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bacterial expression plasmids for cas9 variants  (Addgene inc)


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    Addgene inc bacterial expression plasmids for cas9 variants
    (A) A model of RNA-guided DNA targeting by <t>Cas9.</t> The two-step target capture process that differentiates PAM-relaxed Cas9 variants from WT Spy Cas9 is highlighted. (B) The three Spy Cas9 proteins used in this study with their respective PAM recognition: WT Spy Cas9 (gray), SpG (pink) and SpRY (purple). (C) Quantifications of DNA double-stranded breaks (DSB) by droplet digital PCR (ddPCR) on the target site at 8 h post plasmid transfection (n=3 replicates). See also for more data. (D) Quantifications of indels on the target site at 72 h post RNP nucleofection (n=3). See also for more data. (E) Time-course analysis of average DNA cleavage products (n=3) for sgRNA2 in 5 mM Mg2+ at 24°C for TS (see ). The average rate constants, with the amplitudes from the observed double-exponential decay, are provided in figure legends. See also for NTS cleavage and - for other sgRNAs and conditions. (F) Time-course analysis of the average fluorescence signal (n=3) in the 2AP assay for the same sequence in similar conditions as (E) (see ). For WT Spy Cas9, catalytically dead Cas9 (dCas9) is used here. The rate constants of the observed mono-exponential decay are provided in figure legends. See also for the 2AP-labeling position (15th nt from PAM) and intercept determination (using non-targeting sgRNA). All error bars in (C-F) represent the standard deviation of n = 3 replicates.
    Bacterial Expression Plasmids For Cas9 Variants, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/bacterial+expression+plasmids+for+cas9+variants/px330+u6+chimeric+bb+cbh+hspcas9/pmc12258621-62-0-11
    Average 90 stars, based on 1 article reviews
    bacterial expression plasmids for cas9 variants - by Bioz Stars, 2026-10
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    1) Product Images from "Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing"

    Article Title: Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing

    Journal: Molecular cell

    doi: 10.1016/j.molcel.2025.03.024

    (A) A model of RNA-guided DNA targeting by Cas9. The two-step target capture process that differentiates PAM-relaxed Cas9 variants from WT Spy Cas9 is highlighted. (B) The three Spy Cas9 proteins used in this study with their respective PAM recognition: WT Spy Cas9 (gray), SpG (pink) and SpRY (purple). (C) Quantifications of DNA double-stranded breaks (DSB) by droplet digital PCR (ddPCR) on the target site at 8 h post plasmid transfection (n=3 replicates). See also for more data. (D) Quantifications of indels on the target site at 72 h post RNP nucleofection (n=3). See also for more data. (E) Time-course analysis of average DNA cleavage products (n=3) for sgRNA2 in 5 mM Mg2+ at 24°C for TS (see ). The average rate constants, with the amplitudes from the observed double-exponential decay, are provided in figure legends. See also for NTS cleavage and - for other sgRNAs and conditions. (F) Time-course analysis of the average fluorescence signal (n=3) in the 2AP assay for the same sequence in similar conditions as (E) (see ). For WT Spy Cas9, catalytically dead Cas9 (dCas9) is used here. The rate constants of the observed mono-exponential decay are provided in figure legends. See also for the 2AP-labeling position (15th nt from PAM) and intercept determination (using non-targeting sgRNA). All error bars in (C-F) represent the standard deviation of n = 3 replicates.
    Figure Legend Snippet: (A) A model of RNA-guided DNA targeting by Cas9. The two-step target capture process that differentiates PAM-relaxed Cas9 variants from WT Spy Cas9 is highlighted. (B) The three Spy Cas9 proteins used in this study with their respective PAM recognition: WT Spy Cas9 (gray), SpG (pink) and SpRY (purple). (C) Quantifications of DNA double-stranded breaks (DSB) by droplet digital PCR (ddPCR) on the target site at 8 h post plasmid transfection (n=3 replicates). See also for more data. (D) Quantifications of indels on the target site at 72 h post RNP nucleofection (n=3). See also for more data. (E) Time-course analysis of average DNA cleavage products (n=3) for sgRNA2 in 5 mM Mg2+ at 24°C for TS (see ). The average rate constants, with the amplitudes from the observed double-exponential decay, are provided in figure legends. See also for NTS cleavage and - for other sgRNAs and conditions. (F) Time-course analysis of the average fluorescence signal (n=3) in the 2AP assay for the same sequence in similar conditions as (E) (see ). For WT Spy Cas9, catalytically dead Cas9 (dCas9) is used here. The rate constants of the observed mono-exponential decay are provided in figure legends. See also for the 2AP-labeling position (15th nt from PAM) and intercept determination (using non-targeting sgRNA). All error bars in (C-F) represent the standard deviation of n = 3 replicates.

    Techniques Used: Digital PCR, Plasmid Preparation, Transfection, Fluorescence, Sequencing, Labeling, Standard Deviation

    (A, C, E) Example traces of time-resolved measurements of equilibrium twist change Δθ over time for (A and C) WT Spy Cas9 (50 nM) and (E) SpRY (50 nM), each paired with sgRNA4.1, containing a 3-bp match to the Target2 sequence flanking (A and E) an NGG site or (C) an NCG site on the DNA tether. 250-ms averaged traces are shown in black. For (E) , the vertical dashed lines (--) and dash-dot lines (−.) indicate the start and end, respectively, of the flow of SpRY RNP into the chamber. (B, D) Scatter plots of unwinding lifetime and Δθ 0 for merged Steppi-scored states corresponding to experiments described in (A, C) . The total collection time ( T total ) and number of DNA tethers (n) are provided in the legend. See also – for more data with different sequences and conditions. (F) Fit of average Δθ baseline shift and [RNP] for SpRY to a binding equation yielded an apparent K d ~ 14 nM for sgRNA4.1 on the Target2 sequence flanking an NGG site. See also – for raw traces. (G) Schematic overview of the permanganate DNA footprinting assay. The UREA-PAGE gel image used to generate the graph in (I) highlights KMnO 4 -dependent enriched bands for WT Spy Cas9 (gray), SpG (pink) and SpRY (purple) in colored dashed boxes. The gel image was rendered in ImageLab 6.1 (BioRad) and cropped to exclude irrelevant neighboring lanes. (H) Sequence of the sgRNA (sgRNA3) and the dsDNA substrate with all the reactive thymines on the TS underlined. (I) Oxidation probabilities of thymines across the dsDNA substrate.
    Figure Legend Snippet: (A, C, E) Example traces of time-resolved measurements of equilibrium twist change Δθ over time for (A and C) WT Spy Cas9 (50 nM) and (E) SpRY (50 nM), each paired with sgRNA4.1, containing a 3-bp match to the Target2 sequence flanking (A and E) an NGG site or (C) an NCG site on the DNA tether. 250-ms averaged traces are shown in black. For (E) , the vertical dashed lines (--) and dash-dot lines (−.) indicate the start and end, respectively, of the flow of SpRY RNP into the chamber. (B, D) Scatter plots of unwinding lifetime and Δθ 0 for merged Steppi-scored states corresponding to experiments described in (A, C) . The total collection time ( T total ) and number of DNA tethers (n) are provided in the legend. See also – for more data with different sequences and conditions. (F) Fit of average Δθ baseline shift and [RNP] for SpRY to a binding equation yielded an apparent K d ~ 14 nM for sgRNA4.1 on the Target2 sequence flanking an NGG site. See also – for raw traces. (G) Schematic overview of the permanganate DNA footprinting assay. The UREA-PAGE gel image used to generate the graph in (I) highlights KMnO 4 -dependent enriched bands for WT Spy Cas9 (gray), SpG (pink) and SpRY (purple) in colored dashed boxes. The gel image was rendered in ImageLab 6.1 (BioRad) and cropped to exclude irrelevant neighboring lanes. (H) Sequence of the sgRNA (sgRNA3) and the dsDNA substrate with all the reactive thymines on the TS underlined. (I) Oxidation probabilities of thymines across the dsDNA substrate.

    Techniques Used: Sequencing, Binding Assay, DNA Footprinting

    The reduced genome editing efficiencies of PAM-relaxed Cas9 variants (e.g. SpRY) are explained by two factors: (Top) PAM-relaxed Cas9 variants display prolonged target search times, largely due to kinetic trapping at non-specific binding sites, which reduces the pool of free RNPs available for on-target identification. (Bottom) Upon reaching the target site, these variants also become kinetically trapped in a low-energy initial binding complex and unwind DNA slowly.
    Figure Legend Snippet: The reduced genome editing efficiencies of PAM-relaxed Cas9 variants (e.g. SpRY) are explained by two factors: (Top) PAM-relaxed Cas9 variants display prolonged target search times, largely due to kinetic trapping at non-specific binding sites, which reduces the pool of free RNPs available for on-target identification. (Bottom) Upon reaching the target site, these variants also become kinetically trapped in a low-energy initial binding complex and unwind DNA slowly.

