在玉米中对CRISPR gRNAs进行快速评估,以优化原生质细胞转化
Lauren Higa1, Max Blank1, Ella Hampson2
1Department of Molecular Biosciences and Bioengineering, University of Hawai'i at Mānoa, Honolulu, HI, 96822, USA.
Plant cell reports
|September 12, 2025
概括
研究人员优化了玉米原生质中的CRISPR/Cas9基因编辑,用于快速导向RNA (gRNA) 测试. 这种系统提高了效率和可行性,加速了对热带玉米中控制开花时间的基因的评估.
科学领域:
- 植物生物技术 植物生物技术
- 分子生物学分子生物学
- 农业科学 农业科学
背景情况:
- 热带玉米表现出光周期敏感性,阻碍其进入温带繁殖计划.
- 有效的基因编辑需要强大的方法来评估导向RNA (gRNA) 活动在不同的玉米基因型.
- 目前的gRNA验证方法可能耗时,延迟了繁殖的进展.
研究的目的:
- 为快速评估gRNA活动,开发和优化玉米半原体中的CRISPR/Cas9基因编辑系统.
- 改善原生细胞隔离,转染效率和热带玉米的转染后活力.
- 评估针对多种玉米基因型中的花抑制基因的gRNA编辑效率.
主要方法:
- 优化了使用热带同胞品系Tzi8.8进行玉米中原细胞分离和转染的协议.
- 利用化苗和垂直叶子切割来增强原生质的恢复和生存能力.
- 在四种玉米基因型中通过原生质检测评估了针对ZmCCT9,ZmCCT10和ZmRap2.7的九种gRNA的CRISPR/Cas9编辑效率.
主要成果:
- 实现了可活原生质的高产量 (新鲜重量每克17.88 × 106),可延长可活性长达七天.
- 使用10μg的等离子体DNA达到约50%的转染效率,证明了资源效率.
- 观察到基因编辑效率在不同的gRNA和玉米基因型中从0.4%到23.7%不等.
结论:
- 优化的玉米原质细胞系统为体内gRNA验证提供了快速有效的平台,大大缩短了测试时间.
- 该系统扩展了热带玉米的基因编辑工具包,有助于克服与开花时间相关的繁殖障碍.
- 促进更快地评估基因编辑策略,以改善热带玉米适应温带气候.
相关概念视频
01:59CRISPR
Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced Short...
02:53CRISPR and crRNAs
Bacteria and archaea are susceptible to viral infections just like eukaryotes; therefore, they have developed a unique adaptive immune system to protect themselves. Clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins (CRISPR-Cas) are present in more than 45% of known bacteria and 90% of known archaea.
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
The CRISPR-Cas system stores a copy of foreign DNA in the host genome and uses it to identify the foreign DNA upon reinfection. CRISPR-Cas has three different...
01:59CRISPR
Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced Short...
01:28CRISPR/Cas9 Genome Editing
The CRISPR-Cas system serves as a bacterial defense mechanism against invading genetic elements such as viruses and plasmids, forming the foundation for its adaptation as a powerful genome-editing tool. Originally discovered in prokaryotes, this system has been repurposed to revolutionize genetic engineering across a wide range of organisms, including plants, animals, and humans. The core component, Cas9, is an endonuclease derived from Streptococcus pyogenes, capable of introducing...