Dual‐line Genome‐scale CRISPR Screening Enables Robust Target Gene Discovery
- 1National Key Laboratory For Germplasm Innovation and Utilization for Fruit and Vegetable Horticultural Crops Hubei Hongshan Laboratory Institute of Urban and Horticultural Entomology College of Plant Science and Technology Huazhong Agricultural University Wuhan People's Republic of China
- 2Hubei Insect Resources Utilization and Sustainable Pest Management Key Laboratory College of Plant Science and Technology Huazhong Agricultural University Wuhan People's Republic of China
Abstract
Pooled CRISPR screening enables genome‐scale functional gene discovery by linking genetic perturbations to cellular phenotypes. However, its application in insects remains limited, as systematic differences often obscure the universal functional signals and compromise the reliable identification of genetic regulators. We found that Cas9 genomic integration was associated with distinct transcriptional states, editing activity, and screening outcomes. To address this limitation, we developed a dual‐line CRISPR screening framework that prioritizes genes with concordant effects across independently derived Cas9‐expressing insect cell lines. In Bactrocera dorsalis, dual‐line consensus screening increased in vivo validation of candidate genes from 60% in single‐line screens to 90%. Under cold stress, this framework identified candidate regulators of cold tolerance, including a previously uncharacterized gene whose perturbation altered embryonic survival at low temperature. These results show that dual‐line screening can filter cell line‐specific artifacts and improve functional genomic screening for reliable genetic target discovery in insects.
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BibTeX
@article{li2026dualline,
title = {{Dual‐line Genome‐scale CRISPR Screening Enables Robust Target Gene Discovery}},
author = {Ziniu Li and Jiao Qiao and Qiuyuan Zhang and Peipei Liu and Chenjun Zheng and Qi Zhou and Mengqi Ji and Zhihui Zhu and Xiaoxue Li and Hongyu Zhang},
journal = {Advanced Science},
year = {2026},
month = aug,
doi = {10.1002/advs.77130},
url = {https://doi.org/10.1002/advs.77130},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Dual‐line Genome‐scale CRISPR Screening Enables Robust Target Gene Discovery
AU - Ziniu Li
AU - Jiao Qiao
AU - Qiuyuan Zhang
AU - Peipei Liu
AU - Chenjun Zheng
AU - Qi Zhou
AU - Mengqi Ji
AU - Zhihui Zhu
AU - Xiaoxue Li
AU - Hongyu Zhang
PY - 2026
DA - 2026/08/11
JO - Advanced Science
DO - 10.1002/advs.77130
UR - https://doi.org/10.1002/advs.77130
AB - Pooled CRISPR screening enables genome‐scale functional gene discovery by linking genetic perturbations to cellular phenotypes. However, its application in insects remains limited, as systematic differences often obscure the universal functional signals and compromise the reliable identification of genetic regulators. We found that Cas9 genomic integration was associated with distinct transcriptional states, editing activity, and screening outcomes. To address this limitation, we developed a dual‐line CRISPR screening framework that prioritizes genes with concordant effects across independently derived Cas9‐expressing insect cell lines. In Bactrocera dorsalis, dual‐line consensus screening increased in vivo validation of candidate genes from 60% in single‐line screens to 90%. Under cold stress, this framework identified candidate regulators of cold tolerance, including a previously uncharacterized gene whose perturbation altered embryonic survival at low temperature. These results show that dual‐line screening can filter cell line‐specific artifacts and improve functional genomic screening for reliable genetic target discovery in insects.
SN - 2198-3844
ER -