Protocol for precise large-region haplotype modification for isogenic human iPSC generation using CRISPR-Cas9

Large genomic region modification, such as precise haplotype reconstitution, remains challenging due to the low efficiency of homology-directed repair (HDR) and difficulties in generating HDR templates. Here, we present a two-stage protocol for genome engineering in human induced pluripotent stem cells (hiPSCs) using CRISPR-Cas9 to generate homozygous, multi-kilobase isogenic haplotypes. We describe steps for in silico experimental design, large haplotype deletion and reconstruction, followed by edited cell line validation. This optimized protocol improves both editing efficiency and screening throughput while reducing turnaround time. For complete details on the use and execution of this protocol, please refer to Xue et al. 1 1. Xue, H. ∙ Mishra, M.K. ∙ Liu, Y. ... Physiological role and mechanisms of action for a long noncoding haplotype region Cell Rep. 2025; 44 , 115805 Full Text Full Text (PDF)