CRISPR/Cas9系统在传染病诊疗中的应用进展北大核心CSCD

CRISPR/Cas系统是一种源自细菌和古细菌的适应性免疫系统,经人工改造后发展成为一种强大的基因编辑工具,使基础科学研究发生了革命性的变化。其中,CRISPR/Cas9系统是研究最为深入、发展最快的一种。随着CRISPR/Cas9系统的不断发展,越来越多关于CRISPR/Cas9系统的疗法进入临床试验。本文简要介绍CRISPR/Cas9系统的结构和作用机制,并对其在传染病诊疗中的应用进行综述。     

CRISPR/Cas9介导下高效敲除SQSTM1/p62基因的Hela细胞模型的建立

目的 :探讨针对p62双sgRNA向导CRISPR/Cas9基因编辑效率。方法 :采用CRISPR/Cas9基因编辑技术成功敲除p62基因,通过细胞有限稀释法和嘌呤霉素筛选,免疫印迹法验证双sgRNA和单sgRNA向导的基因编辑成功率;流式细胞仪验证p62缺失对Hela细胞凋亡的影响。结果 :免疫印迹分析得出双sgRNA导向的p62敲除效率高于单sgRNA导向的敲除,靶序列测序比对分析确认p62编码基因发生大片段缺失突变。H_2O_2处理稳定敲除的Hela细胞系显示,p62基因敲除能明显抑制Hela细胞H_2O_2诱导的早期细胞凋亡。结论 :成功建立了p62敲除的Hela细胞系,双sgRNA向导的CRISPR/Cas9基因编辑体系可能是一种更有效的编辑工具。     

光控CRISPR基因编辑技术的研究进展

自CRISPR/Cas9问世以来, 基因编辑技术愈发受到关注, 尤其是核酸酶仅由单一蛋白组成的Ⅱ类系统, 其功能蛋白包括Ⅱ型Cas9、Ⅴ型Cas12和Ⅵ型Cas13等, 可以精准编辑基因组DNA或RNA。失活型CRISPR/Cas9突变体还能够将转录因子、表观遗传因子或碱基修饰酶等效应元件特异性地募集至目标基因位点发挥功能。此外, 光遗传学技术能够从时间和空间水平精确地控制生物反应。结合CRISPR和光遗传学技术可实现细胞和动物体内的动态化时空特异性基因编辑, 在生物学和医学领域发挥巨大的应用潜力。本文综述了光控CRISPR系统的研究进展, 介绍其设计方法和应用策略, 讨论其局限性, 为这一新兴领域的发展提供参考。     

应用CRISPR/Cas9系统构建敲除APE1基因的AC16心肌细胞株

目的应用CRISPR/Cas9系统构建敲除APE1基因的AC16心肌细胞株,为研究APE1在心肌细胞的功能提供研究基础。方法根据RISPR/Cas9靶向原理设计人APE1基因的导向RNA(sgRNA),构建sgRNA-LentiCRISPRV2重组质粒并转入293T细胞制备sgRNA-Cas9慢病毒;该病毒浸染AC16心肌细胞,嘌呤霉素筛选出阳性细胞并稀释至单克隆。免疫印迹法测定单克隆细胞中APE1蛋白表达。结果免疫印迹法检测的结果显示,筛选出的单克隆细胞的APE1蛋白表达完全缺失;PCR产物测序结果表明,靶向敲除APE1的心肌细胞,不存在sgRNA介导CRISPR-Cas9随机的核苷酸插入。结论应用CRISPR/Cas9系统成功构建了敲除APE1基因的AC16心肌细胞株。     

CRISPR/Cas9技术在宫颈癌治疗中的应用进展

宫颈癌是世界上女性癌症排名第4位的恶性肿瘤, 严重威胁女性的健康。宫颈癌患者的主要治疗方案为手术或同步放化疗, 随着医学研究的发展, 研究者们致力于探究更为有效、特异的治疗方案, 以期增加宫颈癌的治疗策略和提高治疗效果。簇状规则间隔短回文重复序列(CRISPR)/CRISPR相关蛋白9(Cas9)技术是Cas9蛋白利用向导RNA(gRNA)的引导进而靶向目标基因, 实现对目标基因精准编辑的方法。目前, CRISPR/Cas9技术已成为一种很有前途的强大基因编辑工具, 是一种新的有效的靶向治疗方法, 且被应用于多种肿瘤的治疗中。主要从作用靶点、联合治疗策略以及相关耐药基因筛选等方面对CRISPR/Cas9技术在宫颈癌治疗中的研究进展进行综述, 以期为宫颈癌的治疗提供新的策略。     

