CRISPR/Cas9基因编辑技术在蚊媒研究中的应用

蚊虫不仅吸血骚扰,而且传播多种疾病,迄今依然是世界性的严重的公共卫生问题。近年来,新型虫媒病不断出现,传统的虫媒病死灰复燃,给蚊媒控制带来了新的挑战。随着基因编辑技术的出现与发展,特别是成簇的规律间隔短回文重复序列系统(Clustered regularly interspaced palindromic repeats-CRISPR associated sequences 9, CRISPR/Cas9)的出现,为开展蚊虫生理、生化、发育、宿主与病原体关系等诸多方面分子生物基础研究提供了靶标特异性的修饰工具,给蚊媒控制技术的发展带来了新的契机。本文主要针对CRISPR/Cas9技术在蚊媒研究领域的应用现状及进展进行综述,并探讨CRISPR/Cas9技术在蚊媒传染病防治的实际应用中所面临的问题,为蚊媒防治措施的应用及改进提供理论参考。     

CRISPR/Cas9在人类疾病治疗中的研究进展

成簇规律间隔短回文重复序列（clustered regularly interspaced short palindromic repeats,CRISPR）/ CRISPR相关蛋白9（CRISPR-associated protein 9,Cas9）是利用向导RNA（guide RNA,gRNA）引导Cas9蛋白对靶...     

CRISPR/Cas9基因编辑技术在心血管领域中的研究进展

CRISPR/Cas9基因编辑技术以其独特的优势已经在生物医学领域中被广泛应用。利用该技术在人类心血管疾病的相关研究如:心肌细胞水平、先天性心脏病动物模型建立、冠心病高危因素、心律失常、心力衰竭和心肌病等方面已经取得了很大进步,但目前的基因编辑技术在实际运用中仍然面临着许多问题。     

CRISPR / Cas9 基因编辑技术在肿瘤免疫治疗中的应用

近年来肿瘤免疫治疗因其显著疗效和创新性备受关注,是肿瘤治疗中最具有前景的研究方向之一。CRISPR基因编辑系统的发展以及其在多领域的延伸应用让研究者看到了它的巨大潜力,在肿瘤免疫治疗研究中也显示出广泛的应用前景。在过继性细胞治疗中,CRISPR/ Cas9 可对TCR-T 和CAR-T 细胞的内源性TCR 以及HLA-Ⅰ分子的基因进行敲除得到通用型效应细胞;在基于免疫检查点阻断的治疗中,其为免疫检查点的抑制和阻断提供了一种新的方法;同时在抗体靶向疗法中,CRISPR/ Cas9 技术在候选靶点筛选及简化抗体制备流程中有着重要应用。CRISPR 从各方面极大地推动了肿瘤免疫治疗的研究,本文将对CRISPR/ Cas9 基因编辑系统在肿瘤免疫治疗方面的应用进行介绍。     

基于CRISPR/Cas9系统高效编辑小鼠miR let-7a

目的 构建靶向小鼠miR let-7a的高效CRISPR/Cas9系统。方法 根据小鼠miR let-7a的两个编码位点let-7a-1和let-7a-2的编码序列各设计了两对dual-sgRNAs导向序列,通过sgRNA表达载体的构建、小鼠N2a细胞的共转染、药物筛选、阳性细胞PCR产物测序以及TA克隆测序分析等实验步骤完成了4对dual-sgRNAs导向序列的打靶效力分析。结果 所构建的4对打靶载体在细胞中发挥了作用,打靶位点均出现了碱基插入或缺失,药物筛选阳性细胞打靶位点RCR产物Sanger法测序峰图在4个靶点位置均有套峰出现。TA克隆测序结果表明本研究设计的4对dual-sgRNAs导向序列的打靶效率分别为42.10%、62.50%、52.63%和47.37%。结论 本研究所设计的4对dual-sgRNAs导向序列均可以有效介导Cas9蛋白切割小鼠miR let-7a,形成突变效应。为后续miR let-7a作用靶点的深入挖掘,阐明其发挥抑癌功能的作用机制提供了依据。     

CRISPR/Cas9技术研究进展

CRISPR/Cas9系统可以很容易的改写多种生物体的基因组,这一技术很快席卷了整个基因组工程领域.在此基础上的实验方法在各个领域都具有相当大的潜力.因而了解以CRISPR/Cas9为基础的基因功能研究,探讨这一技术开发新一代疾病模型的各种途径,疾病治疗以及基因调控等方面的研究进展很有必要.     

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。     

利用CRISPR/Cas9基因编辑靶向下调FGFR2缓解Apert综合征小鼠颅缝早闭

Apert综合征 (Apert Syndrome, AS) 是一类常染色体显性遗传疾病,主要表现为颅缝早闭、中面部发育不良、 并指 (趾)、智力发育障碍等,多由FGFR2分子功能增强型点突变引起。目前,此疾病主要靠手术矫形来恢复功能,其中大脑等病变部位手术难度大,难以实施,且通常需要多次手术。CRISPR/Cas9基因编辑技术在人类遗传病治疗中具有巨大潜力。本研究筛选到靶向FGFR2基因的gRNA,与Cas9经慢病毒包装后作用于Apert小鼠 (FGFR2^+/P253R) 的原代颅骨细胞、体外培养的颅骨以及活体小鼠颅骨中,可敲低FGFR2的表达,改善Apert原代颅骨细胞的分化程度。CRISPR/Cas9处理体外培养的颅骨及注射至 Apert 小鼠头颅,颅骨冠状缝早闭状态有效缓解,异常降低的颅骨骨量、骨密度等也显著改善,表明 CRISPR/Cas9基因编辑可缓解 Apert小鼠头颅异常。本研究为 Apert综合征的基因治疗提供了实验依据,有望为其它骨骼遗传病治疗提供新的策略与借鉴。     

