CRISPR Del/Rei: A simple, flexible and efficient pipeline for scarless genome editing

Scarless genome editing is an important tool for the accurate recapitulation of genetic variation in human disease models. Various CRISPR/Cas9-based scarless editing methods have been reported. However, some of these methods have low editing efficiency (1-5%) and require manual selection of hundreds of clones to reach the desired number. Other protocols use large selection cassettes with laborious vector assembly and specialized reagents and equipment, or have poorly understood off-target effects. To address these limitations, we developed a simple, highly efficient scarless editing strategy to edit DNA sequences in induced pluripotent stem cells, which we call CRISPR Del/Rei. This novel editing strategy consists of a two-step deletion-reinsertion strategy that produces isogenic clones in ~8 weeks using accessible, user-friendly reagents. The editing efficiency ranges from ~15–100% for Step 1 and ~5–20% for Step 2 after selection, which greatly reduces the amount of required manual clone isolation. Screening the transfected bulk cells and the individual clones is rapid and simple, consisting of PCR and gel electrophoresis. Despite the two editing steps, off-target effects are rare. Additionally, the experiment is well-controlled because the same protocol generates isogenic clones carrying all variant alleles. In this way, CRISPR Del/Rei serves as a valuable addition to the evolving CRISPR/Cas9 gene-editing toolset. ### Competing Interest Statement The authors have declared no competing interest.