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吕宝北赵鹏翔张鑫马雪梅谢飞 《生物技术进展》2018,8(3):191-196
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。 相似文献
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成簇规律间隔短回文重复(clustered regularly interspaced short palindromic repeats, CRISPR),是细菌或古菌在与噬菌体长期生存进化获得的一种免疫系统. 根据Cas蛋白(CRISPR-associated protein)的不同,CRISPR系统可分为3种. 其中II型CRISPR/Cas9已被改造成为一种有效的基因编辑工具,并运用于多种物种基因的改造. 作为1种基因编辑的手段,CRISPR/Cas9技术通过诱导DNA双链断裂损伤,进一步干扰基因的表达. 与传统的基因编辑技术相比,CRISPR/Cas9技术显示出效率高、成本低和易操作等特点. 与此同时,二代测序技术的发展促进全基因组的解析. CRISPR技术结合高通量二代测序手段的使用,在肿瘤的治疗领域中已发挥出了独特的优势. 本文就近年来CRISPR/Cas9高通量筛选技术的发展,及其在肿瘤治疗过程中的应用进行综述. 相似文献
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CRISPR/Cas9核酸酶作为一种新的基因组靶向编辑技术,已成功应用于多种动植物基因组修饰研究. CRISPR/Cas9作用后的阳性细胞筛选和富集是该技术的关键之一. 本研究以鸡EAV-HP(endogenous avian retrovirus-HP)基因和MSTN(myostatin)基因为例,从靶位点的选择、表达载体构建、双基因报告载体构建和核酸酶活性验证4个方面,系统研究了CRISPR/Cas9核酸酶技术平台. 结果表明,利用寡聚核苷酸直接退火方法,构建表达载体和报告载体的阳性率分别高达100%和89.5%. 报告载体的PuroR(puromycin resistant gene)和eGFP(enhanced green fluorescent protein)基因的成功表达表明,构建的CRISPR/Cas9系统能有效切割靶序列,并用于后续阳性克隆的筛选和富集. 本方法摒弃了传统分子克隆的PCR扩增和酶切处理目标基因的方法,而是利用寡聚核苷酸直接退火获得含有黏性末端的目标DNA,简化了载体构建过程,低成本且快速获得CRISPR/Cas9基因组靶向编辑系统. 相似文献
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基于CRISPR/Cas9系统介导的第三代基因组定点编辑技术,已被广泛应用于基因编辑和基因表达调控等研究领域。如何提高该技术对基因组编辑的效率与特异性、最大限度降低脱靶风险一直是该领域的难点。近年来,机器学习为解决CRISPR/Cas9系统所面临的问题提供了新思路,基于机器学习的CRISPR/Cas9系统已逐渐成为研究热点。本文阐述了CRISPR/Cas9的作用机理,总结了现阶段该技术面临的基因组编辑效率低、存在潜在的脱靶效应、前间区序列邻近基序(PAM)限制识别序列等问题,最后对机器学习应用于优化设计高效向导RNA (sgRNA)序列、预测sgRNA的活性、脱靶效应评估、基因敲除、高通量功能基因筛选等领域的研究现状与发展前景进行了展望,以期为基因组编辑领域的研究提供参考。 相似文献
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CRISPR/Cas9基因编辑技术已成为基因治疗领域最有前景的工具。在临床应用中,对CRISPR/Cas9进行安全有效的递送一直是亟待解决的问题。纳米粒子,如脂基纳米粒子、聚合物纳米粒子、纳米金颗粒以及生物膜类纳米粒子等,因其生物相容性、安全性和可设计性等特点有望为基因治疗带来新的突破。文中首先对纳米粒子的特性和基因治疗中CRISPR/Cas9的发展进行了概述,然后详细归纳了纳米粒子在递送不同形式的CRISPR/Cas9中的应用,最后对纳米粒子介导的基因治疗的递送在未来面临的挑战和安全性等方面作出总结论述。 相似文献
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从喂养方式、喂养过程、营养成分、护理重点等多方面综述新生儿短肠综合征肠内营养的研究进展,并结合患儿的实际病情以及实验室检查结果等指标,提出对患儿进行持续性的肠内营养支持的具体做法:(1)在现实情况允许的条件下,保证用母乳喂养患儿,无法提供母乳的情况下合理配比奶粉,并根据实际需要添加一些纤维和脂类的补充剂,保障患儿健康发育;(2)在给予肠内营养的过程中全程无菌化处理,调节适宜的温度,合理选择药物;(3)应用大规模对照试验方式,通过大数据对比总结出持续肠内营养对短肠综合征患儿的影响,为儿科护理工作提供科学依据。 相似文献
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《Biochimica et Biophysica Acta (BBA)/General Subjects》2023,1867(8):130385
Colorectal cancer (CRC) treatment is dramatically hampered by resistance to oxaliplatin alone or in the combination of irinotecan or 5-fluorouracil and leucovorin. This study aims to design and assess Chitosan/Hyaluronic Acid/Protamine sulfate (CS/HA/PS) polyplexes loaded with CRISPR plasmid for targeting a key gene in cancer drug resistance. Here, recent findings were considered to validate oxaliplatin-resistant CRC-related genes and systems biology approaches employed to detect the critical gene. The polyplexes were characterized according to particle size, zeta potential, and stability. Moreover, carrier toxicity and transfection efficiency were assessed on oxaliplatin-resistant HT-29 cells. The post-transfection evaluations were performed to confirm gene disruption-mediated CRISPR. Eventually, excision cross complementation group 1(ERCC1), a crucial member of the nucleotide excision repair pathway, was selected to be targeted using CRISPR/Cas9 to reverse oxaliplatin resistance in HT-29 cells. CS/HA/PS polyplexes containing CRISPR/Cas9 plasmid exhibited negligible toxicity and comparable transfection efficiency with Lipofectamine™. Following the efficient gene delivery, sequences in CRISPR/Cas9 target sites were altered, ERCC1 was downregulated, and drug sensitivity was successfully restored in oxaliplatin-resistant cells. Findings indicate that CS/HA/PS/CRISPR polyplexes provide a potential strategy for delivering cargo and targeting oxaliplatin resistance-related gene to manipulate drug resistance as a rising concern in cancer therapeutic approaches. 相似文献