    Techniques Used: Binding Assay, Drug discovery

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    Amplification:

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    Sequencing:

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    Chromatin Immunoprecipitation:

    Article Title: Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-human b2-microglobulin (2M2)-APC BioLegend Cat#316312; RRID_AB_10641281 Monoclonal ANTI-FLAG M2 antibody produced in mouse Millipore sigma Cat#F1804; RRID: AB_262044 CRISPR/Cas9 antibody Diagenode Cat#C15310258; RRID: AB_2715516 Histone H3 (D1H2) XP Rabbit mAb Cell Signaling Technologies Cat#4499; RRID: AB_10544537 Fluorescein/Oregon Green Polyclonal Antibody ThermoFisher Cat#A-889; RRID: AB_221561 Anti-Digoxigenin from sheep MilliporeSigma (Roche) Cat#11333089001; RRID: AB_514496 Bacterial and virus strains E. coli Rosetta 2 (DE3) Competent Cells EMD Millipore Cat#71397 Chemicals, peptides, and recombinant proteins GTP Sigma-Aldrich G8877-1G ATP Sigma-Aldrich A8937-1G UTP Sigma-Aldrich U6625-1G CTP Sigma-Aldrich C1506-1G Spermidine Sigma-Aldrich S2626-1G T7 RNA polymerase N/A N/A Ni-NTA Superflow Qiagen Cat#30430 TEV protease N/A N/A Mini-PROTEAN TGX Precast Protein Gel Bio-Rad Cat#4561033 Piperidine Sigma-Aldrich 411027-100ML Potassium permanganate Sigma-Aldrich 223468-25G dUTP-digoxigenin MilliporeSigma (Roche) Cat#11093088910 Streptavidin-coated gold nanospheres Cytodiagnostics Cat#ACC-60-04-15 Dynabead MyOne Carboxylic Acid ThermoFisher Cat#65012 Proteinase K NEB Cat#P8107S Dynabeads Protein A for Immunoprecipitation Invitrogen Cat#10002D Salmon Sperm DNA ThermoFisher AM9680 Dulbecco’s Modified Eagle Medium Corning Cat#10-013-CV Fetal Bovine Serum VWR Cat#97068-085 Penicillin/streptomcyin Gibco Cat#15140122 Lipofectamine 3000 Transfection Reagent Invitrogen Cat#L3000015 QuickExtract DNA Extraction Solution Biosearch Technologies Cat#QE09050 Q5 High-Fidelity 2x Master Mix NEB Cat#M0492L ddPCR Supermix for Probes (No dUTP) Bio-Rad Cat#1863024 RIPA Lysis and Extraction Buffer Fisher Scientific Cat#PI89900 Protease Inhibitor Cocktail (100X) Thermo Scientific Cat#78429 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat#4368814 Power SYBR Green PCR Master Mix Applied Biosystems Cat#4367659 SPRIselect Beckman Coulter Cat#B23317 cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Roche Cat#11836170001 Isopropyl b-d-1-thiogalactopyranoside (IPTG) Gold Biotechnology Cat#I2481C Critical commercial assays SF Cell Line 96-well Nucleofector Kit Lonza Cat#V4SC-2096 NEBNext Ultra II DNA Library Prep Kit for Illumina NEB Cat#E7645L (Continued on next page) e1 Molecular Cell 85, 1–13.e1–e9, May 1, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Qubit dsDNA Quantification Assay Invitrogen Cat#Q32854 Pierce BCA Protein Assay Kit Thermo Scientific Cat#23225 miRNeasy Mini Kit Qiagen Cat#217004 Deposited data Amplicon sequencing data from genome-editing experiments This study NCBI SRA BioProject ID: PRJNA1239632 ChIP-seq data This study NCBI SRA BioProject ID: PRJNA1239632 Unprocessed gel images & raw data from cell & single molecule experiments This study Mendeley data: https://doi.org/ 10.17632/hzr4fnvc88.1 Experimental models: Cell lines Human embryonic kidney 293T cells UC Berkeley Cell Culture Facility N/A Oligonucleotides Fluorescently labeled DNA substrates IDT Table S13 Cas9 sgRNAs IDT Tables S5, S8, and S9 Primers and oligonucleotides for AuRBT tether construction IDT Tables S15 and S16 RT-qPCR primers IDT Table S12 ddPCR primers/probes IDT Table S11 NGS primers IDT Table S10 Recombinant DNA Mammalian Cas9 or sgRNA expression plasmids This study, modified from Addgene#62988 Tables S4–S7 Bacterial expression plasmids for Cas9 variants This study, modified from Addgene#179525 Tables S4, S6, and S7 pGGASelect NEB Cat#195714; Table S14 Software and algorithms FlowJo v10.10.0 FlowJo https://www.flowjo.com/solutions/downloads/ Image Lab 6.1 Bio-Rad Laboratories, Inc. https://www.bio-rad.com/en-us/product/ image-lab-software?ID=KRE6P5E8Z MATLAB 9.6.0.1472908 (R2019a) Update 9 The MathWorks Inc. https://www.mathworks.com MEMLET Woody et al.44 https://michaelswoody.github.io/MEMLET/ Bowtie2 version 2.5.2 Langmead and Salzberg45 https://github.com/BenLangmead/bowtie2 deepTools2 version 3.5.1 Ramı́rez et al.46 https://github.com/deeptools/deepTools IGV version 2.19.1 Robinson et al.47 https://igv.org/ Seqtk version 1.3 N/A https://github.com/lh3/seqtk MACS2 version 2.2.9.1 Zhang et al.48 https://github.com/macs3-project/MACS IDR version 2.0.4.2 Li et al.49 https://github.com/kundajelab/idr Picard version 2.21.9 Broad Institute https://github.com/broadinstitute/picard BEDTools version 2.29.2 Quinlan and Hall50 https://github.com/arq5x/bedtools2 Python version 3.9.12 Python Software Foundation https://www.python.org/ GraphPad Prism version 10.4.1 GraphPad https://www.graphpad.com/ ..

    Cell Culture:

    Article Title: Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-human b2-microglobulin (2M2)-APC BioLegend Cat#316312; RRID_AB_10641281 Monoclonal ANTI-FLAG M2 antibody produced in mouse Millipore sigma Cat#F1804; RRID: AB_262044 CRISPR/Cas9 antibody Diagenode Cat#C15310258; RRID: AB_2715516 Histone H3 (D1H2) XP Rabbit mAb Cell Signaling Technologies Cat#4499; RRID: AB_10544537 Fluorescein/Oregon Green Polyclonal Antibody ThermoFisher Cat#A-889; RRID: AB_221561 Anti-Digoxigenin from sheep MilliporeSigma (Roche) Cat#11333089001; RRID: AB_514496 Bacterial and virus strains E. coli Rosetta 2 (DE3) Competent Cells EMD Millipore Cat#71397 Chemicals, peptides, and recombinant proteins GTP Sigma-Aldrich G8877-1G ATP Sigma-Aldrich A8937-1G UTP Sigma-Aldrich U6625-1G CTP Sigma-Aldrich C1506-1G Spermidine Sigma-Aldrich S2626-1G T7 RNA polymerase N/A N/A Ni-NTA Superflow Qiagen Cat#30430 TEV protease N/A N/A Mini-PROTEAN TGX Precast Protein Gel Bio-Rad Cat#4561033 Piperidine Sigma-Aldrich 411027-100ML Potassium permanganate Sigma-Aldrich 223468-25G dUTP-digoxigenin MilliporeSigma (Roche) Cat#11093088910 Streptavidin-coated gold nanospheres Cytodiagnostics Cat#ACC-60-04-15 Dynabead MyOne Carboxylic Acid ThermoFisher Cat#65012 Proteinase K NEB Cat#P8107S Dynabeads Protein A for Immunoprecipitation Invitrogen Cat#10002D Salmon Sperm DNA ThermoFisher AM9680 Dulbecco’s Modified Eagle Medium Corning Cat#10-013-CV Fetal Bovine Serum VWR Cat#97068-085 Penicillin/streptomcyin Gibco Cat#15140122 Lipofectamine 3000 Transfection Reagent Invitrogen Cat#L3000015 QuickExtract DNA Extraction Solution Biosearch Technologies Cat#QE09050 Q5 High-Fidelity 2x Master Mix NEB Cat#M0492L ddPCR Supermix for Probes (No dUTP) Bio-Rad Cat#1863024 RIPA Lysis and Extraction Buffer Fisher Scientific Cat#PI89900 Protease Inhibitor Cocktail (100X) Thermo Scientific Cat#78429 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat#4368814 Power SYBR Green PCR Master Mix Applied Biosystems Cat#4367659 SPRIselect Beckman Coulter Cat#B23317 cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Roche Cat#11836170001 Isopropyl b-d-1-thiogalactopyranoside (IPTG) Gold Biotechnology Cat#I2481C Critical commercial assays SF Cell Line 96-well Nucleofector Kit Lonza Cat#V4SC-2096 NEBNext Ultra II DNA Library Prep Kit for Illumina NEB Cat#E7645L (Continued on next page) e1 Molecular Cell 85, 1–13.e1–e9, May 1, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Qubit dsDNA Quantification Assay Invitrogen Cat#Q32854 Pierce BCA Protein Assay Kit Thermo Scientific Cat#23225 miRNeasy Mini Kit Qiagen Cat#217004 Deposited data Amplicon sequencing data from genome-editing experiments This study NCBI SRA BioProject ID: PRJNA1239632 ChIP-seq data This study NCBI SRA BioProject ID: PRJNA1239632 Unprocessed gel images & raw data from cell & single molecule experiments This study Mendeley data: https://doi.org/ 10.17632/hzr4fnvc88.1 Experimental models: Cell lines Human embryonic kidney 293T cells UC Berkeley Cell Culture Facility N/A Oligonucleotides Fluorescently labeled DNA substrates IDT Table S13 Cas9 sgRNAs IDT Tables S5, S8, and S9 Primers and oligonucleotides for AuRBT tether construction IDT Tables S15 and S16 RT-qPCR primers IDT Table S12 ddPCR primers/probes IDT Table S11 NGS primers IDT Table S10 Recombinant DNA Mammalian Cas9 or sgRNA expression plasmids This study, modified from Addgene#62988 Tables S4–S7 Bacterial expression plasmids for Cas9 variants This study, modified from Addgene#179525 Tables S4, S6, and S7 pGGASelect NEB Cat#195714; Table S14 Software and algorithms FlowJo v10.10.0 FlowJo https://www.flowjo.com/solutions/downloads/ Image Lab 6.1 Bio-Rad Laboratories, Inc. https://www.bio-rad.com/en-us/product/ image-lab-software?ID=KRE6P5E8Z MATLAB 9.6.0.1472908 (R2019a) Update 9 The MathWorks Inc. https://www.mathworks.com MEMLET Woody et al.44 https://michaelswoody.github.io/MEMLET/ Bowtie2 version 2.5.2 Langmead and Salzberg45 https://github.com/BenLangmead/bowtie2 deepTools2 version 3.5.1 Ramı́rez et al.46 https://github.com/deeptools/deepTools IGV version 2.19.1 Robinson et al.47 https://igv.org/ Seqtk version 1.3 N/A https://github.com/lh3/seqtk MACS2 version 2.2.9.1 Zhang et al.48 https://github.com/macs3-project/MACS IDR version 2.0.4.2 Li et al.49 https://github.com/kundajelab/idr Picard version 2.21.9 Broad Institute https://github.com/broadinstitute/picard BEDTools version 2.29.2 Quinlan and Hall50 https://github.com/arq5x/bedtools2 Python version 3.9.12 Python Software Foundation https://www.python.org/ GraphPad Prism version 10.4.1 GraphPad https://www.graphpad.com/ ..

