1.23 Å. The defocus value was set between −1.0 μm to −2.5 μm.

Cryo-EM data processing

Motion correction, CTF-estimation, blob particle picking, 2D classification, 3D classification and non-uniform 3D refinement were performed in cryoSPARC v.2 ( Punjani et al., 2017 ). Refinements followed the standard procedure, a series of 2D and 3D classifications with C1 symmetry were performed as shown in Fig. S2 to generate the final maps. A solvent mask was generated and was used for all subsequent local refinement steps. CTF post refinement was conducted to refine the beam-induced motion of the particle set, resulting in the final maps. The detailed data processing and refinement statistics for all cryo-EM structures are summarized in Fig. S2 and Table 1 for the data acquisition and structure refinement.

Materials availability

All unique/stable reagents generated in this study are available from the lead contact with a completed Materials Transfer Agreement.

EXPERIMENTAL MODEL AND SUBJECT DETAILS Cell lines and culture

HAP1 cell culture This GFP-tagged diploid HAP1 cell line was a gift from Yan Zhang’s lab ( Tan et al., 2022 ). Cells were cultured in DMDM (Gibco) supplemented with 10% FBS (Gibco) at 37 °C and 5% CO2 in a humidified incubator. Cells were suspended using Trypsin-EDTA solution (GIBCO) and split every 2 to 3 days.

HEK293 cell culture

HEK293 cells were cultured in DMDM (Gibco) supplemented with 10% FBS (Gibco) at 37 °C and 5% CO2 in a humidified incubator. Cells were suspended using Trypsin-EDTA solution (GIBCO) and split every 2 to 3 days. Bacterial strain Escherichia coli BL21 (DE3) E. coli BL21 (DE3) cells were used for protein production. Cells were grown in Lysogeny Broth (LB) supplemented with appropriate antibiotics. Escherichia coli BL21 AI E. coli BL21 AI cells were used for assaying for CRISPR interference by the Pfu I-A system. Cells were grown in Lysogeny Broth (LB) supplemented with appropriate antibiotics. Escherichia coli DH5 alpha E. coli DH5α was used for cloning. Cells were grown at 37 C in LB supplemented with appropriate antibiotics.

Plasmids and cloning

Plasmids, primers, and RNA guides used in this work are listed in Supplementary Tables 1 , 2 , and 3 , respectively. Cloning was performed in E. coli DH5α. The Type I-A cas operon from Pyrococcus furiosus DSM 3638 was PCR-amplified using the iproof Polymerase (BioRad) and cloned into the pCDFDuet vector, giving rise to Plasmid pCascade/Cas3. For plasmid pCRISPR (or pcrRNA), a series of synthetic constructs composed of T7 promotor, CRISPR array (repeat-spacer-repeat with Cas6 or ribozyme derived 5’ handle-spacer) were introduced into the high copy number vector pRSFDuet. For purification, cas8a was cloned into pCDFduet with a N-terminal Twin-strep tag. cas11-cas7-cas5 operon was inserted into pETDuet with a C-terminal His tag on Cas5a. Cas3 HD and Cas3 HEL were expressed individually from the pRSFDuet vector with a N-terminal His tag. All plasmids were verified by Sanger sequencing. Bacterial transformations were carried out using chemically competent cells, and transformants were selected on LB agar plate supplemented with the appropriate antibiotics.

