,1,4,5, 宝颖1, 马华峥1, 伊宗裔1,4,5, 周卓1,2,3,6, 魏文胜,1,2,3,4,6Gene editing technology and its research progress in China
Yiou Chen,1,4,5, Ying Bao1, Huazheng Ma1, Zongyi Yi1,4,5, Zhuo Zhou1,2,3,6, Wensheng Wei,1,2,3,4,6通讯作者:
编委: 高彩霞
收稿日期:2018-07-10修回日期:2018-09-7网络出版日期:2018-10-20
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Received:2018-07-10Revised:2018-09-7Online:2018-10-20
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陈一欧,博士研究生,专业方向：生物化学与分子生物学E-mail:
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陈一欧, 宝颖, 马华峥, 伊宗裔, 周卓, 魏文胜. 基因编辑技术及其在中国的研究发展[J]. 遗传, 2018, 40(10): 900-915 doi:10.16288/j.yczz.18-195
Yiou Chen, Ying Bao, Huazheng Ma, Zongyi Yi, Zhuo Zhou, Wensheng Wei.
随着人类基因组计划的完成和高通量测序技术的发展,人们读取大量序列信息的能力得到飞速提升。“读”取能力的提高进一步激发了人们改写生物体内遗传序列信息的需求,而近年来发展迅速的基因编辑技术则大幅提高了这种“写”的能力。这种特殊的生物技术能够让研究者对基因组序列或者基因转录产物进行人为编辑,以改变目的基因或调控元件的序列、表达量或功能。这一革命性技术一经问世就冲击到生命科学的各个研究领域,必将在未来相当长时间内对人类健康、疾病治疗、新药研发、物种改良以及生命科学基础研究等众多方面产生广泛而深远的影响,也是世界范围内竞争最为激烈的下一代核心生物技术。本文在介绍基因编辑技术发展的基础上,对我国在该领域的研究进行了回顾性总结,并对该领域的前沿研究进展和应用发展进行了综述,旨在为了解基因编辑技术的发展历程及利用该技术进行深入研究提供理论依据。
1 基因编辑技术的发展
早期基因编辑技术包括归巢内切酶(homing endonuclease, HEs)、锌指核酸内切酶(zinc finger endonuclease, ZFN)和类转录激活因子效应物(transcription activator-like effector nucleases, TALENs),但脱靶效应或组装复杂性限制了这些技术在基因编辑领域中的应用。近年来,以CRISPR/Cas9系统为代表的新型基因编辑技术飞速发展,并开始在诸多生物学领域中得到广泛应用。1.1 早期基因编辑技术
1.1.1 归巢内切酶早期的基因编辑技术依赖于细胞内同源重组途径(homologous recombination, HR)将外源DNA序列插入基因组[1]。通过在外源DNA序列两端加入同源臂,能够实现外源序列的精确整合。然而真核生物中同源重组发生频率极低,约为1/106~1/109 [2];并且相对于靶位点而言,外源序列更容易随机整合到基因组上其他位点,造成脱靶效应,从而限制了该技术的应用[3]。
研究表明,利用归巢内切酶在目的位点附近引入双链断裂(double-strand break, DSB)能够激活损伤修复机制参与断裂修复[4, 5],进而提高基因编辑效率。真核生物中产生DNA双链断裂后的修复途径除HR外,主要为非同源末端连接(non-homologous end joining, NHEJ)。与同源介导的修复(homology-directed repair, HDR)相比,NHEJ发生频率更高,且直接对断裂位点进行修复而不依赖于模板,但容易引起DNA接口处碱基的插入或缺失,造成移码突变,从而导致基因的敲除[2, 6]。
1.1.2 ZFNs和TALENs技术
由于归巢内切酶的DNA识别和切割功能位于同一结构域,使其编辑位点受到序列的限制[7],因此人们把目光转向了锌指核酸内切酶[8,9,10]和类转录激活因子效应物[11, 12]。
ZFN和TALEN均为人工构建的工程核酸酶,DNA结合结构域与FokⅠ核酸内切酶的切割结构域分开,使得人们可以对DNA结合结构域进行设计,改变其对DNA序列的识别特异性,实现对目的位点的精确编辑[13, 14]。ZFN对于DNA序列的特异性识别主要依赖于锌指蛋白(zinc finger protein, ZFP),但ZFN设计成本较高,且序列的上下文依赖效应会降低编辑效率[15, 16]。
TALEN对于DNA序列的特异性识别依赖于TALE蛋白(transcription activator-like effectors, TALEs)中的RVDs (repeat variable diresidues)。由于TALE/ TALEN的模块化和构建的优势,人工编码TALE蛋白比ZFN在基因编辑和转录调控中有着更为广泛的应用[17],如Yang等[18]首次破解了全部400种可能的RVD碱基识别偏好性,Zhang等[19]解码了TALE蛋白对DNA表观修饰5-甲基胞嘧啶和5-羟甲基胞嘧啶的特异性识别。当然,ZFN和TALEN技术均依赖蛋白质对DNA序列的特异性识别,组装的复杂性是限制它们在基因编辑中应用的主要障碍。
1.2 CRISPR/Cas9系统
1.2.1 CRISPR/Cas9系统的发现CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins)系统是目前应用最为广泛的基因编辑工具。1987年,日本大阪大学Nakata研究组首次在大肠杆菌iap基因的3′端侧翼序列中发现了5段长为29nt的重复回文序列,它们由32nt的非重复序列隔开,且这5段重复序列不与任何已知的原核生物序列同源[20]。2000年,西班牙阿利坎特大学Mojica研究组在多种原核生物中均发现了该类重复序列[21]。2002年,荷兰乌得勒支大学Jansen研究组将这种成簇的规律间断短重复序列命名为CRISPR,将位于CRISPR位点侧翼的基因命名为Cas (Cas 1~Cas 4),并发现其具有与解旋酶和内切酶相似的结构[22],但其作用机制仍未知。随后的一些研究猜测其可能参与细菌的免疫机制[23, 24]。2007年,美国丹尼斯克公司Horvath等[25]首次通过病毒侵染实验确定了CRISPR/Cas在细菌中起到抵抗病毒侵染的功能。