    Labeling:

    Article Title: Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-human b2-microglobulin (2M2)-APC BioLegend Cat#316312; RRID_AB_10641281 Monoclonal ANTI-FLAG M2 antibody produced in mouse Millipore sigma Cat#F1804; RRID: AB_262044 CRISPR/Cas9 antibody Diagenode Cat#C15310258; RRID: AB_2715516 Histone H3 (D1H2) XP Rabbit mAb Cell Signaling Technologies Cat#4499; RRID: AB_10544537 Fluorescein/Oregon Green Polyclonal Antibody ThermoFisher Cat#A-889; RRID: AB_221561 Anti-Digoxigenin from sheep MilliporeSigma (Roche) Cat#11333089001; RRID: AB_514496 Bacterial and virus strains E. coli Rosetta 2 (DE3) Competent Cells EMD Millipore Cat#71397 Chemicals, peptides, and recombinant proteins GTP Sigma-Aldrich G8877-1G ATP Sigma-Aldrich A8937-1G UTP Sigma-Aldrich U6625-1G CTP Sigma-Aldrich C1506-1G Spermidine Sigma-Aldrich S2626-1G T7 RNA polymerase N/A N/A Ni-NTA Superflow Qiagen Cat#30430 TEV protease N/A N/A Mini-PROTEAN TGX Precast Protein Gel Bio-Rad Cat#4561033 Piperidine Sigma-Aldrich 411027-100ML Potassium permanganate Sigma-Aldrich 223468-25G dUTP-digoxigenin MilliporeSigma (Roche) Cat#11093088910 Streptavidin-coated gold nanospheres Cytodiagnostics Cat#ACC-60-04-15 Dynabead MyOne Carboxylic Acid ThermoFisher Cat#65012 Proteinase K NEB Cat#P8107S Dynabeads Protein A for Immunoprecipitation Invitrogen Cat#10002D Salmon Sperm DNA ThermoFisher AM9680 Dulbecco’s Modified Eagle Medium Corning Cat#10-013-CV Fetal Bovine Serum VWR Cat#97068-085 Penicillin/streptomcyin Gibco Cat#15140122 Lipofectamine 3000 Transfection Reagent Invitrogen Cat#L3000015 QuickExtract DNA Extraction Solution Biosearch Technologies Cat#QE09050 Q5 High-Fidelity 2x Master Mix NEB Cat#M0492L ddPCR Supermix for Probes (No dUTP) Bio-Rad Cat#1863024 RIPA Lysis and Extraction Buffer Fisher Scientific Cat#PI89900 Protease Inhibitor Cocktail (100X) Thermo Scientific Cat#78429 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat#4368814 Power SYBR Green PCR Master Mix Applied Biosystems Cat#4367659 SPRIselect Beckman Coulter Cat#B23317 cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Roche Cat#11836170001 Isopropyl b-d-1-thiogalactopyranoside (IPTG) Gold Biotechnology Cat#I2481C Critical commercial assays SF Cell Line 96-well Nucleofector Kit Lonza Cat#V4SC-2096 NEBNext Ultra II DNA Library Prep Kit for Illumina NEB Cat#E7645L (Continued on next page) e1 Molecular Cell 85, 1–13.e1–e9, May 1, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Qubit dsDNA Quantification Assay Invitrogen Cat#Q32854 Pierce BCA Protein Assay Kit Thermo Scientific Cat#23225 miRNeasy Mini Kit Qiagen Cat#217004 Deposited data Amplicon sequencing data from genome-editing experiments This study NCBI SRA BioProject ID: PRJNA1239632 ChIP-seq data This study NCBI SRA BioProject ID: PRJNA1239632 Unprocessed gel images & raw data from cell & single molecule experiments This study Mendeley data: https://doi.org/ 10.17632/hzr4fnvc88.1 Experimental models: Cell lines Human embryonic kidney 293T cells UC Berkeley Cell Culture Facility N/A Oligonucleotides Fluorescently labeled DNA substrates IDT Table S13 Cas9 sgRNAs IDT Tables S5, S8, and S9 Primers and oligonucleotides for AuRBT tether construction IDT Tables S15 and S16 RT-qPCR primers IDT Table S12 ddPCR primers/probes IDT Table S11 NGS primers IDT Table S10 Recombinant DNA Mammalian Cas9 or sgRNA expression plasmids This study, modified from Addgene#62988 Tables S4–S7 Bacterial expression plasmids for Cas9 variants This study, modified from Addgene#179525 Tables S4, S6, and S7 pGGASelect NEB Cat#195714; Table S14 Software and algorithms FlowJo v10.10.0 FlowJo https://www.flowjo.com/solutions/downloads/ Image Lab 6.1 Bio-Rad Laboratories, Inc. https://www.bio-rad.com/en-us/product/ image-lab-software?ID=KRE6P5E8Z MATLAB 9.6.0.1472908 (R2019a) Update 9 The MathWorks Inc. https://www.mathworks.com MEMLET Woody et al.44 https://michaelswoody.github.io/MEMLET/ Bowtie2 version 2.5.2 Langmead and Salzberg45 https://github.com/BenLangmead/bowtie2 deepTools2 version 3.5.1 Ramı́rez et al.46 https://github.com/deeptools/deepTools IGV version 2.19.1 Robinson et al.47 https://igv.org/ Seqtk version 1.3 N/A https://github.com/lh3/seqtk MACS2 version 2.2.9.1 Zhang et al.48 https://github.com/macs3-project/MACS IDR version 2.0.4.2 Li et al.49 https://github.com/kundajelab/idr Picard version 2.21.9 Broad Institute https://github.com/broadinstitute/picard BEDTools version 2.29.2 Quinlan and Hall50 https://github.com/arq5x/bedtools2 Python version 3.9.12 Python Software Foundation https://www.python.org/ GraphPad Prism version 10.4.1 GraphPad https://www.graphpad.com/ ..

    Quantitative RT-PCR:

    Article Title: Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-human b2-microglobulin (2M2)-APC BioLegend Cat#316312; RRID_AB_10641281 Monoclonal ANTI-FLAG M2 antibody produced in mouse Millipore sigma Cat#F1804; RRID: AB_262044 CRISPR/Cas9 antibody Diagenode Cat#C15310258; RRID: AB_2715516 Histone H3 (D1H2) XP Rabbit mAb Cell Signaling Technologies Cat#4499; RRID: AB_10544537 Fluorescein/Oregon Green Polyclonal Antibody ThermoFisher Cat#A-889; RRID: AB_221561 Anti-Digoxigenin from sheep MilliporeSigma (Roche) Cat#11333089001; RRID: AB_514496 Bacterial and virus strains E. coli Rosetta 2 (DE3) Competent Cells EMD Millipore Cat#71397 Chemicals, peptides, and recombinant proteins GTP Sigma-Aldrich G8877-1G ATP Sigma-Aldrich A8937-1G UTP Sigma-Aldrich U6625-1G CTP Sigma-Aldrich C1506-1G Spermidine Sigma-Aldrich S2626-1G T7 RNA polymerase N/A N/A Ni-NTA Superflow Qiagen Cat#30430 TEV protease N/A N/A Mini-PROTEAN TGX Precast Protein Gel Bio-Rad Cat#4561033 Piperidine Sigma-Aldrich 411027-100ML Potassium permanganate Sigma-Aldrich 223468-25G dUTP-digoxigenin MilliporeSigma (Roche) Cat#11093088910 Streptavidin-coated gold nanospheres Cytodiagnostics Cat#ACC-60-04-15 Dynabead MyOne Carboxylic Acid ThermoFisher Cat#65012 Proteinase K NEB Cat#P8107S Dynabeads Protein A for Immunoprecipitation Invitrogen Cat#10002D Salmon Sperm DNA ThermoFisher AM9680 Dulbecco’s Modified Eagle Medium Corning Cat#10-013-CV Fetal Bovine Serum VWR Cat#97068-085 Penicillin/streptomcyin Gibco Cat#15140122 Lipofectamine 3000 Transfection Reagent Invitrogen Cat#L3000015 QuickExtract DNA Extraction Solution Biosearch Technologies Cat#QE09050 Q5 High-Fidelity 2x Master Mix NEB Cat#M0492L ddPCR Supermix for Probes (No dUTP) Bio-Rad Cat#1863024 RIPA Lysis and Extraction Buffer Fisher Scientific Cat#PI89900 Protease Inhibitor Cocktail (100X) Thermo Scientific Cat#78429 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat#4368814 Power SYBR Green PCR Master Mix Applied Biosystems Cat#4367659 SPRIselect Beckman Coulter Cat#B23317 cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Roche Cat#11836170001 Isopropyl b-d-1-thiogalactopyranoside (IPTG) Gold Biotechnology Cat#I2481C Critical commercial assays SF Cell Line 96-well Nucleofector Kit Lonza Cat#V4SC-2096 NEBNext Ultra II DNA Library Prep Kit for Illumina NEB Cat#E7645L (Continued on next page) e1 Molecular Cell 85, 1–13.e1–e9, May 1, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Qubit dsDNA Quantification Assay Invitrogen Cat#Q32854 Pierce BCA Protein Assay Kit Thermo Scientific Cat#23225 miRNeasy Mini Kit Qiagen Cat#217004 Deposited data Amplicon sequencing data from genome-editing experiments This study NCBI SRA BioProject ID: PRJNA1239632 ChIP-seq data This study NCBI SRA BioProject ID: PRJNA1239632 Unprocessed gel images & raw data from cell & single molecule experiments This study Mendeley data: https://doi.org/ 10.17632/hzr4fnvc88.1 Experimental models: Cell lines Human embryonic kidney 293T cells UC Berkeley Cell Culture Facility N/A Oligonucleotides Fluorescently labeled DNA substrates IDT Table S13 Cas9 sgRNAs IDT Tables S5, S8, and S9 Primers and oligonucleotides for AuRBT tether construction IDT Tables S15 and S16 RT-qPCR primers IDT Table S12 ddPCR primers/probes IDT Table S11 NGS primers IDT Table S10 Recombinant DNA Mammalian Cas9 or sgRNA expression plasmids This study, modified from Addgene#62988 Tables S4–S7 Bacterial expression plasmids for Cas9 variants This study, modified from Addgene#179525 Tables S4, S6, and S7 pGGASelect NEB Cat#195714; Table S14 Software and algorithms FlowJo v10.10.0 FlowJo https://www.flowjo.com/solutions/downloads/ Image Lab 6.1 Bio-Rad Laboratories, Inc. https://www.bio-rad.com/en-us/product/ image-lab-software?ID=KRE6P5E8Z MATLAB 9.6.0.1472908 (R2019a) Update 9 The MathWorks Inc. https://www.mathworks.com MEMLET Woody et al.44 https://michaelswoody.github.io/MEMLET/ Bowtie2 version 2.5.2 Langmead and Salzberg45 https://github.com/BenLangmead/bowtie2 deepTools2 version 3.5.1 Ramı́rez et al.46 https://github.com/deeptools/deepTools IGV version 2.19.1 Robinson et al.47 https://igv.org/ Seqtk version 1.3 N/A https://github.com/lh3/seqtk MACS2 version 2.2.9.1 Zhang et al.48 https://github.com/macs3-project/MACS IDR version 2.0.4.2 Li et al.49 https://github.com/kundajelab/idr Picard version 2.21.9 Broad Institute https://github.com/broadinstitute/picard BEDTools version 2.29.2 Quinlan and Hall50 https://github.com/arq5x/bedtools2 Python version 3.9.12 Python Software Foundation https://www.python.org/ GraphPad Prism version 10.4.1 GraphPad https://www.graphpad.com/ ..

