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| BMC前沿视角 | 来自陈玲玲实验室的热点解读（二） |
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本期BMC前沿视角我们有幸邀请到了Genome Biology编委陈玲玲研究员及其实验室成员为我们解读最近发表在Genome Biology，Molecular Cancer，Nature biotechnology，Genome Medicine，BMC Biology的八篇重要文献，以飨读者。
该期内容涵盖基于CRISPRi的放疗筛选鉴定胶质瘤中潜在的可作为治疗靶点的长链非编码RNA；通过对表观基因组进行靶向去甲基化，成功建立表观遗传疾病模型小鼠; 对150个RNA结合蛋白的增强CLIP数据分析系统性地揭示了RNA结合蛋白调控RNA加工规律；2F1和EIF4A3介导的circRNA circSEPT9促进三阴性乳腺癌的发生和发展；基于CRISPR–Cas12的SARS-CoV-2检测方法；双相情感障碍患者大脑类器官的全转录组分析及功能研究；Hi-D：纳米水平勾勒单个活细胞的细胞核动力学；细胞分化过程中细胞核大小及DNA的可及性参与调控旁斑的形成。
图1
陈玲玲实验室简介
陈玲玲的实验室主要研究长非编码RNA（long noncoding RNAs，lncRNAs），这是一类长度大于 200 个核苷酸的非编码 RNA分子。她的团队通过创建无poly(A) 转录组纯化和分析体系，发现了小核仁RNA结尾的具有特殊结构的长非编码RNA家族，也和国际上其他实验室相继报道了环形RNA在哺乳动物细胞基因组中普遍存在。更重要的是，这些具有特殊结构的编码RNA在基因调控和人类疾病中有着密切的相关作用。她的团队将继续深入研究这些长非编码RNA的生成加工以及它们在人类健康和疾病中作用的机理，关于陈玲玲研究组的更多内容请见http://www.chenlab-ncrna.com/。
陈玲玲研究员目前是Cell、Cell Chem Cell、Genome Biol、RNA、Trends Cell Biol、Trends Genet 等期刊的编辑委员会的成员。她在2017年被选为霍华德·休斯医学研究所（Howard Hughes Medical Institute，HHMI）国际研究员。
感谢陈玲玲实验室以下成员对本期内容作出主要贡献：
前排：郭纯洁，陈玲玲，李响（PhD），刘楚霄（PhD）
后排：姚润文，王洋，蒋望（PhD）
点击链接 了解陈玲玲实验室更多介绍
本期文章解读
#5. Title: CRISPR–Cas12-based detection of SARS-CoV-2
First Author: James P. Broughton
Correspondence Author: Janice S. Chen & Charles Y. Chiu
Letter|Nature biotechnology
Date: 2020-04-16
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由SARS-CoV-2感染造成的COVID-19已经迅速传播并造成全球大流行。截至2020年5月3日，全球共报告感染病例超过330万例，死亡病例超过24万例。目前，疾病控制与预防中心（CDC）确诊感染病例的金标准是通过定量RT-PCR（qRT-PCR）检测SARS-CoV-2。但是，该方法耗时4?6 小时，且需要进行多步操作，包括RNA提取、逆转录、扩增等。此外，体积庞大且笨重的检测仪器也限制了qRT-PCR方法在SARS-CoV-2检测上的应用。Broughton团队提出了一种基于CRISPR–Cas12的SARS-CoV-2检测方法，可用于对提取的患者样本RNA进行初步验证，称为靶向SARS-CoV-2 DNA的核酸内切酶CRISPR反式报告系统（DETECTR）。DETECTR首先对提取的RNA同时进行逆转录和环介导等温扩增（RT-LAMP），随后使用Cas12通过切割的方式检测样本RNA中是否含有已公布的的冠状病毒序列，该过程大约耗时40 分钟。最后，结合侧向层析试纸，DETECTR可以提供高精度的定性结果。这种基于CRISPR–Cas12的检测方法能够简单、快速地确认是否感染SARS-CoV-2，将为实验室以外的即时检验提供便利。
COVID-19, the disease associated with SARS-CoV-2 infection has rapidly spread as a global pandemic. There are more than 3.3 million reported cases and over 240,000 death worldwide as of 3rd May 2020. Till now, quantitative RT–PCR (qRT–PCR) for detection of the virus in 4~6 h has become the golden standard for infection confirmation by Centers for Disease Control and Prevention (CDC). However, a series of steps are needed including RNA extraction, reverse transcription, amplification for acquiring quantitative results. Additionally, bulky instruments also limit the application of qRT-PCR method in populated areas. Broughton et al. have provided a CRISPR–Cas12-based SARS-CoV-2 detection for initial validation of extracted patient sample RNA, called SARS-CoV-2 DNA Endonuclease-Targeted CRISPR Trans Reporter (DETECTR). DETECTR takes about 40 min to perform simultaneous reverse transcription and isothermal amplification using loop-mediated amplification (RT–LAMP) with extracted RNA, followed by Cas12 detection of predefined coronavirus sequences, after which cleavage of a reporter molecule confirms detection of the virus. Coupled with lateral flow visual readout, DETECTR could provide qualitative results with high accuracy. This CRISPR–Cas12-based detection method enables a simple and fast confirmation of SARS-CoV-2 infection, which will likely benefit point-of-care testing in clinics beyond laboratories.