随着研究的深入,科学家们发现了多种CRISPR/ Cas系统,根据Cas蛋白的数量可以分为两类(ClassⅠ和Class Ⅱ),跟据Cas的结构和功能可分为6种(TypeⅠ~VI),并可进一步分为多个亚型(Subtype)[26, 27]。相比ClassⅠ,ClassⅡ仅需一个Cas蛋白,因此目前基因编辑中常用的系统均为ClassⅡ,比如Cas9,以及不需要tracrRNA的Cpf1 (Cas12a)[28]和具有RNA切割活性的Cas13[29]。
1.2.2 CRISPR/Cas9系统的作用机制
经过20多年的研究,人们对CRISPR/Cas系统的作用机制有了相对清晰的了解。以CRISPR/Cas9为例,细菌对外来病毒的入侵分为3步：(1) 病毒入侵时,CRISPR/Cas系统将病毒的DNA切成短片段,并插入重复序列之间,作为“记忆”储存[30,31,32,33];(2) 同种病毒再次入侵时,CRISPR阵列及Cas9基因转录,Cas9翻译为蛋白,转录出的tracrRNA与pre-crRNA互补配对,经过内源核糖核酸酶(RNase)加工成熟,最后形成Cas9-crRNA-tracrRNA的三聚体[34];(3) 在crRNA与病毒DNA互补配对之前,Cas9需要与特定的PAM (protospacer adjacent motif)序列结合以区别病毒和自身基因组,Cas识别并结合PAM后将DNA双链解旋,crRNA在PAM上游与目标序列互补配对。在PAM和靶点序列均匹配时,Cas9构象发生改变,其双链内切酶的活性被激活,在PAM上游的特定位置将病毒的双链DNA切断[35,36,37]。
1.2.3 CRISPR/Cas9系统的发展
这种特异性识别并切割DNA产生DSB的特性十分适合基因编辑工具的要求。2012年,美国加州大学伯克利分校Doudna和Charpentier研究组首次在体外证明了CRISPR/Cas9特异性切割靶标DNA的功能,并将crRNA-tracrRNA改造为sgRNA (single guide RNA)[38] (图1)。2013年,美国麻省理工学院Feng Zhang和哈佛大学George Church研究组首次在哺乳动物细胞系中利用CRISPR/Cas9实现了基因编辑[39, 40]。自此,全球各地的实验室开始投入到对这一新型基因编辑工具的研究中。目前CRISPR/Cas9系统已应用于多种领域,如调控体内基因表达[41]、构建动物的疾病模型[42]、研究各种细胞内基因调控网络[43]、基因的高通量筛选[44,45,46,47]等;同时,人们对CRISPR/Cas系统进行了深入探索,细菌中具有种类丰富的Cas,不同的Cas具有各自的特性,包括PAM序列、蛋白大小以及切割活性,扩展了CRISPR/Cas系统的应用范围[29, 48]。除了目前常用的SpCas9 (来自化脓性链球菌Streptococcus pyogenes的Cas9)和SaCas9 (来自金黄色葡萄球菌Staphylococcus aureus 的Cas9),研究者对野生型Cas蛋白进行人工改造,如dCas9 (dead Cas9)[49]和xCas9(一种SpCas9突变体)[50]等,进一步提高了人们对此系统的操作和应用水平。由于其作用机制灵活、易于操作、种类多样的优点,CRISPR/Cas的应用和方法学的研究得以迅速发展。
1.3 CRISPR/Cpf1 (Cas12a)系统
Cpf1与Cas9同属第二类CRISPR系统,但在pre-crRNA的加工上,Cpf1系统没有tracrRNA,而是由其本身的RNase结构域完成整个加工过程[28]。加工成熟的crRNA与Cpf1结合后,能激活其核酸内切酶的活性以切割靶标DNA片段[51]。Cpf1发挥核酸内切酶作用的是Ruv-C样核酸内切酶结构域和特有的Nuc结构域,二者分别切割靶标片段的非互补链和互补链[52]。Cpf1-crRNA可在不依赖于PAM的情况下切割单链DNA,产生特定长度片段和随机小片段[53, 54]。相比Cas9,Cpf1系统无需tracrRNA且crRNA更短,所以目前其主要应用为多位点基因编辑和大片段的删除。利用Cpf1系统则可以在一个质粒上用一个启动子串联多个pre-crRNA,在Cpf1的作用下加工出单独的成熟crRNA[55],大大缩减了片段合成的长度[56]。此外,由于Cpf1能够识别富含T碱基片段,扩充了CRISPR/Cas系统基因编辑范围,包括单碱基编辑[57]和基因表达调控[58]。然而,5°-TTTN-3°的PAM也限制了Cpf1的应用空间,因此研究者们对Cpf1识别PAM的结构域进行了改造,扩大了PAM识别范围[59, 60]。
2 中国在基因编辑领域的研究进展
近年来,我国科学家在基因编辑领域取得了令人鼓舞的进展,在基因编辑系统发展、机制研究、构建基因编辑动植物模型和基因治疗等方面取得了突出的成绩。2.1 基因编辑系统机制研究与发展
中国科学院生物物理所王艳丽研究组和哈尔滨工业大学黄志伟研究组对Cpf1-crRNA复合物、SpyCas-sgRNA-AcrllA4复合物、Cas1-Cas2-DNA复合物、Cas13a蛋白、Cas13a-crRNA复合物以及CRISPR系统Cascade复合物等进行了结构解析及机制分析[61,62,63,64,65],为理解基因编辑工作原理及其调控机制提供了重要依据 。中国科学院健康科学研究所常兴研究组利用靶向性胞嘧啶脱氨酶在体内实现了高效、高通量DNA碱基编辑新方法;上海科技大学陈佳研究组发展了一系列基于CRISPR/Cpf1的新型碱基编辑器(Cpf1- BE)[66, 67]。北京大学魏文胜研究组建立了基于CRISPR系统的高通量筛选方法,并在全基因组范围内实现了对编码基因和非编码基因的功能性筛选[44, 68]。
2.2 动植物模型构建
