Volume: 17, Issue: 2

Review Article

Progress and Challenges for Live-cell Imaging of Genomic Loci Using CRISPR-based Platforms

Xiaotian Wu, Shiqi Mao, Yachen Ying, Christopher J. Krueger, Antony K. Chen

Chromatin conformation, localization, and dynamics are crucial regulators of cellular behaviors. Although fluorescence in situ hybridization-based techniques have been widely utilized for investigating chromatin architectures in healthy and diseased states, the requirement for cell fixation precludes the comprehensive dynamic analysis necessary to fully understand chromatin activities. This has spurred the development and application of a variety of imaging methodologies for visualizing single chromosomal loci in the native cellular context. In this review, we describe currently-available approaches for imaging single genomic loci in cells, with special focus on clustered regularly interspaced short palindromic repeats (CRISPR)-based imaging approaches. In addition, we discuss some of the challenges that limit the application of CRISPR-based genomic imaging approaches, and potential solutions to address these challenges. We anticipate that, with continued refinement of CRISPR-based imaging techniques, significant understanding can be gained to help decipher chromatin activities and their relevance to cellular physiology and pathogenesis.
染色质的三维空间构象、核内定位以及动力学特征在调控真核细胞基因表达进而影响细胞行为方面发挥着重要作用，例如：染色质空间构象的变化可以促使增强子等调控元件与靶基因相互靠近,从而促进基因表达；染色质从核质迁移至核膜处会降低其运动速率及基因表达水平等。 尽管荧光原位杂交技术已经被广泛运用于特定染色质的标记，进而研究染色质在发育过程中或病理条件下的行为变化，但是由于需要进行细胞固定、探针清洗等操作，该技术难以帮助研究者全面、真实地了解染色质在活细胞中的动态活动信息。因此，开发并优化在活细胞环境中标记特定染色质位点的荧光成像方法具有重要的意义。本文综述了目前用于在活细胞中标记特定染色质位点的成像方法，重点介绍了基于Clustered regularly interspaced short palindromic repeats (CRISPR)系统的荧光成像方法。本文分别阐述了基于荧光蛋白、有机荧光染料以及纳米颗粒等不同发光元素的CRISPR 成像体系的工作原理，以及现阶段每个体系在活细胞标记特定染色质位点，观测其时程变化，并获取其动力学信息等方面的应用。本文最后列举了CRISPR 成像领域存在的挑战，并针对这些挑战提出了一些建议。我们期待，基于CRISPR系统的染色质标记技术的不断完善能够为破译染色质活动与细胞生理及病理的关系提供有力的支持。

Page 119-128

Original Research

Single-cell Analysis of CAR-T Cell Activation Reveals A Mixed TH1/TH2 Response Independent of Differentiation

Iva Xhangolli, Burak Dura, GeeHee Lee, Dongjoo Kim, Yang Xiao, Rong Fan

The activation mechanism of chimeric antigen receptor (CAR)-engineered T cells may differ substantially from T cells carrying native T cell receptor, but this difference remains poorly understood. We present the first comprehensive portrait of single-cell level transcriptional and cytokine signatures of anti-CD19/4-1BB/CD28/CD3ζ CAR-T cells upon antigen-specific stimulation. Both CD4+ helper T (TH) cells and CD8+ cytotoxic CAR-T cells are equally effective in directly killing target tumor cells and their cytotoxic activity is associated with the elevation of a range of TH1 and TH2 signature cytokines, e.g., interferon γ, tumor necrotic factor α, interleukin 5 (IL5), and IL13, as confirmed by the expression of master transcription factor genes TBX21 and GATA3. However, rather than conforming to stringent TH1 or TH2 subtypes, single-cell analysis reveals that the predominant response is a highly mixed TH1/TH2 function in the same cell. The regulatory T cell activity, although observed in a small fraction of activated cells, emerges from this hybrid TH1/TH2 population. Granulocyte-macrophage colony stimulating factor (GM-CSF) is produced from the majority of cells regardless of the polarization states, further contrasting CAR-T to classic T cells. Surprisingly, the cytokine response is minimally associated with differentiation status, although all major differentiation subsets such as naïve, central memory, effector memory, and effector are detected. All these suggest that the activation of CAR-engineered T cells is a canonical process that leads to a highly mixed response combining both type 1 and type 2 cytokines together with GM-CSF, supporting the notion that polyfunctional CAR-T cells correlate with objective response of patients in clinical trials. This work provides new insights into the mechanism of CAR activation and implies the necessity for cellular function assays to characterize the quality of CAR-T infusion products and monitor therapeutic responses in patients.

