Photoswitchable gRNAs for Spatiotemporally Controlled CRISPR-Cas-Based Genomic Regulation
Photoswitchable gRNAs for Spatiotemporally Controlled CRISPR-Cas-Based Genomic Regulation
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DOI：
10.1021/acscentsci.9b01093
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发表时间：
2020-05-27
影响因子：
18.2
通讯作者：
Stevens, Molly M.
中科院分区：
文献类型：
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作者：
Moroz-Omori, Elena, V;Satyapertiwi, Dwiantari;Stevens, Molly M.
最近发现的CRISPR-Cas基因编辑系统及其衍生物在基础生物学研究和制药科学中有许多应用。对基因编辑和调控事件的精确外部控制的需求推动了诱导型CRISPR-Cas系统的发展。虽然大多数光可控CRISPR-Cas系统都是基于蛋白质工程，但我们开发了一种替代合成方法，该方法基于用光笼化基团修饰crRNA/tracrRNA双链体（向导RNA或gRNA），防止gRNA识别其基因组靶序列，直到在光照的几秒钟内诱导其脱保护。这种方法依赖于光笼化gRNA的直接固相合成，与工程化光响应蛋白相比，具有更简单的纯化和表征过程。我们已经证明了在体外短暂暴露于光时gRNA的光笼化和光介导的DNA切割的可行性。我们已经实现了光介导的时空分辨基因编辑以及细胞中的基因激活，而光笼化的gRNA在没有光照射的情况下几乎没有显示出可检测的基因编辑或激活。最后，我们已经将该系统应用于斑马鱼胚胎体内时空控制基因编辑，使该策略能够用于发育生物学和组织工程应用。
The recently discovered CRISPR-Cas gene editing system and its derivatives have found numerous applications in fundamental biology research and pharmaceutical sciences. The need for precise external control over the gene editing and regulatory events has driven the development of inducible CRISPR-Cas systems. While most of the light-controllable CRISPR-Cas systems are based on protein engineering, we developed an alternative synthetic approach based on modification of crRNA/tracrRNA duplex (guide RNA or gRNA) with photocaging groups, preventing the gRNA from recognizing its genome target sequence until its deprotection is induced within seconds of illumination. This approach relies on a straightforward solid-phase synthesis of the photocaged gRNAs, with simpler purification and characterization processes in comparison to engineering a light-responsive protein. We have demonstrated the feasibility of photocaging of gRNAs and light-mediated DNA cleavage upon brief exposure to light in vitro. We have achieved light-mediated spatiotemporally resolved gene editing as well as gene activation in cells, whereas photocaged gRNAs showed virtually no detectable gene editing or activation in the absence of light irradiation. Finally, we have applied this system to spatiotemporally control gene editing in zebrafish embryos in vivo, enabling the use of this strategy for developmental biology and tissue engineering applications.