2013年,中国科学院动物研究所周琪研究组首次利用CRISPR/Cas9系统实现了大鼠多基因快速同时敲除[69],并建立了多种基因编辑动物模型。近期,中国科学院广州生物医药与健康研究院赖良学研 究组利用CRISPR技术成功构建了亨廷顿病疾病猪模型[70]。在遗传信息与生理特性上,食蟹猴、猕猴等灵长类动物与人类具有更高的相似性,是作为人类疾病研究的理想模型。2016年,中山大学中山医学院项鹏、华南农业大学兽医学院杨世华和中国科学院动物研究所周琪研究组在食蟹猴中应用TALEN技术实现了MCPH1基因(最早被鉴定出来的小头症相关基因)突变,成功获得了人类头小畸形症的疾病模型猴[71]。昆明理工大学季维智等利用基因编辑技术构建了多种灵长类动物疾病模型,如利用TALEN技术构建了瑞特综合征食蟹猴模型[72],该成果入选了“2017年中国生命科学十大进展”。2018年初,Cell Research杂志连续发表两篇分别来自中国科学院神经科学研究所杨辉和季维智等研究组的论文,报道了世界首例表达荧光蛋白基因敲入的食蟹猴[73, 74]。
在植物基因编辑方面,中国科学院遗传与发育生物学研究所高彩霞、中国科学院上海植物逆境生物学研究中心朱健康和华南农业大学刘耀光等研究组分别利用CRISPR-Cas9系统在拟南芥、水稻、小麦、玉米等多种植物中成功实现了基因编辑,为植物基因研究及农作物遗传改良提供了平台[75,76,77,78,79]。
2.3 基因治疗
2016年,Nature杂志报道了中国率先开展的世界首个人类CRISPR临床试验。研究者计划将PD-1 (Programmed cell death protein 1,一种重要免疫抑制蛋白)敲除的T细胞注射到化疗、放疗和其他治疗均无效的转移性非小细胞肺癌患者体内,以促进机体针对癌细胞的免疫反应[80]。中山大学黄军就研究组首次在不能存活的人类三核受精卵中应用CRISPR/ Cas9技术对β-地中海贫血症的致病基因进行了基因编辑[67]。由于涉及人类胚胎编辑,这项研究在世界范围内引起了广泛讨论,使科学界和全社会对其应用前景和可能涉及的伦理风险产生了关注。2.4 交流平台
为推动我国基因编辑技术研发及其在生命科学基础研究、农业畜牧业育种以及基因治疗等医学领域的应用,防范其可能带来的生物安全风险和伦理争议,提升我国在基因编辑相关科学前沿、重大应用及下游产业化等领域的国际竞争力,2016年6月,国家科技部发起召开了 “香山科学会议—基因编辑技术的研究与应用”,针对我国基因编辑研究现状和所面临的机遇和挑战进行了探讨。2017年9月,中国首个基因组编辑学术团体—中国遗传学会基因组编辑分会在北京成立,中国遗传学会名誉理事长李家洋担任大会主席(图2),该学会的成立为从事基因编辑研究的****提供了良好的学术交流平台。2018年4月,第一届基因编辑技术方向的冷泉港国际会议：“冷泉港亚洲会议—基因组编辑全面讨论”于苏州召开,来自世界各地的科学家就基因编辑的最新研究进展开展了广泛而深入的讨论。图2
新窗口打开|下载原图ZIP|生成PPT图2李家洋在中国遗传学会基因组编辑分会成立大会上发言
图片转载自中国遗传学会网站
Fig. 2Jiayang Li spoke at the inaugural meeting of the genome editing branch of the genetics society of China
3 基因编辑技术最新研究进展
随着CRISPR/Cas系统的发现,新一代基因编辑技术在全世界范围引起了研究者的广泛关注。研究者们对CRISPR/Cas系统进行了深入研究,包括对CRISPR/Cas9系统进行优化、拓展CRISPR/Cas系统应用范围、开发RNA编辑系统和利用CRISPR/Cas系统进行高通量筛选。3.1 CRISPR/Cas9系统的优化
对CRISPR/Cas9系统的优化包括探索未知的CRISPR家族蛋白外以及对已知的Cas蛋白进行改造。针对大家较为熟知的Cas9蛋白,改造主要包括以下3个方面。3.1.1 减少Cas9蛋白体积,使其更易于通过病毒载体系统进行体内表达
目前最常用的SpCas9和SaCas9蛋白分别由1368和1053个氨基酸组成,研究者在2017年发现了已知体积最小的Cas9蛋白,CjCas9,仅有984个氨基酸[81],而对已发现的Cas9蛋白的体积减少尚无有效的方法。
3.1.2 减少Cas9蛋白脱靶效应,提高专一性和保真度
在检测Cas9蛋白脱靶效应的研究中,研究者通过对Cas9蛋白在全基因组造成的DSB进行分析来确定其脱靶位点,如GUIDE-seq[82]和Digenome- Seq[83]分析等。每种分析方法各有利弊,目前对于确定Cas9造成的基因组DSB位点仍具有挑战。
在减少Cas9蛋白脱靶效应方面,研究者通过 对SpCas9蛋白部分位点进行突变降低了其脱靶效 应[84, 85]。如将SpCas9的Asn 497、Arg 661、Gln 695、Gln 926氨基酸突变为Ala,得到SpCas9-HF,研究人员通过GUIDE-seq方法对整个基因组进行脱靶分析,发现与SpCas9相比,SpCas9-HF的脱靶显著减少[85]。另一方面,Cas9蛋白具有多个负责不同功能的结构域,研究人员针对SpCas9的REC3结构域(负责识别sgRNA和靶向序列形成的互补链并控制HNH核酸酶以调节总体催化能力)进行突变也获得了更高特异性的HypaCas9[86]。部分研究表明通过结构导向的设计来优化Cas9也能增强其特异性[84]。
除了针对Cas9蛋白本身进行优化外,有研究表明降低Cas9在体内活化的时间也可以降低其脱靶效应,如直接向细胞内递送sgRNA和Cas9蛋白的核糖核苷酸蛋白(RNP)复合物,或将Cas9蛋白拆分为两部分再利用小分子诱导使之在细胞内重新结合成完整的Cas9蛋白[87, 88]。
3.1.3 增加Cas9蛋白的靶向范围,即拓展PAM识别范围
研究者利用筛选或蛋白质进化的手段获得了具有较普遍PAM的SaCas9和SpCas9的变体[50, 89, 90],如Johnny等[50]通过对SpCas9进行突变体筛选得到了可以广泛增加靶向范围的xCas9,其不仅可以识别传统的PAM序列,还可以识别NG,GAA和GAT,大大增加了Cas9的靶向范围。除上述手段外,也有研究者从蛋白质结构的角度优化FnCas9 (Francisella novicida Cas9)从而改变其PAM序列[91]。
3.2 拓展CRISPR/Cas系统的应用范围
除了对目标基因进行切割编辑,CRISPR/Cas系统还被用于其他方面。通过dCas9融合或招募其他酶或者蛋白,即可利用dCas9定位靶向DNA序列的特性将酶或蛋白带到靶向区域,从而进行基因的单碱基编辑、基因表达的激活与抑制、特定位点的表观遗传、特定位点的成像和染色体三维结构等研究[92]。下面以基因的单碱基编辑和基因表达激活与抑制为例进行介绍。3.2.1 碱基编辑系统
