Oh Youngbin, Nagalakshmi Ugrappa, Dahlbeck Douglas, Koehler Naio, Cho Myeong-Je, Dinesh-Kumar Savithramma P, Staskawicz Brian J
Innovative Genomics Institute, University of California, Berkeley, California, 94720, USA.
Department of Plant Biology and The Genome Center, The College of Biological Sciences, University of California, Davis, California, USA.
Plant J. 2025 Apr;122(1):e70115. doi: 10.1111/tpj.70115.
Here, we report the successful implementation of heritable virus-induced genome editing (VIGE) in tomato (Solanum lycopersicum). We generated three transgenic tomato lines expressing Streptococcus pyogenes Cas9 (SpCas9) under the control of Cauliflower mosaic virus 35S (35S), S. lycopersicum ribosomal protein S5A (SlRPS5A), or S. lycopersicum YAO promoters (SlYAO). These three lines were tested for somatic and heritable editing using the tobacco rattle virus (TRV)-based system carrying guide RNAs (gRNAs) fused with mobile RNA sequences. TRV with gRNA targeted to Phytoene desaturase (SlPDS) and Downy mildew resistance 6 (SlDMR6) genes fused to mobile RNA sequences showed significant somatic editing efficiency in all three tomato lines expressing SpCas9. However, the progenies from the SlYAO promoter-driven SpCas9 tomato infected with TRV with gRNA targeted to SlDMR6 fused to the mobile RNA sequence resulted in monoallelic mutations with a frequency of 3%. Optimization of environmental conditions, such as reduced light intensity, significantly increased heritable editing frequencies, from 0% to 86% at the SlPDS and from 3% to 100% at the SlDMR6, including biallelic mutations. These findings underscore the use of appropriate promoters to express Cas nucleases and optimized environmental conditions to enhance heritable genome editing efficiency in tomato using VIGE. Furthermore, our method enables the generation of mutants without additional tissue culture or transformation once a SpCas9-expressing tomato line is established.
在此，我们报告了在番茄（Solanum lycopersicum）中成功实施可遗传的病毒诱导基因组编辑（VIGE）。我们生成了三个转基因番茄品系，它们在花椰菜花叶病毒35S（35S）、番茄核糖体蛋白S5A（SlRPS5A）或番茄YAO启动子（SlYAO）的控制下表达化脓性链球菌Cas9（SpCas9）。使用携带与移动RNA序列融合的引导RNA（gRNA）的烟草脆裂病毒（TRV）系统对这三个品系进行了体细胞和可遗传编辑测试。靶向八氢番茄红素去饱和酶（SlPDS）和霜霉病抗性6（SlDMR6）基因并与移动RNA序列融合的带有gRNA的TRV在所有三个表达SpCas9的番茄品系中均显示出显著的体细胞编辑效率。然而，用靶向与移动RNA序列融合的SlDMR6的gRNA感染的由SlYAO启动子驱动的SpCas9番茄的后代产生了单等位基因突变，频率为3%。优化环境条件，如降低光照强度，可显著提高可遗传编辑频率，在SlPDS处从0%提高到86%，在SlDMR6处从3%提高到100%，包括双等位基因突变。这些发现强调了使用合适的启动子来表达Cas核酸酶以及优化环境条件以提高番茄中使用VIGE进行可遗传基因组编辑的效率。此外，一旦建立了表达SpCas9的番茄品系，我们的方法无需额外的组织培养或转化就能产生突变体。
