A single-chain and fast-responding light-inducible Cre recombinase as a novel optogenetic switch.
A single-chain and fast-responding light-inducible Cre recombinase as a novel optogenetic switch.
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DOI：
10.7554/elife.61268
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发表时间：
2021-02-23
期刊：
影响因子：
7.7
通讯作者：
Yvert G
中科院分区：
文献类型：
--
作者：
Duplus-Bottin H;Spichty M;Triqueneaux G;Place C;Mangeot PE;Ohlmann T;Vittoz F;Yvert G
光遗传学使基因组操作具有高时空分辨率，为基础和应用生物学研究开辟了令人兴奋的可能性。在这里，我们报告的发展LiCre，一种新的光诱导Cre重组酶。LiCre由单一的含黄素蛋白质制成，其包含与在其N-末端和C-末端结构域中携带去稳定化突变的Cre变体融合的燕麦的AsLOV 2光感受器结构域。LiCre可以在蓝光照射的几分钟内被激活，而不需要额外的化学品。当与现有的基于两个分裂单元的光活化Cre重组酶相比时，LiCre显示出更快和更强的光活化以及在黑暗中更低的残留活性。LiCre在酵母和人类细胞中都是有效的，在酵母中，它允许我们用光控制β-胡萝卜素的生产。由于其简单性和性能，LiCre特别适合基础和生物医学研究，以及控制工业生物过程。在生物学家的工具包中，Cre蛋白占有特殊的位置。这种酶在某些病毒中自然存在，它识别并修改特定的基因序列，产生改变，打开或关闭附近的任何基因。通过基因工程改造细胞或生物体，使它们携带Cre及其靶序列，使科学家能够控制特定基因的激活，通常是在单个组织或器官中。然而，这依赖于一旦Cre蛋白在感兴趣的细胞中就“按需”激活Cre蛋白的能力。一种方法是将酶分成两部分，然后在暴露于蓝光时重新组装。然而，这涉及将两个部分分别引入组织中的挑战性步骤。相反，Duplus-Bottin等人设计了LiCre，这是一种新系统，其中Cre蛋白的大部分与燕麦用于检测其环境的光传感器融合。LiCre在黑暗中是关闭的，但当暴露在蓝光下时，它开始识别和修改Cre靶序列。Duplus-Bottin等人随后评估了LiCre与面包酵母和人类肾细胞中的两部分Cre系统的比较情况。这表明，这种新蛋白在黑暗中的“不正确”活动较少，并且在蓝光下可以更快地打开。这种改进的方法可以为科学家提供一种更好的工具来研究某些基因在精确位置和时间点的作用，同时也有助于他们利用基因序列进行工业或基因治疗。
Optogenetics enables genome manipulations with high spatiotemporal resolution, opening exciting possibilities for fundamental and applied biological research. Here, we report the development of LiCre, a novel light-inducible Cre recombinase. LiCre is made of a single flavin-containing protein comprising the AsLOV2 photoreceptor domain of Avena sativa fused to a Cre variant carrying destabilizing mutations in its N-terminal and C-terminal domains. LiCre can be activated within minutes of illumination with blue light without the need of additional chemicals. When compared to existing photoactivatable Cre recombinases based on two split units, LiCre displayed faster and stronger activation by light as well as a lower residual activity in the dark. LiCre was efficient both in yeast, where it allowed us to control the production of β-carotene with light, and human cells. Given its simplicity and performances, LiCre is particularly suited for fundamental and biomedical research, as well as for controlling industrial bioprocesses. In a biologist’s toolkit, the Cre protein holds a special place. Naturally found in certain viruses, this enzyme recognises and modifies specific genetic sequences, creating changes that switch on or off whatever gene is close by. Genetically engineering cells or organisms so that they carry Cre and its target sequences allows scientists to control the activation of a given gene, often in a single tissue or organ. However, this relies on the ability to activate the Cre protein ‘on demand’ once it is in the cells of interest. One way to do so is to split the enzyme into two pieces, which can then reassemble when exposed to blue light. Yet, this involves the challenging step of introducing both parts separately into a tissue. Instead, Duplus-Bottin et al. engineered LiCre, a new system where a large section of the Cre protein is fused to a light sensor used by oats to detect their environment. LiCre is off in the dark, but it starts to recognize and modify Cre target sequences when exposed to blue light. Duplus-Bottin et al. then assessed how LiCre compares to the two-part Cre system in baker's yeast and human kidney cells. This showed that the new protein is less ‘incorrectly’ active in the dark, and can switch on faster under blue light. The improved approach could give scientists a better tool to study the role of certain genes at precise locations and time points, but also help them to harness genetic sequences for industry or during gene therapy.