Komor等[93]最近报道将nCas9 (nickase Cas9)与APOBEC1脱氨酶和尿嘧啶糖基酶抑制蛋白(uracyl glycosylase inhibitor, UGI)融合,可在靶位点有效地将胞嘧啶(C)转化为胸腺嘧啶(T),这个过程不会引起双链DNA断裂。另外,通过将RNA腺苷脱氨酶进行蛋白质功能进化使之可以使其识别DNA底物,再将其融合至nCas9可以实现靶位点的腺嘌呤(A)到鸟嘌呤(G)的转化[94]。这些单位点的碱基编辑技术极大的拓展了基因编辑的应用范围,实现了对基因的定点编辑。除了APOBEC1蛋白外,激活诱导腺苷脱氨酶(AID)也可以被融合到dCas9后实现定点的碱基编。值得注意的是,在复合物中没有UGI的情况下,dCas9-AID复合物可作为一种强大的局部诱变剂,成为功能获得性筛选的新工具[66, 95, 96]。
3.2.2 CRISPR介导的基因表达调控
在基因表达调控方面,CRISPR系统也有广泛的应用前景。dCas9与DNA有很强的结合能力,因此会阻止其他DNA蛋白与DNA结合从而对基因的表达造成影响[97]。将转录抑制复合物如KRAB与dCas9融合会抑制该位点附近基因的表达,即形成CRISPRi (CRISPR interference)系统[98]。与之相反,将单纯疱疹病毒16-氨基酸长反式激活结构域(VP16)的4个串联拷贝组成的VP64与dCas9融合会介导基因表达的上调,即形成CRISPRa (CRISPR activation)系统[99]。利用上述两套CRISPR介导基因表达调控的方法,研究者可以对感兴趣的基因进行功能研究,或者在全基因组范围内进行基因功能筛选。
3.3 RNA编辑系统
除了针对DNA进行基因编辑,CRISPR蛋白家族中的Cas13可以利用gRNA (guide RNA)靶向RNA。目前已经发现Cas13a (也被称为C2c2)[100]、Cas13b[101]、Cas13c[102]和Cas13d[103]这4种蛋白都具有该功能。上述蛋白由于具有RNA结合特性,从而被发展成为核糖核酸的检测器[104]。在将Cas13蛋白连接作用于RNA腺嘌呤脱氨酶后,通过对gRNA的优化即可针对RNA上特定位点的腺嘌呤进行脱氨,从而使腺嘌呤转化为次黄嘌呤(Inosine, I),生物体内的翻译系统会将I识别为鸟嘌呤,实现腺嘌呤到鸟嘌呤的转变,但目前该技术仍存在脱靶较严重的问题[29]。虽然这项技术仅限于腺嘌呤到鸟嘌呤的转变,但是将Cas13蛋白融合其他脱氨酶或者表观修饰酶后可以大大拓展该技术在RNA编辑、调控中的应用范围。3.4 利用CRISPR/Cas9系统进行高通量筛选
遗传分析的重要目的之一即鉴别引起生物特定表型的基因。其对应的基因筛选方法包括反向(假说法)和正向遗传筛选。反向遗传筛选利用已知研究结果来检测特定基因变异的表型。而正向遗传筛选通过“表型到基因型”的研究方法大范围修饰或调节基因的表达,从而筛选出产生特殊表型的细胞或生物体,进而分析其对应的基因突变,是发现和注释功能性遗传元件的有力工具[105]。通过基因编辑技术在细胞、组织或动物模型水平删除、插入以及改造DNA序列,能够帮助研究者进一步研究基因或调控原件在生物中的功能和机制。目前,CRISPR/Cas9系统已经用于大规模构建基因敲除文库,并可在人类全基因组水平上进行大规模基因或调控原件的筛选及相关研究[44, 45, 106~109]。
在哺乳动物细胞中,CRISPR/Cas9文库筛选是通过一系列步骤进行的,包括gRNA库的构建、慢病毒转导、细胞筛选和数据分析等。针对蛋白质编码基因,最常见的CPISPR筛选为直接利用Cas9对靶基因进行敲除从而对细胞的表型进行筛选。这类筛选主要用于功能丧失型的筛选[44~46, 110]。针对非编码基因,由于单纯的通过Cas9蛋白破坏其结构并不能造成其功能的完全缺失,因此对非编码基因的筛选较蛋白质编码基因的筛选更困难。对于lncRNA (long non-coding RNA)筛选,通过配对gRNA即pgRNA (paired-gRNA)对lncRNA进行大片段的敲除也是lncRNA筛选的手段之一,如Zhu等[68]利用该方法筛选出51个对癌症细胞生长起到促进或者抑制作用的lncRNA。
近期,Han等[107]开发了能够与21 321个药物靶基因组合的包含了490 000对gRNA的配对RNA文库,并在K562细胞中鉴定了几种发挥协同作用的药物组合。Najam等[108]使用两种正交的Cas9酶,即SaCas9和SpCas9,进行正交组合遗传筛选,并通过该方法对基因之间的相互作用进行了大规模分析。
除此之外,CRISPRa和CRISPRi也是非常有效的工具。如Joung等[109]利用CRISPRa对编码基因附近的lncRNA进行筛选;Liu等[106]利用CRISPRi针对7种不同细胞系中的16 401个lncRNA位点进行筛选,发现lncRNA具有很高的细胞特异性。
4 基因编辑技术的应用
基因编辑技术在基因功能研究、药物开发、疾病治疗和作物育种等方面有着重要意义和广阔的应用前景。如前文所述,基因编辑技术可以在全基因组范围进行基因功能研究,除此之外,该技术也被广泛应用于生物治疗以及药物研究等领域。4.1 疾病模型构建
很多疾病的致病机制十分复杂,比如癌症,通常涉及多种抑癌基因或致癌基因的遗传改变。因此构建适合的疾病模型对探索疾病的发生和进展以及抗癌药物的筛选有着重要的意义。对于已知致病基因的疾病,研究人员可以运用CRISPR/Cas9等基因编辑技术,构建对应的基因突变动物或细胞模型,从而进一步进行药物或其他治疗方式的研究。对于功能未知或者部分未知的基因,研究人员可以通过构建疾病模型从而进一步明确疾病与基因之间的 关系。研究人员应用基因编辑技术,已经实现了多种疾病的体内和体外疾病模型的构建。目前,科学家们在鼠科动物上实现了多种疾病模型的构建,如利用CRISPR技术靶向Pten和p53 (两种抑癌基因)构造的肝癌模型[111]以及诱导CD74-ROS1,EML4-ALK 和 KIF5B-RET融合导致的肺癌模型等[112]。对于一些由多基因突变导致的人类复杂疾病,CRISPR/Cas9技术可以同时进行多基因编辑,如Zuckermann等[113]通过在小鼠大脑中单基因敲除(Ptch1)和多基因敲除(Trp53, Pten, Nf1),成功构建了成神经管细胞瘤和成胶质细胞瘤疾病模型。此外,研究人员还应用CRISPR/Cas9技术在小鼠中构建了心肌病和心力衰竭模型[114]。Paquet等[115]利用CRISPR/Cas9技术将特定点突变引入人诱导多能干细胞中,实现了基因单拷贝和多拷贝编辑,从而得到携带阿尔茨海默病相关突变的细胞。
