ABSTRACT

Advances in genome editing technologies have tremendous potential to address the limitations of classical resistance breeding. CRISPR-Cas9 based gene editing has been applied successfully in plants to tolerate virus infections. In this study, we successfully tested CRISPR-Cas9 system to counteract cotton leaf curl disease (CLCuD) caused by whitefly transmitted cotton leaf curl viruses (CLCuVs). We also analyzed the ability of CLCuV to escape the Cas9 endonuclease activity. Targeting overlapping genes of most prevalent CLCuVs with three gRNAs resulted in virus interference, as validated by low virus titer. Furthermore, multiplex CRISPR-Cas9 construct simultaneously targeting six genes of CLCuV, was found more effective to interfere with virus proliferation compared to targeting single region individually. Additionally, transgenic N. benthamiana plants expressing multiple gRNAs simultaneously showed enhanced tolerance against CLCuV infection when compared to wild-type plants. T7 Endonuclease-I (T7EI) assay, showing indels in the CLCuV genome, confirmed the occurrence of double strand breaks (DSBs) in DNA at target sequence induced by Cas9 endonuclease. We observed that targeting CLCuV genome at multiple sites simultaneously resulted in better interference, also with inefficient recovery of altered virus molecules. Next, we tested multiplex construct in cotton to interfere CLCuV infection. We found significant decrease in virus accumulation in cotton leaves co-infiltrated with multiplex cassette and virus compared to cotton leaves infiltrated with virus only. The results demonstrate future use of CRISPR-Cas9 system for engineering virus resistance in crops. Moreover, our results also advocate that resistance to mixed virus infections can be engineered using multiplex genome editing.

摘要

基因组编辑技术的进步具有解决经典抗性育种局限性的巨大潜力。基于 CRISPR-Cas9 的基因编辑已成功应用于植物以耐受病毒感染。在这项研究中，我们成功测试了 CRISPR-Cas9 系统来对抗由粉虱传播的棉花卷叶病毒 (CLCuVs) 引起的棉花卷叶病 (CLCuD)。我们还分析了 CLCuV 逃避 Cas9 核酸内切酶活性的能力。用三个 gRNA 靶向最流行的 CLCuV 的重叠基因会导致病毒干扰，正如低病毒滴度所证实的那样。此外，与单独靶向单个区域相比，发现同时靶向六个 CLCuV 基因的多重 CRISPR-Cas9 构建体更有效地干扰病毒增殖。此外，转基因本塞姆氏烟草与野生型植物相比，同时表达多种 gRNA 的植物对 CLCuV 感染的耐受性增强。T7 核酸内切酶-I (T7EI) 测定显示 CLCuV 基因组中的插入缺失，证实了 Cas9 核酸内切酶诱导的靶序列 DNA 中双链断裂 (DSB) 的发生。我们观察到，在多个位点同时靶向 CLCuV 基因组会导致更好的干扰，同时改变的病毒分子的恢复效率也很低。接下来，我们在棉花中测试了多重构建体以干扰 CLCuV 感染。我们发现与仅用病毒浸润的棉叶相比，用多重盒和病毒共同浸润的棉叶中病毒积累显着减少。结果证明了 CRISPR-Cas9 系统在农作物中工程抗病毒性的未来用途。而且，