Hu Nan, Tian Honglin, Li Yanhua, Li Xu, Li Daozheng, Li Lijie, Wang Siting, Zhang Yaolong, Shi Xin, Huang Baowen, Lu Quanwei, Wang Tao, Pan Xiaoping, Tu Liqin, Dai Dongyang, Zhang Baohong, Peng Renhai, Yan Fang
College of Biology and Food Engineering, Anyang Institute of Technology, Anyang, Henan 455000, China; College of Horticulture and Forestry, Tarim University, Alar, Xinjiang 843300, China.
College of Biology and Food Engineering, Anyang Institute of Technology, Anyang, Henan 455000, China.
Trends Biotechnol. 2025 Jul;43(7):1788-1808. doi: 10.1016/j.tibtech.2025.03.016. Epub 2025 Apr 24.
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing (GEd) technology has revolutionized plant science, facilitating gene function studies and crop improvement. Despite its success, plant-specific CRISPR/Cas9 systems require further optimization. This study aims to boost plant GEd efficiency by revamping the CRISPR/Cas9 system. We addressed large fragment deletions in T-DNA (transfer DNA) postgenomic insertion by developing a binary expression vector, pHNR, which maintains T-DNA integrity using protective sequences. We discovered an artificial promoter, P, effective in tobacco, Arabidopsis, and tomato transformation, and designed a dual-component dual-transcription unit CRISPR/Cas9 system (DDS) with optimal gene expression at a poly(A) length of ~150 base pairs. Enhancing the poly(A) tail length of Cas9 mRNA significantly boosted plant GEd efficiency. We also identified compatible hCas9 versions through transitory expression in tobacco leaves. Utilizing pHNRhCas9NG, we efficiently knocked out ten genes in tomato, achieving almost 100% gene-editing efficiency. Our system offers a novel, scalable tool for plant GEd, advancing CRISPR/Cas9 capabilities.
成簇规律间隔短回文重复序列（CRISPR）/Cas9基因组编辑（GEd）技术彻底改变了植物科学，推动了基因功能研究和作物改良。尽管取得了成功，但植物特异性CRISPR/Cas9系统仍需进一步优化。本研究旨在通过改进CRISPR/Cas9系统提高植物GEd效率。我们通过开发二元表达载体pHNR解决了基因组插入后T-DNA（转移DNA）中的大片段缺失问题，该载体使用保护序列维持T-DNA完整性。我们发现了一种在烟草、拟南芥和番茄转化中有效的人工启动子P，并设计了一种双组分双转录单元CRISPR/Cas9系统（DDS），在聚腺苷酸长度约为150个碱基对时具有最佳基因表达。增加Cas9 mRNA的聚腺苷酸尾长度显著提高了植物GEd效率。我们还通过在烟草叶片中的瞬时表达鉴定了兼容的hCas9版本。利用pHNRhCas9NG，我们在番茄中高效敲除了十个基因，实现了近100%的基因编辑效率。我们的系统为植物GEd提供了一种新颖、可扩展的工具，提升了CRISPR/Cas9的能力。
