CRISPR/Cas9

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Editor:XiaoleiXu,MayoClinic,UNITEDSTATES

Received:June4,2015;Accepted:July29,2015;Published:August18,2015

DataAvailability:AllrelevantdataarewithinthepaperanditsSupportingInformationfiles.

Funding:ThisworkwassupportedbyMajorScienceandTechnologyProjectsofChina(2014ZX08010-004)andtheCAAS(ChineseAcademyofAgricultureSciences)InnovationProject.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.

Competinginterests:Theauthorshavedeclaredthatnocompetinginterestsexist.

TheT6generationtransgenicsoybeanline(genotype,Zigongdongdou)harboringahomozygousbartransgeneandawildtypesoybeanline(genotype,Williams82)wereused.

TargetSiteSelectionandConstructionoftheSgRNA:Cas9ExpressionVector

(A)Schematicillustratingthetargetedbarsequence(blue)andcorrespondingPAM(red).PCRamplificationspanningthetargetlociwasconductedusingtheprimersbar-Fandbar-R.RestrictionenzymeApaIattheCas9cleavagesiteisunderlined.(B)PCR/REassaytodetectCRISPR/Cas9-inducedmutationsintargetloci.Lanes1–30,PCRproductsofsamplestreatedwithsgRNA:Cas9weredigestedwithApaI.LanesWTandWT*,undigestedanddigestedwild-typecontrols,respectively.Theredarrowheadindicatestheundigestedbands.Thenumbersatthebottomofthegelsindicatemutationfrequenciesmeasuredaccordingtobandintensities.M,DL2000ladderDNAmarker.(C)Cloningandsequencingoftheundigestedbands.Deletionsandinsertionsareindicatedasdashesandredlowercaseletters,respectively.Basesubstitutionsareindicatedwithgreencapitalletters.Thetypesofindelsareindicatedintherightcolumn.

(A)SchematicillustratingtheGmFEI2-SP2targetsequence(blue)andcorrespondingPAM(red).PCRamplificationspanningthetargetlociwasconductedusingtheprimersGmFEI2-SP2-FandGmFEI2-SP2-R.TherestrictionenzymeBslIattheCas9cleavesiteisunderlined.(B)PCR/REassaytodetectCRISPR/Cas9-inducedmutationsintargetloci.Lanes1–30,PCRproductsofsamplestreatedwithsgRNA:Cas9weredigestedwithBslI.LanesWTandWT*,undigestedanddigestedwild-typecontrols,respectively.Theredarrowheadindicatestheundigestedbands.Thenumbersatthebottomofthegelsindicatemutationfrequenciesmeasuredaccordingtobandintensities.M,DL2000ladderDNAmarker.(C)Cloningandsequencingoftheundigestedbands.Deletionsandinsertionsareindicatedasdashesandredlowercaseletters,respectively.Basesubstitutionsareindicatedwithgreencapitalletters.Thetypesofindelsareindicatedintherightcolumn.

(A)Schematicillustratingthetargetsequence(blue)andcorrespondingPAM(red).PCRamplificationsspanningthetargetlociwereconductedfromanidenticalDNAtemplateusingthespecificpairprimersGmFEI2-F/GmFEI2-RandGmFEI1-F/GmFEI1-R,respectively.RestrictionenzymeBslIattheCas9cleavagesiteisunderlined.(B)PCR/REassaytodetectCRISPR/Cas9-inducedmutationsintargetsitesofthetwogenes.Lanes1–30,PCRproductsfromthesamplestreatedwithsgRNA:Cas9weredigestedwithBslI.LanesWTandWT*,undigestedanddigestedwild-typecontrols,respectively.Theredarrowheadindicatestheundigestedbands.Thenumbersatthebottomofthegelsindicatemutationfrequenciesmeasuredaccordingtobandintensities.M,DL2000ladderDNAmarker.Theredframeindicatestwosimultaneousgene-editingeventsoccurringinthesamehairyroot.(C)Cloningandsequencingoftheundigestedbands.Deletionsandinsertionsareindicatedasdashesandredlowercaseletters,respectively.Thetypesofindelsareindicatedintherightcolumn.Thenumbersbeforebracketscorrespondtothenumbersofthelanes.