#6. Title: Transcriptome analysis and functional characterization of cerebral organoids in bipolar disorder
First Author: Annie Kathuria
Correspondence Author: Rakesh Karmacharya
Research article|Genome Medicine
Date: 2020-04-19
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研究精神疾病的主要障碍之一是无法获取患者的活神经元组织。对人类体细胞来源诱导的多能干细胞（iPSCs）进行重编程，使其生成大脑类器官，可以作为替代手段模拟具有疾病特异性遗传背景的人类活体神经元组织。在这项研究中，Kathuria团队对8例双向情感障碍（BPI）患者和8名健康对照者的iPSCs产生的大脑类器官进行全转录组RNA测序。通过深入分析RNA测序结果的基因表达谱，研究人员发现BPI患者中参与细胞黏附、神经发育和突触生物学通路的基因表达明显下调，而参与免疫信号传导的基因表达则明显上调。Gene Ontology（GO）分析表明BPI患者存在内质网生物学特性相关的缺陷。内质网-线粒体相互作用的细胞特征研究及BPI患者大脑类器官的功能研究均支持了这一发现。这是首个比较从BPI患者和健康个体获取的大脑类器官的综合性研究，研究结果表明利用患者来源的iPSCs建立的体外三维细胞模型，对于研究神经精神疾病的发病机制具有潜在的应用价值。基于患者的iPSCs生成的体外类器官模型为发现具有治疗应用潜力的新分子靶点提供了一种有价值的方法。
A major barrier to study psychiatric disorders has been the infeasibility of accessing live neuronal tissues from patients. Reprogramming human induced pluripotent stem cells (iPSCs) from somatic cells and generating brain organoids from these iPSCs provide a mean to mimic live human neuronal tissues with disease-specific genetic backgrounds. In this study, Kathuria et al. performed RNA-seq of cerebral organoids generated from iPSCs of eight bipolar disorder (BPI) patients and eight healthy control individuals. Comparing gene expression profiles of these RNA-seq data showed downregulation of pathways involved in cell adhesion, neurodevelopment, and synaptic biology in BPI along with upregulation of genes involved in immune signaling. Gene ontology (GO) analyses suggested deficits related to endoplasmic reticulum biology, which was supported by cellular characterization of ER–mitochondria interactions and by functional studies in BPI cerebral organoids. It is the first comprehensive study of comparing cerebral organoids generated from BPI patients and healthy individuals, and their results showed the potential utility of using patient-derived iPSCs to generate ex vivo three-dimensional cellular models to study mechanisms of neuropsychiatric disorders. Generating ex vivo organoid models from patient-derived iPSCs provides a valuable way to identify new molecular targets that have the potential for therapeutic applications.
