基于CRISPR/dCas9技术活细胞成像基因组位点的研究进展

基因组的三维空间组织在调节其功能、维持表观遗传和稳定性方面起着重要作用.虽然基于荧光原位杂交的标记技术或单细胞显微观察等方法对此进行了广泛研究,但是细胞内部的时空动态在很大程度上仍难以捉摸.目前标记和观察活细胞中特定内源基因组位点的方法操作难度大且对生物的侵入性伤害较大.CRISPR的发现彻底改变了基因组工程领域.除了...     

Construction of a CRISPR-Cas9 System for Pig Genome Targeting

A Cas9/sgRNA RNA-guided endonuclease expression system including a codon-optimized Streptococcus pyogenes A20 Cas9 recombinant protein expression vector and a spacer-guide chimeric RNA expression vector using the porcine U6 promoter was constructed for application in pigs. Only the Flag₂-NLS₁-Cas9-NLS₂ recombinant protein in complex with sgRNA was translocated into the nucleus; the Flag₂-NLS₁-Cas9-NLS₂ protein alone was excluded from the nucleus. Up to 13% of porcine PK1 cells targeted in vitro were observed, regardless of transfection efficiency.     

CRISPR/Cas9的应用及脱靶效应研究进展

CRISPR/Cas9基因编辑技术在生命科学领域掀起了一场全新的技术革命,该技术可以对基因组特定位点进行靶向编辑,包括缺失、插入、修复等。CRISPR/Cas9比锌指核酸酶 (ZFNs)和转录激活因子样效应物核酸酶(TALENs)技术更易于操作,而且更高效。CRISPR/Cas9系统中的向导RNA(Single guide RNA, sgRNA)是一段与目标DNA片段匹配的RNA序列,指导Cas9蛋白对基因组进行识别。研究发现,设计的sgRNA会与非靶点DNA序列错配,引入非预期的基因突变,即脱靶效应(Off-target effects)。脱靶效应严重制约了CRISPR/Cas9基因编辑技术的广泛应用。为了避免脱靶效应,研究者对影响脱靶效应的因素进行了系统研究并提出了许多降低脱靶效应的方法。文章总结了CRISPR/Cas9系统的应用及脱靶效应研究进展,以期为相关领域的工作提供参考。     

CRISPR/Cas9在肿瘤治疗中的研究进展

CRISPR/Cas9技术自从出现以来便迅速应用于肿瘤研究。在肿瘤发生的机理研究中,CRISPR/Cas9可用于研究单核苷酸突变、染色体异位等因素在肿瘤发生中的作用机制,同时也可以用于肿瘤细胞中功能缺陷基因的筛选。在肿瘤治疗方法的研究中,CRISPR/Cas9主要用于诱发机制比较清晰且诱因为病毒的肿瘤类型,例如鼻咽癌、宫颈癌等,通过对相应病毒的基因进行编辑从而抑制其致癌作用。利用CRISPR/Cas9技术还可以加速新肿瘤治疗靶点基因的发现。尽管发展和应用十分迅速,但是CRISPR/Cas9在肿瘤研究和治疗中的作用仍然受多种因素的限制,包括Cas9和sgRNA的输送效率、脱靶效应以及安全性和成本等。对CRISPR/Cas9在肿瘤研究中的应用进展进行了综述,以期为肿瘤发生、转移机制和肿瘤治疗等方面的研究提供参考。     

CRISPR/Cas9介导的疾病模型构建与基因修复研究进展

近年来,可编程核酸酶介导的基因编辑技术迅猛发展。CRISPR/Cas9技术源于细菌和古生菌的适应性免疫系统,主要由Cas9内切酶和向导RNA（guide RNA,gRNA）组成。Cas9内切酶在gRNA的指导下造成DNA的双链断裂,从而使研究人员能够精准高效地操纵特定基因组位点。同时,该系统可以揭示基因在疾病进程中所扮演的未知角色,在临床治疗中有应用潜能。现总结了CRISPR/Cas9技术在疾病模型构建与基因修复领域应用的研究进展。     