METHOD DETAILS Protein expression and purification pCDFDuet-Twin-Strep-Cas8a ( Strep R ), pETDuet- Cas11-Cas7-Cas5 ( Amp R )and pcrRNA-Cas6 ( Kan R ) were co-transformed into E. coli BL21 (DE3) cell under the appropriate antibiotic selection. A 4 liter cell culture was grown in LB medium at 37 °C until an optical density of 0.6 at 600 nm. The culture temperature was then reduced to 20°C and incubated for an additional 1 hour. Expression was induced with 0.5 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 20°C overnight. Cells were harvested by centrifugation and resuspended in 100 mL buffer A containing 50 mM HEPES pH 7.5, and 300 mM NaCl, 10% glycerol, and 2 mM β-ME. Cells were lysed by sonication, and the debris was cleared using centrifugation at 12,000 rpm for 50 min at 4 °C. The supernatant was applied onto a pre-equilibrated 4 mL streptavidin column (Twin-strep purification). After washing with 50 ml of buffer A, the protein was eluted with 20 ml buffer B (50 mM HEPES pH 7.5, 300 mM NaCl, 10% glycerol, 2.5 mM desthiobiotin). The sample was then concentrated to 1 ml and loaded onto a Superdex 200 16/60 size-exclusion column (GE Healthcare) equilibrated with buffer C (10 mM HEPES pH 7.5, 300 mM NaCl), the peak fractions of Cascade complex were pooled and snap-frozen in liquid nitrogen for later usage. For Cas3 HD and Cas3 HEL purification, pRSFDuet-Cas3HD or Cas3HEL (Kan R ) was individually transformed into E. coli BL21 (DE3), expressed using the same procedure as described above. Cells were harvested by centrifugation and lysed by sonication in 80 ml of buffer A containing 50 mM HEPES pH 7.5, 500 mM NaCl and 20 mM imidazole, 10% glycerol, and 2 mM β-ME. The lysate was centrifuged at 12,000 rpm for 50 min at 4 °C, and the supernatant was applied to a pre-equilibrated 4 mL Ni-NTA column. After washing with 100 ml of buffer A, the protein was eluted with 20 ml buffer B (50 mM HEPES pH 7.5, 500 mM NaCl, 10% glycerol, 300 mM imidazole, and 2 mM β-ME), concentrated to 1.5 mL and further purified on Superdex 200 16/60 equilibrated with buffer C (10 mM HEPES pH 7.5, 300 mM NaCl), the peak fractions were pooled and snap-frozen in liquid nitrogen for later usage. RNA extraction and ureal gel running 20 μL of Cascade sample at 2 μM and 20 μL phenol-chloroform solution was mixed together and vortexed vigorously for 2 min at room temperature. The aqueous and organic phases were separated by 13,000 rpm centrifuge for 15 min at room temperature. 10 μL sample was taken from the aqueous phase (top layer), mixed with 15 μL of formamide loading dye, heat-denatured at 95 °C for 10 min, and immediately loaded to a 12% ureal-polyacrylamide (PAGE) gel. After 50 minutes of electrophoresis at 25 watts, the gel was stained with EtBr to for 10 min, destained in water for 10 minutes, and scanned with the ChemiDoc imaging system (Bio-Rad) at appropriate wavelength. In vivo assay for Cascade and Cas3–mediated interference pCDFduet-Cascade/Cas3 (StrepR), pRSFDuet-crRNA-Cas6 (KanaR)and pETDuet-Targets (AmpR)with different PAM sequence were co-transformed into the E. coli BL21AI cell line and grown on LB agarose plates containing kanamycin (50 μg/ml), ampicillin (100 μg/ml), streptomycin (30 μg/ml). After transformation, a single colony were cultured at 37°C in nonselective LB medium (Strep + Kana) to O.D.600 of 0.3, at which point the expression of Pfu Cascade, Cas3, and pre-crRNA was induced for 12 h by the addition of 0.5% L-arabinose and 1 mM IPTG. Each cell culture was then divided into two equal volumes and plated onto Strep + Kana + Amp LB plates (selective for pETDuet-target) and Strep + Kan plates (non-selective for the target plasmid) in a series of dilutions. The number of colonies on each plate was counted after overnight incubation at 37°C. The CRISPR interference efficiency was reflected in the ratio of colony-forming units on the nonselective over selective plates. Each experiment was repeated three times to calculate the s.d. The experimental procedure is illustrated in Supplementary Figure 1K and Table 1 .

Fluorescently labeled prespacer substrate preparation Fluorescent

DNA oligos ( Supplementary Table 2 ) for biochemistry were synthesized (Integrated DNA Technologies) with either a 5AmMC6 or 3AmMO label, fluorescently labeled in-house by Cy3 or Cy5-NHS dye (Lumiprobe), and annealed at equimolar amount, and native PAGE purified to remove unannealed ssDNA. Electrophoretic mobility shift assay 5 nM final concentration of fluorescently labeled target DNA was incubated with titrations of Pfu Cascade or Cas3/Cascade complex in a 20 μL total reaction volume containing 50 mM Tris pH 8.0, 150 mM NaCl, and 10% glycerol. After a 15-minute incubation at 42°C or specified condition in the figure legends,10 μL of each sample was loaded onto 1% agarose gel equilibrated in 0.5 × TBE buffer. Electrophoresis was performed at 60 V for 40 min in cold room. The fluorescent signals from the gel were recorded using a ChemiDoc imaging system (Bio-Rad). For the fluorescence labeled protein EMSA assay in Fig. 1C ., the proteins were labeled using corresponding fluorophore (Sulfo-Cy3 or Cy5, Lumiprobe) following our previously published protocol ( Xiao et al., 2017 ). The 20 μL binding experiments used 300 nM Cy3-Cas3, 100 nM Cy5-Cascade, and 20 nM FAM-DNA in single tube or combined two or three components in one tube. Incubation and electrophoresis were carried out as above.