CRISPR-Cas9研究及应用进展

规律成簇间隔短回文重复（ clustered regularly interspaced short palindromic repeats , CRISPR ）是细菌的一种适应性免疫防御机制,与 CRISPR 相关（ CRISPR associated , Cas ）蛋白共同形成CRISPR-Cas 系统,抵御外源DNA 入侵。Ⅱ型CRISPR-Cas ,又叫CRISPR-Cas 9,因其简单的结构与靶向编辑 DNA 的性质,目前被用于开发针对单基因疾病、病毒性疾病、肿瘤等疾病的新型治疗模式研究中。该文简述了 CRISPR-Cas 9的作用机制以及其在疾病研究中的应用。     

靶向猪基因组单链向导RNA快速筛选以及利用图案微阵列收获单克隆细胞的研究

规律性短重复回文序列簇(CRISPR)和CRISPR辅助蛋白9(Cas9)构成的CRISPR/Cas9基因编辑技术快速推进了基因修饰猪作为医学研究模式动物的广泛应用。而高效的靶基因单链向导RNA(sgRNA)是利用CRISPR/Cas9技术进行基因编辑成功的关键,对于猪等繁殖周期较长的大动物,则需要在实施动物实验前,在体外筛选出高效的sgRNA以避免时间和资源成本浪费。另外,如何高效获得阳性基因编辑单克隆细胞是目前尚待解决的难题。本研究建立了靶向猪基因组的sgRNA快速筛选方法,利用荧光载体富集基因编辑细胞,同时探索利用图案微阵列培养技术快速获得单克隆细胞的方法,在此基础上高效获得延胡索酰乙酰乙酸酶(Fah)基因编辑细胞,为后续生产作为人类肝细胞生物反应器的Fah基因敲除猪奠定基础。     

基于CRISPR/Cas9系统的HEK293T细胞DMD基因第51号外显子的靶向敲除

目的应用CRISPR/Cas9基因编辑技术,完成HEK293T细胞中DMD基因第51号外显子(exon51)高效的靶向敲除。方法设计靶向人DMD基因exon51 5'端及3'端的sgRNA并克隆至CRISPR/Cas9载体质粒PX459中,转染至HEK293T细胞后,提取基因组DNA并使用Surveyor法检测切割活性;使用目标外显子两端切割活性最高的sgRNA构建PX459-2sgRNA质粒,转染至HEK293T细胞后用PCR及T载体测序检测靶向外显子切除情况。结果50%的HEK293T细胞中DMD基因exon51被定向切除,编辑效率较高。结论建立使用CRISPR/Cas9单质粒敲除人DMD基因exon51的平台,为DMD及其他遗传病的基因治疗研究奠定实验基础。     

CRISPR/Cas9在免疫细胞基因编辑中的应用

CRISPR/Cas系统是最近几年才被发现的,它是基于RNA来控制细菌与古细菌中病毒与质粒的入侵.在有CRISPR/Cas免疫系统的原核生物基因中发现了短的重复序列和来自于CRISPR基因座的小RNA,它能引导Cas9蛋白去识别与降解入侵的核苷酸序列.目前,CRISPR/Cas9系统已迅速革新基因工程这片领域,让研究者可以相对轻松地改变多种生物的基因组,而且在免疫系统中能通过编辑免疫细胞达到缓解疾病的程度.     