Expansion of CRISPR targeting sites in Bombyx mori

The CRISPR/Cas9 system has been proven as a revolutionary genome engineering tool. In most cases, single guide RNA (sgRNA) targeting sites have been designed as GN19NGG or GGN18NGG, because of restriction of the initiation nucleotide for RNA Pol III promoters. Here, we demonstrate that the U6 promoter from a lepidopteran model insect, Bombyx mori, effectively expressed the sgRNA initiated with any nucleotide bases (adenine, thymine, guanine or cytosine), which further expands the CRISPR targeting space. A detailed expansion index in the genome was analysed when N20NGG was set as the CRISPR targeting site instead of GN19NGG, and revealed a significant increase of suitable targets, with the highest increase occurring on the Z sex chromosome. Transfection of different types of N20NGG sgRNAs targeting the enhanced green fluorescent protein (EGFP) combined with Cas9, significantly reduced EGFP expression in the BmN cells. An endogenous gene, BmBLOS2, was also disrupted by using various types of N20NGG sgRNAs, and the cleavage efficiency of N20NGG sgRNAs with different initial nucleotides and GC contents was evaluated in vitro. Furthermore, transgenic silkworms expressing Cas9 and sgRNAs targeting the BmBLOS2 gene were generated with many types of mutagenesis. The typical transparent skin phenotype in knock-out silkworms was stable and inheritable, suggesting that N20NGG sgRNAs function sufficiently in vivo. Our findings represent a renewal of CRISPR/Cas9 target design and will greatly facilitate insect functional genetics research.     

靶向miRNA前体不同类型sgRNA的丰度及特异性评估

CRISPR/Cas技术能高效进行基因组定点编辑,但不同细菌来源或人工改造的Cas9以及Cpf1等核酸酶识别的PAM (protospacer adjacent motif)有差异,因此不同的基因编辑核酸酶可能采用不同类型的sgRNAs(small guide RNAs)。MicroRNAs (miRNAs)是一类调控性的小分子非编码RNAs,为了研究miRNA前体中是否可能存在特异性高的sgRNAs靶点,本文利用本课题组前期开发的生物信息学软件CRISPR-offinder,对靶向28 645条miRNA前体的11种不同类型sgRNA的丰度及特异性进行了分析,并利用CRISPR/Cas9慢病毒技术构建了猪miR-302/367基因簇敲除细胞系,对构建的猪miRNA敲除细胞系的效率进行了检测。结果表明,每个miRNA前体中平均存在约8种不同类型sgRNA的靶点;通过评估靶向猪miRNA前体sgRNA的脱靶效应,发现其中特异性高的sgRNA仅占18.2%;通过CRISPR/Cas9慢病毒技术成功构建了猪miR-302/367基因簇敲除细胞系,发现通过该技术构建miRNA敲除细胞系的效率为40%。本研究为利用CRISPR/Cas技术靶向敲除miRNA提供了重要资源。     

CRISPR/Cas9系统中sgRNA设计与脱靶效应评估

基于CRISPR/Cas9系统介导的第三代基因组编辑技术,已成功应用于动物、植物和微生物等诸多物种的基因组改造。如何提高CRISPR/Cas9技术的基因组编辑效率和最大限度降低脱靶风险一直是本领域的研究热点,而使用高效且特异的sgRNA(Small guide RNA)是基因组改造成功的关键性因素之一。目前,已有多款针对CRISPR/Cas9技术的sgRNA设计和/或脱靶效应评估软件,但不同的软件各有优缺点。本文重点对16款sgRNA 设计和脱靶效应评估在线和单机版软件的特点进行了阐述,通过制定38项评估指标对不同软件进行了比较分析,最后对11种用于检测基因组编辑效率和脱靶的实验方法,以及如何筛选高效且特异的sgRNA进行了归纳总结。     

Developing CRISPR-based sex-ratio distorters for the genetic control of fruit fly pests: A how to manual

Agricultural pest control using genetic-based methods provides a species-specific and environmentally harmless way for population suppression of fruit flies. One way to improve the efficiency of such methods is through self-limiting, female-eliminating approaches that can alter an insect populations' sex ratio toward males. In this microreview, we summarize recent advances in synthetic sex ratio distorters based on X-chromosome shredding that can induce male-biased progeny. We outline the basic principles to guide the efficient design of an X-shredding system in an XY heterogametic fruit fly species of interest using CRISPR/Cas gene editing, newly developed computational tools, and insect genetic engineering. We also discuss technical aspects and challenges associated with the efficient transferability of this technology in fruit fly pest populations, toward the potential use of this new class of genetic control approaches for pest management purposes.     

BEguider:一个单碱基编辑器sgRNA设计与编辑效率预测工具

单碱基编辑器是实用且高效的基因编辑工具,其编辑效率与单向导RNA(single guide RNA, sgRNA)序列的设计密切相关。目前单碱基编辑器sgRNA序列的设计缺少特定的法则,主要依靠经验和大量尝试完成。本研究基于卷积神经网络,开发了一个单碱基编辑器sgRNA序列设计工具BEguider。BEguider利用TensorFlow 2深度学习框架建立编辑效率预测模型,能够在人基因组范围内针对NGG PAM序列依赖的单碱基编辑器ABE7.10-NGG和BE4-NGG批量设计sgRNA序列,预测编辑效率。此外,通过整合Cas-OFFinder, BEguider能够提供对sgRNA脱靶情况的评估。利用BEguider设计sgRNA序列,有助于研究人员提高实验效率,节约实验成本。     

A Toolkit of CRISPR-Based Genome Editing Systems in Drosophila

The last couple of years have witnessed an explosion in development of CRISPR-based genome editing technologies in cell lines as well as in model organisms. In this review, we focus on the applications of this popular system in Drosophila. We discuss the effectiveness of the CRISPR/Cas9 systems in terms of delivery, mutagenesis detection, parameters affecting efficiency, and off-target issues, with an emphasis on how to apply this powerful tool to characterize gene functions.     

