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新型CRISPR/SpRY系统用于快速检测水中CRISPR/Cas介导的基因编辑

Zhixun Su¹, Xiaofu Wang², Xiaoyun Chen²

¹College of Food Science and Technology, Ningbo University, Ningbo, 315800, China; State Key Laboratory for Managing Biotic and Chemical Threats to the Quality and Safety of Agro-products, Key Laboratory of Traceability for Agricultural Genetically Modified Organisms, Ministry of Agriculture and Rural Affairs, Zhejiang Academy of Agricultural Sciences, Hangzhou, 310021, China.

Analytica chimica acta

|April 12, 2024

概括

一个新的CRISPR/SpRY检测平台提供了快速的,无需仪器的基因编辑作物的选. 这种具有成本效益的方法可以在不到一个小时内有效地识别各种突变,从而推进生物技术应用.

科学领域:

生物技术是生物技术.
植物科学植物科学
分子生物学分子生物学

背景情况:

聚类正规间隔短平行体重复 (CRISPR/Cas9) 是商业化植物产品中关键的基因编辑工具.
对基因编辑产品的快速,现场和无仪器检测仍然是一个重大挑战.
现有的检测方法通常依赖于特定的序列动图,限制了它们的多功能性.

研究的目的:

开发一种新的CRISPR/SpRY检测平台,用于快速选CRISPR/Cas9诱导的突变.
建立一种高效和多功能方法,用于检测基因编辑植物中的各种突变类型.
在基于CRISPR的检测中克服原空间子相邻动机 (PAM) 序列依赖性的局限性.

主要方法:

设计和评估了CRISPR/SpRY检测平台的工作流程,使用在TGW位置编辑的米 (Oryza sativa L.).
优化了CRISPR/SpRY反应系统,并将其与复合酶聚合酶放大 (RPA) 集成到一个单测定中.
经过验证的方法灵敏度使用重组等离子体和验证的适用性与不同的大米样本.

主要成果:

CRISPR/SpRY检测平台成功地识别了各种突变,包括插入,删除 (indels) 和核酸替代,具有高灵敏度.
一测试显示检测极限低至1%,并在不到一个小时内完成了整个过程.
与以前的技术相比,该方法被证明是简单的,无需仪器的,成本高效的和时间高效的.

结论:

开发的CRISPR/SpRY检测平台为基因编辑生物体的快速视觉查提供了开创性的解决方案.
这种PAM序列释放系统显著扩大了基于CRISPR的突变检测的范围和多功能性.
该平台对推动生物技术和确保基因编辑产品的质量具有巨大的前景.

关键词:

这就是CRISPR/SpRY.基因编辑基因编辑在体外切割裂变.在 RPA RPA 中使用.视觉检测视觉检测是一种视觉检测.

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CRISPR and crRNAs

CRISPR 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...