Plasmid transformation assay in P. furiosus

P. furiosus strains were generated via the previously described pop-in/pop-out marker technique ( Elmore et al. 2016 ). Cultures and media were prepared as described previously (Lipscomb et al. 2011). Incubations were performed under anaerobic conditions at 95°C in defined P. furiosus media. Liquid cultures were grown to mid to late log phase, and 200 μL of culture was combined with 100 ng of plasmid DNA (in 4.0 μL). The mixtures were plated on solid defined media and incubated for ~62 h. Following incubation, colonies on each plate were enumerated, and transformation efficiency (Colony Formation Units/μg plasmid DNA) was calculated and plotted logarithmically. This assay was carried out with a minimum of three replicates. Affinity pull-down assay 15 μg of strep-tagged Pfu Cascade and 15 μg of untagged Pfu Cas3 complex were mixed and incubated with 10 μL of strep resin at 4°C for 30 min in a binding buffer (50 mM HEPES pH7.5, 10% glycerol, 300 mM NaCl), in a total assay volume of 50 μL. The strep resin was pelleted by centrifugation at ~100 g for 30 seconds, washed 3 times with 200 μL of the corresponding binding buffer, then eluted with 70 μL of elution buffer (50 mM HEPES pH7.5, 300 mM NaCl, 2.5 mM desthiobiotin and 10% glycerol). Eluted proteins were separated on 12% SDS-PAGE and stained by Coomassie blue. Cascade-mediated Cas3 DNA cleavage assay and collateral activity assay The 127 bp dsDNA substrate was produced from PCR using 5′-fluorescently labeled primers (Cy3 at NTS and Cy5 at TS). The reaction mixture was prepared from 100 nM final concentration of Pfu Cascade, 100 nM Pfu Cas3 (HEL+HD) and 10 nM substrate in a cleavage buffer containing 10 mM HEPES pH 7.5, 150 mM NaCl, 10 mM MgCl 2 and 100 μM CoCl 2 . The reaction was incubated at 58°C (or indicated temperatures in the figures) for 30 min. The collateral cleavage activity of pfu Cas3 used 10 nM FAM-ssDNA reporter instead. After incubation, the nucleic acids were phenol-chloroform extracted and separated on a 12% denaturing polyacrylamide gel. Fluorescent signals were recorded using a Typhoon™ scanner (Amersham). HASTE detection assay 22 samples with the corresponding concentration of target plasmid were mixed with the PCR system by introducing primers and iProof polymerase in 50 μl total reaction volume. After 25 cycles (this step is taken for 15 min), 1 μl of each sample was combined with 20 μl HASTE tool (100 nM final concentration of Pfu Cascade, 100 nM Pfu Cas3 and 100 nM ssDNA-FQ reporter) respectively, and incubated at 60 °C for 15 min. Finally, all reaction tubes were scanned using a BioRad ChemiDoc imager. For the comparison, all samples were also resolved after electrophoresis on 1% agarose gels. Lateral flow assay for Pfu Cascade-Cas3 based nucleic acid detection 20 μL cleavage reactions were prepared and incubated at 58°C for 30 min with 1 μM final concentration of FAM-ssDNA-biotin reporter. Afterwards, 10 μL 30% PEG 6k and 30 μL of HybriDetect 1 assay buffer (Milenia) was added to the reaction and allowed to diffuse on a HybriDetect 1 lateral flow strip (Milenia) for 5 min. RNP electroporation of GFP-HAP1 and GFP-HEK293 cells The GFP-tagged diploid HAP1 cell line was a gift from Yan Zhang’s lab ( Tan et al., 2022 ). The GFP-tagged HEK293 cell line was purchased from GenTarget. The two cell lines were maintained in similar fashion, in DMDM (Gibco) supplemented with 10% FBS (Gibco) at 37 °C and 5% CO2 in a humidified incubator. The cells were electroporated using the Neon Transfection system (ThermoFisher) according to the manufacturer’s instructions. Briefly, HAP1 cells were individualized with 0.05% Trypsin-EDTA solution (GIBCO), washed once with DMDM (*give description), 10% FBS and resuspended in Neon buffer R to a concentration of 5×10 6 cells/mL. 20–40 pmol of NLS- Pfu Cascade/NLS- Pfu Cas3 complex were mixed with approximately 5×10 4 cells in buffer R (Neon Transfection system) in a total volume of 14 μL. Each mixture was electroporated using a 10 μL Neon tip (1450 V, 13 ms, 4 pulses) and plated in 6-well tissue culture plates containing 2 mL IMDM, 10% FBS. Flow cytometry analysis, FACS sorting and single cell isolation Cells were individualized with 0.05% Trypsin-EDTA solution (GIBCO) 5 days after electroporation and resuspended in 1× PBS before experiments. For analysis, individualized cells were analyzed on a BD Biosciences FACSAria Fusion using the 488nm laser for EGFP. Data analysis was performed using FlowJo® v10.4.1 in Flow Cytometry Facility of Cornell university. Sorted cells were then cultured in tissue culture incubator with 5% CO2 at 37°C for other usage.