DNA Unwinding Is the Primary Determinant of CRISPR-Cas9 Activity

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CRISPR系统用于昆虫基因表达调控的研究进展与展望

昆虫基因功能研究因缺少相应的工具而受到明显限制,但CRISPR/Cas9系统的出现为昆虫基因编辑及转录调控研究提供巨大助力。将Cas9核酸酶的RuvC和HNH剪切结构域失活改造得到的dCas9系统近年来在基因转录调控方面得到了广泛应用,同时CRISPR/dCpf1和最新发现的CRISPR/Cas13(a/b)系统为基因功能研究提供更多选择。本文综述了dCas9, dCpf1及Cas13(a/b)系统作用机理及在果蝇中的转录调控研究进展,以期为相关昆虫研究提供参考。     

Recent Advances in CRISPR‐Cas9 Genome Editing Technology for Biological and Biomedical Investigations

The Type II CRISPR‐Cas9 system is a simple, efficient, and versatile tool for targeted genome editing in a wide range of organisms and cell types. It continues to gain more scientific interest and has established itself as an extremely powerful technology within our synthetic biology toolkit. It works upon a targeted site and generates a double strand breaks that become repaired by either the NHEJ or the HDR pathway, modifying or permanently replacing the genomic target sequences of interest. These can include viral targets, single‐mutation genetic diseases, and multiple‐site corrections for wide scale disease states, offering the potential to manage and cure some of mankind's most persistent biomedical menaces. Here, we present the developing progress and future potential of CRISPR‐Cas9 in biological and biomedical investigations, toward numerous therapeutic, biomedical, and biotechnological applications, as well as some of the challenges within. J. Cell. Biochem. 119: 81–94, 2018. © 2017 Wiley Periodicals, Inc.     

基于CRISPR/Cas9系统构建绵羊VASA基因敲入载体及验证

本研究旨在探索VASA基因在绵羊睾丸发育中的表达变化,并通过构建VASA基因敲入载体,为下一步进行绵羊生殖细胞体外诱导分化研究提供基础。采集性成熟前后即3月龄（3-month-old,3M）和9月龄（9-month-old,9M）绵羊睾丸组织,利用实时荧光定量PCR （quantitative real-time PCR,qPCR）和Western blotting技术分析VASA基因的差异表达,并利用免疫组织化学技术对VASA基因的表达定位进行分析。设计靶向VASA基因的向导RNA （guide RNA,gRNA）,并构建同源重组载体,进行质粒转染绵羊耳成纤维细胞。结合CRISPR/dCas9技术对VASA基因进行激活,进一步验证载体效率。结果表明,VASA基因随着绵羊睾丸发育,表达水平极显著增加（P<0.01）,且主要定位在精母细胞和圆形精子细胞中。利用CRISPR/Cas9系统构建了VASA基因敲入载体,联合pEGFP-PGK puro-VASA载体转染耳成纤维细胞,CRISPR/dCas9系统激活后,耳成纤维细胞成功表达VASA基因。结果提示,VASA基因在绵羊睾丸发育和精子发生中发挥潜在功能,且通过CRISPR/Cas9系统可在体外构建VASA基因敲入载体,为下一步探究VASA基因对绵羊雄性生殖细胞的发育和分化提供有效的研究手段。     

基于CRISPR/Cas9技术的高通量筛选平台:发掘病毒复制相关宿主分子的新途径

病毒作为严格的细胞内寄生生物,需要多种宿主蛋白辅助其完成生命周期。寻找与病毒复制相关的宿主因子并揭示其作用机制,将有助于阐明病毒的感染机制,为疫病的防治提供新靶标。与RNA干扰技术相比,近年来兴起的CRISPR/Cas9技术能更特异、高效、准确地实现基因组编辑,因而在功能基因研究中得到更广泛应用。而基于CRISPR/Cas9系统的宿主全基因组sgRNA文库高通量筛选技术平台,可快速发现参与病毒侵入、复制等生物学过程的关键宿主因子,通过明确病毒-宿主分子相互作用进而揭示病毒的生命周期,为分子病毒学和免疫学提供了强大的研究工具。本文主要总结了基于CRISPR/Cas9技术的高通量筛选平台的具体筛选流程,归纳和讨论了该平台在筛选调控病毒复制相关宿主因子中的应用现状和发展前景。