T7e1 endonuclease

We adapted the T7E1 assay method from previous publications with minor modifications [21 (link)]. Genomic DNA was extracted from DF-1 cells after G418 selection. Genomic regions encompassing the CRISPR/Cas9 target sites were amplified using specific primer sets (Table 1). The amplicons were reannealed to form a heteroduplex DNA structure after denaturation. Subsequently, the heteroduplex amplicons were treated with 5 units T7E1 endonuclease (New England Biolabs) for 20 min at 37 °C and then analyzed by 1% agarose gel electrophoresis.

Genomic DNA was extracted from DF-1 cells after puromycin selection. Genomic regions encompassing the CRISPR/Cas9 target sites were amplified using specific primer sets. PCR analysis of the targeted loci was examined in a total volume of 20 μL containing 100 ng genomic DNA, 10× PCR buffer (BioFACT, Daejeon, Korea), 0.4 μL dNTPs (10 mmol/L each), 10 pmol of each primer, and 0.5 U Taq polymerase (BioFACT) under the following thermocycling conditions: 2 min at 95 °C, followed by 35 cycles of 20 s at 95 °C, 40 s at 65 °C, and 30 s at 72 °C, and a final 5 min at 72 °C. Primers are listed in Table 1. The PCR amplicons were re-annealed to form a heteroduplex DNA structure after denaturation. Subsequently, the heteroduplex amplicons were treated with 5 units T7E1 endonuclease (New England Biolabs) for 20 min at 37 °C and then analyzed by 1% agarose gel electrophoresis.

RNP nucleofection was performed with 2 × 10⁵ K562 or Jurkat cells using Lonza 4D nucleofector (Lonza) following the machine’s pre-set K562 and Jurkat transfection programs [22 (link)]. Briefly, Cas9 proteins and sgRNA were pre-assembled at indicated concentrations at room temperature for 10 min, mixed with suspended cells and then subjected to electroporation according to manufacturer’s instructions. Unless noted otherwise, cells were harvested at 48 h post nucleofection and subjected to T7E1 assay using T7E1 endonuclease (NEB) as described [22 (link)]. For sequence analysis, CRISPR-Cas9 target sites were PCR amplified, cloned into pEASY-Blunt-Zero Cloning Kit (Beijing TransGen Biotech, Beijing, China) and then sequenced with M13 primer.
For detection of nucleus localized Cas9 proteins, 2 × 10⁶ of K562 cells were nucleofected with 100 μg Cas9 proteins and then harvested at indicated time points. Collected cells were fractionated using nuclear and cytoplasmic extraction kit (Beyotime Biotechnology, Shanghai, China) for western blot (WB) analysis. Cas9 proteins were detected using monoclonal anti-HA antibody (Cell Signaling Technology, Danvers, MA, USA, 2999) at 1:5000 dilutions. Nuclear internal control lamin B1 was detected using anti-Lamin B1 antibody (Cell Signaling Technology, 13435S) at 1:5000 dilutions.

293T cells in 6-well dishes were cultured to 50–60% confluence as mentioned above. The cells were transfected with 1 μg of KLHDC4 single-guide RNA (sgRNA) plasmid (ligated into the pX330 vector; sgRNA#1: 5’-GGCGGACAGCTGTGGGTCTT-3’; sgRNA#2: 5’-CACAGCTGTCCGCCACCTTG-3’; both sgRNAs targeting the exon 5 of KLHDC4 gene were selected from a published database [20 (link)] of predicted high-specificity protospacer adjacent motif target sites in the human exome) and 5 μl of Lipofectamine 2000 per well. As a control, cells were also tranfected with the pX330 empty vector. The cells were harvested at 72 hours post-transfection, and genomic DNA was extracted (Qiagen DNeasy Blood & Tissue Kit). A region of exon 5 of the KLHDC4 gene was amplified with genomic DNA-specific primers (forward: 5’-TGACTGAGGACGTGCTTTCC-3’; reverse: 5’-CCACAGGAGAAGAGCTGCAA-3’). The homoduplex PCR products were denatured and rehybridized using stepdown annealing conditions to generate homo- and heteroduplexes. The mixture of duplexes was treated with T7E1 endonuclease for 20 minutes at 37°C (New England Biolabs); the reaction was stopped using 1.5 μl of 0.25 M EDTA, and the products were analyzed on a 3% agarose gel.

sgRNA design: sgRNAs were designed (http://crispor.tefor.net/, accessed on 22 June 2022) and their efficacies were validated in NIH/3T3 cells. sgRNA was synthesized in vitro (IDT DNA, Leuven, Belgium) and used for electroporation into mES cells.
Electroporation protocol: NIH/3T3 or WT mES cells were mixed with CRISPR-Cas9 components (IDT-DNA, Leuven, Belgium) and electroporated via Amaxa^TM 4D-Nucleofector^TM (Lonza, Cologne, Germany) using the manufacturer’s guide. The extended electroporation protocol is included in the Supplementary Methods.
Surveyor nuclease assay: sgRNAs were first validated in NIH/3T3 cells via hetero-duplexes formed by CRISPR-Cas9 mediated gene editing, reaction buffer, and T7E1 endonuclease (NEB, Frankfurt, Germany). sgRNA with the highest efficacy was used for WT mES cell gene edit.
After electroporation, mES cells were seeded, single colonies were picked, expanded, and further assessed via PCR and restriction digestion. This was done by the Hind III restriction enzyme (NEB, Frankfurt, Germany). Positive clones were sent for Sanger sequencing (LGC genomics, Berlin, Germany).