Page 129-139

Original Research

Global Quantitative Mapping of Enhancers in Rice by STARR-seq

Jialei Sun, Na He, Longjian Niu, Yingzhang Huang, Wei Shen, Yuedong Zhang, Li Li, Chunhui Hou

Enhancers activate transcription in a distance-, orientation-, and position-independent manner, which makes them difficult to be identified. Self-transcribing active regulatory region sequencing (STARR-seq) measures the enhancer activity of millions of DNA fragments in parallel. Here we used STARR-seq to generate a quantitative global map of rice enhancers. Most enhancers were mapped within genes, especially at the 5′ untranslated regions (5′UTR) and in coding sequences. Enhancers were also frequently mapped proximal to silent and lowly-expressed genes in transposable element (TE)-rich regions. Analysis of the epigenetic features of enhancers at their endogenous loci revealed that most enhancers do not co-localize with DNase I hypersensitive sites (DHSs) and lack the enhancer mark of histone modification H3K4me1. Clustering analysis of enhancers according to their epigenetic marks revealed that about 40% of identified enhancers carried one or more epigenetic marks. Repressive H3K27me3 was frequently enriched with positive marks, H3K4me3 and/or H3K27ac, which together label enhancers. Intergenic enhancers were also predicted based on the location of DHS regions relative to genes, which overlap poorly with STARR-seq enhancers. In summary, we quantitatively identified enhancers by functional analysis in the genome of rice, an important model plant. This work provides a valuable resource for further mechanistic studies in different biological contexts.
细胞状态、功能等特性的形成是生物学的基本问题之一，而最终决定细胞类型的因素是基因表达，在细胞分化和对内外信号产生响应的过程中，基因表达的调控往往是最终的结果，基因表达调控机制本身也是生物学研究的核心问题之一。基因表达调控在线性DNA水平主要表现为调控元件的选择和对靶基因调控的参与，其中研究较多的调控元件为增强子。增强子的功能不依赖在基因组中和靶基因间的距离、方向和位置，这些特点很大程度上增加了增强子鉴定的难度。调控元件自转录测序方法（STARR-seq）通过同时测量千百万条DNA片段的自转录水平从而可以达到鉴定增强子的目的。 我们使用该技术在国际上首次实现了对水稻基因组的分析实现定量确定全基因组范围内增强子活性的分布图谱，并揭示了多种水稻增强子特点。绝大多数增强子位于基因序列内，尤其是在5'端非翻译区和编码的外显子区，这和哺乳动物中增强子一般被认为分布在远离基因的位置不同。相当数量的增强子位于转座子富集区域内沉默和非转录基因的附近，一定程度上近年来转座序列可以演化为新的增强子的发现相吻合。我们对水稻增强子的表观遗传特征进行了多层次的分析，发现功能性增强子大多数并不和DNA酶超敏感点相重叠，同时和动物体系里增强子的组蛋白修饰H3K4me1重叠不多。通过聚类分析进一步发现仅有约40%增强子具有一种及一种以上的表观特征，有趣的是H3K4me3和H3K27ac的组合而不是H3K4me1和H3K27me3的组合更富集在功能性鉴定的增强子上。同时，H3K27me3和H3K4me3和H3K27ac可以同时富集，可能是细胞和不同染色体的差异性。 我们还借鉴基于超敏感点位置预测增强子的方法预测增强子，并与STARR-seq鉴定的增强子相比较，我们的结果显示基于纯粹超敏感点位置的增强子预测结果和功能性鉴定获得的结果具有巨大的差异，几乎不重叠。 鉴定增强子的方法有多种，各种方法的结果间可能可以互补，我们的工作不仅提供了一个新的增强子数据，更重要的是揭示了不同方法鉴定的增强子具有完全不同的特点，为进一步推动增强子鉴定的研究和对增强子功能和作用机制的研究提供了资源基础