    Next-Generation Sequencing:

    Article Title: Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-human b2-microglobulin (2M2)-APC BioLegend Cat#316312; RRID_AB_10641281 Monoclonal ANTI-FLAG M2 antibody produced in mouse Millipore sigma Cat#F1804; RRID: AB_262044 CRISPR/Cas9 antibody Diagenode Cat#C15310258; RRID: AB_2715516 Histone H3 (D1H2) XP Rabbit mAb Cell Signaling Technologies Cat#4499; RRID: AB_10544537 Fluorescein/Oregon Green Polyclonal Antibody ThermoFisher Cat#A-889; RRID: AB_221561 Anti-Digoxigenin from sheep MilliporeSigma (Roche) Cat#11333089001; RRID: AB_514496 Bacterial and virus strains E. coli Rosetta 2 (DE3) Competent Cells EMD Millipore Cat#71397 Chemicals, peptides, and recombinant proteins GTP Sigma-Aldrich G8877-1G ATP Sigma-Aldrich A8937-1G UTP Sigma-Aldrich U6625-1G CTP Sigma-Aldrich C1506-1G Spermidine Sigma-Aldrich S2626-1G T7 RNA polymerase N/A N/A Ni-NTA Superflow Qiagen Cat#30430 TEV protease N/A N/A Mini-PROTEAN TGX Precast Protein Gel Bio-Rad Cat#4561033 Piperidine Sigma-Aldrich 411027-100ML Potassium permanganate Sigma-Aldrich 223468-25G dUTP-digoxigenin MilliporeSigma (Roche) Cat#11093088910 Streptavidin-coated gold nanospheres Cytodiagnostics Cat#ACC-60-04-15 Dynabead MyOne Carboxylic Acid ThermoFisher Cat#65012 Proteinase K NEB Cat#P8107S Dynabeads Protein A for Immunoprecipitation Invitrogen Cat#10002D Salmon Sperm DNA ThermoFisher AM9680 Dulbecco’s Modified Eagle Medium Corning Cat#10-013-CV Fetal Bovine Serum VWR Cat#97068-085 Penicillin/streptomcyin Gibco Cat#15140122 Lipofectamine 3000 Transfection Reagent Invitrogen Cat#L3000015 QuickExtract DNA Extraction Solution Biosearch Technologies Cat#QE09050 Q5 High-Fidelity 2x Master Mix NEB Cat#M0492L ddPCR Supermix for Probes (No dUTP) Bio-Rad Cat#1863024 RIPA Lysis and Extraction Buffer Fisher Scientific Cat#PI89900 Protease Inhibitor Cocktail (100X) Thermo Scientific Cat#78429 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat#4368814 Power SYBR Green PCR Master Mix Applied Biosystems Cat#4367659 SPRIselect Beckman Coulter Cat#B23317 cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Roche Cat#11836170001 Isopropyl b-d-1-thiogalactopyranoside (IPTG) Gold Biotechnology Cat#I2481C Critical commercial assays SF Cell Line 96-well Nucleofector Kit Lonza Cat#V4SC-2096 NEBNext Ultra II DNA Library Prep Kit for Illumina NEB Cat#E7645L (Continued on next page) e1 Molecular Cell 85, 1–13.e1–e9, May 1, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Qubit dsDNA Quantification Assay Invitrogen Cat#Q32854 Pierce BCA Protein Assay Kit Thermo Scientific Cat#23225 miRNeasy Mini Kit Qiagen Cat#217004 Deposited data Amplicon sequencing data from genome-editing experiments This study NCBI SRA BioProject ID: PRJNA1239632 ChIP-seq data This study NCBI SRA BioProject ID: PRJNA1239632 Unprocessed gel images & raw data from cell & single molecule experiments This study Mendeley data: https://doi.org/ 10.17632/hzr4fnvc88.1 Experimental models: Cell lines Human embryonic kidney 293T cells UC Berkeley Cell Culture Facility N/A Oligonucleotides Fluorescently labeled DNA substrates IDT Table S13 Cas9 sgRNAs IDT Tables S5, S8, and S9 Primers and oligonucleotides for AuRBT tether construction IDT Tables S15 and S16 RT-qPCR primers IDT Table S12 ddPCR primers/probes IDT Table S11 NGS primers IDT Table S10 Recombinant DNA Mammalian Cas9 or sgRNA expression plasmids This study, modified from Addgene#62988 Tables S4–S7 Bacterial expression plasmids for Cas9 variants This study, modified from Addgene#179525 Tables S4, S6, and S7 pGGASelect NEB Cat#195714; Table S14 Software and algorithms FlowJo v10.10.0 FlowJo https://www.flowjo.com/solutions/downloads/ Image Lab 6.1 Bio-Rad Laboratories, Inc. https://www.bio-rad.com/en-us/product/ image-lab-software?ID=KRE6P5E8Z MATLAB 9.6.0.1472908 (R2019a) Update 9 The MathWorks Inc. https://www.mathworks.com MEMLET Woody et al.44 https://michaelswoody.github.io/MEMLET/ Bowtie2 version 2.5.2 Langmead and Salzberg45 https://github.com/BenLangmead/bowtie2 deepTools2 version 3.5.1 Ramı́rez et al.46 https://github.com/deeptools/deepTools IGV version 2.19.1 Robinson et al.47 https://igv.org/ Seqtk version 1.3 N/A https://github.com/lh3/seqtk MACS2 version 2.2.9.1 Zhang et al.48 https://github.com/macs3-project/MACS IDR version 2.0.4.2 Li et al.49 https://github.com/kundajelab/idr Picard version 2.21.9 Broad Institute https://github.com/broadinstitute/picard BEDTools version 2.29.2 Quinlan and Hall50 https://github.com/arq5x/bedtools2 Python version 3.9.12 Python Software Foundation https://www.python.org/ GraphPad Prism version 10.4.1 GraphPad https://www.graphpad.com/ ..

    Recombinant:

