人工核酸酶介导的基因组编辑技术

传统的基因组编辑技术是基于胚胎干细胞和同源重组实现生物基因组定向改造,但是该技术打靶效率低,严重制约了生命科学以及医学的研究.因此,研究新的基因组编辑技术十分重要.人工核酸酶介导的基因组编辑技术是通过特异性识别靶位点造成DNA双链断裂,引起细胞内源性的修复机制实现靶基因的修饰.与传统的基因组编辑技术相比,人工核酸酶技术打靶效率高,这对于基因功能的研究、构建人类疾病动物模型以及探索新型疾病治疗方案有着重要的意义.人工核酸酶技术有3种类型：锌指核酸酶(ZFN)、类转录激活因子核酸酶(TALEN)及规律成簇的间隔短回文重复序列(CRISPR).本文将对以上3种人工核酸酶技术的原理以及在生命科学和医学研究的应用进行综述.     

基因编辑技术

基因编辑是指通过核酸酶对靶基因进行定点改造,实现特定DNA的定点敲除、敲入以及突变等,最终下调或上调基因的表达,以使细胞获得新表型的一种新型技术。基因编辑技术已被广泛运用于基因结构与功能的研究和多种细胞的基因工程改造,为疾病模型的建立、动植物新品种的培育及基因治疗等的研究提供新的手段。基因编辑技术主要包括锌指核酸酶技术(ZFN)、转录激活子样效应因子技术(TALEN)和成簇的规律间隔的短回文重复序列/CRISPR相关蛋白 (CRISPR/Cas) 系统等。本文将对3种基因编辑技术的原理、运用及其最新进展进行综述,以期为相关技术及其运用的研究提供参考。     

CRISPR/Cas9基因编辑技术在致瘤病毒感染研究中的应用

成簇的规律间隔的短回文重复序列及其相关蛋白9〔clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9),CRISPR/Cas9〕基因编辑技术的发现源于真细菌和古细菌中CRISPR/Cas系统介导的适应性免疫机制研究。该技术利用特异性向导RNA识别靶点基因,引导核酸内切酶Cas9对其切割,并通过同源重组或非同源末端连接完成对目的DNA的编辑。某些病毒感染机体后,可将其基因组整合到宿主细胞基因组中或潜伏于组织中而无法被彻底清除,从而引起持续性感染。本文参考2013年以来CRISPR/Cas9基因组编辑技术的最新相关研究报道,重点综述其在人类免疫缺陷病毒1型(human immunodeficiency virus type 1,HIV-1)、人乳头瘤病毒(human papillomavirus,HPV )、乙型肝炎病毒(hepatitis B virus, HBV)、 Epstein-Barr病毒(Epstein-Barr virus,EBV)等致瘤病毒感染相关疾病研究中的应用,并概括其作用于这些病毒的有效靶点。     

CRISPR/Cas9技术在感染性疾病中的应用

成簇的规律间隔的短回文重复序列及其相关蛋白9〔clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9),CRISPR/Cas9〕是一种新兴的基因编辑技术,与以前的三大基因编辑技术——归巢核酸内切酶、锌指核酸酶和转录激活因子样效应物核酸酶技术相比,其在靶向特异性、操作简便性、治疗彻底性、应用广泛性等方面具有更大的优势和发展潜力。艾滋病、乙型肝炎、疟疾等感染性疾病的治疗一直是医学上的重大难题,科学家正努力尝试利用CRISPR/Cas9技术解决这些医学难题。本文主要综述了CRISPR/Cas9技术在这些感染性疾病中应用的研究进展。     

新型靶向基因组编辑技术研究进展

传统的靶向基因组编辑技术改造基因效率非常低,严重制约了基础研究和临床应用。因此,新的靶向基因组编辑工具的研究显得非常重要,以此来提高基因原位修复、定点整合及高通量基因敲除的效率。主要论述了近年来发现的新型靶向基因组编辑技术即锌指核酸酶(ZFN)、转录激活子样效应因子核酸酶(TALENs)、规律成簇间隔短回文重复(CRISPR)/Cas系统。从它们的发现、结构和研究进展及应用前景等方面进行了总结;通过比较三者的优缺点,发现规律成簇间隔短回文重复(CRISPRs)具有明显的优点。     

