Engen mutation detection kit

The Monarch Genomic DNA Purification Kit and the EnGen Mutation Detection Kit were purchased from New England Biolabs (Ipswich, MA), and the assays were carried out according to the manufacture’s protocols. Briefly, 150,000 cells were transfected with Cas9-g82, Cas9-g165, Cas9-g82/165, or Cas9-g82/165 + HDR using lipofectamine and were allowed to incubate for 2 days. Cells were harvested and the genomic DNA was isolated. The target site of interest was PCR amplified using primers ordered from IDT (Coralville, Iowa) with the Q5 Hot Start High Fidelity 2X Master Mix supplied with the EnGen Mutation Detection Kit. Primer sequences used in this study for cloning human (Hu) g82 and g165 and mouse (Ms) g70 and g166 into the multiplex CRISPR/Cas9 assembly system, the repair templates, and primers for the T7E1 assay are shown in Table 1. The PCR products were checked on an agarose gel, and then the samples were denatured for 5 min at 95 °C and allowed to anneal at room temperature for 1 h. Finally, the sample was digested with the T7 endonuclease 1 enzyme for 30 min at 37 °C followed by Proteinase K digestion for 5 min at 37 °C, and the resulting products were analyzed by agarose gel electrophoresis.

Potential off-target hits were identified with the CRISPOR tool. We analyzed only the off-target sites located on chromosome 9. We amplified 800 bp to 1 kb template fragments by PCR from the gDNA of G1 and G2 males for each selected off-target sequence and analyzed them with the EnGen® Mutation Detection kit (New England Biolabs #E3321). None of these fragments presented an digestions not displayed by the positive control from the kit. We also generated F4 homozygous mice, from which we amplified the same genomic DNA fragment, and sequenced it (Eurofins Genomics). All fragments had the expected WT sequence (C57BL/6J).

To analyze the genomic region of CD46, a 609 bp region spanning the CRISPR/Cas target site was amplified from isolated genomic DNA using the primer pair T7EI CD46 fw (AAT GAC AGC CAG CCA AGT TTT C) and T7EI CD46 rev (TTC GTG TCA TTC ATC TGG CA). The same approach was used for the CAR region. Here, an 829 bp region spanning the CRISPR/Cas target site was amplified using the primer pair T7EI CAR fw (CTT AAA GGA GGG CGC CAA CG) and T7EI CAR rev (AGC AAA GAA AGG CAA GCC CG). All oligonucleotides were synthesized by Integrated DNA Technologies (IDT, Leuven, Belgium). The resulting PCR products were then subjected to T7EI mutation detection analysis using the EnGen^® Mutation Detection Kit (New England Biolabs, Frankfurt am Main, Germany) according to the manufacturer’s instructions. The resulting digests were analyzed using the CRISPR Discovery Gel Kit (Agilent, Santa Clara, CA, USA) in combination with a 5200 Fragment Analyzer System (Agilent) according to the manufacturer’s instructions.