TheCRISPR/Cas9systemhasbeensuccessfullyusedforgenomeengineeringinmanyimportantcrops.However,thismethodhasnotbeenwidelyusedinsoybean.Inthepresentstudy,wedescribedarapidandhighlyspecificmethodforgeneratinganddetectingCRISPR/Cas9-mediatedgenomeeditingindesiredtargetgenesinsoybean.Toimprovetransformationefficiency,thesgRNAandCas9cassetteswereassembledononevector.TheCas9wascodon-optimizedfordicotyledons.Onceadesiredtargetsequencewasselected,onlytheDNAsequenceencodingsgRNAneedstoreplace.ThisvectorcontainedaGFPfluorescentlabelandgreatlyimprovedtheefficiencyofscreeningforpositivehairroots.TheresultsshowedthattheCRISPR/Cas9systemcouldeditbothendogenousandexogenousgenesinsoybeanhairyroots.Indeed,weconsideredthatthistechnologymightbeusefulinsoybeanwhole-planttransformation.InducedhairyrootisarapidmodelsystemforstudyingtheCRISPR/Cas9systeminsoybean.Testingtheeffectivenessoftargetsitesinsoybeanhairyrootsbeforegeneratingtransgenicplantscanbetimesaving,lesslaborintensiveandmorecosteffective.

(A)SchematicillustratingtheGmFEI2-SP1targetsequence(blue)andcorrespondingPAM(red).PCRamplificationspanningthetargetlociwasconductedusingtheprimersGmFEI2-SP1-FandGmFEI2-SP1-R.TherestrictionenzymeAluIattheCas9cleavesiteisunderlined.(B)PCR/REassaytodetectCRISPR/Cas9-inducedmutationsintargetloci.Lanes1–30,PCRproductsofsamplestreatedwithsgRNA:Cas9weredigestedwithAluI.LanesWTandWT*,undigestedanddigestedwild-typecontrols,respectively.Theredarrowheadindicatestheundigestedbands.Thenumbersatthebottomofthegelsindicatemutationfrequenciesmeasuredaccordingtobandintensities.M,DL2000ladderDNAmarker.(C)Cloningandsequencingoftheundigestedbands.Deletionsandinsertionsareindicatedasdashesandredlowercaseletters,respectively.Thetypesofmutationsareindicatedintherightcolumn.

(TIF)

(A)SchematicillustratingtheGmSHR-SP1targetsequence(blue)andcorrespondingPAM(red).PCRamplificationspanningthetargetlociwasconductedusingtheprimersGmSHR-SP1-FandGmSHR-SP1-R.(B)T7EIassaytodetectCRISPR/Cas9-inducedmutationsintargetloci.Lanes1–30,PCRproductsofsamplestreatedwithsgRNA:Cas9wereannealedanddigestedwithT7EI.LanesWTandWT*,undigestedanddigestedwild-typecontrols,respectively.Theredarrowheadindicatesthedigestedbands.Thenumbersatthebottomofthegelsindicatemutationfrequenciesmeasuredaccordingtobandintensities.M,DL2000ladderDNAmarker.(C)CloningandsequencingofPCRproductswhichhavethedigestedbands.Deletionsandinsertionsareindicatedasdashesandredlowercaseletters,respectively.Thetypesofmutationsareindicatedintherightcolumn.

(A)SchematicillustratingtheGmSHR-SP2targetsequence(blue)andcorrespondingPAM(red).PCRamplificationspanningthetargetlociwasconductedusingtheprimersGmSHR-SP2-FandGmSHR-SP2-R.(B)T7EIassaytodetectCRISPR/Cas9-inducedmutationsintargetloci.Lanes1–22,PCRproductsofsamplestreatedwithsgRNA:Cas9wereannealedanddigestedwithT7EI.LanesWTandWT*,undigestedanddigestedwild-typecontrols,respectively.Theredarrowheadindicatesthedigestedbands.Thenumbersatthebottomofthegelsindicatemutationfrequenciesmeasuredaccordingtobandintensities.M,DL2000ladderDNAmarker.(C)CloningandsequencingofPCRproductswhichhavethedigestedbands.Deletionsandinsertionsareindicatedasdashesandredlowercaseletters,respectively.Basesubstitutionsareindicatedwithgreencapitalletters.Thetypesofmutationsareindicatedintherightcolumn.