成簇的规律间隔的短回文重复序列(CRISPR)介导的基因组编辑技术研究进展

成簇的规律间隔的短回文重复序列(CRISPR)介导的基因组编辑技术已成为生物医学领域中研究基因功能及其表达调控的重要手段之一。随着CRISPR技术的拓展,各种基于CRISPR技术的新型研究、诊断和治疗等工具得到进一步的开发,并在新型动物疾病模型的建立、疾病快速诊断、肿瘤及免疫相关性疾病的治疗等方面取得了重大进展和突破。我们重点对CRISPR/CRISPR相关蛋白(Cas)技术的发展现状及其在生物医学领域的应用进展进行综述,并对其未来应用前景和发展方向进行讨论和展望。     

Application of CRISPR/Cas9 gene editing technique in the study of cancer treatment

In recent years, gene editing, especially that using clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9, has made great progress in the field of gene function. Rapid development of gene editing techniques has contributed to their significance in the field of medicine. Because the CRISPR/Cas9 gene editing tool is not only powerful but also has features such as strong specificity and high efficiency, it can accurately and rapidly screen the whole genome, facilitating the administration of gene therapy for specific diseases. In the field of tumor research, CRISPR/Cas9 can be used to edit genomes to explore the mechanisms of tumor occurrence, development, and metastasis. In these years, this system has been increasingly applied in tumor treatment research. CRISPR/Cas9 can be used to treat tumors by repairing mutations or knocking out specific genes. To date, numerous preliminary studies have been conducted on tumor treatment in related fields. CRISPR/Cas9 holds great promise for gene-level tumor treatment. Personalized and targeted therapy based on CRISPR/Cas9 will possibly shape the development of tumor therapy in the future. In this study, we review the findings of CRISPR/Cas9 for tumor treatment research to provide references for related future studies on the pathogenesis and clinical treatment of tumors.     

CRISPR/Cas9 and CAR-T cell,collaboration of two revolutionary technologies in cancer immunotherapy,an instruction for successful cancer treatment

Clustered regularly interspaced short palindromic repeats/CRISPR associated nuclease9 (CRISPR/Cas9) technology, an acquired immune system in bacteria and archaea, has provided a new tool for accurately genome editing. Using only a single nuclease protein in complex with 2 short RNA as a site-specific endonuclease made it a simple and flexible genome editing tool to target nearly any genomic locus. Due to recent developments in therapeutic engineered T cell and effective responses of CD19-directed chimeric antigen receptor T cells (CART19) in patients with B-cell leukemia and lymphoma, adoptive T cell immunotherapy, particularly CAR-T cell therapy became a rapidly growing field in cancer therapy and recently Kymriah and Yescarta (CD19-directed CAR-T cells) were approved by FDA. Therefore, the combination of CRISPR/Cas9 technology as a genome engineering tool and CAR-T cell therapy (engineered T cells that express chimeric antigen receptors) may lead to further improvement in efficiency and safety of CAR-T cells. This article reviews mechanism and therapeutic application of CRISPR/Cas9 technology, accuracy of this technology, cancer immunotherapy by CAR T cells, the application of CRISPR technology for the production of universal CAR T cells, improving their antitumor efficacy, and biotech companies that invested in CRISPR technology for CAR-T cell therapy.     

The landscape of CRISPR/Cas9 for inborn errors of metabolism

Since its discovery as a genome editing tool, the clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9) system has opened new horizons in the diagnosis, research, and treatment of genetic diseases. CRISPR/Cas9 can rewrite the genome at any region with outstanding precision to modify it and further instructions for gene expression. Inborn Errors of Metabolism (IEM) are a group of more than 1500 diseases produced by mutations in genes encoding for proteins that participate in metabolic pathways. IEM involves small molecules, energetic deficits, or complex molecules diseases, which may be susceptible to be treated with this novel tool. In recent years, potential therapeutic approaches have been attempted, and new models have been developed using CRISPR/Cas9. In this review, we summarize the most relevant findings in the scientific literature about the implementation of CRISPR/Cas9 in IEM and discuss the future use of CRISPR/Cas9 to modify epigenetic markers, which seem to play a critical role in the context of IEM. The current delivery strategies of CRISPR/Cas9 are also discussed.     

