CRISPR-based pipeline for generating and validating multi-kb deletions and indels in iPSCs

Here, we present a CRISPR-Cas9 protocol for generating both small indels and multi-kilobase deletions to enable genome editing in human induced pluripotent stem cells (iPSCs). We describe steps for a ribonucleoprotein (RNP)-based approach that integrates guide RNA selection, electroporation, and multi-tiered screening to accurately identify indels, deletions, inversions, and mixed genotypes. Human iPSCs provide a versatile genome editing platform due to their capacity for clonal expansion and differentiation. This protocol provides a scalable framework for genome editing and quality control. For complete details on the use and execution of this protocol, please refer to Hamerlinck et al. 1 1. Hamerlinck, L. ∙ D’haene, E. ∙ Vaughan, M.B. ... Non-coding structural variants disrupt FOXG1 transcriptional regulation in early neurodevelopment Nat. Commun. 2026; 17 , 7197 Crossref Scopus (2) PubMed Google Scholar