CRISPR/Cas9基因编辑技术敲除HuT-78细胞PD-1分子对其增殖能力的影响

利用CRISPR/Cas9技术成功敲除人T细胞系HuT-78细胞中编码PD-1分子的基因PDCD1,并比较敲除了PDCD1基因的及对照组的HuT-78细胞增殖能力的差异。实验结果显示敲除PDCD1基因的HuT-78细胞的增殖能力不受PD-L1的影响,为将此技术应用于肿瘤免疫治疗提供了实验依据。     

The implication of CRISPR/Cas9 genome editing technology in combating human oncoviruses

It is evidenced that 20% of all tumors in humans are caused by oncoviruses, including human papilloma viruses, Epstein-Barr virus, Kaposi sarcoma virus, human polyomaviruses, human T-lymphotrophic virus-1, and hepatitis B and C viruses. Human immunodeficiency virus is also involved in carcinogenesis, although not directly, but by facilitating the infection of many oncoviruses through compromising the immune system. Being intracellular parasites with the property of establishing latency and integrating into the host genome, these viruses are a therapeutic challenge for biomedical researchers. Therefore, strategies able to target nucleotide sequences within episomal or integrated viral genomes are of prime importance in antiviral or anticancerous armamentarium. Recently, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) has emerged as a powerful genome editing tool. Standing out as a precise and efficient oncoviruses method, it has been extensively applied in recent experimental ventures in the field of molecular medicine, particularly in combating infections including tumor inducing viruses. This review is aimed at collating the experimental and clinical advances in CRISPR/Cas9 technology in terms of its applications against oncoviruses. Primarily, it will focus on the application of CRISPR/Cas9 in combating tumor viruses, types of mechanisms targeted, and the significant outcomes till date. The technical pitfalls of the CRISPR/Cas9 and the comparative approaches in evaluating this technique with respect to other available alternatives are also described briefly. Furthermore, the review also discussed the clinical aspects and the ethical, legal, and social issues associated with the use of CRISPR/Cas9.     

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.     

利用CRISPR/Cas9系统构建水通道蛋白9基因敲除小鼠

目的 利用CRISPR/Cas9系统敲除小鼠水通道蛋白9(AQP-9)基因,构建稳定敲除AQP-9基因的纯合子AQP-9-/-小鼠.方法 根据CRISPR/Cas9靶点设计原则,在Ensembl数据库上找到AQP-9基因序列的外显子区域,综合分析选定AQP-9-202的公共外显子2,前期在其两边设计7个小向导RNA (...     

Harnessing CRISPR/Cas 9 System for manipulation of DNA virus genome

The recent development of the Clustered Regularly Interspaced Palindromic Repeat (CRISPR)/CRISPR‐associated protein 9 (Cas9) system, a genome editing system, has many potential applications in virology. The possibility of introducing site specific breaks has provided new possibilities to precisely manipulate viral genomics. Here, we provide diagrams to summarize the steps involved in the process. We also systematically review recent applications of the CRISPR/Cas9 system for manipulation of DNA virus genomics and discuss the therapeutic potential of the system to treat viral diseases.     

Pancreatic cancer modeling using retrograde viral vector delivery and in vivo CRISPR/Cas9-mediated somatic genome editing

Pancreatic ductal adenocarcinoma (PDAC) is a genomically diverse, prevalent, and almost invariably fatal malignancy. Although conventional genetically engineered mouse models of human PDAC have been instrumental in understanding pancreatic cancer development, these models are much too labor-intensive, expensive, and slow to perform the extensive molecular analyses needed to adequately understand this disease. Here we demonstrate that retrograde pancreatic ductal injection of either adenoviral-Cre or lentiviral-Cre vectors allows titratable initiation of pancreatic neoplasias that progress into invasive and metastatic PDAC. To enable in vivo CRISPR/Cas9-mediated gene inactivation in the pancreas, we generated a Cre-regulated Cas9 allele and lentiviral vectors that express Cre and a single-guide RNA. CRISPR-mediated targeting of Lkb1 in combination with oncogenic Kras expression led to selection for inactivating genomic alterations, absence of Lkb1 protein, and rapid tumor growth that phenocopied Cre-mediated genetic deletion of Lkb1. This method will transform our ability to rapidly interrogate gene function during the development of this recalcitrant cancer.     

Repurposing CRISPR/Cas9 for in situ functional assays

RNAi combined with next-generation sequencing has proven to be a powerful and cost-effective genetic screening platform in mammalian cells. Still, this technology has its limitations and is incompatible with in situ mutagenesis screens on a genome-wide scale. Using p53 as a proof-of-principle target, we readapted the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 (CRISPR associated 9) genome-editing system to demonstrate the feasibility of this methodology for targeted gene disruption positive selection assays. By using novel “all-in-one” lentiviral and retroviral delivery vectors heterologously expressing both a codon-optimized Cas9 and its synthetic guide RNA (sgRNA), we show robust selection for the CRISPR-modified Trp53 locus following drug treatment. Furthermore, by linking Cas9 expression to GFP fluorescence, we use an “all-in-one” system to track disrupted Trp53 in chemoresistant lymphomas in the Eμ-myc mouse model. Deep sequencing analysis of the tumor-derived endogenous Cas9-modified Trp53 locus revealed a wide spectrum of mutants that were enriched with seemingly limited off-target effects. Taken together, these results establish Cas9 genome editing as a powerful and practical approach for positive in situ genetic screens.