    Article Title: Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-human b2-microglobulin (2M2)-APC BioLegend Cat#316312; RRID_AB_10641281 Monoclonal ANTI-FLAG M2 antibody produced in mouse Millipore sigma Cat#F1804; RRID: AB_262044 CRISPR/Cas9 antibody Diagenode Cat#C15310258; RRID: AB_2715516 Histone H3 (D1H2) XP Rabbit mAb Cell Signaling Technologies Cat#4499; RRID: AB_10544537 Fluorescein/Oregon Green Polyclonal Antibody ThermoFisher Cat#A-889; RRID: AB_221561 Anti-Digoxigenin from sheep MilliporeSigma (Roche) Cat#11333089001; RRID: AB_514496 Bacterial and virus strains E. coli Rosetta 2 (DE3) Competent Cells EMD Millipore Cat#71397 Chemicals, peptides, and recombinant proteins GTP Sigma-Aldrich G8877-1G ATP Sigma-Aldrich A8937-1G UTP Sigma-Aldrich U6625-1G CTP Sigma-Aldrich C1506-1G Spermidine Sigma-Aldrich S2626-1G T7 RNA polymerase N/A N/A Ni-NTA Superflow Qiagen Cat#30430 TEV protease N/A N/A Mini-PROTEAN TGX Precast Protein Gel Bio-Rad Cat#4561033 Piperidine Sigma-Aldrich 411027-100ML Potassium permanganate Sigma-Aldrich 223468-25G dUTP-digoxigenin MilliporeSigma (Roche) Cat#11093088910 Streptavidin-coated gold nanospheres Cytodiagnostics Cat#ACC-60-04-15 Dynabead MyOne Carboxylic Acid ThermoFisher Cat#65012 Proteinase K NEB Cat#P8107S Dynabeads Protein A for Immunoprecipitation Invitrogen Cat#10002D Salmon Sperm DNA ThermoFisher AM9680 Dulbecco’s Modified Eagle Medium Corning Cat#10-013-CV Fetal Bovine Serum VWR Cat#97068-085 Penicillin/streptomcyin Gibco Cat#15140122 Lipofectamine 3000 Transfection Reagent Invitrogen Cat#L3000015 QuickExtract DNA Extraction Solution Biosearch Technologies Cat#QE09050 Q5 High-Fidelity 2x Master Mix NEB Cat#M0492L ddPCR Supermix for Probes (No dUTP) Bio-Rad Cat#1863024 RIPA Lysis and Extraction Buffer Fisher Scientific Cat#PI89900 Protease Inhibitor Cocktail (100X) Thermo Scientific Cat#78429 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat#4368814 Power SYBR Green PCR Master Mix Applied Biosystems Cat#4367659 SPRIselect Beckman Coulter Cat#B23317 cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Roche Cat#11836170001 Isopropyl b-d-1-thiogalactopyranoside (IPTG) Gold Biotechnology Cat#I2481C Critical commercial assays SF Cell Line 96-well Nucleofector Kit Lonza Cat#V4SC-2096 NEBNext Ultra II DNA Library Prep Kit for Illumina NEB Cat#E7645L (Continued on next page) e1 Molecular Cell 85, 1–13.e1–e9, May 1, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Qubit dsDNA Quantification Assay Invitrogen Cat#Q32854 Pierce BCA Protein Assay Kit Thermo Scientific Cat#23225 miRNeasy Mini Kit Qiagen Cat#217004 Deposited data Amplicon sequencing data from genome-editing experiments This study NCBI SRA BioProject ID: PRJNA1239632 ChIP-seq data This study NCBI SRA BioProject ID: PRJNA1239632 Unprocessed gel images & raw data from cell & single molecule experiments This study Mendeley data: https://doi.org/ 10.17632/hzr4fnvc88.1 Experimental models: Cell lines Human embryonic kidney 293T cells UC Berkeley Cell Culture Facility N/A Oligonucleotides Fluorescently labeled DNA substrates IDT Table S13 Cas9 sgRNAs IDT Tables S5, S8, and S9 Primers and oligonucleotides for AuRBT tether construction IDT Tables S15 and S16 RT-qPCR primers IDT Table S12 ddPCR primers/probes IDT Table S11 NGS primers IDT Table S10 Recombinant DNA Mammalian Cas9 or sgRNA expression plasmids This study, modified from Addgene#62988 Tables S4–S7 Bacterial expression plasmids for Cas9 variants This study, modified from Addgene#179525 Tables S4, S6, and S7 pGGASelect NEB Cat#195714; Table S14 Software and algorithms FlowJo v10.10.0 FlowJo https://www.flowjo.com/solutions/downloads/ Image Lab 6.1 Bio-Rad Laboratories, Inc. https://www.bio-rad.com/en-us/product/ image-lab-software?ID=KRE6P5E8Z MATLAB 9.6.0.1472908 (R2019a) Update 9 The MathWorks Inc. https://www.mathworks.com MEMLET Woody et al.44 https://michaelswoody.github.io/MEMLET/ Bowtie2 version 2.5.2 Langmead and Salzberg45 https://github.com/BenLangmead/bowtie2 deepTools2 version 3.5.1 Ramı́rez et al.46 https://github.com/deeptools/deepTools IGV version 2.19.1 Robinson et al.47 https://igv.org/ Seqtk version 1.3 N/A https://github.com/lh3/seqtk MACS2 version 2.2.9.1 Zhang et al.48 https://github.com/macs3-project/MACS IDR version 2.0.4.2 Li et al.49 https://github.com/kundajelab/idr Picard version 2.21.9 Broad Institute https://github.com/broadinstitute/picard BEDTools version 2.29.2 Quinlan and Hall50 https://github.com/arq5x/bedtools2 Python version 3.9.12 Python Software Foundation https://www.python.org/ GraphPad Prism version 10.4.1 GraphPad https://www.graphpad.com/ ..

    Expressing:

    Article Title: Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-human b2-microglobulin (2M2)-APC BioLegend Cat#316312; RRID_AB_10641281 Monoclonal ANTI-FLAG M2 antibody produced in mouse Millipore sigma Cat#F1804; RRID: AB_262044 CRISPR/Cas9 antibody Diagenode Cat#C15310258; RRID: AB_2715516 Histone H3 (D1H2) XP Rabbit mAb Cell Signaling Technologies Cat#4499; RRID: AB_10544537 Fluorescein/Oregon Green Polyclonal Antibody ThermoFisher Cat#A-889; RRID: AB_221561 Anti-Digoxigenin from sheep MilliporeSigma (Roche) Cat#11333089001; RRID: AB_514496 Bacterial and virus strains E. coli Rosetta 2 (DE3) Competent Cells EMD Millipore Cat#71397 Chemicals, peptides, and recombinant proteins GTP Sigma-Aldrich G8877-1G ATP Sigma-Aldrich A8937-1G UTP Sigma-Aldrich U6625-1G CTP Sigma-Aldrich C1506-1G Spermidine Sigma-Aldrich S2626-1G T7 RNA polymerase N/A N/A Ni-NTA Superflow Qiagen Cat#30430 TEV protease N/A N/A Mini-PROTEAN TGX Precast Protein Gel Bio-Rad Cat#4561033 Piperidine Sigma-Aldrich 411027-100ML Potassium permanganate Sigma-Aldrich 223468-25G dUTP-digoxigenin MilliporeSigma (Roche) Cat#11093088910 Streptavidin-coated gold nanospheres Cytodiagnostics Cat#ACC-60-04-15 Dynabead MyOne Carboxylic Acid ThermoFisher Cat#65012 Proteinase K NEB Cat#P8107S Dynabeads Protein A for Immunoprecipitation Invitrogen Cat#10002D Salmon Sperm DNA ThermoFisher AM9680 Dulbecco’s Modified Eagle Medium Corning Cat#10-013-CV Fetal Bovine Serum VWR Cat#97068-085 Penicillin/streptomcyin Gibco Cat#15140122 Lipofectamine 3000 Transfection Reagent Invitrogen Cat#L3000015 QuickExtract DNA Extraction Solution Biosearch Technologies Cat#QE09050 Q5 High-Fidelity 2x Master Mix NEB Cat#M0492L ddPCR Supermix for Probes (No dUTP) Bio-Rad Cat#1863024 RIPA Lysis and Extraction Buffer Fisher Scientific Cat#PI89900 Protease Inhibitor Cocktail (100X) Thermo Scientific Cat#78429 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat#4368814 Power SYBR Green PCR Master Mix Applied Biosystems Cat#4367659 SPRIselect Beckman Coulter Cat#B23317 cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Roche Cat#11836170001 Isopropyl b-d-1-thiogalactopyranoside (IPTG) Gold Biotechnology Cat#I2481C Critical commercial assays SF Cell Line 96-well Nucleofector Kit Lonza Cat#V4SC-2096 NEBNext Ultra II DNA Library Prep Kit for Illumina NEB Cat#E7645L (Continued on next page) e1 Molecular Cell 85, 1–13.e1–e9, May 1, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Qubit dsDNA Quantification Assay Invitrogen Cat#Q32854 Pierce BCA Protein Assay Kit Thermo Scientific Cat#23225 miRNeasy Mini Kit Qiagen Cat#217004 Deposited data Amplicon sequencing data from genome-editing experiments This study NCBI SRA BioProject ID: PRJNA1239632 ChIP-seq data This study NCBI SRA BioProject ID: PRJNA1239632 Unprocessed gel images & raw data from cell & single molecule experiments This study Mendeley data: https://doi.org/ 10.17632/hzr4fnvc88.1 Experimental models: Cell lines Human embryonic kidney 293T cells UC Berkeley Cell Culture Facility N/A Oligonucleotides Fluorescently labeled DNA substrates IDT Table S13 Cas9 sgRNAs IDT Tables S5, S8, and S9 Primers and oligonucleotides for AuRBT tether construction IDT Tables S15 and S16 RT-qPCR primers IDT Table S12 ddPCR primers/probes IDT Table S11 NGS primers IDT Table S10 Recombinant DNA Mammalian Cas9 or sgRNA expression plasmids This study, modified from Addgene#62988 Tables S4–S7 Bacterial expression plasmids for Cas9 variants This study, modified from Addgene#179525 Tables S4, S6, and S7 pGGASelect NEB Cat#195714; Table S14 Software and algorithms FlowJo v10.10.0 FlowJo https://www.flowjo.com/solutions/downloads/ Image Lab 6.1 Bio-Rad Laboratories, Inc. https://www.bio-rad.com/en-us/product/ image-lab-software?ID=KRE6P5E8Z MATLAB 9.6.0.1472908 (R2019a) Update 9 The MathWorks Inc. https://www.mathworks.com MEMLET Woody et al.44 https://michaelswoody.github.io/MEMLET/ Bowtie2 version 2.5.2 Langmead and Salzberg45 https://github.com/BenLangmead/bowtie2 deepTools2 version 3.5.1 Ramı́rez et al.46 https://github.com/deeptools/deepTools IGV version 2.19.1 Robinson et al.47 https://igv.org/ Seqtk version 1.3 N/A https://github.com/lh3/seqtk MACS2 version 2.2.9.1 Zhang et al.48 https://github.com/macs3-project/MACS IDR version 2.0.4.2 Li et al.49 https://github.com/kundajelab/idr Picard version 2.21.9 Broad Institute https://github.com/broadinstitute/picard BEDTools version 2.29.2 Quinlan and Hall50 https://github.com/arq5x/bedtools2 Python version 3.9.12 Python Software Foundation https://www.python.org/ GraphPad Prism version 10.4.1 GraphPad https://www.graphpad.com/ ..

    Article Title: Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing
    Article Snippet: Mammalian Cas9 or sgRNA expression plasmids , This study, modified from Addgene#62988 , – . .. Bacterial expression plasmids for Cas9 variants , This study, modified from Addgene#179525 , , and . .. pGGASelect , NEB , Cat#195714 .