4.2 基因诊断与核酸检测
CRISPR系统具有用一条sgRNA靶向DNA或者RNA的特性,利用该特点研究者开发了一系列的工具用于检测样品中是否存在某种特定的核酸,从而实现即时检测病原体、基因分型和疾病监测等功能。以SHERLOCK (specific high-sensitivity enzymatic reporter unlocking)系统为例,Cas13蛋白识别目标RNA后会被激活从而切割其周围RNA,利用这个特性,可对特定病原体的核酸进行检测。目前该系统已成功用于寨卡病毒和登革热病毒不同菌株的检测,具有灵敏度高、便利廉价的优势[116]。除了针对RNA,研究者还利用Cas12a和Csm6等蛋白,开发了升级版的SHERLOCK系统[104],可以检测出肺癌患者血液样本中的游离肿瘤DNA,并能够在单一反应中同时检测寨卡病毒和登革热病毒。4.3 靶向基因治疗
广义上的基因治疗是指在DNA水平上,通过特定的技术手段用正常的基因来替换或者补偿致病基因突变,从而达到治疗目的。常用的基因治疗方法包括用非病毒载体方法、慢病毒载体或腺病毒载体向体内注射正常基因,或者利用近几年崛起的基因编辑技术纠正致病突变等[117]。传统的基因治疗手段可将正常基因导入细胞,但致病突变依然存在,不能从根本上治愈疾病。而基因编辑技术可以对基因进行精准编辑,从而修复或修饰内源致病突变[117,118,119]。因此,以CRISPR/Cas9系统为代表的基因编辑技术在临床治疗上具有广阔的应用前景。
目前,国内外已经发表了数篇应用基因编辑技术在模式动物中纠正遗传疾病致病基因的报道[120]。2014年,Yin等[121]首次应用CRISPR/Cas9技术实现了体内致病基因编辑。研究人员将CRISPR/Cas9系统注射到患有人类Ⅰ型酪氨酸血症(一种致死遗传病)的疾病模型小鼠肝脏中,纠正了致病基因Fah的突变,并回复了小鼠体重减轻等疾病表型。Dever等[122]利用CRISPR/Cas9技术修复了人体造血干细胞中导致镰刀型细胞贫血症的基因β-globin,并成功注射到小鼠体内,改造的造血干细胞在小鼠体内16周后仍保持正常分化。同年,Science报道了研究人员通过CRISPR/Cas9技术恢复了患杜氏肌营养不良症(Duchenne muscular dystrophy, DMD)小鼠的肌肉功能[123,124,125]。2018年初,研究者应用CRISPR/Cas9技术成功修复了小鼠致聋基因TMC1突变[126]。Hawksworth等[127]利用CRISPR/Cas9技术在人类成体红细胞系中实现了5个血型相关基因的同时敲除,增强其输血相容性,为长期输血的患者的同种免疫以及稀有血型输血时难以匹配血型的难题带来了新的解决方法。
4.4 动植物品系培育
应用CRISPR/Cas9等技术在农作物或动物基因中进行基因编辑,与传统杂交等育种方法相比,可以精确、快速培育出新品种[128]。在植物育种方面,研究者利用基因编辑技术可以加速作物培育过程或获得利于其生长的抗性[129]。如在水稻中对OsEPSPS基因(编码与草甘膦具亲和能力的5-烯醇式丙酮酰-莽草酸-3-磷酸合成酶)进行定点替换,可获得抗草甘膦除草剂的表型[130],以及通过基因敲除、改造启动子等方法加速水稻培育过程、获得抗旱和抗其他类型除草剂的农作物品种等[131,132,133,134]。在动物品系培养方面,研究者同样可以通过基因编辑技术得到具有优良性状的猪、牛、羊等家 畜[135,136,137,138,139,140]。如研究者利用ZFNs[140]和CRISPR/Cas9[141]技术通过MSTN基因敲除获得了高瘦肉率转基因猪。在疾病防治方面,Whitworth等[142]利用CRISPR/ Cas9技术得到的CD163 (清道夫受体,参与机体免疫过程)敲除猪可以对猪繁殖与呼吸综合征（PRRS）病毒耐受。
5 基因编辑技术展望
CRISPR/Cas系统作为近几年来发展起来的新一代基因编辑技术,自问世以来就获得了极大的关注。由于可以精确改变内源致病基因,基因编辑技术有望从根本上治愈某些遗传疾病;通过基因编辑技术得到的新品种中不引入外源基因,可以使作物改良的过程更加迅速和安全。基因编辑技术在临床上的应用已经在世界范围得到重视,如美国国立卫生研究院(NIH)于2018年年初宣布,将启动1.9亿美元用于体细胞基因编辑研究计划。基因编辑技术正逐步改变生命科学和医学研究的面貌。虽然研究者们利用CRISPR/Cas系统开发了很多强有力的基因编辑工具,但是这些工具仍存着一些在问题。首先是递送方式的问题,由于AAV病毒(adeno-associated virus)递送至细胞的DNA片段容量有限,而Cas蛋白过大,因此有必要寻找一个新的较小的Cas蛋白或者人工改造生成较小的Cas蛋白。其次,Cas9蛋白本身及最新开发的碱基编辑技术仍存在脱靶效应,因此开发精准的基因编辑技术十分重要。最后,由于Cas9蛋白是细菌来源的蛋白,近期有报道称大部分人可能存在对Cas9蛋白的适应性免疫反应。因此,上述技术距离临床应用还有很长的路要走。
同时,基因编辑技术的应用上也存在着一些亟待解决的问题。如在靶向基因治疗中,目前该技术应用于人体的案例还鲜有报道,并且追踪时间有限,安全性评估信息不够全面;在靶向基因治疗技术广泛应用于人类之前,科学家们还需要在更多的动物上进行安全性和有效性测试;基因编辑技术的效率和精准性还需要进一步的提高,确保避免脱靶效应可能带来的副作用;在胚胎中进行基因编辑仍面临着伦理方面的争议,在单细胞阶段进行基因编辑还存在遗传嵌合等问题,因此,相应的政策法规还需要进一步完善。
随着研究者的不断深入探索,基因编辑技术正在逐步发展成熟,具有极大的研究潜力和广阔的应用前景。相信在不久的将来,基因编辑技术会在人类生产和临床疾病治疗中发挥其无可替代的价值。
(责任编委: 高彩霞)
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URLPMID:2660260 [本文引用: 2]