基于CRISPR/Cas系统的多重基因编辑与调控技术

规律成簇的间隔短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)及其相关Cas蛋白所构建的CRISPR/Cas系统是古细菌或细菌中特有的一种获得性免疫系统。研究人员将其开发成基因编辑工具之后,凭借其高效、精准和通用性强等优点迅速成为合成生物学领域的热门研究方向,在生命科学、生物工程技术、食品科学及农作物育种等多个领域引发了革命性的影响。目前基于CRISPR/Cas系统单基因编辑与调控技术日益完善,但在多重基因编辑和调控方面仍存在挑战。本文聚焦基于CRISPR/Cas系统的多重基因编辑与调控技术开发及应用,针对单个细胞内实现多位点基因编辑或调控和细胞群体内实现多位点基因编辑或调控技术,依据作用原理对其进行了系统总结和阐述,包括基于CRISPR/Cas系统的双链断裂、单链断裂以及多重基因调控技术等。这些工作丰富了多重基因编辑与调控的工具,为CRISPR/Cas系统在多领域的应用作出了贡献。     

Use of designer nucleases for targeted gene and genome editing in plants

The ability to efficiently inactivate or replace genes in model organisms allowed a rapid expansion of our understanding of many of the genetic, biochemical, molecular and cellular mechanisms that support life. With the advent of new techniques for manipulating genes and genomes that are applicable not only to single‐celled organisms, but also to more complex organisms such as animals and plants, the speed with which scientists and biotechnologists can expand fundamental knowledge and apply that knowledge to improvements in medicine, industry and agriculture is set to expand in an exponential fashion. At the heart of these advancements will be the use of gene editing tools such as zinc finger nucleases, modified meganucleases, hybrid DNA/RNA oligonucleotides, TAL effector nucleases and modified CRISPR/Cas9. Each of these tools has the ability to precisely target one specific DNA sequence within a genome and (except for DNA/RNA oligonucleotides) to create a double‐stranded DNA break. DNA repair to such breaks sometimes leads to gene knockouts or gene replacement by homologous recombination if exogenously supplied homologous DNA fragments are made available. Genome rearrangements are also possible to engineer. Creation and use of such genome rearrangements, gene knockouts and gene replacements by the plant science community is gaining significant momentum. To document some of this progress and to explore the technology's longer term potential, this review highlights present and future uses of designer nucleases to greatly expedite research with model plant systems and to engineer genes and genomes in major and minor crop species for enhanced food production.     

CRISPR/Cas基因编辑技术研究进展及其在斑马鱼中的应用

规律成簇间隔的短回文序列(Clustered regularly interspaced short palindromic repeats,CRISPR)是细菌和古菌中的获得性免疫系统,利用该系统能定点进行基因编辑。最近,科学家发现了新的CRISPR-associated (Cas)蛋白,其中由Cas12a介导的基因编辑能显著降低脱靶率。文中对CRISPR/Cas系统的发现历史、组成和分类、工作原理进行概述,并总结了该系统的最新研究进展及在斑马鱼Danio rerio中的应用。     

CRISPR/Cas9基因组编辑技术在癌症研究中的应用

CRISPR/cas9基因组编辑技术因其设计简单以及操作容易,使其在基因编辑的研究中越来越受到欢迎。利用该技术,科研人员可以实现在碱基的水平对基因组进行定点修饰。CRISPR系统现已经被广泛地应用到多个物种的基因组编辑以及癌症的相关研究中。本文在最新研究进展的基础上,结合对癌症研究及基因组编辑技术的理解,对CRISPR/Cas9技术在癌症研究中的应用进行了综述。     

毕赤酵母基因编辑技术研究进展

巴斯德毕赤酵母是一种重要的蛋白表达系统,基因编辑技术作为代谢工程的基本工具,对于毕赤酵母的代谢改造十分重要。近十年基因编辑技术发展迅速,除传统的同源重组和Cre/loxP重组外,相继出现了许多新的基因编辑技术,例如ZFN、TALEN和CRISPR/Cas9等,这些技术的出现使基因编辑更加简便高效。本文对毕赤酵母中传统和新型基因编辑技术的原理应用和研究进展进行了简要综述,并结合相关领域的发展对毕赤酵母基因编辑技术的发展进行了展望。     

