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: Figure 4. Promiscuous DNA binding by PAM-relaxed Cas9 variants (A, C, and E) Example traces of time-resolved measurements of equilibrium twist change Dq over time for (A) and (C) WT SpyCas9 (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 and D) Scatter plots of unwinding lifetime and Dq0 for merged Steppi-scored states corresponding to experiments described in (A) and (C). The total collection time (Ttotal) and number of DNA tethers (n) are provided in the legend. See also Figures S4A–S4C for more data with different sequences and conditions. (F) Fit of average Dq baseline shift and [RNP] for SpRY to a binding equation yielded an apparent Kd 14 nM for sgRNA4.1 on the Target2 sequence flanking an NGG site. See also Figures S4F–S4H 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 KMnO4-dependent enriched bands for WT SpyCas9 (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: 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/

Techniques: Binding Assay, Sequencing, DNA Footprinting