#7. Title: Hi-D: nanoscale mapping of nuclear dynamics in single living cells
First Author: Haitham A. Shaban
Correspondence Author: Kerstin Bystricky
Method|Genome Biology
Date: 2020-04-20
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在哺乳动物细胞中，染色体占据着特定的位置，构成具有动态变化的染色质域。由于细胞核具有高度致密性的特点并富含精细结构，我们对活细胞核的纳米级动力学特征的了解一直以来受到限制。Shaban团队开发了一种被称之为high-resolution diffusion mapping（Hi-D）的方法，能够在单细胞水平以亚像素的精度直接对致密结构（例如染色质和其他各种核成分）的动力学进行分析。Hi-D应用密集光流法（dense optical flow）从时间序列的常规共聚焦荧光显微镜图像中定量重建密集分布的荧光分子（或染料标记的DNA）的运动，从而能够以准确、可靠、快速、简单的方式追踪和量化大分子的活动。Shaban团队使用Hi-D技术描绘了单细胞的基因组构象和动态变化的生物物理特性图谱。研究人员发现，细胞核中的DNA可以根据其动力学在空间上被划分成大小在0.3-3 μm之间的结构域亚群，并且这些结构域亚群会随着转录活动的变化发生变化。已知相比常染色质，异染色质相对不活跃，Hi-D进一步发现了染色质的活跃程度与染色质致密度没有关系。总而言之，Hi-D技术可在不牺牲活性荧光团标记密度、不需要标记物制备的特殊经验、不使用高端显微镜的条件下，实时描绘大量密集的细胞核因子在受到刺激时的动态图谱。
In mammalian cells, chromosomes occupy into dynamic nuclear territories. Due to the highly compact nature and the abundant fine structure of the nucleus, our understanding of the nuclear dynamics at the nanoscale in live cells has been limited. Shaban et al. developed a high-resolution diffusion mapping (Hi-D) to analyze the dynamics of dense structures such as chromatin and other abundant nuclear constituents directly in single cells at sub-pixel accuracy. Hi-D applied the dense optical flow method to quantitatively reconstruct the motion of densely distributed fluorescent molecules (or dye-labeled DNA) from a series of conventional confocal fluorescence microscopy images, which enabled tracking and quantification of macromolecule behaviors in an accurate, robust, fast, and easy way. Using Hi-D, Shaban et al. presented the single-cell biophysical property maps of genome conformation and behavior. They found that DNA dynamics in the nucleus were spatially partitioned into subpopulations domains with a size between 0.3–3-μm, which could be remodeled in response to transcriptional activities. Consistent with the view that heterochromatin was less dynamic than euchromatin, Hi-D revealed that chromatin dynamics was uncoupled from chromatin compaction. Collectively, Hi-D is a useful tool in obtaining comprehensive and dynamic maps of dense and bulk nuclear factors at real-time in responses to stimuli without losing active fluorophore density or need for prior experience in labelling preparation or advanced microscopy.
#8. Title: Nucleus size and DNA accessibility are linked to the regulation of paraspeckle formation in cellular differentiation
First Author: Markus Grosch
Correspondence Author: Micha Drukker
Research article |BMC Biology
Date: 2020-04-22
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旁斑（paraspeckles）是一种广泛存在于哺乳动物细胞核中的亚结构小体，由长非编码RNA NEAT1 和40余种蛋白质组装而成，对多种细胞生物学过程具有调控作用。NEAT1是维持旁斑完整性和功能所必须的结构性长链非编码RNA（lncRNA）。细胞系分化过程中旁斑是如何动态变化的？Grosch团队应用靶向NEAT1的单分子荧光原位杂交（smFISH）技术来观察人类多能干细胞（hPSC）向不同细胞系分化过程中旁斑数量和大小的变化。研究人员发现，旁斑的数量与具体的细胞谱系或分化的时间无关，而是与细胞核大小呈正相关。值得注意的是，他们还发现在分化过程中旁斑会插入DNA双螺旋结构中，且这种结合对旁斑组装也是必需的。他们进而通过smFISH靶标NEAT1的方法，绘制了20多种细胞类型在hPSC分化过程中旁斑动态变化蓝图，进而为研究hPSC分化过程中旁斑动力学和形态学提供了宝贵的资源库。总体而言，这项非常有趣的研究揭示了在hPSC分化过程中细胞核大小与旁斑数量之间存在正相关。
Paraspeckles are a type of nuclear membrane-less structures, which sequester RNAs and proteins and regulate multiple cellular progresses. NEAT1 is the architecture long noncoding RNA (lncRNA) that is required for paraspeckle integrity and function. What is the dynamic of paraspeckles during cell lineage differentiation? Grosch et al. used single molecule Florescence in Situ Hybridization (smFISH) targeting NEAT1 to visualize the number and size of paraspeckles during human pluripotent stem cell (hPSC) differentiation to different cell lineages. They found that the number of paraspeckles was not determined by cell lineages or the timing of differentiation but was rather positively correlated with the nucleus size. Remarkably, they found that paraspeckles were intercalated in dsDNA helix and that the binding of dsDNA was also required for the assembly of paraspeckles. They further drew a dynamic atlas of paraspeckles, shown by NEAT1 smFISH, in more than 20 cell types upon hPSC differentiation, thereby providing a resource for studying paraspeckle dynamics and morphologies during hPSC differentiation. Overall, it is an interesting study that uncovers a positive correlation between the size of the nucleus and the number of paraspeckles during hPSC differentiation.
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（来源：科学网）
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