The Generation of Zebrafish Mariner Model Using the CRISPR/Cas9 System

Targeted genome editing mediated by clustered, regularly interspaced, short palindromic repeat (CRISPR)/CRISPR-associated nuclease 9 (Cas9) technology has emerged as a powerful tool for gene function studies and has great potential for gene therapy. Although CRISPR/Cas9 has been widely used in many research fields, only a few successful zebrafish models have been established using this technology in hearing research. In this study, we successfully created zebrafish mariner mutants by targeting the motor head domain of Myo7aa using CRISPR/Cas9. The CRISPR/Cas9-generated mutants showed unbalanced swimming behavior and disorganized sterocilia of inner ear hair cells, which resemble the phenotype of the zebrafish mariner mutants. In addition, we found that CRISPR/Cas9-generated mutants have reduced number of stereociliary bundles of inner ear hair cells and have significant hearing loss. Furthermore, phenotypic analysis was performed on F0 larvae within the first week post fertilization, which dramatically shortens data collection period. Therefore, results of this study showed that CRISPR/Cas9 is a quick and effective method to generate zebrafish mutants as a model for studying human genetic deafness. Anat Rec, 303:556–562, 2020. © 2019 American Association for Anatomy     

Critical cancer vulnerabilities identified by unbiased CRISPR/Cas9 screens inform on efficient cancer Immunotherapy

The mutational landscape of human cancers is highly complex. While next generation sequencing aims to comprehensively catalogue somatic alterations in tumor cells, it fails to delineate driver from passenger mutations. Functional genomic approaches, particularly CRISPR/Cas9, enable both gene discovery, and annotation of gene function. Indeed, recent CRISPR/Cas9 technologies have flourished with the development of more sophisticated and versatile platforms capable of gene knockouts to high throughput genome wide editing of a single nucleotide base. With new platforms constantly emerging, it can be challenging to navigate what CRISPR tools are available and how they can be effectively applied to understand cancer biology. This review provides an overview of current and emerging CRISPR technologies and their power to model cancer and identify novel treatments. Specifically, how CRISPR screening approaches have been exploited to enhance immunotherapies through the identification of tumor intrinsic and extrinsic mechanisms to escape immune recognition will be discussed.     

Developmental progress of CRISPR/Cas9 and its therapeutic applications for HIV‐1 infection

The CRISPR/Cas9 system has been developed as a powerful tool for targeted gene editing. As a result of technical enhancements in recent years, this technology has become the method of choice for efficiently modifying targeted HIV‐1 genome efficiently as part of HIV therapy. CRISPR can be modified to target specific sequences that Cas9 then cuts. In this article, we outline the development of the CRISPR/Cas9 system. We also show how this technology can be used for the prevention and treatment of HIV‐1 infection. Optimistically, this technology promises to make a significant impact on the fight against HIV‐1 in the future.     

CRISPR系统及其在遗传病治疗中应用的研究进展

CRISPR(clustered regularly interspaced short palindromic repeats)系统是细菌和古生菌用于抵抗外源性病毒和质粒入侵的适应性免疫防御系统,近年来遗传工程迅猛发展,CRISPR系统中的Cas9、Cpf1等多种蛋白已被改造为基因组编辑的关键工具.对于由遗传物质发生改变而引起的或者由致病基因突变所控制的遗传病,CRISPR系统已被证实是编辑修正致病基因的有力手段,并有望成为人类遗传病治疗的新策略.本文主要从CRISPR系统的研究进展及其在遗传病治疗中的应用进行综述.     

Single‐nucleotide editing: From principle,optimization to application

Cytosine base editors (CBEs) and adenine base editors (ABEs), which are generally composed of an engineered deaminase and a catalytically impaired CRISPR‐Cas9 variant, are new favorite tools for single base substitution in cells and organisms. In this review, we summarize the principle of base‐editing systems and elaborate on the evolution of different platforms of CBEs and ABEs, including their deaminase, Cas9 variants, and editing outcomes. Moreover, we highlight their applications in mouse and human cells and discuss the challenges and prospects of base editors. The ABE‐ and CBE systems have been used in gene silencing, pathogenic gene correction, and functional genetic screening. Single base editing is becoming a new promising genetic tool in biomedical research and gene therapy.