Plant-Based Biosensors for Detecting CRISPR-Mediated Genome Engineering
Plant-Based Biosensors for Detecting CRISPR-Mediated Genome Engineering
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DOI：
10.1021/acssynbio.1c00455
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发表时间：
2021-12-08
影响因子：
4.7
通讯作者：
Yang, Xiaohan
中科院分区：
文献类型：
--
作者：
Yuan, Guoliang;Hassan, Md Mahmudul;Yang, Xiaohan
CRISPR/Cas最近成为各种物种基因组工程最可靠的系统。然而，人们对CRISPR/Cas技术相关风险的担忧正在增加，因为CRISPR基因编辑可能会意外引起潜在的非预期DNA变化。因此，开发一种能够检测和报告生物系统中活性CRISPR/Cas工具存在的系统是非常必要的。在这里，我们开发了四种实时检测系统，可以自发地指示植物中用于基因组编辑和基因调控的活性CRISPR-Cas工具的存在，包括CRISPR/Cas9核酸酶，碱基编辑，引物编辑和CRISPRa。使用基于荧光的分子生物传感器，我们证明了CRISPR/Cas9核酸酶、碱基编辑、引物编辑和CRISPRa的活性可以在通过原生质体转化和叶片渗透（在拟南芥、白杨和烟草中）的瞬时表达和在拟南芥中的稳定转化中有效地检测。
CRISPR/Cas has recently emerged as the most reliable system for genome engineering in various species. However, concerns about risks associated with the CRISPR/Cas technology are increasing on potential unintended DNA changes that might accidentally arise from CRISPR gene editing. Developing a system that can detect and report the presence of active CRISPR/Cas tools in biological systems is therefore very necessary. Here, we developed four real-time detection systems that can spontaneously indicate the presence of active CRISPR-Cas tools for genome editing and gene regulation including CRISPR/Cas9 nuclease, base editing, prime editing, and CRISPRa in plants. Using the fluorescence-based molecular biosensors, we demonstrated that the activities of CRISPR/Cas9 nuclease, base editing, prime editing, and CRISPRa can be effectively detected in transient expression via protoplast transformation and leaf infiltration (in Arabidopsis, poplar, and tobacco) and stable transformation in Arabidopsis.