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Peer-reviewedCC BY-NC 4.0STAR Protocols

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

  • Manoj K. Mishra1,2
  • Rajan Pandey1,2
  • Bhavika Therani1,2
  • Aron M. Geurts3
  • Mingyu Liang1,2
  • Yong Liu1,2
  1. 1Department of Physiology, University of Arizona College of Medicine-Tucson, Tucson, AZ, USA
  2. 2Molecular Systems Medicine Initiative, University of Arizona Health Sciences, Tucson, AZ, USA
  3. 3Department of Physiology, Medical College of Wisconsin, Milwaukee, WI, USA
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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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Cite

BibTeXRIS
BibTeX
@article{mishra2026protocol,
  title = {{Protocol for precise large-region haplotype modification for isogenic human iPSC generation using CRISPR-Cas9}},
  author = {Manoj K. Mishra and Rajan Pandey and Bhavika Therani and Aron M. Geurts and Mingyu Liang and Yong Liu},
  journal = {STAR Protocols},
  year = {2026},
  month = oct,
  doi = {10.1016/j.xpro.2026.104927},
  url = {https://doi.org/10.1016/j.xpro.2026.104927},
  note = {CC BY-NC 4.0},
}
RIS
TY  - JOUR
TI  - Protocol for precise large-region haplotype modification for isogenic human iPSC generation using CRISPR-Cas9
AU  - Manoj K. Mishra
AU  - Rajan Pandey
AU  - Bhavika Therani
AU  - Aron M. Geurts
AU  - Mingyu Liang
AU  - Yong Liu
PY  - 2026
DA  - 2026/10/29
JO  - STAR Protocols
DO  - 10.1016/j.xpro.2026.104927
UR  - https://doi.org/10.1016/j.xpro.2026.104927
AB  - 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
SN  - 2666-1667
ER  -