Page 140-153

Original Research

m6A Regulates Neurogenesis and Neuronal Development by Modulating Histone Methyltransferase Ezh2

Junchen Chen, Yi-Chang Zhang, Chunmin Huang, Hui Shen, Baofa Sun, Xuejun Cheng, Yu-Jie Zhang, Yun-Gui Yang, Qiang Shu, Ying Yang, Xuekun Li

N6-methyladenosine (m6A), catalyzed by the methyltransferase complex consisting of Mettl3 and Mettl14, is the most abundant RNA modification in mRNAs and participates in diverse biological processes. However, the roles and precise mechanisms of m6A modification in regulating neuronal development and adult neurogenesis remain unclear. Here, we examined the function of Mettl3, the key component of the complex, in neuronal development and adult neurogenesis of mice. We found that the depletion of Mettl3 significantly reduced m6A levels in adult neural stem cells (aNSCs) and inhibited the proliferation of aNSCs. Mettl3 depletion not only inhibited neuronal development and skewed the differentiation of aNSCs more toward glial lineage, but also affected the morphological maturation of newborn neurons in the adult brain. m6A immunoprecipitation combined with deep sequencing (MeRIP-seq) revealed that m6A was predominantly enriched in transcripts related to neurogenesis and neuronal development. Mechanistically, m6A was present on the transcripts of histone methyltransferase Ezh2, and its reduction upon Mettl3 knockdown decreased both Ezh2 protein expression and consequent H3K27me3 levels. The defects of neurogenesis and neuronal development induced by Mettl3 depletion could be rescued by Ezh2 overexpression. Collectively, our results uncover a crosstalk between RNA and histone modifications and indicate that Mettl3-mediated m6A modification plays an important role in regulating neurogenesis and neuronal development through modulating Ezh2.
与DNA相比，RNA的修饰形式更加丰富和多样化，其中研究最为深入、含量最高的是N6-甲基腺嘌呤（N6-methyladenosine ，m6A）。甲基转移酶Mettl3和Mettl14催化甲基转移到腺嘌呤而产生m6A，后者又可以在去甲基化转移酶FTO和ALKBH5的作用下实现去甲基化。研究发现m6A修饰参与了多种生物学过程，与多种疾病密切相关。我们实验室和其他研究团队之前的工作都揭示了m6A修饰与神经发育和神经系统功能密切相关，但是关于Mettl3介导的m6A修饰在成体神经发生中的功能和机制尚不明确。基于此，我们采用分离培养的小鼠成体神经干细胞进行了本研究。我们的研究发现，Mettl3敲低可以显著降低神经干细胞中m6A修饰的水平，抑制神经干细胞的增殖，使得神经干细胞在分化时更倾向于进入胶质细胞谱系。Mettl3敲低后显著改变了与细胞增殖、神经发育等基因的表达，特别是组蛋白甲基化转移酶Ezh2的表达，而过表达Ezh2可以拯救Mettl3缺失而导致的神经干细胞增殖和分化能力的异常。我们的研究结果揭示了RNA修饰和组蛋白修饰相互作用，从而精细调控成体神经发生，为表观遗传修饰在成体神经发生的调控作用提供了新的证据。

Page 154-168

Original Research

Integrating Culture-based Antibiotic Resistance Profiles with Whole-genome Sequencing Data for 11,087 Clinical Isolates

Valentina Galata, Cédric C. Laczny, Christina Backes, Georg Hemmrich-Stanisak, Susanne Schmolke, Andre Franke, Eckart Meese, Mathias Herrmann, Lutz von Müller, Achim Plum, Rolf Müller, Cord Stähler, Andreas E. Posch, Andreas Keller