Homologous recombination between DNA sequences residing in the chromosome and newly introduced, cloned DNA sequences (gene targeting) allows the transfer of any modification of the cloned gene into the genome of a living cell. This article discusses the current status of gene targeting with particular emphasis on germ line modification of the mouse genome, and describes the different methods so far employed to identify those rare embryonic stem cells in which the desired targeting event has occurred.
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URLPMID:8928227 [本文引用: 1]
Abstract DNA double-strand breaks (DSBs) pose a threat to the genomic integrity of a cell. The failure to heal a break or the inappropriate repair of a break can result in the loss of genetic information and other potentially deleterious consequences, such as chromosomal translocations. Recent developments using rare-cutting endonucleases have allowed investigators to introduce one or a few DSBs into complex genomes. Such studies have begun to elucidate the complex mechanisms of nonhomologous and homologous repair used by mammalian cells to repair these lesions. A key finding is that gene targeting is stimulated two to three orders of magnitude by a DSB at the target locus. Thus, the use of rare-cutting endonucleases and the co-opting of cellular repair mechanisms might provide scientists with another tool for engineering changes into genomes.
URL [本文引用: 1]
[本文引用: 1]
URLPMID:17130168 [本文引用: 1]
Abstract Meganucleases, or homing endonucleases (HEs) are sequence-specific endonucleases with large (>14 bp) cleavage sites that can be used to induce efficient homologous gene targeting in cultured cells and plants. These findings have opened novel perspectives for genome engineering in a wide range of fields, including gene therapy. However, the number of identified HEs does not match the diversity of genomic sequences, and the probability of finding a homing site in a chosen gene is extremely low. Therefore, the design of artificial endonucleases with chosen specificities is under intense investigation. In this report, we describe the first artificial HEs whose specificity has been entirely redesigned to cleave a naturally occurring sequence. First, hundreds of novel endonucleases with locally altered substrate specificity were derived from I-CreI, a Chlamydomonas reinhardti protein belonging to the LAGLIDADG family of HEs. Second, distinct DNA-binding subdomains were identified within the protein. Third, we used these findings to assemble four sets of mutations into heterodimeric endonucleases cleaving a model target or a sequence from the human RAG1 gene. These results demonstrate that the plasticity of LAGLIDADG endonucleases allows extensive engineering, and provide a general method to create novel endonucleases with tailored specificities.