    Modification:

    Article Title: Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-human b2-microglobulin (2M2)-APC BioLegend Cat#316312; RRID_AB_10641281 Monoclonal ANTI-FLAG M2 antibody produced in mouse Millipore sigma Cat#F1804; RRID: AB_262044 CRISPR/Cas9 antibody Diagenode Cat#C15310258; RRID: AB_2715516 Histone H3 (D1H2) XP Rabbit mAb Cell Signaling Technologies Cat#4499; RRID: AB_10544537 Fluorescein/Oregon Green Polyclonal Antibody ThermoFisher Cat#A-889; RRID: AB_221561 Anti-Digoxigenin from sheep MilliporeSigma (Roche) Cat#11333089001; RRID: AB_514496 Bacterial and virus strains E. coli Rosetta 2 (DE3) Competent Cells EMD Millipore Cat#71397 Chemicals, peptides, and recombinant proteins GTP Sigma-Aldrich G8877-1G ATP Sigma-Aldrich A8937-1G UTP Sigma-Aldrich U6625-1G CTP Sigma-Aldrich C1506-1G Spermidine Sigma-Aldrich S2626-1G T7 RNA polymerase N/A N/A Ni-NTA Superflow Qiagen Cat#30430 TEV protease N/A N/A Mini-PROTEAN TGX Precast Protein Gel Bio-Rad Cat#4561033 Piperidine Sigma-Aldrich 411027-100ML Potassium permanganate Sigma-Aldrich 223468-25G dUTP-digoxigenin MilliporeSigma (Roche) Cat#11093088910 Streptavidin-coated gold nanospheres Cytodiagnostics Cat#ACC-60-04-15 Dynabead MyOne Carboxylic Acid ThermoFisher Cat#65012 Proteinase K NEB Cat#P8107S Dynabeads Protein A for Immunoprecipitation Invitrogen Cat#10002D Salmon Sperm DNA ThermoFisher AM9680 Dulbecco’s Modified Eagle Medium Corning Cat#10-013-CV Fetal Bovine Serum VWR Cat#97068-085 Penicillin/streptomcyin Gibco Cat#15140122 Lipofectamine 3000 Transfection Reagent Invitrogen Cat#L3000015 QuickExtract DNA Extraction Solution Biosearch Technologies Cat#QE09050 Q5 High-Fidelity 2x Master Mix NEB Cat#M0492L ddPCR Supermix for Probes (No dUTP) Bio-Rad Cat#1863024 RIPA Lysis and Extraction Buffer Fisher Scientific Cat#PI89900 Protease Inhibitor Cocktail (100X) Thermo Scientific Cat#78429 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat#4368814 Power SYBR Green PCR Master Mix Applied Biosystems Cat#4367659 SPRIselect Beckman Coulter Cat#B23317 cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Roche Cat#11836170001 Isopropyl b-d-1-thiogalactopyranoside (IPTG) Gold Biotechnology Cat#I2481C Critical commercial assays SF Cell Line 96-well Nucleofector Kit Lonza Cat#V4SC-2096 NEBNext Ultra II DNA Library Prep Kit for Illumina NEB Cat#E7645L (Continued on next page) e1 Molecular Cell 85, 1–13.e1–e9, May 1, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Qubit dsDNA Quantification Assay Invitrogen Cat#Q32854 Pierce BCA Protein Assay Kit Thermo Scientific Cat#23225 miRNeasy Mini Kit Qiagen Cat#217004 Deposited data Amplicon sequencing data from genome-editing experiments This study NCBI SRA BioProject ID: PRJNA1239632 ChIP-seq data This study NCBI SRA BioProject ID: PRJNA1239632 Unprocessed gel images & raw data from cell & single molecule experiments This study Mendeley data: https://doi.org/ 10.17632/hzr4fnvc88.1 Experimental models: Cell lines Human embryonic kidney 293T cells UC Berkeley Cell Culture Facility N/A Oligonucleotides Fluorescently labeled DNA substrates IDT Table S13 Cas9 sgRNAs IDT Tables S5, S8, and S9 Primers and oligonucleotides for AuRBT tether construction IDT Tables S15 and S16 RT-qPCR primers IDT Table S12 ddPCR primers/probes IDT Table S11 NGS primers IDT Table S10 Recombinant DNA Mammalian Cas9 or sgRNA expression plasmids This study, modified from Addgene#62988 Tables S4–S7 Bacterial expression plasmids for Cas9 variants This study, modified from Addgene#179525 Tables S4, S6, and S7 pGGASelect NEB Cat#195714; Table S14 Software and algorithms FlowJo v10.10.0 FlowJo https://www.flowjo.com/solutions/downloads/ Image Lab 6.1 Bio-Rad Laboratories, Inc. https://www.bio-rad.com/en-us/product/ image-lab-software?ID=KRE6P5E8Z MATLAB 9.6.0.1472908 (R2019a) Update 9 The MathWorks Inc. https://www.mathworks.com MEMLET Woody et al.44 https://michaelswoody.github.io/MEMLET/ Bowtie2 version 2.5.2 Langmead and Salzberg45 https://github.com/BenLangmead/bowtie2 deepTools2 version 3.5.1 Ramı́rez et al.46 https://github.com/deeptools/deepTools IGV version 2.19.1 Robinson et al.47 https://igv.org/ Seqtk version 1.3 N/A https://github.com/lh3/seqtk MACS2 version 2.2.9.1 Zhang et al.48 https://github.com/macs3-project/MACS IDR version 2.0.4.2 Li et al.49 https://github.com/kundajelab/idr Picard version 2.21.9 Broad Institute https://github.com/broadinstitute/picard BEDTools version 2.29.2 Quinlan and Hall50 https://github.com/arq5x/bedtools2 Python version 3.9.12 Python Software Foundation https://www.python.org/ GraphPad Prism version 10.4.1 GraphPad https://www.graphpad.com/ ..

    Article Title: Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing
    Article Snippet: Mammalian Cas9 or sgRNA expression plasmids , This study, modified from Addgene#62988 , – . .. Bacterial expression plasmids for Cas9 variants , This study, modified from Addgene#179525 , , and . .. pGGASelect , NEB , Cat#195714 .

    Software:

    Article Title: Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing.
    Article Snippet: REAGENT or RESOURCE SOURCE IDENTIFIER Antibodies Anti-human b2-microglobulin (2M2)-APC BioLegend Cat#316312; RRID_AB_10641281 Monoclonal ANTI-FLAG M2 antibody produced in mouse Millipore sigma Cat#F1804; RRID: AB_262044 CRISPR/Cas9 antibody Diagenode Cat#C15310258; RRID: AB_2715516 Histone H3 (D1H2) XP Rabbit mAb Cell Signaling Technologies Cat#4499; RRID: AB_10544537 Fluorescein/Oregon Green Polyclonal Antibody ThermoFisher Cat#A-889; RRID: AB_221561 Anti-Digoxigenin from sheep MilliporeSigma (Roche) Cat#11333089001; RRID: AB_514496 Bacterial and virus strains E. coli Rosetta 2 (DE3) Competent Cells EMD Millipore Cat#71397 Chemicals, peptides, and recombinant proteins GTP Sigma-Aldrich G8877-1G ATP Sigma-Aldrich A8937-1G UTP Sigma-Aldrich U6625-1G CTP Sigma-Aldrich C1506-1G Spermidine Sigma-Aldrich S2626-1G T7 RNA polymerase N/A N/A Ni-NTA Superflow Qiagen Cat#30430 TEV protease N/A N/A Mini-PROTEAN TGX Precast Protein Gel Bio-Rad Cat#4561033 Piperidine Sigma-Aldrich 411027-100ML Potassium permanganate Sigma-Aldrich 223468-25G dUTP-digoxigenin MilliporeSigma (Roche) Cat#11093088910 Streptavidin-coated gold nanospheres Cytodiagnostics Cat#ACC-60-04-15 Dynabead MyOne Carboxylic Acid ThermoFisher Cat#65012 Proteinase K NEB Cat#P8107S Dynabeads Protein A for Immunoprecipitation Invitrogen Cat#10002D Salmon Sperm DNA ThermoFisher AM9680 Dulbecco’s Modified Eagle Medium Corning Cat#10-013-CV Fetal Bovine Serum VWR Cat#97068-085 Penicillin/streptomcyin Gibco Cat#15140122 Lipofectamine 3000 Transfection Reagent Invitrogen Cat#L3000015 QuickExtract DNA Extraction Solution Biosearch Technologies Cat#QE09050 Q5 High-Fidelity 2x Master Mix NEB Cat#M0492L ddPCR Supermix for Probes (No dUTP) Bio-Rad Cat#1863024 RIPA Lysis and Extraction Buffer Fisher Scientific Cat#PI89900 Protease Inhibitor Cocktail (100X) Thermo Scientific Cat#78429 High-Capacity cDNA Reverse Transcription Kit Applied Biosystems Cat#4368814 Power SYBR Green PCR Master Mix Applied Biosystems Cat#4367659 SPRIselect Beckman Coulter Cat#B23317 cOmplete, Mini, EDTA-free Protease Inhibitor Cocktail Roche Cat#11836170001 Isopropyl b-d-1-thiogalactopyranoside (IPTG) Gold Biotechnology Cat#I2481C Critical commercial assays SF Cell Line 96-well Nucleofector Kit Lonza Cat#V4SC-2096 NEBNext Ultra II DNA Library Prep Kit for Illumina NEB Cat#E7645L (Continued on next page) e1 Molecular Cell 85, 1–13.e1–e9, May 1, 2025 .. REAGENT or RESOURCE SOURCE IDENTIFIER Qubit dsDNA Quantification Assay Invitrogen Cat#Q32854 Pierce BCA Protein Assay Kit Thermo Scientific Cat#23225 miRNeasy Mini Kit Qiagen Cat#217004 Deposited data Amplicon sequencing data from genome-editing experiments This study NCBI SRA BioProject ID: PRJNA1239632 ChIP-seq data This study NCBI SRA BioProject ID: PRJNA1239632 Unprocessed gel images & raw data from cell & single molecule experiments This study Mendeley data: https://doi.org/ 10.17632/hzr4fnvc88.1 Experimental models: Cell lines Human embryonic kidney 293T cells UC Berkeley Cell Culture Facility N/A Oligonucleotides Fluorescently labeled DNA substrates IDT Table S13 Cas9 sgRNAs IDT Tables S5, S8, and S9 Primers and oligonucleotides for AuRBT tether construction IDT Tables S15 and S16 RT-qPCR primers IDT Table S12 ddPCR primers/probes IDT Table S11 NGS primers IDT Table S10 Recombinant DNA Mammalian Cas9 or sgRNA expression plasmids This study, modified from Addgene#62988 Tables S4–S7 Bacterial expression plasmids for Cas9 variants This study, modified from Addgene#179525 Tables S4, S6, and S7 pGGASelect NEB Cat#195714; Table S14 Software and algorithms FlowJo v10.10.0 FlowJo https://www.flowjo.com/solutions/downloads/ Image Lab 6.1 Bio-Rad Laboratories, Inc. https://www.bio-rad.com/en-us/product/ image-lab-software?ID=KRE6P5E8Z MATLAB 9.6.0.1472908 (R2019a) Update 9 The MathWorks Inc. https://www.mathworks.com MEMLET Woody et al.44 https://michaelswoody.github.io/MEMLET/ Bowtie2 version 2.5.2 Langmead and Salzberg45 https://github.com/BenLangmead/bowtie2 deepTools2 version 3.5.1 Ramı́rez et al.46 https://github.com/deeptools/deepTools IGV version 2.19.1 Robinson et al.47 https://igv.org/ Seqtk version 1.3 N/A https://github.com/lh3/seqtk MACS2 version 2.2.9.1 Zhang et al.48 https://github.com/macs3-project/MACS IDR version 2.0.4.2 Li et al.49 https://github.com/kundajelab/idr Picard version 2.21.9 Broad Institute https://github.com/broadinstitute/picard BEDTools version 2.29.2 Quinlan and Hall50 https://github.com/arq5x/bedtools2 Python version 3.9.12 Python Software Foundation https://www.python.org/ GraphPad Prism version 10.4.1 GraphPad https://www.graphpad.com/ ..