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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。 相似文献
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自2012年首次证明了CRISPR/Cas9可以在体外进行DNA切割试验以来,CRISPR技术逐渐在基因编辑研究中获得了迅速的发展,除了应用于基因编辑领域之外,它在基因表达调控、基因成像、基因分析等方面也展现出了巨大的应用潜力。尤其在基因分析领域,CRISPR技术由于其精确的基因识别、室温的反应条件、易设计性和操作性等特色,使得一系列新型的基因检测技术得以发展,并取得了超越常规技术的一些检测参数。本文以Cas9蛋白为对象,综述了近些年来在该领域取得的研究进展。主要论述Cas9蛋白的功能、改造、引导RNA(sgRNA)的设计及其在基因分析方法上的应用。 相似文献
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在CRISPR/Cas9系统介导的基因编辑中,借助于双链DNA (double-stranded DNA,dsDNA)供体模板的重组效应能够实现对目标基因组靶位点的精确编辑和基因敲入,然而高等真核生物细胞中同源重组的低效性限制了该基因编辑策略的发展和应用。为提高CRISPR/Cas9系统介导dsDNA供体模板的同源重组效率,本研究利用大肠杆菌(Escherichia coli)乳糖操纵子阻遏蛋白LacI与操纵序列LacO特异性结合的特点,通过重组DNA技术将密码子人源化优化的阻遏蛋白基因LacI分别与脓链球菌(Streptococcus pyogenes)源的SpCas9和路邓葡萄球菌(Staphylococcus lugdunensis)源的SlugCas9-HF融合表达,通过PCR将操纵序列LacO与dsDNA供体嵌合,构建了新型的CRISPR/Cas9-hLacI供体适配系统(donor adapting system,DAS)。首先在报告载体水平上对Cas9核酸酶活性、DAS介导的同源引导修复(homology-directed repair,HDR)效率进行了验证和优化,其次在基因组水平对其介导的基因精确编辑进行了检测,并最终利用CRISPR/SlugCas9-hLacI DAS在HEK293T细胞中实现了VEGFA位点的精确编辑,效率高达30.5%,显著高于野生型。综上所述,本研究开发了新型的CRISPR/Cas9-hLacI供体适配基因编辑系统,丰富了CRISPR/Cas9基因编辑技术种类,为以后的基因编辑及分子设计育种研究提供了新的工具。 相似文献
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Maryam Mehravar Abolfazl Shirazi Mahboobeh Nazari Mehdi Banan 《Developmental biology》2019,445(2):156-162
The CRISPR/Cas9 system is a rapid, simple, and often extremely efficient gene editing method. This method has been used in a variety of organisms and cell types over the past several years. However, using this technology for generating gene-edited animals involves a number of obstacles. One such obstacle is mosaicism, which is common in founder animals. This is especially the case when the CRISPR/Cas9 system is used in embryos. Here we review the pros and cons of mosaic mutations of gene-edited animals caused by using the CRISPR/Cas9 system in embryos. Furthermore, we will discuss the mechanisms underlying mosaic mutations resulting from the CRISPR/Cas9 system, as well as the possible strategies for reducing mosaicism. By developing ways to overcome mosaic mutations when using CRISPR/Cas9, genotyping for germline gene disruptions should become more reliable. This achievement will pave the way for using the CRISPR technology in the research and clinical applications where mosaicism is an issue. 相似文献
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Qun Zhou Hengji Zhan Xinhui Liao Lan Fang Yuhan Liu Haibiao Xie Kang Yang Qunjun Gao Mengting Ding Zhiming Cai Weiren Huang Yuchen Liu 《Cell proliferation》2019,52(2)
With the development of synthetic biology, synthetic gene circuits have shown great applied potential in medicine, biology, and as commodity chemicals. An ultimate challenge in the construction of gene circuits is the lack of effective, programmable, secure and sequence‐specific gene editing tools. The clustered regularly interspaced short palindromic repeat (CRISPR) system, a CRISPR‐associated RNA‐guided endonuclease Cas9 (CRISPR‐associated protein 9)‐targeted genome editing tool, has recently been applied in engineering gene circuits for its unique properties‐operability, high efficiency and programmability. The traditional single‐targeted therapy cannot effectively distinguish tumour cells from normal cells, and gene therapy for single targets has poor anti‐tumour effects, which severely limits the application of gene therapy. Currently, the design of gene circuits using tumour‐specific targets based on CRISPR/Cas systems provides a new way for precision cancer therapy. Hence, the application of intelligentized gene circuits based on CRISPR technology effectively guarantees the safety, efficiency and specificity of cancer therapy. Here, we assessed the use of synthetic gene circuits and if the CRISPR system could be used, especially artificial switch‐inducible Cas9, to more effectively target and treat tumour cells. Moreover, we also discussed recent advances, prospectives and underlying challenges in CRISPR‐based gene circuit development. 相似文献
