Generation of a multiplex mutagenesis population via pooledCRISPR ‐Cas9 in soya bean
| Autor: | Jiafeng Sun, Huaqin Kuang, Yuefeng Guan, Maoxiang Yang, Lan Yang, Juehui Yuan, Shikui Song, Zhihui Zhang, Dong Wang, Pingping Gong, Suning Li, Bo Liu, Mengyan Bai |
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| Rok vydání: | 2019 |
| Předmět: |
0106 biological sciences
0301 basic medicine Candidate gene Population Mutant Gene redundancy Plant Science Biology 01 natural sciences Genome 03 medical and health sciences Genome editing nodulation Multiplex education Research Articles soya bean Gene Editing CRISPR population Genetics education.field_of_study food and beverages 030104 developmental biology Mutagenesis multiplex mutagenesis Soybeans CRISPR-Cas Systems Root Nodules Plant CRISPR‐Cas9 Agronomy and Crop Science Research Article 010606 plant biology & botany Biotechnology Transformation efficiency |
| Zdroj: | Plant Biotechnology Journal |
| ISSN: | 1467-7652 1467-7644 |
| Popis: | Summary The output of genetic mutant screenings in soya bean [Glycine max (L.) Merr.] has been limited by its paleopolypoid genome. CRISPR‐Cas9 can generate multiplex mutants in crops with complex genomes. Nevertheless, the transformation efficiency of soya bean remains low and, hence, remains the major obstacle in the application of CRISPR‐Cas9 as a mutant screening tool. Here, we report a pooled CRISPR‐Cas9 platform to generate soya bean multiplex mutagenesis populations. We optimized the key steps in the screening protocol, including vector construction, sgRNA assessment, pooled transformation, sgRNA identification and gene editing verification. We constructed 70 CRISPR‐Cas9 vectors to target 102 candidate genes and their paralogs which were subjected to pooled transformation in 16 batches. A population consisting of 407 T0 lines was obtained containing all sgRNAs at an average mutagenesis frequency of 59.2%, including 35.6% lines carrying multiplex mutations. The mutation frequency in the T1 progeny could be increased further despite obtaining a transgenic chimera. In this population, we characterized gmric1/gmric2 double mutants with increased nodule numbers and gmrdn1‐1/1‐2/1‐3 triple mutant lines with decreased nodulation. Our study provides an advanced strategy for the generation of a targeted multiplex mutant population to overcome the gene redundancy problem in soya bean as well as in other major crops. |
| Databáze: | OpenAIRE |
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