Construction of multi-targeted CRISPR libraries in tomato to overcome functional redundancy at genome-scale level

Amichai Berman, Ning Su, Zhuorong Li, Udi Landau, Joydeep Chakraborty, Natali Gerbi, Jia Liu, Yuntai Qin, Boxi Yuan, Wei Wei, Osnat Yanai, Itay Mayrose, Yuqin Zhang () and Eilon Shani ()
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Amichai Berman: Tel Aviv University
Ning Su: University of Chinese Academy of Sciences
Zhuorong Li: University of Chinese Academy of Sciences
Udi Landau: Tel Aviv University
Joydeep Chakraborty: Tel Aviv University
Natali Gerbi: Tel Aviv University
Jia Liu: University of Chinese Academy of Sciences
Yuntai Qin: University of Chinese Academy of Sciences
Boxi Yuan: University of Chinese Academy of Sciences
Wei Wei: University of Chinese Academy of Sciences
Osnat Yanai: Netter Center
Itay Mayrose: Tel Aviv University
Yuqin Zhang: University of Chinese Academy of Sciences
Eilon Shani: Tel Aviv University

Nature Communications, 2025, vol. 16, issue 1, 1-14

Abstract: Abstract Genetic variance is vital for breeding programs and mutant screening, yet traditional mutagenesis methods wrestle with genetic redundancy and a lack of specificity in gene targeting. CRISPR-Cas9 offers precise, site-specific gene editing, but its application in crop improvement has been limited by scalability challenges. In this study, we develop genome-wide multi-targeted CRISPR libraries in tomato, enhancing the scalability of CRISPR gene editing in crops and addressing the challenges of redundancy while maintaining its precision. We design 15,804 unique single guide RNAs (sgRNAs), each targeting multiple genes within the same gene families. These sgRNAs are classified into 10 sub-libraries based on gene function. We generate approximately 1300 independent CRISPR lines and successfully identify mutants with distinct phenotypes related to fruit development, fruit flavor, nutrient uptake, and pathogen response. Additionally, we develop CRISPR-GuideMap, a double-barcode tagging system to enable large-scale sgRNA tracking in generated plants. Our results demonstrate that multi-targeted CRISPR libraries are scalable and effective for large-scale gene editing and offer an approach to overcome gene functional redundancy in basic plant research and crop breeding.

Date: 2025
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DOI: 10.1038/s41467-025-59280-6

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