Emerging antibiotic resistance is a major global health threat. The analysis of nucleic acid sequences linked to susceptibility phenotypes facilitates the study of genetic antibiotic resistance determinants to inform molecular diagnostics and drug development. We collected genetic data (11,087 newly-sequenced whole genomes) and culture-based resistance profiles (10,991 out of the 11,087 isolates comprehensively tested against 22 antibiotics in total) of clinical isolates including 18 main species spanning a time period of 30 years. Species and drug specific resistance patterns were observed including increased resistance rates for Acinetobacter baumannii to carbapenems and for Escherichia coli to fluoroquinolones. Species-level pan-genomes were constructed to reflect the genetic repertoire of the respective species, including conserved essential genes and known resistance factors. Integrating phenotypes and genotypes through species-level pan-genomes allowed to infer gene–drug resistance associations using statistical testing. The isolate collection and the analysis results have been integrated into GEAR-base, a resource available for academic research use free of charge at https://gear-base.com.

Page 169-182

Original Research

Chronic Food Antigen-specific IgG-mediated Hypersensitivity Reaction as A Risk Factor for Adolescent Depressive Disorder

Ran Tao, Zhicheng Fu, Lijun Xiao

Major depressive disorder (MDD) is the most common nonfatal disease burden worldwide. Systemic chronic low-grade inflammation has been reported to be associated with MDD progression by affecting monoaminergic and glutamatergic neurotransmission. However, whether various proinflammatory cytokines are abnormally elevated before the first episode of depression is still largely unclear. Here, we evaluated 184 adolescent patients who were experiencing their first episode of depressive disorder, and the same number of healthy individuals was included as controls. We tested the serum levels of high-sensitivity C-reactive protein (hs-CRP), tumor necrosis factor-α (TNF-α), IgE, 14 different types of food antigen-specific IgG, histamine, homocysteine, S100 calcium-binding protein B, and diamine oxidase. We were not able to find any significant differences in the serum levels of hs-CRP or TNF-α between the two groups. However, the histamine level of the patients (12.35 μM) was significantly higher than that of the controls (9.73 μM, P < 0.001, Mann–Whitney U test). Moreover, significantly higher serum food antigen-specific IgG positive rates were also found in the patient group. Furthermore, over 80% of patients exhibited prolonged food intolerance with elevated levels of serum histamine, leading to hyperpermeability of the blood–brain barrier, which has previously been implicated in the pathogenesis of MDD. Hence, prolonged high levels of serum histamine could be a risk factor for depressive disorders, and antihistamine release might represent a novel therapeutic strategy for depression treatment.
作为全球最主要的非致命疾病负担，抑郁症（MDD）早已成为医学研究的热点领域，但其发病机制至今尚不明确，这直接影响到抑郁症的防治策略选择。在细胞因子学说中，认为促炎症性细胞因子会通过血脑屏障影响脑内的单胺类神经递质和谷氨酸能神经传递，表明慢性低度炎症与抑郁症关联性较强。然而，在抑郁症的首次发作之前，促炎细胞因子是否已经异常升高却并无报道证实。因此本文将184名首次抑郁障碍发作的青少年患者作为研究对象，并将相同数量的同期健康个体作为对照。所有被试均检测8项指标：血清超敏C反应蛋白（hs-CRP）、肿瘤坏死因子-α（TNF-α）、IgE、14种不同类型的食物抗原特异性IgG、组胺、同型半胱氨酸（Hcy）、S100钙结合蛋白B（S100B）和二胺氧化酶（DAO）等。结果两组血清hs-CRP、TNF-α水平差异无统计学意义，这与已有的研究结果不同，表明炎症因子可能不是抑郁症首次发作的原因。与此同时我们发现的是，病例组胺水平（12.35μm）明显高于对照组（9.73μm，P＜0.001），同时反映血脑屏障通透性的指标S100B也升高，证实高水平的血清组胺导致血脑屏障的通透性增加，进而引发与抑郁症相关的一系列精神症状的可能性。对病例组组织胺水平升高的原因进行分析，IgE（病例组37.87% vs 对照组22.3%）与IgG（病例组89.67% vs 对照组13.04%）的阳性率病例组均高于对照组，但是IgG两组之间差异更加显著，表明食物特异性免疫球蛋白G是组织胺升高的主要原因。因此本研究认为：长期慢性的食物不耐受导致的组胺升高是抑郁症发病的危险因素。抑郁症早期的发病机制可能不同于其他阶段，在抑郁症的早期发病机制中，应强调慢性食物抗原特异性IgG介导的超敏反应或慢性食物不耐受的作用，而不是慢性低度炎症。相应地，在防治策略上，我们提出了组织胺释放抑制剂对抑郁症治疗的可能性。

