Photocontrol of CRISPR/Cas9 function by site-specific chemical modification of guide RNA.
Photocontrol of CRISPR/Cas9 function by site-specific chemical modification of guide RNA.
复制标题
通过向导RNA的位点特异性化学修饰来光控CRISPR/Cas9功能
DOI：
10.1039/d0sc04343e
复制
发表时间：
2020-09-25
期刊：
影响因子：
8.4
通讯作者：
Xu L
中科院分区：
文献类型：
--
作者：
Wang Y;Liu Y;Xie F;Lin J;Xu L
CRISPR/Cas9的功能可以通过指导RNA（gRNA）的合理工程化来有条件地控制，以靶向所选择的基因，从而精确操纵基因组。特别地，可以通过使用特定刺激激活的化学修饰的gRNA提供了一种独特的工具来扩展条件控制的多功能性。在本文中，与以前的gRNA工程化通常仅关注RNA部分而忽略RNA-蛋白质相互作用不同，我们瞄准了gRNA种子区中核糖的2′-OH与Cas9蛋白之间的相互作用位点，并鉴定了特定位点处的化学修饰可用于调节Cas9活性。通过在这些特定位点引入光不稳定基团，我们实现了Cas9活性的光学控制，而不破坏沃森-克里克碱基配对。我们通过活细胞中CRISPR介导的基因激活和核酸酶切割进一步检查了我们的设计，并通过使用光照射成功操纵了基因表达。我们的位点特异性修饰策略表现出高效和动态的光学响应，并为基于RNA-蛋白质相互作用而不是RNA本身结构操纵gRNA提供了新的视角。此外，这些特定位点也可能用于修饰其他刺激响应基团，这将进一步丰富CRISPR/Cas9功能条件控制的工具箱。
The function of CRISPR/Cas9 can be conditionally controlled by the rational engineering of guide RNA (gRNA) to target the gene of choice for precise manipulation of the genome. Particularly, chemically modified gRNA that can be activated by using specific stimuli provides a unique tool to expand the versatility of conditional control. Herein, unlike previous engineering of gRNA that generally focused on the RNA part only but neglected RNA–protein interactions, we aimed at the interactive sites between 2′-OH of ribose in the seed region of gRNA and the Cas9 protein and identified that chemical modifications at specific sites could be utilized to regulate the Cas9 activity. By introducing a photolabile group at these specific sites, we achieved optical control of Cas9 activity without disrupting the Watson–Crick base pairing. We further examined our design through CRISPR-mediated gene activation and nuclease cleavage in living cells and successfully manipulated the gene expression by using light irradiation. Our site-specific modification strategy exhibited a highly efficient and dynamic optical response and presented a new perspective for manipulating gRNA based on the RNA–protein interaction rather than the structure of RNA itself. In addition, these specific sites could also be potentially utilized for modification of other stimuli-responsive groups, which would further enrich the toolbox for conditional control of CRISPR/Cas9 function.
登录
查看更多内容
影响因子：
46.9
作者：
通讯作者：
--
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
影响因子：
15
作者：
Hemphill J;Borchardt EK;Brown K;Asokan A;Deiters A
通讯作者：
Deiters A
DOI：
10.1002/anie.201606123
发表时间：
2016-09-26
期刊：
Angewandte Chemie (International ed. in English)
影响因子：
--
作者：
Jain PK;Ramanan V;Schepers AG;Dalvie NS;Panda A;Fleming HE;Bhatia SN
通讯作者：
Bhatia SN
影响因子：
14.8
作者：
Davis, Kevin M.;Pattanayak, Vikram;Thompson, David B.;Zuris, John A.;Liu, David R.
通讯作者：
Liu, David R.