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Abstract Permanent modification of the human genome in vivo is impractical owing to the low frequency of homologous recombination in human cells, a fact that hampers biomedical research and progress towards safe and effective gene therapy. Here we report a general solution using two fundamental biological processes: DNA recognition by C2H2 zinc-finger proteins and homology-directed repair of DNA double-strand breaks. Zinc-finger proteins engineered to recognize a unique chromosomal site can be fused to a nuclease domain, and a double-strand break induced by the resulting zinc-finger nuclease can create specific sequence alterations by stimulating homologous recombination between the chromosome and an extrachromosomal DNA donor. We show that zinc-finger nucleases designed against an X-linked severe combined immune deficiency (SCID) mutation in the IL2Rgamma gene yielded more than 18% gene-modified human cells without selection. Remarkably, about 7% of the cells acquired the desired genetic modification on both X chromosomes, with cell genotype accurately reflected at the messenger RNA and protein levels. We observe comparably high frequencies in human T cells, raising the possibility of strategies based on zinc-finger nucleases for the treatment of disease.
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A long-term goal in the field of restriction-modification enzymes has been to generate restriction endonucleases with novel sequence specificities by mutating or engineering existing enzymes. This will avoid the increasingly arduous task of extensive screening of bacteria and other microorganisms for new enzymes. Here, we report the deliberate creation of novel site-specific endonucleases by linking two different zinc finger proteins to the cleavage domain of Fok I endonuclease. Both fusion proteins are active and under optimal conditions cleave DNA in a sequence-specific manner. Thus, the modular structure of Fok I endonuclease and the zinc finger motifs makes it possible to create ``artificial'' nucleases that will cut DNA near a predetermined site. This opens the way to generate many new enzymes with tailor-made sequence specificities desirable for various applications.
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Abstract Reverse genetics in model organisms such as Drosophila melanogaster, Arabidopsis thaliana, zebrafish and rats, efficient genome engineering in human embryonic stem and induced pluripotent stem cells, targeted integration in crop plants, and HIV resistance in immune cells - this broad range of outcomes has resulted from the application of the same core technology: targeted genome cleavage by engineered, sequence-specific zinc finger nucleases followed by gene modification during subsequent repair. Such 'genome editing' is now established in human cells and a number of model organisms, thus opening the door to a range of new experimental and therapeutic possibilities.
URLPMID:3017587 [本文引用: 1]
DNA double-strand breaks enhance homologous recombination in cells and have been exploited for targeted genome editing through use of engineered endonucleases. Here we report the creation and initial characterization of a group of rare-cutting, site-specific DNA nucleases produced by fusion of the restriction enzyme FokI endonuclease domain (FN) with the high-specificity DNA-binding domains of AvrXa7 and PthXo1. AvrXa7 and PthXo1 are members of the transcription activator-like (TAL) effector family whose central repeat units dictate target DNA recognition and can be modularly constructed to create novel DNA specificity. The hybrid FN-AvrXa7, AvrXa7-FN and PthXo1-FN proteins retain both recognition specificity for their target DNA (a 26 p sequence for AvrXa7 and 24 p for PthXo1) and the double-stranded DNA cleaving activity of FokI and, thus, are called TAL nucleases (TALNs). With all three TALNs, DNA is cleaved adjacent to the TAL-binding site under optimal conditions in vitro. When expressed in yeast, the TALNs promote DNA homologous recombination of a LacZ gene containing paired AvrXa7 or asymmetric AvrXa7/PthXo1 target sequences. Our results demonstrate the feasibility of creating a tool box of novel TALNs with potential for targeted genome modification in organisms lacking facile mechanisms for targeted gene knockout and homologous recombination.
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URLPMID:20660643 [本文引用: 1]
Abstract Engineered nucleases that cleave specific DNA sequences in vivo are valuable reagents for targeted mutagenesis. Here we report a new class of sequence-specific nucleases created by fusing transcription activator-like effectors (TALEs) to the catalytic domain of the FokI endonuclease. Both native and custom TALE-nuclease fusions direct DNA double-strand breaks to specific, targeted sites.
URLPMID:21179091 [本文引用: 1]
Nucleases that cleave unique genomic sequences in living cells can be used for targeted gene editing and mutagenesis. Here we develop a strategy for generating such reagents based on transcription activator-like effector (TALE) proteins from Xanthomonas. We identify TALE truncation variants that efficiently cleave DNA when linked to the catalytic domain of FokI and use these nucleases to generate discrete edits or small deletions within endogenous human NTF3 and CCR5 genes at efficiencies of up to 25%. We further show that designed TALEs can regulate endogenous mammalian genes. These studies demonstrate the effective application of designed TALE transcription factors and nucleases for the targeted regulation and modification of endogenous genes.
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URLPMID:22484455 [本文引用: 1]
Engineered transcription activator–like effector nucleases (TALENs) have shown promise as facile and broadly applicable genome editing tools. However, no publicly available high-throughput method for constructing TALENs has been published, and large-scale assessments of the success rate and targeting range of the technology remain lacking. Here we describe the fast ligation-based automatable solid-phase high-throughput (FLASH) system, a rapid and cost-effective method for large-scale assembly of TALENs. We tested 48 FLASH-assembled TALEN pairs in a human cell–based EGFP reporter system and found that all 48 possessed efficient gene-modification activities. We also used FLASH to assemble TALENs for 96 endogenous human genes implicated in cancer and/or epigenetic regulation and found that 84 pairs were able to efficiently introduce targeted alterations. Our results establish the robustness of TALEN technology and demonstrate that FLASH facilitates high-throughput genome editing at a scale not currently possible with other genome modification technologies.
URLPMID:26000843 [本文引用: 1]
Boettcher and McManus provide a practical guide to choosing the right tool for your gene silencing experiment—RNAi, TALEN, or CRISPR.
URLPMID:4011339 [本文引用: 1]
Cell death and differentiation is a monthly research journal focused on the exciting field of programmed cell death and apoptosis. It provides a single accessible source of information for both scientists and clinicians, keeping them up-to-date with advances in the field. It encompasses programmed cell death, cell death induced by toxic agents, differentiation and the interrelation of these with cell proliferation.