(A)SchematicillustratingtheGmSHR-SP3targetsequence(blue)andcorrespondingPAM(red).PCRamplificationspanningthetargetlociwasconductedusingtheprimersGmSHR-SP3-FandGmSHR-SP3-R.TherestrictionenzymeEco47IattheCas9cleavesiteisunderlined.(B)PCR/REassaytodetectCRISPR/Cas9-inducedmutationsintargetloci.Lanes1–28,PCRproductsofsamplestreatedwithsgRNA:Cas9weredigestedwithEco47I.LanesWTandWT*,undigestedanddigestedwild-typecontrols,respectively.Theredarrowheadindicatestheundigestedbands.Thenumbersatthebottomofthegelsindicatemutationfrequenciesmeasuredaccordingtobandintensities.M,DL2000ladderDNAmarker.(C)Cloningandsequencingoftheundigestedbands.Deletionsandinsertionsareindicatedasdashesandredlowercaseletters,respectively.Basesubstitutionsareindicatedwithgreencapitalletters.Thetypesofmutationsareindicatedintherightcolumn.

GFP,greenfluorescentprotein.atU6,ArabidopsisU6promoter.gRNA,guide-RNA.dpCas9,Cas9codon-optimizedfordicotyledons.Ubipromoter,maizeubiquitinpromoter.

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Conceivedanddesignedtheexperiments:WH.Performedtheexperiments:YCLCXL.Analyzedthedata:YCLCWH.Contributedreagents/materials/analysistools:SSCWBJWHTH.Wrotethepaper:YCLC.Revisedthemanuscript:WHTH.

