Journal: Nature protocols

Article Title: CRISPR off-target detection with DISCOVER-seq.

doi: 10.1038/s41596-020-0309-5

Figure Lengend Snippet: Fig. 1 | Overview of DISCOVER-seq workflow and quality controls. (i) Cells of interest are edited with Cas9 and gRNA to introduce DNA DSBs at the target site. Successful genome editing should be confirmed (Boxes 1 and 2). (ii) Edited cells are crosslinked and lysed, and chromatin is sheared to 150–500 bp by sonication. The correct size of sheared chromatin should be confirmed by agarose gel electrophoresis (Box 3). (iii) MRE11-bound chromatin fragments are enriched by immunoprecipitation (IP). Enriched DNA is then purified and reverse crosslinked. Successful MRE11 pulldown should be confirmed by qPCR (Box 2). (iv) If ChIP was successful, purified DNA is prepared for NGS by adaptor ligation and PCR amplification of ChIP-seq libraries. The quality of the amplified libraries should be confirmed by size separation on a fragment analyzer. (v) Libraries are sequenced as 100-bp paired-end (PE) reads on a next-generation sequencer. Data are then analyzed using BLENDER software to identify off-target sites. Off-target sites can be validated with targeted amplicon sequencing (Box 1).

Article Snippet: Chromatin IP ● Dynabeads protein A (Thermo Fisher Scientific, cat. no. 10002D) ● MRE11 antibody Option 1: rabbit polyclonal, cross-reactive with mouse and human MRE11 (Novus, cat. no. NB 100-142, RRID: AB_350079) Option 2: recombinant rabbit monoclonal, cross-reactive with mouse and human MRE11 (Abcam, cat. no. ab208020, RRID: AB_2814655) c CRITICAL We tested several antibodies and these two performed the best.

Techniques: Introduce, Sonication, Agarose Gel Electrophoresis, Immunoprecipitation, Ligation, ChIP-sequencing, Software, Amplification, Sequencing

Journal: Nature protocols

Article Title: CRISPR off-target detection with DISCOVER-seq.

doi: 10.1038/s41596-020-0309-5

Figure Lengend Snippet: Fig. 2 | Comparison of MRE11 antibodies. a, Cross-reactivity between human and mouse of five different commercially available MRE11 antibodies as determined by western blot. Whole-cell extracts (30 μg) from human K562 cells and murine B16-F10 cells were used for the analysis. NB 100-142, ab208020 and MA5-17261 are cross-reactive. Black box indicates antibody used by Wienert et al. (ref. 7). b, Performance of MRE11 antibodies in ChIP. K562 cells were edited with RNPs targeting EMX1. ChIP-qPCR was performed 12 h after nucleofection. Shown is the enrichment of the EMX1 region compared to an unedited negative-control region (RNF2). NB 100-142 and ab208020 performed best in this assay. c, Increasing amounts of antibody led to more MRE11 depletion from cell lysate, as determined by western blot. However, increasing pulldown efficiency with larger amounts of antibody is not directly reflected in the levels of eluted MRE11, possibly because of non-linearity of western blots. d, Increasing the amount of antibody increases the on- and off-target signal by qPCR. K562 cells were edited with RNPs targeting VEGFA. ChIP-qPCR was performed 12 h after nucleofection. Shown is the enrichment of the VEGFA region and a known off-target region (PAPD7) compared to an unaffected negative-control region (RNF2). FT, flow-through; IP, immunoprecipitation.

Article Snippet: Chromatin IP ● Dynabeads protein A (Thermo Fisher Scientific, cat. no. 10002D) ● MRE11 antibody Option 1: rabbit polyclonal, cross-reactive with mouse and human MRE11 (Novus, cat. no. NB 100-142, RRID: AB_350079) Option 2: recombinant rabbit monoclonal, cross-reactive with mouse and human MRE11 (Abcam, cat. no. ab208020, RRID: AB_2814655) c CRITICAL We tested several antibodies and these two performed the best.

Techniques: Comparison, Western Blot, ChIP-qPCR, Negative Control, Immunoprecipitation

Journal: Nature protocols

Article Title: CRISPR off-target detection with DISCOVER-seq.

doi: 10.1038/s41596-020-0309-5

Figure Lengend Snippet: Fig. 3 | Quality controls of a DISCOVER-seq experiment. a, Example of a ChIP-qPCR time course to determine the ideal time point for DISCOVER-seq using Cas9 RNP (Box 2). K562 cells were nucleofected with Cas9 and gRNA targeting the HBB locus. Cells were then crosslinked at indicated time points and MRE11 ChIP performed. Shown is the fold enrichment of the on-target region (HBB, gray boxes) over a negative-control region for each sample (left y axis) and the indel frequencies (red circles, right y axis). For this experiment, 12 h after nucleofection was chosen as the ideal time point for DISCOVER-seq (arrow). b, Agarose gel electrophoresis of insufficiently (4 cycles) and optimally (12 cycles) sheared chromatin. Sheared chromatin size (150–500 bp) should be checked after sonication before proceeding with the protocol (Box 3). c, Example of quality control by qPCR for successful isolation of an on-target locus by ChIP before proceeding to ChIP-seq library preparation. B16-F10 cells were nucleofected with a non-targeting gRNA (non-targ.) or a gRNA targeting Pcsk9. After 12 h, cells were crosslinked and MRE11 ChIP was performed. Shown is the fold enrichment of the on-target site Pcsk9 (gray boxes) over a negative control region (left y axis) and the indel frequencies after 4 d (red circles, right y axis). d, Example electropherogram of a ChIP-seq library before sequencing. Although the mean library size is optimal, this sample contains adaptor dimers. If adaptor dimers are present, DNA size selection of the library should be performed (see Troubleshooting). RFUs, relative fluorescence units.

Article Snippet: Chromatin IP ● Dynabeads protein A (Thermo Fisher Scientific, cat. no. 10002D) ● MRE11 antibody Option 1: rabbit polyclonal, cross-reactive with mouse and human MRE11 (Novus, cat. no. NB 100-142, RRID: AB_350079) Option 2: recombinant rabbit monoclonal, cross-reactive with mouse and human MRE11 (Abcam, cat. no. ab208020, RRID: AB_2814655) c CRITICAL We tested several antibodies and these two performed the best.

Techniques: ChIP-qPCR, Negative Control, Agarose Gel Electrophoresis, Sonication, Control, Isolation, ChIP-sequencing, Sequencing, Size Selection