Droogers Wouter J, Willems Jelmer, MacGillavry Harold D, de Jong Arthur P H
Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands.
Cell Biology, Neurobiology and Biophysics, Department of Biology, Faculty of Science, Utrecht University, Utrecht, The Netherlands
eNeuro. 2022 Jul 18;9(4). doi: 10.1523/ENEURO.0056-22.2022.
CRISPR/Cas9-mediated knock-in methods enable the labeling of individual endogenous proteins to faithfully determine their spatiotemporal distribution in cells. However, reliable multiplexing of knock-in events in neurons remains challenging because of cross talk between editing events. To overcome this, we developed conditional activation of knock-in expression (CAKE), allowing efficient, flexible, and accurate multiplex genome editing in rat neurons. To diminish cross talk, CAKE is based on sequential, recombinase-driven guide RNA (gRNA) expression to control the timing of genomic integration of each donor sequence. We show that CAKE is broadly applicable to co-label various endogenous proteins, including cytoskeletal proteins, synaptic scaffolds, ion channels and neurotransmitter receptor subunits. To take full advantage of CAKE, we resolved the nanoscale co-distribution of endogenous synaptic proteins using super-resolution microscopy, demonstrating that their co-organization depends on synapse size. Finally, we introduced inducible dimerization modules, providing acute control over synaptic receptor dynamics in living neurons. These experiments highlight the potential of CAKE to reveal new biological insight. Altogether, CAKE is a versatile method for multiplex protein labeling that enables the detection, localization, and manipulation of endogenous proteins in neurons.Accurate localization and manipulation of endogenous proteins is essential to unravel neuronal function. While labeling of individual proteins is achievable with existing gene editing techniques, methods to label multiple proteins in neurons are limiting. We introduce a new CRISPR/Cas9 strategy, CAKE, achieving faithful duplex protein labeling using sequential editing of genes. We use CAKE to visualize the co-localization of essential neuronal proteins, including cytoskeleton components, ion channels and synaptic scaffolds. Using super-resolution microscopy, we demonstrate that the co-organization of synaptic scaffolds and neurotransmitter receptors scales with synapse size. Finally, we acutely modulate the dynamics of synaptic receptors using labeling with inducible dimerization domains. Thus, CAKE mediates accurate duplex endogenous protein labeling and manipulation to address biological questions in neurons.
CRISPR/Cas9介导的敲入方法能够对单个内源性蛋白质进行标记，从而准确确定其在细胞中的时空分布。然而，由于编辑事件之间的串扰，在神经元中可靠地进行多重敲入事件仍然具有挑战性。为了克服这一问题，我们开发了敲入表达的条件激活（CAKE）技术，该技术能够在大鼠神经元中实现高效、灵活且准确的多重基因组编辑。为了减少串扰，CAKE基于序列性、重组酶驱动的引导RNA（gRNA）表达来控制每个供体序列基因组整合的时间。我们表明，CAKE广泛适用于共标记各种内源性蛋白质，包括细胞骨架蛋白、突触支架、离子通道和神经递质受体亚基。为了充分利用CAKE，我们使用超分辨率显微镜解析了内源性突触蛋白的纳米级共分布，证明它们的共组织依赖于突触大小。最后，我们引入了可诱导二聚化模块，能够对活神经元中的突触受体动力学进行急性调控。这些实验突出了CAKE揭示新生物学见解的潜力。总之，CAKE是一种用于多重蛋白质标记的通用方法，能够检测、定位和操纵神经元中的内源性蛋白质。
准确地定位和操纵内源性蛋白质对于揭示神经元功能至关重要。虽然使用现有的基因编辑技术可以实现对单个蛋白质的标记，但在神经元中标记多种蛋白质的方法仍然有限。我们引入了一种新的CRISPR/Cas9策略——CAKE，通过对基因的顺序编辑实现可靠的双链蛋白质标记。我们使用CAKE来可视化重要神经元蛋白质的共定位，包括细胞骨架成分、离子通道和突触支架。使用超分辨率显微镜，我们证明突触支架和神经递质受体的共组织与突触大小成比例。最后，我们使用可诱导二聚化结构域的标记对突触受体的动力学进行急性调节。因此，CAKE介导了准确的双链内源性蛋白质标记和操纵，以解决神经元中的生物学问题。