Page 183-189

Letter

Transcriptome and Regulatory Network Analyses of CD19-CAR-T Immunotherapy for B-ALL

Qiong Zhang, Hui Hu, Si-Yi Chen, Chun-Jie Liu, Fei-Fei Hu, Jianming Yu, Yaohui Wu, An-Yuan Guo

Chimeric antigen receptor (CAR) T cell therapy has exhibited dramatic anti-tumor efficacy in clinical trials. In this study, we reported the transcriptome profiles of bone marrow cells in four B cell acute lymphoblastic leukemia (B-ALL) patients before and after CD19-specific CAR-T therapy. CD19-CAR-T therapy remarkably reduced the number of leukemia cells, and three patients achieved bone marrow remission (minimal residual disease negative). The efficacy of CD19-CAR-T therapy on B-ALL was positively correlated with the abundance of CAR and immune cell subpopulations, e.g., CD8+ T cells and natural killer (NK) cells, in the bone marrow. Additionally, CD19-CAR-T therapy mainly influenced the expression of genes linked to cell cycle and immune response pathways, including the NK cell mediated cytotoxicity and NOD-like receptor signaling pathways. The regulatory network analyses revealed that microRNAs (e.g., miR-148a-3p and miR-375), acting as oncogenes or tumor suppressors, could regulate the crosstalk between the genes encoding transcription factors (TFs; e.g., JUN and FOS) and histones (e.g., HIST1H4A and HIST2H4A) involved in CD19-CAR-T therapy. Furthermore, many long non-coding RNAs showed a high degree of co-expression with TFs or histones (e.g., FOS and HIST1H4B) and were associated with immune processes. These transcriptome analyses provided important clues for further understanding the gene expression and related mechanisms underlying the efficacy of CAR-T immunotherapy.
B细胞急性淋巴细胞白血病（B-ALL）是由于B细发育异常，细胞凋亡逃避和不受控制的细胞增殖所引起的。在传统放化疗疗效不佳的情况下，通过改造后靶向B细胞恶性肿瘤特异性抗原CD19的嵌合抗原受体（CAR）T细胞（CD19-CAR-T）被认为是B-ALL免疫疗法中最有希望的方法。CD19-CAR-T可以识别和消除肿瘤细胞，并且已证实对复发/难治性B-ALL患者的缓解有显着功效。目前大多数关于CAR-T免疫疗法的研究都集中在临床疗效评估和新抗原的发展上，缺少对CAR-T治疗后患者基因表达和调控机制的研究。 本研究报道了4例B-ALL患者接受CD19-CAR-T治疗前后骨髓细胞的转录组图谱。临床检测显示CD19-CAR-T治疗后，患者白血病细胞的数量显著减少，并且实现了3名患者的骨髓缓解（微小残留病阴性）。研究发现，CD19-CAR-T治疗B-ALL的疗效与骨髓中CAR细胞和部分免疫细胞亚群（例如CD8+ T细胞和自然杀伤（NK）细胞）的丰度成正相关。另外，CD19-CAR-T疗法影响了大量与细胞周期和免疫应答通路相关基因的表达，包括参与NK细胞介导的细胞毒性和NOD样受体信号传导等通路基因。调控网络分析显示，重要microRNA（如miR-148a-3p和miR-375）可以通过调控转录因子基因（如JUN和FOS）以及组蛋白基因（如HIST1H4A和HIST2H4A）的表达而参与CD19-CAR-T治疗调控。此外，许多长非编码RNA与转录因子或组蛋白基因高度共表达而参与免疫过程。这些转录组表达和调控分析为进一步了解CAR-T免疫治疗疗效的相关机制提供了重要线索。