DNA lesion analysis by long-range PCR genotyping Genomic

DNA of HAP1 cells were isolated using Gentra Puregene Cell Kit (QIAGEN) per manufacturer protocol. Long-range PCRs were all done using iProof DNA Polymerase (Biorad). Products were resolved on 1% agarose gels, stained by EtBr dye and visualized with Chemidoc MP imager (Biorad). See Supplementary Table 2 for all primers used for long-range PCRs. To define lesion junctions shown in Figures 7 , lesion PCR reactions were purified using QIAquick PCR Purification Kit (QIAGEN), cloned into pJET vector (Thermo fisher), and transformed into DH5α cells. Plasmids from randomly picked single colonies were Sanger-sequenced to define the deletion boundaries. Cryo-EM data acquisition 4 μL of 0.6 mg/mL SEC-purified complexes were applied to a Quantifoil holey carbon grid (1.2/1.3, 400 mesh), which had been glow-discharged for 30 sec at 30 mA current. Grids were blotted at 8°C for 4 seconds with zero force setting, 100% humidity and plunge-frozen in liquid ethane using a Mark IV FEI/Thermo Fisher Vitrobot. Cryo-EM images were collected on a 200 kV Talos Arctica transmission microscope (Thermo Fisher) equipped with a K3 direct electron detector (Gatan). The total exposure time of each movie stack was ~ 3.5 s, leading to a total accumulated dose of 50 electrons per Å^2 which fractionated into 50 frames. Dose fractionated super-resolution movie stacks collected from the K3 direct electron detector were 2× binned to a pixel size of 1.23 Å. The defocus value was set between −1.0 μm to −2.5 μm.

Cryo-EM data processing

Motion correction, CTF-estimation, blob particle picking, 2D classification, 3D classification and non-uniform 3D refinement were performed in cryoSPARC v.2 ( Punjani et al., 2017 ). Refinements followed the standard procedure, a series of 2D and 3D classifications with C1 symmetry were performed as shown in Fig. S2 to generate the final maps. A solvent mask was generated and was used for all subsequent local refinement steps. CTF post refinement was conducted to refine the beam-induced motion of the particle set, resulting in the final maps. The detailed data processing and refinement statistics for all cryo-EM structures are summarized in Fig. S2 and Table 1 for the data acquisition and structure refinement.

Supplementary Material 2 Supplementary Movie S1. Rotating views of four cryo-EM snapshots of Pfu Type I-A CRISPR effector complexes. Related to Figure 2 . 3 Supplementary Movie S2. 3D variant display of the apo Cascade complex and the Cascade-Cas3 complex, analyzed by Program CryoSPARC. Related to Figure 3 . This analysis reveals the extent of the inherent conformational dynamics in each complex. The Cas8 NTD and the entire ‘inner belly’ of Cascade are significantly more dynamic in the apo Cascade complex. 4 Supplementary Movie S3. PAM recognition mechanism illustrated from different views of the Cascade-Cas3/partial R-loop structure. Related to Figure 4 . 5 Supplementary Movie S4. Morphing between partial and full R-loop Cascade-Cas3 structures revealing the concerted conformational changes in the ‘inner belly’ of the Cascade upon full R-loop formation. Related to Figure 5 . This conformational change is further transcended from the Cas8a subunit of Cascade to Cas3, causing a rigid-body movement in Cas3 and also triggering the allosteric activation of its HD nuclease. 6 Supplementary Movie S5. Animation of the envisioned partial-to-full R-loop transition and the allosteric regulation at Cas3 HD nuclease center. Related to Figure 5 . 7 Supplementary Movie S6.

Time lapse of the RNA-guided

GFP knock-out editing by Pfu I-A Cascade-Cas3 (right panel), done in the GFP-tagged, diploid form of the HAP1 cells. Related to Figure 7 . The negative control is displayed on the left-side panel, where the non-targeting Pfu I-A Cascade-Cas3 was electroporated into the cells. 8 Supplementary Movie S7.

Time lapse of the RNA-guided

GFP knock-out editing by Pfu I-A Cascade-Cas3 (right panel), done in the GFP-tagged HEK293 cells. Related to Figure 7 . The negative control is displayed on the left-side panel, where the non-targeting Pfu I-A Cascade-Cas3 was electroporated into the cells. Note that the GFP intensities among HEK293 cells are not as homogenous as those among the HAP1 cells. This property is inherent to the HEK293 cell line, and the GFP tagging information is not available from the vendor. 9