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Verónica Pazmiño-Ibarra Adrià Mengual-Martí Alexandra Marisa Targovnik Salvador Herrero 《Biotechnology and bioengineering》2019,116(11):2823-2833
The clustered regularly interspaced short palindromic repeats (CRISPR) system–associated Cas9 endonuclease is a molecular tool that enables specific sequence editing with high efficiency. In this study, we have explored the use of CRISPR/Cas9 system for the engineering of baculovirus. We have shown that the delivering of Cas9-single guide RNA ribonucleoprotein (RNP) complex with or without DNA repair template into Sf21 insect cells through lipofection might be efficient to produce knockouts as well as knock-ins into the baculovirus. To evaluate potential application of our CRISPR/Cas9 method to improve baculovirus as protein expression vector and as biopesticide, we attempted to knockout several genes from a recombinant AcMNPV form used in the baculovirus expression system as well as in a natural occurring viral isolate from the same virus. We have additionally confirmed the adaptation of this methodology for the generation of viral knock-ins in specific regions of the viral genome. Analysis of the generated mutants revealed that the editing efficiency and the type of changes was variable but relatively high. Depending on the targeted gene, the editing rate ranged from 10% to 40%. This study established the first report revealing the potential of CRISPR/Cas9 for genome editing in baculovirus, contributing to the engineering of baculovirus as a protein expression vector as well as a biological control agent. 相似文献
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Hui Zhang Jinshan Zhang Pengliang Wei Botao Zhang Feng Gou Zhengyan Feng Yanfei Mao Lan Yang Heng Zhang Nanfei Xu Jian‐Kang Zhu 《Plant biotechnology journal》2014,12(6):797-807
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. 相似文献
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CRISPR/Cas基因编辑技术在植物基因功能研究和作物遗传改良方面具有重要应用价值,其主要依赖gRNA引导核酸内切酶在目标基因组位置产生双链断裂(DSBs),DSBs在通过非同源末端连接(NHEJ)或同源重组(HDR)方式进行修复时,会引起靶标位置核苷酸序列的缺失、插入或者替换,从而实现基因编辑。介绍了CRISPR/Cas基因编辑技术的作用机理及发展趋势,并对CRISPR/Cas技术在主要粮食及经济作物育种中的应用进展进行了总结,以期为农作物育种提供有益的参考。 相似文献
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Niaz Ahmad Mehboob-ur Rahman Zahid Mukhtar Yusuf Zafar Baohong Zhang 《Journal of cellular physiology》2020,235(2):666-682
Clustered regularly interspaced short palindromic repeats (CRISPR)-based genome editing, derived from prokaryotic immunity system, is rapidly emerging as an alternative platform for introducing targeted alterations in genomes. The CRISPR-based tools have been deployed for several other applications including gene expression studies, detection of mutation patterns in genomes, epigenetic regulation, chromatin imaging, etc. Unlike the traditional genetic engineering approaches, it is simple, cost-effective, and highly specific in inducing genetic variations. Despite its popularity, the technology has limitations such as off-targets, low mutagenesis efficiency, and its dependency on in-vitro regeneration protocols for the recovery of stable plant lines. Several other issues such as persisted CRISPR activity in subsequent generations, the potential for transferring to its wild type population, the risk of reversion of edited version to its original phenotype particularly in cross-pollinated plant species when released into the environment and the scarcity of validated targets have been overlooked. This article briefly highlights these undermined aspects, which may challenge the wider applications of this platform for improving crop genetics. 相似文献
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成簇规律间隔短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR)系统在近年来得到越来越广泛的应用。相比传统的基因组编辑技术,CRISPR/Cas系统具有编辑效率高、特异性强、花费低、对实验技术要求低等优势。其中Ⅱ型和Ⅴ型CRISPR/Cas系统分别仅需要单个Cas9蛋白和单个Cpf1蛋白作为切割双链DNA的工具,因此特别受到研究者们的青睐。目前CRISPR/Cas9技术已成功应用于斑马鱼、小鼠、人类细胞等真核生物的基因组编辑中,并取得一系列重要成果,但在细菌领域进行的相关研究并不多。文中将简单描述CRISPR/Cas系统及其作用机制,重点介绍该系统的优化以及近年来其在细菌学领域所取得的进展。 相似文献
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Lu-Feng Hu Yu-Xuan Li Jia-Zhen Wang Yu-Ting Zhao Yangming Wang 《Wiley interdisciplinary reviews. RNA》2023,14(1):e1731
The clustered regularly interspaced short palindromic repeats (CRISPR) system is a product of million years of evolution by microbes to fight against invading genetic materials. Around 10 years ago, scientists started to repurpose the CRISPR as genetic tools by molecular engineering approaches. The guide RNA provides a versatile and unique platform for the innovation to improve and expand the application of CRISPR-Cas9 system. In this review, we will first introduce the basic sequence and structure of guide RNA and its role during the function of CRISPR-Cas9. We will then summarize recent progress on the development of various guide RNA engineering strategies. These strategies have been dedicated to improve the performance of CRISPR-Cas9, to achieve precise spatiotemporal control of CRISPR-Cas9, and to broaden the application of CRISPR-Cas9. Finally, we will briefly discuss the uniqueness and advantage of guide RNA-engineering based systems versus those with engineered Cas9 proteins and speculate potential future directions in guide RNA engineering. This article is categorized under:
- RNA Methods > RNA Analyses In Vitro and In Silico
- RNA Methods > RNA Nanotechnology
- Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs
- RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes
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Plant viruses are known to infect most economically important crops and pose a major threat to global food security. Currently, few resistant host phenotypes have been delineated, and while chemicals are used for crop protection against insect pests and bacterial or fungal diseases, these are inefficient against viral diseases. Genetic engineering emerged as a way of modifying the plant genome by introducing functional genes in plants to improve crop productivity under adverse environmental conditions. Recently, new breeding technologies, and in particular the exciting CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR‐associated proteins) technology, was shown to be a powerful alternative to engineer resistance against plant viruses, thus has great potential for reducing crop losses and improving plant productivity to directly contribute to food security. Indeed, it could circumvent the “Genetic modification” issues because it allows for genome editing without the integration of foreign DNA or RNA into the genome of the host plant, and it is simpler and more versatile than other new breeding technologies. In this review, we describe the predominant features of the major CRISPR/Cas systems and outline strategies for the delivery of CRISPR/Cas reagents to plant cells. We also provide an overview of recent advances that have engineered CRISPR/Cas‐based resistance against DNA and RNA viruses in plants through the targeted manipulation of either the viral genome or susceptibility factors of the host plant genome. Finally, we provide insight into the limitations and challenges that CRISPR/Cas technology currently faces and discuss a few alternative applications of the technology in virus research. 相似文献
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The oleaginous yeast Rhodosporidium toruloides is considered a promising candidate for production of chemicals and biofuels thanks to its ability to grow on lignocellulosic biomass, and its high production of lipids and carotenoids. However, efforts to engineer this organism are hindered by a lack of suitable genetic tools. Here we report the development of a CRISPR/Cas9 system for genome editing in R. toruloides based on a fusion 5S rRNA–tRNA promoter for guide RNA (gRNA) expression, capable of greater than 95% gene knockout for various genetic targets. Additionally, multiplexed double-gene knockout mutants were obtained using this method with an efficiency of 78%. This tool can be used to accelerate future metabolic engineering work in this yeast. 相似文献
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Jingying Li Chen Zhang Yubing He Shaoya Li Lei Yan Yucai Li Ziwei Zhu Lanqin Xia 《植物学报(英文版)》2023,65(2):444-467
Precise replacement of an allele with an elite allele controlling an important agronomic trait in a predefined manner by gene editing technologies is highly desirable in crop improvement.Base editing and prime editing are two newly developed precision gene editing systems which can introduce the substitution of a single base and install the desired short indels to the target loci in the absence of double-strand breaks and donor repair templates,respectively.Since their discoveries,various strate... 相似文献
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Viral diseases severely affect crop yield and quality, thereby threatening global food security. Genetic improvement of plant virus resistance is essential for sustainable agriculture. In the last decades, several modern technologies were applied in plant antiviral engineering. Here we summarized breakthroughs of the two major antiviral strategies, RNA silencing and genome editing. RNA silencing strategy has been used in antiviral breeding for more than thirty years, and many crops engineered to stably express small RNAs targeting various viruses have been approved for commercial release. Genome editing technology has emerged in the past decade, especially CRISPR/Cas, which provides new methods for genetic improvement of plant virus resistance and accelerates resistance breeding. Finally, we discuss the potential of these technologies for breeding crops, and the challenges and solutions they may face in the future. 相似文献
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基因治疗是将外源正常基因通过一定方式导入人体靶细胞以纠正或补偿因基因缺陷和异常引起的疾病,从而达到治疗目的。因此,基因治疗的技术方法在研究持续感染HIV-1或潜伏感染HIV-1原病毒患者的治疗中具有重大的现实意义。目前,现有的基因治疗方法存在识别靶向位点有限及脱靶几率大等主要问题。最新研究表明来源于细菌和古菌的规律间隔成簇短回文重复序列及其相关核酸酶9系统[Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9), CRISPR/Cas9]已被成功改造成基因组定点编辑工具。因此,如何利用CRISPR/Cas9系统实现对HIV-1病毒基因组进行高效靶向修饰,从而达到治疗HIV-1感染病患的目的已经成为当前研究的热点。本文参考最新国内外研究成果,重点介绍了 CRISPR/Cas9基因组编辑技术在HIV-1感染疾病治疗中的应用,主要包括CCR5基因编辑、清除HIV-1原病毒以及活化HIV-1原病毒,以期为HIV-1感染疾病的预防与治疗提供重要研究参考。 相似文献
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CRISPR/Cas9(clustered regularly interspaced short palindromic repeat/CRISPR-associated nuclease 9)技术是一种RNA引导的基因组靶向编辑技术,能对基因组序列进行精确编辑,在探究基因功能、修复受损基因、沉默有害基因、改良品质性状等方面具有广阔的应用前景。近年来,随着对CRISPR/Cas9系统研究的不断深入和改造,该系统以其操作简易、省时、高效等优点在生物学研究的众多领域中得以推广和应用,特别是在果蝇(Bombyx mori)、家蚕(silkworm)、埃及伊蚊(Aedes aegypti)和蝴蝶(butterfly)等多种昆虫中。本文概述了CRISPR/Cas9的结构、作用原理及发展优化,总结了CRISPR/Cas9导入昆虫的策略和在昆虫中的应用,以及对CRISPR/Cas9系统产生脱靶问题的应对策略,以期对经济昆虫和有益昆虫的分子育种、害虫的生物技术防控等研究提供参考。 相似文献