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1.CellCRISPR基因编辑技术的过去现在和未来CRISPR技术不仅适用于基因组编辑,而且还能够对基因表达进行瞬时操作,如CRISPR干扰(CRISPRi)和CRISPR激活(CRISPRa)转录调节技术,通过靶向失活的Cas9(dCas9)与转录调节结构域(如VP64或KRAB)融合到基因启动子实现RNA引导的基因转录控制。 Cas9的催化失活突变体最初https://mp.weixin.qq.com/s?__biz=MzA3MTg1OTM3MQ==&mid=2649990125&idx=4&sn=a5ccc014731240b04e5429f599c913f3&chksm=869c0c2eca634ac43bbf05435c313cebf5bd1943e0ce826bf0dce02045f9341abdfe7fa14796&scene=27
2.Addgene:CRISPRGuideRead more about Cas9 multiplexing. Browse Plasmids: Multiplex gRNA Vectors Engineering the Cas9 Endonuclease CRISPR specificity is determined by both the gRNA sequence and the Cas9 enzyme. Ideally, a gRNA targeting sequence will have perfect homology to the target DNA with no homology elsewhere in http://www.addgene.org/guides/crispr/
3.生物工程的突破:CRISPR技术如何改变遗传疾病治疗免疫crispr然而,CRISPR技术的出现,为我们带来了新的曙光。CRISPR技术的核心在于一种名为“Cas9”的酶。它能够精准定位到基因组中的特定位置,进行切割并进行基因的修复或替换。凭借这一能力,科学家们能够在实验室中模拟出基因突变的过程,并对其进行纠正。更为重要的是,这项技术的操作相对简单,成本也较低,使得以往高不可攀的https://www.163.com/dy/article/JJH7I8AM0556AUYG.html
4.HowdoesCRISPRwork?LiveScienceCRISPR, short for CRISPR-Cas9, is a genome-editing tool that allows scientists to precisely cut and modify DNA sequences. It has revolutionized the study of genes, helped to enhance crops and improved health care. The gene-editing system was originally discovered inbacteria, where it limits infhttps://www.livescience.com/58790-crispr-explained.html
5.2016年11月全球CRISPR/Cas9取得重大进展梳理资讯中心近日,来自四川大学华西生物治疗国家重点实验室的魏于全院士课题组首次采用人工病毒进行CRISPR-Cas9基因编辑系统输送,成功在小鼠肿瘤模型中完成了靶基因编辑,达到了较好的肿瘤治疗效果,相关成果发表在《美国化学学会·纳米》杂志上。 。虽然目前为止已经有不少研究用CRISPR进行肿瘤基因编辑,但是这些研究存在着不少问题,使用的https://www.instrument.com.cn/news/20161130/207646.shtml
6.CRISPR科学家为了让Cas蛋白定向剪切DNA序列,在CRISPR工作机理的基础上,人为重组了一段目的基因的sgRNA(small-guide RNA),在sgRNA的引导下,Cas9蛋白可以实现对目的基因的定向切割。本文主要介绍,目的基因sgRNA设计和如何将sgRNA构建到CRISPR-Cas相关载体。 “ 1. 目的基因sgRNA设计http://www.dentalearner.com/archives/3855
7.文献分享—crisprcas9技术诱导拟南芥同源染色体易位大家好,本周给大家分享的是一篇发表在nature plants上的关于crispr-cas9技术诱导拟南芥同源染色体易位的文章。 图1.png 文章题目:CRISPR–Cas9-mediated induction of heritable chromosomal translocations inArabidopsis(CRISPR-Cas9 介导的拟南芥遗传染色体易位诱导) https://www.jianshu.com/p/cb8d37aa8abf
8.用于基因工程的CRISPRCRISPR-Cas9 系统正迅速成为应用最广泛的技术之一,不断推动基因工程、合成生物学和功能基因组学取得新进展。CRISPR/Cas 之所以被广为接受,是由于其具有无与伦比的特异性,并且我们可以在不降低活性或保真度的条件下轻松操纵这种特异性。研究人员一开始认为细菌免疫系统是一个不太可能的假设,而现在它即将成为基因组工程和https://www.agilent.com/zh-cn/crispr-cas9-for-genetic-engineering-details-specifications
9.CRISPRCRISPR Cas9 is a genome editing technique that is revolutionising the synthetic and engineering biology industry as well as academic research.http://www.synbicite.com/synthetic-biology/CRISPR/
10.CRISPR/Cas9技术及其在药物研发中的应用摘要: CRISPR/Cas9系统是在细菌和古细菌中发现的一种为抵御病毒和质粒的不断攻击而演化来的获得性免疫防御机制,由规律成簇的间隔短回文重复(clustered regularly interspaced short palindromic repeats,CRISPR)和Cas(CRISPR-associated)蛋白组成。通过改造最简单的Ⅱ型CRISPR系统,将特殊小向导RNA(small guide RNA,sgRNA)和https://html.rhhz.net/YXXB/html/20180102.htm
11.CRISPRTransfusion-dependent β-thalassemia (TDT) and sickle cell disease (SCD) are severe monogenic diseases with severe and potentially life-threatening manifestations. BCL11A is a transcription factor thttps://www.nejm.org/doi/full/10.1056/NEJMoa2031054
