Construction of multi-targeted CRISPR libraries in tomato to overcome functional redundancy at genome-scale level
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 lim...
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| Format: | Article |
| Language: | English |
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Nature Portfolio
2025-05-01
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| Series: | Nature Communications |
| Online Access: | https://doi.org/10.1038/s41467-025-59280-6 |
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| _version_ | 1850204019114377216 |
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| author | 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 Eilon Shani |
| author_facet | 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 Eilon Shani |
| author_sort | Amichai Berman |
| collection | DOAJ |
| description | 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. |
| format | Article |
| id | doaj-art-beb25279d602496ea1e83f9eb5eb44fb |
| institution | OA Journals |
| issn | 2041-1723 |
| language | English |
| publishDate | 2025-05-01 |
| publisher | Nature Portfolio |
| record_format | Article |
| series | Nature Communications |
| spelling | doaj-art-beb25279d602496ea1e83f9eb5eb44fb2025-08-20T02:11:22ZengNature PortfolioNature Communications2041-17232025-05-0116111410.1038/s41467-025-59280-6Construction of multi-targeted CRISPR libraries in tomato to overcome functional redundancy at genome-scale levelAmichai Berman0Ning Su1Zhuorong Li2Udi Landau3Joydeep Chakraborty4Natali Gerbi5Jia Liu6Yuntai Qin7Boxi Yuan8Wei Wei9Osnat Yanai10Itay Mayrose11Yuqin Zhang12Eilon Shani13School of Plant Sciences and Food Security, Tel Aviv UniversityCollege of Advanced Agricultural Sciences, University of Chinese Academy of SciencesCollege of Advanced Agricultural Sciences, University of Chinese Academy of SciencesSchool of Plant Sciences and Food Security, Tel Aviv UniversitySchool of Plant Sciences and Food Security, Tel Aviv UniversitySchool of Plant Sciences and Food Security, Tel Aviv UniversityCollege of Advanced Agricultural Sciences, University of Chinese Academy of SciencesCollege of Advanced Agricultural Sciences, University of Chinese Academy of SciencesCollege of Advanced Agricultural Sciences, University of Chinese Academy of SciencesCollege of Advanced Agricultural Sciences, University of Chinese Academy of SciencesNetaGenomiX, Netter CenterSchool of Plant Sciences and Food Security, Tel Aviv UniversityCollege of Advanced Agricultural Sciences, University of Chinese Academy of SciencesSchool of Plant Sciences and Food Security, Tel Aviv UniversityAbstract 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.https://doi.org/10.1038/s41467-025-59280-6 |
| spellingShingle | 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 Eilon Shani Construction of multi-targeted CRISPR libraries in tomato to overcome functional redundancy at genome-scale level Nature Communications |
| title | Construction of multi-targeted CRISPR libraries in tomato to overcome functional redundancy at genome-scale level |
| title_full | Construction of multi-targeted CRISPR libraries in tomato to overcome functional redundancy at genome-scale level |
| title_fullStr | Construction of multi-targeted CRISPR libraries in tomato to overcome functional redundancy at genome-scale level |
| title_full_unstemmed | Construction of multi-targeted CRISPR libraries in tomato to overcome functional redundancy at genome-scale level |
| title_short | Construction of multi-targeted CRISPR libraries in tomato to overcome functional redundancy at genome-scale level |
| title_sort | construction of multi targeted crispr libraries in tomato to overcome functional redundancy at genome scale level |
| url | https://doi.org/10.1038/s41467-025-59280-6 |
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