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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。 相似文献
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新兴的CRISPR/Cas9基因编辑技术可实现在分子水平上对基因进行操作,具有设计简单、易于操作、特异性好、效率高等优点,广泛应用于肿瘤发生、发展和转移的潜在机制以及临床治疗的研究.利用纳米技术研发的非病毒纳米载体可以将CRISPR/Cas9系统高效递送到体内,为CRISPR/Cas9技术在临床领域的应用提供新途径.本文介绍CRISPR/Cas9的作用原理,简要概括目前CRISPR/Cas9系统的递送形式和常用的纳米递送载体,总结在部分肿瘤治疗中应用该技术的研究进展,并进一步对此进行展望. 相似文献
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Marilyn S. Baffoe-Bonnie 《Bioethics》2019,33(6):661-668
CRISPR/Cas9 is quickly becoming one of the most influential biotechnologies of the last five years. Clinical trials will soon be underway to test whether CRISPR/Cas9 can edit away the genetic mutations that cause sickle cell disease (SCD). This article will present the background of CRISPR/Cas9 gene editing and SCD, highlighting research that supports the application of CRISPR/Cas9 to SCD. While much has been written on why SCD is a good biological candidate for CRISPR/Cas9, less has been written on the ethical implications of including SCD in CRISPR/Cas9 research. This article will argue that there is a strong case in favor of including SCD. Three benefits are achieving distributive justice in research, continuing to repair the negative relationship between patients with SCD and the health‐care system, and benefit‐sharing for those who do not directly participate in CRISPR/Cas9 research. Opponents will argue that SCD is a risky candidate, that researchers will not find willing participants, and that the burden of SCD is low. Of this set of arguments, the first gives pause. However, on balance, the case in favor of including SCD in CRISPR/Cas9 research is stronger than the case against. Ultimately, this article will show that the historic and sociopolitical injustices that impede progress in treating and curing SCD can be alleviated through biotechnology. 相似文献
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Dong Zhanqi Qin Qi Hu Zhigang Chen Peng Huang Liang Zhang Xinling Tian Ting Lu Cheng Pan Minhui 《中国病毒学》2019,34(4):444-453
Recently the developed single guide(sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease(CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously. However, there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses. To address the need for sustained delivery of a multiplex CRISPR/Cas9-based genome-editing vehicle against insect viruses, we developed a one-vector(pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus(BmNPV) in insect cells.We screened the immediate-early-1 gene(ie-1), the major envelope glycoprotein gene(gp64), and the late expression factor gene(lef-11), and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis. In addition, we constructed a multiplex editing vector(PSL1180-Cas9-sgIE1-sgLEF11-sgGP64, sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection. This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication. This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus. 相似文献
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近年来,CRISPR/Cas系统因其效率高、靶向性强、易操作等优势,已被广泛应用于多种病毒研究中。本文首先简单介绍了CRISPR/Cas系统的分类,并比较了Cas9和Cas12a与Cas13a的特点;其次重点介绍了CRISPR/Cas9通过靶向破坏病毒基因组,或编辑宿主关于病毒生命周期的关键因子的策略在抗病毒方面的各种应用,CRISPR/Cas13a采用靶向破坏病毒基因组方法在抗病毒中的应用,以及CRISPR/Cas12a和CRISPR/Cas13a在病毒基因检测中的应用。最后讨论了CRISPR/Cas在病毒研究中面临的挑战,并讨论了CRISPR/Cas12a作为抗病毒工具的潜在应用前景。由于CRISPR/Cas系统自身的优势,预计该系统将会给病毒相关的疾病诊断和控制带来革命性的变化。 相似文献