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    bacterial expression plasmids for cas9 variants  (Addgene inc)
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    (A) A model of RNA-guided DNA targeting by <t>Cas9.</t> The two-step target capture process that differentiates PAM-relaxed Cas9 variants from WT Spy Cas9 is highlighted. (B) The three Spy Cas9 proteins used in this study with their respective PAM recognition: WT Spy Cas9 (gray), SpG (pink) and SpRY (purple). (C) Quantifications of DNA double-stranded breaks (DSB) by droplet digital PCR (ddPCR) on the target site at 8 h post plasmid transfection (n=3 replicates). See also for more data. (D) Quantifications of indels on the target site at 72 h post RNP nucleofection (n=3). See also for more data. (E) Time-course analysis of average DNA cleavage products (n=3) for sgRNA2 in 5 mM Mg2+ at 24°C for TS (see ). The average rate constants, with the amplitudes from the observed double-exponential decay, are provided in figure legends. See also for NTS cleavage and - for other sgRNAs and conditions. (F) Time-course analysis of the average fluorescence signal (n=3) in the 2AP assay for the same sequence in similar conditions as (E) (see ). For WT Spy Cas9, catalytically dead Cas9 (dCas9) is used here. The rate constants of the observed mono-exponential decay are provided in figure legends. See also for the 2AP-labeling position (15th nt from PAM) and intercept determination (using non-targeting sgRNA). All error bars in (C-F) represent the standard deviation of n = 3 replicates.
    Bacterial Expression Plasmids For Cas9 Variants, supplied by Addgene inc, used in various techniques. Bioz Stars score: 90/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    Figure 1. Types of engineered <t>Cas9</t> variants and the results of in vitro DNA cleavage assay. (A) Domain organization of type II-A Cas9 from S. Pyogenes (SpCas9) and correspondingly color-coded crystal structure of Cas9:gRNA:DNA ternary complex (PDB ID: 5F9R) (6). (B) Engineered Cas9 variants used in this work and their mutated residues. (C) Two-dimensional plot for cleavage efficiency of wild-type and engineered Cas9 variants toward on-target and off-target DNAs with different degrees of mismatch (denoted by Mi-j for bases mutated from the ith through jth sites counting from PAM).
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    (A) A model of RNA-guided DNA targeting by Cas9. The two-step target capture process that differentiates PAM-relaxed Cas9 variants from WT Spy Cas9 is highlighted. (B) The three Spy Cas9 proteins used in this study with their respective PAM recognition: WT Spy Cas9 (gray), SpG (pink) and SpRY (purple). (C) Quantifications of DNA double-stranded breaks (DSB) by droplet digital PCR (ddPCR) on the target site at 8 h post plasmid transfection (n=3 replicates). See also for more data. (D) Quantifications of indels on the target site at 72 h post RNP nucleofection (n=3). See also for more data. (E) Time-course analysis of average DNA cleavage products (n=3) for sgRNA2 in 5 mM Mg2+ at 24°C for TS (see ). The average rate constants, with the amplitudes from the observed double-exponential decay, are provided in figure legends. See also for NTS cleavage and - for other sgRNAs and conditions. (F) Time-course analysis of the average fluorescence signal (n=3) in the 2AP assay for the same sequence in similar conditions as (E) (see ). For WT Spy Cas9, catalytically dead Cas9 (dCas9) is used here. The rate constants of the observed mono-exponential decay are provided in figure legends. See also for the 2AP-labeling position (15th nt from PAM) and intercept determination (using non-targeting sgRNA). All error bars in (C-F) represent the standard deviation of n = 3 replicates.

    Journal: Molecular cell

    Article Title: Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing

    doi: 10.1016/j.molcel.2025.03.024

    Figure Lengend Snippet: (A) A model of RNA-guided DNA targeting by Cas9. The two-step target capture process that differentiates PAM-relaxed Cas9 variants from WT Spy Cas9 is highlighted. (B) The three Spy Cas9 proteins used in this study with their respective PAM recognition: WT Spy Cas9 (gray), SpG (pink) and SpRY (purple). (C) Quantifications of DNA double-stranded breaks (DSB) by droplet digital PCR (ddPCR) on the target site at 8 h post plasmid transfection (n=3 replicates). See also for more data. (D) Quantifications of indels on the target site at 72 h post RNP nucleofection (n=3). See also for more data. (E) Time-course analysis of average DNA cleavage products (n=3) for sgRNA2 in 5 mM Mg2+ at 24°C for TS (see ). The average rate constants, with the amplitudes from the observed double-exponential decay, are provided in figure legends. See also for NTS cleavage and - for other sgRNAs and conditions. (F) Time-course analysis of the average fluorescence signal (n=3) in the 2AP assay for the same sequence in similar conditions as (E) (see ). For WT Spy Cas9, catalytically dead Cas9 (dCas9) is used here. The rate constants of the observed mono-exponential decay are provided in figure legends. See also for the 2AP-labeling position (15th nt from PAM) and intercept determination (using non-targeting sgRNA). All error bars in (C-F) represent the standard deviation of n = 3 replicates.

    Article Snippet: Bacterial expression plasmids for Cas9 variants , This study, modified from Addgene#179525 , , and .

    Techniques: Digital PCR, Plasmid Preparation, Transfection, Fluorescence, Sequencing, Labeling, Standard Deviation

    (A, C, E) Example traces of time-resolved measurements of equilibrium twist change Δθ over time for (A and C) WT Spy Cas9 (50 nM) and (E) SpRY (50 nM), each paired with sgRNA4.1, containing a 3-bp match to the Target2 sequence flanking (A and E) an NGG site or (C) an NCG site on the DNA tether. 250-ms averaged traces are shown in black. For (E) , the vertical dashed lines (--) and dash-dot lines (−.) indicate the start and end, respectively, of the flow of SpRY RNP into the chamber. (B, D) Scatter plots of unwinding lifetime and Δθ 0 for merged Steppi-scored states corresponding to experiments described in (A, C) . The total collection time ( T total ) and number of DNA tethers (n) are provided in the legend. See also – for more data with different sequences and conditions. (F) Fit of average Δθ baseline shift and [RNP] for SpRY to a binding equation yielded an apparent K d ~ 14 nM for sgRNA4.1 on the Target2 sequence flanking an NGG site. See also – for raw traces. (G) Schematic overview of the permanganate DNA footprinting assay. The UREA-PAGE gel image used to generate the graph in (I) highlights KMnO 4 -dependent enriched bands for WT Spy Cas9 (gray), SpG (pink) and SpRY (purple) in colored dashed boxes. The gel image was rendered in ImageLab 6.1 (BioRad) and cropped to exclude irrelevant neighboring lanes. (H) Sequence of the sgRNA (sgRNA3) and the dsDNA substrate with all the reactive thymines on the TS underlined. (I) Oxidation probabilities of thymines across the dsDNA substrate.

    Journal: Molecular cell

    Article Title: Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing

    doi: 10.1016/j.molcel.2025.03.024

    Figure Lengend Snippet: (A, C, E) Example traces of time-resolved measurements of equilibrium twist change Δθ over time for (A and C) WT Spy Cas9 (50 nM) and (E) SpRY (50 nM), each paired with sgRNA4.1, containing a 3-bp match to the Target2 sequence flanking (A and E) an NGG site or (C) an NCG site on the DNA tether. 250-ms averaged traces are shown in black. For (E) , the vertical dashed lines (--) and dash-dot lines (−.) indicate the start and end, respectively, of the flow of SpRY RNP into the chamber. (B, D) Scatter plots of unwinding lifetime and Δθ 0 for merged Steppi-scored states corresponding to experiments described in (A, C) . The total collection time ( T total ) and number of DNA tethers (n) are provided in the legend. See also – for more data with different sequences and conditions. (F) Fit of average Δθ baseline shift and [RNP] for SpRY to a binding equation yielded an apparent K d ~ 14 nM for sgRNA4.1 on the Target2 sequence flanking an NGG site. See also – for raw traces. (G) Schematic overview of the permanganate DNA footprinting assay. The UREA-PAGE gel image used to generate the graph in (I) highlights KMnO 4 -dependent enriched bands for WT Spy Cas9 (gray), SpG (pink) and SpRY (purple) in colored dashed boxes. The gel image was rendered in ImageLab 6.1 (BioRad) and cropped to exclude irrelevant neighboring lanes. (H) Sequence of the sgRNA (sgRNA3) and the dsDNA substrate with all the reactive thymines on the TS underlined. (I) Oxidation probabilities of thymines across the dsDNA substrate.