Page 190-200

Method

SSCC: A Novel Computational Framework for Rapid and Accurate Clustering Large-scale Single Cell RNA-seq Data

Xianwen Ren, Liangtao Zheng, Zemin Zhang

Clustering is a prevalent analytical means to analyze single cell RNA sequencing (scRNA-seq) data but the rapidly expanding data volume can make this process computationally challenging. New methods for both accurate and efficient clustering are of pressing need. Here we proposed Spearman subsampling-clustering-classification (SSCC), a new clustering framework based on random projection and feature construction, for large-scale scRNA-seq data. SSCC greatly improves clustering accuracy, robustness, and computational efficacy for various state-of-the-art algorithms benchmarked on multiple real datasets. On a dataset with 68,578 human blood cells, SSCC achieved 20% improvement for clustering accuracy and 50-fold acceleration, but only consumed 66% memory usage, compared to the widelyused software package SC3. Compared to k-means, the accuracy improvement of SSCC can reach 3-fold. An R implementation of SSCC is available at https://github.com/Japrin/sscClust.

Page 201-210

Method

SeqSQC: A Bioconductor Package for Evaluating the Sample Quality of Next-generation Sequencing Data

Qian Liu, Qiang Hu, Song Yao, Marilyn L. Kwan, Janise M. Roh, Hua Zhao, Christine B. Ambrosone, Lawrence H. Kushi, Song Liu, Qianqian Zhu

As next-generation sequencing (NGS) technology has become widely used to identify genetic causal variants for various diseases and traits, a number of packages for checking NGS data quality have sprung up in public domains. In addition to the quality of sequencing data, sample quality issues, such as gender mismatch, abnormal inbreeding coefficient, cryptic relatedness, and population outliers, can also have fundamental impact on downstream analysis. However, there is a lack of tools specialized in identifying problematic samples from NGS data, often due to the limitation of sample size and variant counts. We developed SeqSQC, a Bioconductor package, to automate and accelerate sample cleaning in NGS data of any scale. SeqSQC is designed for efficient data storage and access, and equipped with interactive plots for intuitive data visualization to expedite the identification of problematic samples. SeqSQC is available at http://bioconductor.org/packages/SeqSQC.

Page 211-218

Research Highlight

Deep Learning Deepens the Analysis of Alternative Splicing

Xudong Zou, Xin Gao, Wei Chen

基因组学数据在数据量和数据维度上的日益增长，给传统的数据分析方法带来了挑战，同时也为开发新的分析方法提供了机会。近年来，深度学习推动了人工智能和机器学习领域新一波的浪潮。最近，美国加利福尼亚大学洛杉矶分校的邢毅教授（现为费城儿童医院教授）实验室开发了一个全新的计算框架：DARTS[1]。DARTS通过一个贝叶斯框架系统性地整合了人工智能方法和实验数据的优势。其中基于海量序列数据以及RNA结合蛋白mRNA表达水平数据训练出的深度神经网络模型为贝叶斯模型提供一个先验概率，而贝叶斯模型进一步整合RNA-seq实验数据的信息，从而达到对样本间差异可变剪接的准确预测。作者将DARTS应用于上皮-间充质转移（EMT）样本的RNA-seq数据集上发现，DARTS不仅能预测EMT过程中显著差异的可变剪接事件，而且能发现低丰度基因中的差异剪接事件。相比于之前的方法，DARTS有两个方面的重要创新：其一，DARTS模型不仅考虑基因组序列特征，同时考虑RNA结合蛋白的表达水平;其二，DARTS巧妙地结合了统计学习模型的预测能力和实验数据所含的丰富信息，使得最终的模型在高丰度和低丰度基因上都达到准确的预测。 DARTS在上述两个方面的重要创新，一方面提高了样本间差异可变剪接事件的预测准确性，另一方面，通过在模型训练时利用公共的RNA-seq数据，提高了DARTS对于低丰度基因中差异可变剪接事件的预测能力，使得先前因为测序深度偏低而无法用于差异可变剪接分析的RNA-seq数据集的应用成为可能。这一突破扩展了DARTS的应用范围，并为今后类似问题的方法研发提供了可借鉴的思路。

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