CRISPR/Cas基因编辑系统研究进展

规律性成簇间隔的短回文重复序列(clustered regularly interspaced short palindromic repeats, CRISPR)的发现和工程技术对生命科学的发展带来巨大的推动作用。RNA引导的Cas(CRISPR-associated)酶已被用作操纵细胞、动物和植物基因组的工具。这加速了基础研究的步伐,并使其在临床和农业上的应用成为可能。CRISPR/Cas9对在实验系统中进行的功能基因组学的研究有重大影响。CRISPR/Cas9系统自发现以来,因其操作便捷、成本低、特异性高、可同时打靶任意数量基因等优点而被广泛应用。经过近几年研究发现,Cas9变异体(Cas12a、Cas13)有利于突破和克服CRISPR/Cas9应用中的一些限制,Cas12a极大地扩展了基因编辑靶位点的选择范围,同时其介导的多基因编辑具有明显的优势;Cas13等蛋白能特异性结合和编辑RNA,开启了转录组研究的新篇章。本文主要就CRISPR/Cas的研究背景以及Cas9、Cas12a和Cas13系统研究进展和应用进行综述,并对其应用前景和发展方向进行了展望。     

Efficient gene editing in adult mouse livers via adenoviral delivery of CRISPR/Cas9

We developed an adenovirus-based CRISPR/Cas9 system for gene editing in vivo. In the liver, we demonstrated that the system could reach the level of tissue-specific gene knockout, resulting in phenotypic changes. Given the wide spectrum of cell types susceptible to adenoviral infection, and the fact that adenoviral genome rarely integrates into its host cell genome, we believe the adenovirus-based CRISPR/Cas9 system will find applications in a variety of experimental settings.     

Updates on CRISPR-based gene editing in HIV-1/AIDS therapy

Although tremendous efforts have been made to prevent and treat HIV-1 infection, HIV-1/AIDS remains a major threat to global human health. The combination antiretroviral therapy (cART), although able to suppress HIV-1 replication, cannot eliminate the proviral DNA integrated into the human genome and thus requires lifelong treatment that may lead to various side effects. In recent years, clustered regularly interspaced short palindromic repeat (CRISPR)-associated nuclease 9 (Cas9) related gene-editing systems have been developed and designed as effective ways to treat HIV-1 infection. However, new gene-targeting tools derived from or functioning like CRISPR/Cas9, including base editor, prime editing, SHERLOCK, DETECTR, PAC-MAN, ABACAS, pfAGO, have been developed and optimized for pathogens detection and diseases correction. Here, we summarize recent studies on HIV-1/AIDS gene therapy and provide more gene-editing targets based on studies relating to the molecular mechanism of HIV-1 infection. We also identify the strategies and potential applications of these new gene-editing technologies for HIV-1/AIDS treatment in the future. Moreover, we discuss the caveats and problems that should be addressed before the clinical use of these versatile CRISPR-based gene targeting tools. Finally, we offer alternative solutions to improve the practice of gene targeting in HIV-1/AIDS gene therapy.     

The CRISPR/Cas9 system produces specific and homozygous targeted gene editing in rice in one generation

The CRISPR/Cas9 system has been demonstrated to efficiently induce targeted gene editing in a variety of organisms including plants. Recent work showed that CRISPR/Cas9‐induced gene mutations in Arabidopsis were mostly somatic mutations in the early generation, although some mutations could be stably inherited in later generations. However, it remains unclear whether this system will work similarly in crops such as rice. In this study, we tested in two rice subspecies 11 target genes for their amenability to CRISPR/Cas9‐induced editing and determined the patterns, specificity and heritability of the gene modifications. Analysis of the genotypes and frequency of edited genes in the first generation of transformed plants (T0) showed that the CRISPR/Cas9 system was highly efficient in rice, with target genes edited in nearly half of the transformed embryogenic cells before their first cell division. Homozygotes of edited target genes were readily found in T0 plants. The gene mutations were passed to the next generation (T1) following classic Mendelian law, without any detectable new mutation or reversion. Even with extensive searches including whole genome resequencing, we could not find any evidence of large‐scale off‐targeting in rice for any of the many targets tested in this study. By specifically sequencing the putative off‐target sites of a large number of T0 plants, low‐frequency mutations were found in only one off‐target site where the sequence had 1‐bp difference from the intended target. Overall, the data in this study point to the CRISPR/Cas9 system being a powerful tool in crop genome engineering.