    Article Snippet: Bacterial expression plasmids for Cas9 variants , This study, modified from Addgene#179525 , , and .

    Techniques: Sequencing, Binding Assay, DNA Footprinting

    The reduced genome editing efficiencies of PAM-relaxed Cas9 variants (e.g. SpRY) are explained by two factors: (Top) PAM-relaxed Cas9 variants display prolonged target search times, largely due to kinetic trapping at non-specific binding sites, which reduces the pool of free RNPs available for on-target identification. (Bottom) Upon reaching the target site, these variants also become kinetically trapped in a low-energy initial binding complex and unwind DNA slowly.

    Journal: Molecular cell

    Article Title: Rapid two-step target capture ensures efficient CRISPR-Cas9-guided genome editing

    doi: 10.1016/j.molcel.2025.03.024

    Figure Lengend Snippet: The reduced genome editing efficiencies of PAM-relaxed Cas9 variants (e.g. SpRY) are explained by two factors: (Top) PAM-relaxed Cas9 variants display prolonged target search times, largely due to kinetic trapping at non-specific binding sites, which reduces the pool of free RNPs available for on-target identification. (Bottom) Upon reaching the target site, these variants also become kinetically trapped in a low-energy initial binding complex and unwind DNA slowly.

    Article Snippet: Bacterial expression plasmids for Cas9 variants , This study, modified from Addgene#179525 , , and .

    Techniques: Binding Assay, Drug discovery

    Figure 1. Types of engineered Cas9 variants and the results of in vitro DNA cleavage assay. (A) Domain organization of type II-A Cas9 from S. Pyogenes (SpCas9) and correspondingly color-coded crystal structure of Cas9:gRNA:DNA ternary complex (PDB ID: 5F9R) (6). (B) Engineered Cas9 variants used in this work and their mutated residues. (C) Two-dimensional plot for cleavage efficiency of wild-type and engineered Cas9 variants toward on-target and off-target DNAs with different degrees of mismatch (denoted by Mi-j for bases mutated from the ith through jth sites counting from PAM).

    Journal: Nucleic acids research

    Article Title: Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis.

    doi: 10.1093/nar/gkab858

    Figure Lengend Snippet: Figure 1. Types of engineered Cas9 variants and the results of in vitro DNA cleavage assay. (A) Domain organization of type II-A Cas9 from S. Pyogenes (SpCas9) and correspondingly color-coded crystal structure of Cas9:gRNA:DNA ternary complex (PDB ID: 5F9R) (6). (B) Engineered Cas9 variants used in this work and their mutated residues. (C) Two-dimensional plot for cleavage efficiency of wild-type and engineered Cas9 variants toward on-target and off-target DNAs with different degrees of mismatch (denoted by Mi-j for bases mutated from the ith through jth sites counting from PAM).

    Article Snippet: The other Cas9 variants were obtained from Addgene (HypaCas9 #101218, evoCas9 #107550, xCas9(3.7) #108379).

    Techniques: In Vitro, DNA Cleavage Assay

    Figure 2. Single-molecule FRET assay for conformational dynamics of Cas9:gRNA:DNA ternary complex. (A) Experimental scheme for single-molecule FRET detection of two different conformations of the complex (denoted ‘open’ with FRET efficiency Elow ∼0.2 and ‘zipped’ with Ehigh ∼0.8). The two FRET histograms represent when the DNA is on-target (upper) and off-target with M18–20 mismatch (lower). (B) Fraction of Ehigh (= Ehigh/(Ehigh + Elow)) for wild-type and engineered Cas9 variants with respect to on-target and off-target DNAs with different degrees of mismatch (mean ± s.e.m., n = 2 or 3). Relative population was calculated from FRET histograms fitted to Gaussian functions (Supplementary Figure S3A).

    Journal: Nucleic acids research

    Article Title: Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis.

    doi: 10.1093/nar/gkab858

    Figure Lengend Snippet: Figure 2. Single-molecule FRET assay for conformational dynamics of Cas9:gRNA:DNA ternary complex. (A) Experimental scheme for single-molecule FRET detection of two different conformations of the complex (denoted ‘open’ with FRET efficiency Elow ∼0.2 and ‘zipped’ with Ehigh ∼0.8). The two FRET histograms represent when the DNA is on-target (upper) and off-target with M18–20 mismatch (lower). (B) Fraction of Ehigh (= Ehigh/(Ehigh + Elow)) for wild-type and engineered Cas9 variants with respect to on-target and off-target DNAs with different degrees of mismatch (mean ± s.e.m., n = 2 or 3). Relative population was calculated from FRET histograms fitted to Gaussian functions (Supplementary Figure S3A).

    Article Snippet: The other Cas9 variants were obtained from Addgene (HypaCas9 #101218, evoCas9 #107550, xCas9(3.7) #108379).

    Techniques:

    Figure 3. Classification of single-molecule time trajectories and determination of reaction quotients for different stages of Cas9:gRNA:DNA interaction pathway. (A) Representative time trajectories of three different states of Cas9:gRNA:DNA for (#1) docked-open state (DOpen), (#2) transitional state, and (#3) docked-zipped state (DZipped). Transitional state is subcategorized into transitional-open (TOpen) and transitional-zipped (TZipped) states according to our reversible reaction model. Each of the three reversible transitions involving these four states is characterized by its own reaction quotient, QD, QT or QZ. (B) Fraction (in %) of the ternary complexes of wild-type and engineered Cas9 variants in each of the docked-zipped, transitional, and docked-open states when they interact with on-target and off-target DNAs (mean ± s.e.m., n = 3). (C) Representative FRET histograms constructed from the sum of complexes in the docked-open + transitional states (upper), transitional states only (middle), and transitional + docked-zipped states (lower). Population ratio of open (DOpen and/or TOpen) versus zipped (DZipped and/or TZipped) conformations is used to calculate the reaction quotients. (D) The calculated reaction quotients for complexes of wild-type and engineered Cas9 variants toward on-target and off-target DNAs.

    Journal: Nucleic acids research

    Article Title: Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis.

    doi: 10.1093/nar/gkab858

    Figure Lengend Snippet: Figure 3. Classification of single-molecule time trajectories and determination of reaction quotients for different stages of Cas9:gRNA:DNA interaction pathway. (A) Representative time trajectories of three different states of Cas9:gRNA:DNA for (#1) docked-open state (DOpen), (#2) transitional state, and (#3) docked-zipped state (DZipped). Transitional state is subcategorized into transitional-open (TOpen) and transitional-zipped (TZipped) states according to our reversible reaction model. Each of the three reversible transitions involving these four states is characterized by its own reaction quotient, QD, QT or QZ. (B) Fraction (in %) of the ternary complexes of wild-type and engineered Cas9 variants in each of the docked-zipped, transitional, and docked-open states when they interact with on-target and off-target DNAs (mean ± s.e.m., n = 3). (C) Representative FRET histograms constructed from the sum of complexes in the docked-open + transitional states (upper), transitional states only (middle), and transitional + docked-zipped states (lower). Population ratio of open (DOpen and/or TOpen) versus zipped (DZipped and/or TZipped) conformations is used to calculate the reaction quotients. (D) The calculated reaction quotients for complexes of wild-type and engineered Cas9 variants toward on-target and off-target DNAs.

    Article Snippet: The other Cas9 variants were obtained from Addgene (HypaCas9 #101218, evoCas9 #107550, xCas9(3.7) #108379).

    Techniques: Construct

    Figure 4. Specificity values (S) of wild-type and engineered Cas9 variants. (A) Specificity values (ST and SZ) of wild-type and engineered Cas9 variants. (B) WT-normalized specificity values (ST and SZ) of engineered Cas9 variants (mean ± s.e.m., n = 2 or 3).

    Journal: Nucleic acids research

    Article Title: Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis.

    doi: 10.1093/nar/gkab858

    Figure Lengend Snippet: Figure 4. Specificity values (S) of wild-type and engineered Cas9 variants. (A) Specificity values (ST and SZ) of wild-type and engineered Cas9 variants. (B) WT-normalized specificity values (ST and SZ) of engineered Cas9 variants (mean ± s.e.m., n = 2 or 3).

    Article Snippet: The other Cas9 variants were obtained from Addgene (HypaCas9 #101218, evoCas9 #107550, xCas9(3.7) #108379).

    Techniques:

    Figure 5. Schematic representation for different stages of off-target discrimination activity by Cas9:gRNA:DNA ternary complex of different Cas9 vari- ants. Off-target discrimination occurs mainly in the stage of DNA:RNA heteroduplexation for of evoCas9 and HypaCas9, whereas it occurs in the post- heteroduplexation stage for Cas9-HF1, eCas9 and Sniper-Cas9.

    Journal: Nucleic acids research

    Article Title: Quantitative assessment of engineered Cas9 variants for target specificity enhancement by single-molecule reaction pathway analysis.

    doi: 10.1093/nar/gkab858

    Figure Lengend Snippet: Figure 5. Schematic representation for different stages of off-target discrimination activity by Cas9:gRNA:DNA ternary complex of different Cas9 vari- ants. Off-target discrimination occurs mainly in the stage of DNA:RNA heteroduplexation for of evoCas9 and HypaCas9, whereas it occurs in the post- heteroduplexation stage for Cas9-HF1, eCas9 and Sniper-Cas9.

    Article Snippet: The other Cas9 variants were obtained from Addgene (HypaCas9 #101218, evoCas9 #107550, xCas9(3.7) #108379).

    Techniques: Activity Assay