用于时空控制的基于 CRISPR-Cas 的基因组调控的光开关 gRNA。,ACS Central Science

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用于时空控制的基于 CRISPR-Cas 的基因组调控的光开关 gRNA。
ACS Central Science ( IF 12.7 ) Pub Date : 2020-04-01 , DOI: 10.1021/acscentsci.9b01093
Elena V Moroz-Omori _{1,

2} , Dwiantari Satyapertiwi ₁ , Marie-Christine Ramel ₃ , Håkon Høgset ₁ , Ilona K Sunyovszki _{1,

4} , Ziqian Liu ₁ , Jonathan P Wojciechowski ₁ , Yueyun Zhang ₁ , Christopher L Grigsby ₂ , Liliana Brito _{1,

4} , Laurence Bugeon ₃ , Margaret J Dallman ₃ , Molly M Stevens _{1,

2}

Affiliation

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药物科学中得到了大量应用。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。虽然大多数光控 CRISPR-Cas 系统都基于蛋白质工程，但我们开发了一种替代合成方法，该方法基于使用光笼组修饰 crRNA/tracrRNA 双链体（引导 RNA 或 gRNA），防止 gRNA 识别其基因组目标序列直到在光照几秒内诱导其脱保护。这种方法依赖于光笼化 gRNA 的直接固相合成，与改造光响应蛋白相比，具有更简单的纯化和表征过程。我们已经证明了短暂暴露于光线下 gRNAs 和光介导的 DNA 切割的光笼化的可行性体外。我们已经实现了光介导的时空解析基因编辑以及细胞中的基因激活，而光笼式 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，我们已将该系统应用于体内斑马鱼胚胎的时空控制基因编辑，从而使该策略能够用于发育生物学和组织工程应用。

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Photoswitchable gRNAs for Spatiotemporally Controlled CRISPR-Cas-Based Genomic Regulation.

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.

更新日期：2020-04-01

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