12.CRISPR/Cas9系统在植物基因组编辑中技术改进与创新的研究进展植物学报 Chinese Bulletin of Botany 2019, 54 (3): 385–395, www.chinbullbotany.com doi: 10.11983/CBB18151 ·专题论坛· CRISPR/Cas9系统在植物基因组编辑中技术 改进与创新的研究进展 苏钺凯, 邱镜仁, 张晗, 宋振巧, 王建华* 山东农业大学农学院, 泰安 271018 摘要 CRISPR/Cas9基因组编辑技术是一项对https://www.chinbullbotany.com/CN/article/downloadArticleFile.do?attachType=PDF&id=29501
13.GenomeengineeringusingtheCRISPRTargeted nucleases are powerful tools for mediating genome alteration with high precision. The RNA-guided Cas9 nuclease from the microbial clustered regularly interspaced short palindromic repeats (CRISPR) adaptive immune system can be used to facilitatehttps://www.nature.com/articles/nprot.2013.143
14.Cas9mRNAThermoFisherScientific注:CRISPR-Cas9 基因组编辑需要向导 RNA (gRNA),以便切割感兴趣的靶序列处的基因组 DNA。更多信息,请 查阅CRISPR-Cas9 的向导 RNA和CRISPR Cas9 筛选文库。 适用于多重基因编辑和转基因模型系统应用的 Cas9 mRNA 工作流程 通过GeneArt CRISPR 核酸酶 mRNA,您可以在单个孔中同时转染多达 4 个不同的 gRNAs,并同https://www.thermofisher.cn/cn/zh/home/life-science/genome-editing/cas9-nucleases/cas9-mrna.html
15.CRISPRBy 进哥哥Posted on 2024-01-26 CRISPR-Cas9 分子生物学 1. CRISPR/cas9系统 CRISPR/C […] CRISPRi和CRISPRa:基因表达干预的新利器 Read More 同源定向修复简介及CRISPR knock-in原理 By 进哥哥Posted on 2023-11-10 CRISPR-Cas9 分子生物学 DNA损伤是指DNA结构或碱基配对的部位发生损伤。 […] 同https://www.jingege.wang/tag/crispr-cas9/
16.基于CRISPR/Cas9技术的条件性基因敲除方法与流程本发明属于基因修饰技术领域,具体涉及基于CRISPR/Cas9技术的条件性基因敲除方法。背景技术:随着科技的进步和对生命科学领域的不断探索,人们对活体内某一基因在特定组织、细胞及时间内的表达情况的研究显得更为迫切。近年来迅速发展的位点特异性重组技术是适应这种需要而产生的关键基因操作工具,其能够在一定发育阶段或在https://www.xjishu.com/zhuanli/27/201410608382.html
17.综述治疗性CRISPR/cas9技术研究进展研究人员从 β 珠蛋白生成障碍性贫血病人身上取下成纤维细胞,然后诱导成多能干细胞,转染靶向性的 Crispr/cas9 和 DNA 模板进行 HDR 修复,同源重组修复通过抗性基因筛选,筛选后通过转座酶切除,再将这样的多能性干细胞诱导成红细胞的前体细胞,然后用于移植。Hans Clevers 主持的一项研究证明 Crispr/cas9 系统可用于http://www.biotech.org.cn/information/144780/
18.CRISPR天然的CRISPR-Cas9系统由三部分组成:SpCas9 (简称Cas9)、crRNA、tracrRNA。tracrRNA(在CRISPR-Cas9编辑技术中被优化并命名为gRNA scaffold),它负责与Cas9结合,与重复序列具有同源性。crRNA为引导序列,约20个碱基,具有特异性。其中,crRNA和tracrRNA通过局部碱基配对组合并与Cas9结合后,引导Cas9识别切割目标DNA序列 (图1)https://blog.csdn.net/tinygene/article/details/124614426
19.基因编辑CRISPR/Cas系统中Cas9/Cas12/Cas13结构特点在Cas9介导的DNA切割后,通过非同源末端连接(NHEJ)或同源定向修复(HDR)途径使基因编辑产生作用。金黄色葡萄球菌Cas9(SaCas9)是Cas9的一种变体,具有独特的PAM识别能力,可以靶向5′-NNRRT PAM。最近,研究人员发现了CRISPR/CasX,一种Cas9的变体,它更小,在基因编辑方面更有效,被认为是所有变体中最小的。此外,通过将https://m.magigen.com/cn/characteristics-CRISPR-Cas9-Cas12-Cas13.html
20.基因敲除鼠技术系列之三:CRISPR/Cas9系统(基因编辑)CRISPR/Cas9系统(基因编辑) 一、定义 1、CRISPR CRISPR(Clustered Regularly Interspaced Short Palindromic Repeats,成簇规律间隔短回文重复序列),首次由日本科学家于1987年在大肠杆菌染色体上发现,是一种包含短重复序列(20–50 bp)的DNA片段,CRISPR基因存在于超过40%的细菌和90%的古细菌的基因组中[1,2]。 https://3g.dxy.cn/bbs/topic/45352377
21.CRISPR/Cas9靶向基因修饰药物开发平台CRISPR/Cas9靶向基因修饰药物开发Cas9 蛋白是一种源自细菌的蛋白质,该蛋白搭配上一段能够与某段 DNA 特异性结合的RNA序列就可以形成一把“高精度的手术刀”,对基因进行失活、激活或者改造等遗传学操作。 CRISPR–Cas系统自2013年1月兴起后,就在短短的一个月之内,在包括《科学》(Science)和《自然生物技术》(https://www.cqwestern.net/yanjiu.html
22.过表达基因超过病毒载体容量怎么解?CRISPR/dCas9助力实现超大基因CRISPR/Cas9(Clustered Regularly Interspaced Short Palindromic Repeats/Cas9)系统是目前被广泛运用的基因编辑系统,其原理是由CRISPR转录产生的sgRNA介导Cas9核酸酶靶向目标序列,对序列进行切割。Cas9的核酸酶剪切活性取决于两个结构域:RuvC和HNH。当这两个结构域同时处于失活状态时,Cas9将不具有核酸酶活性,成为dCas9(deadhttps://brainvta.biomart.cn/news/3042476.htm