Towards organism-level systems biology by next-generation genetics and whole-organ cell profiling
Towards organism-level systems biology by next-generation genetics and whole-organ cell profiling
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DOI：
10.1007/s12551-021-00859-w
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发表时间：
2021-11
影响因子：
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通讯作者：
Y. Minami;Yufei Yuan;H. Ueda
中科院分区：
文献类型：
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作者：
Y. Minami;Yufei Yuan;H. Ueda
哺乳动物中分子和细胞网络的系统级鉴定和分析可以通过“下一代”遗传学来加速，“下一代”遗传学被定义为可以在单代中实现所需遗传组成而无需任何动物杂交的遗传学。我们最近建立了一种使用“Triple-CRISPR”方法生产敲除（KO）小鼠的高效程序，该方法通过CRISPR/Cas9系统中的三重gRNA靶向单个基因。该程序实现了几乎完美的KO效率（96-100%）。我们还建立了一个高效的程序，“ES-小鼠”的方法，生产敲入（KI）小鼠在一个单一的世代。在这种方法中，ES细胞用三种抑制剂处理以保持其效力，然后注射到8细胞期胚胎中。这些程序大大缩短了生产KO或KI小鼠所需的时间，从数年缩短到约3个月。产生的KO和KI小鼠也可以通过“全器官细胞分析”以单细胞分辨率进行系统分析，该分析通过组织清除方法（如CUBIC）和先进的光片显微镜实现。本文综述了上述技术的建立及其在哺乳动物大脑三种状态（NREM睡眠、REM睡眠和清醒）分析中的应用。它还讨论了钙和毒蕈碱受体在这些状态中的作用，以及当前的挑战和未来的机会，在下一代哺乳动物遗传学和全器官细胞分析生物体水平的系统生物学。
The system-level identification and analysis of molecular and cellular networks in mammals can be accelerated by “next-generation” genetics, which is defined as genetics that can achieve desired genetic makeup in a single generation without any animal crossing. We recently established a highly efficient procedure for producing knock-out (KO) mice using the “Triple-CRISPR” method, which targets a single gene by triple gRNAs in the CRISPR/Cas9 system. This procedure achieved an almost perfect KO efficiency (96–100%). We also established a highly efficient procedure, the “ES-mouse” method, for producing knock-in (KI) mice within a single generation. In this method, ES cells were treated with three inhibitors to keep their potency and then injected into 8-cell-stage embryos. These procedures dramatically shortened the time required to produce KO or KI mice from years down to about 3 months. The produced KO and KI mice can also be systematically profiled at a single-cell resolution by the “whole-organ cell profiling,” which was realized by tissue-clearing methods, such as CUBIC, and an advanced light-sheet microscopy. The review describes the establishment and application of these technologies above in analyzing the three states (NREM sleep, REM sleep, and awake) of mammalian brains. It also discusses the role of calcium and muscarinic receptors in these states as well as the current challenges and future opportunities in the next-generation mammalian genetics and whole-organ cell profiling for organism-level systems biology.