Protocol for precise large-region haplotype modification for isogenic human iPSC generation using CRISPR-Cas9
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Updated: Oct 31, 2026

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells
Published on: September 25, 2019
Manoj K Mishra1, Rajan Pandey1, Bhavika Therani1
1Department of Physiology, University of Arizona College of Medicine-Tucson, Tucson, AZ, USA; Molecular Systems Medicine Initiative, University of Arizona Health Sciences, Tucson, AZ, USA.
Abstract:
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.
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