URLPMID:5638953 [本文引用: 1]
Abstract DNA recognition by transcription activator-like effector (TALE) proteins is mediated by tandem repeats that specify nucleotides through repeat-variable diresidues. These repeat-variable diresidues form direct and sequence-specific contacts to DNA bases; hence, TALE-DNA interaction is sensitive to DNA chemical modifications. Here we conduct a thorough investigation, covering all theoretical repeat-variable diresidue combinations, for their recognition capabilities for 5-methylcytosine and 5-hydroxymethylcytosine, two important epigenetic markers in higher eukaryotes. We identify both specific and degenerate repeat-variable diresidues for 5-methylcytosine and 5-hydroxymethylcytosine. Utilizing these novel repeat-variable diresidues, we achieve methylation-dependent gene activation and genome editing in vivo; we also report base-resolution detection of 5hmC in an in vitro assay. Our work deciphers repeat-variable diresidues for 5-methylcytosine and 5-hydroxymethylcytosine, and provides tools for TALE-dependent epigenome recognition.Transcription activator-like effector proteins recognise specific DNA sequences via tandem repeats. Here the authors demonstrate TALEs can recognise the methylated bases 5mC and 5hmC, enabling them to detect epigenetic modifications.
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Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addition of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with associated cas genes, provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity.
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URLPMID:26593719 [本文引用: 1]
Abstract Microbial CRISPR-Cas systems are divided into Class 1, with multisubunit effector complexes, and Class 2, with single protein effectors. Currently, only two Class 2 effectors, Cas9 and Cpf1, are known. We describe here three distinct Class 2 CRISPR-Cas systems. The effectors of two of the identified systems, C2c1 and C2c3, contain RuvC-like endonuclease domains distantly related to Cpf1. The third system, C2c2, contains an effector with two predicted HEPN RNase domains. Whereas production of mature CRISPR RNA (crRNA) by C2c1 depends on tracrRNA, C2c2 crRNA maturation is tracrRNA independent. We found that C2c1 systems can mediate DNA interference in a 5'-PAM-dependent fashion analogous to Cpf1. However, unlike Cpf1, which is a single-RNA-guided nuclease, C2c1 depends on both crRNA and tracrRNA for DNA cleavage. Finally, comparative analysis indicates that Class 2 CRISPR-Cas systems evolved on multiple occasions through recombination of Class 1 adaptation modules with effector proteins acquired from distinct mobile elements. Copyright 2015 Elsevier Inc. All rights reserved.
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URLPMID:29070703 [本文引用: 3]
Abstract Nucleic acid editing holds promise for treating genetic disease, particularly at the RNA level, where disease-relevant sequences can be rescued to yield functional protein products. Type VI CRISPR-Cas systems contain the programmable single-effector RNA-guided RNases Cas13. Here, we profile Type VI systems to engineer a Cas13 ortholog capable of robust knockdown and demonstrate RNA editing by using catalytically-inactive Cas13 (dCas13) to direct adenosine to inosine deaminase activity by ADAR2 to transcripts in mammalian cells. This system, referred to as RNA Editing for Programmable A to I Replacement (REPAIR), which has no strict sequence constraints, can be used to edit full-length transcripts containing pathogenic mutations. We further engineer this system to create a high specificity variant and minimize the system to facilitate viral delivery. REPAIR presents a promising RNA editing platform with broad applicability for research, therapeutics, and biotechnology. Copyright 2017, American Association for the Advancement of Science.
URLPMID:28729350 [本文引用: 1]
Abstract CRISPR-Cas systems depend on the Cas1-Cas2 integrase to capture and integrate short foreign DNA fragments into the CRISPR locus, enabling adaptation to new viruses. We present crystal structures of Cas1-Cas2 bound to both donor and target DNA in intermediate and product integration complexes, as well as a cryo-electron microscopy structure of the full CRISPR locus integration complex including the accessory protein Integration Host Factor (IHF). The structures show unexpectedly that indirect sequence recognition dictates integration site selection by favoring deformation of the repeat and the flanking sequences. IHF binding bends the DNA sharply, bringing an upstream recognition motif into contact with Cas1 to increase both the specificity and efficiency of integration. These results explain how the Cas1-Cas2 CRISPR integrase recognizes a sequence-dependent DNA structure to ensure site-selective CRISPR array expansion during the initial step of bacterial adaptive immunity. Copyright 2017, American Association for the Advancement of Science.
URLPMID:29590607 [本文引用: 1]
Abstract CRISPR-Cas systems adapt their immunological memory against their invaders by integrating short DNA fragments into clustered regularly interspaced short palindromic repeat (CRISPR) loci. While Cas1 and Cas2 make up the core machinery of the CRISPR integration process, various class I and II CRISPR-Cas systems encode Cas4 proteins for which the role is unknown. Here, we introduced the CRISPR adaptation genes cas1, cas2, and cas4 from the type I-D CRISPR-Cas system of Synechocystis sp. 6803 into Escherichia coli and observed that cas4 is strictly required for the selection of targets with protospacer adjacent motifs (PAMs) conferring I-D CRISPR interference in the native host Synechocystis. We propose a model in which Cas4 assists the CRISPR adaptation complex Cas1-2 by providing DNA substrates tailored for the correct PAM. Introducing functional spacers that target DNA sequences with the correct PAM is key to successful CRISPR interference, providing a better chance of surviving infection by mobile genetic elements.
URLPMID:22402487 [本文引用: 1]
The clustered regularly interspaced short palindromic repeats and their associated proteins (CRISPR/Cas) constitute a recently identified prokaryotic defense mechanism against invading nucleic acids. Activity of the CRISPR/Cas system comprises of three steps: (i) insertion of alien DNA sequences into the CRISPR array to prevent future attacks, in a process called ‘adaptation’, (ii) expression of the relevant proteins, as well as expression and processing of the array, followed by (iii) RNA-mediated interference with the alien nucleic acid. Here we describe a robust assay in Escherichia coli to explore the hitherto least-studied process, adaptation. We identify essential genes and DNA elements in the leader sequence and in the array which are essential for the adaptation step. We also provide mechanistic insights on the insertion of the repeat-spacer unit by showing that the first repeat serves as the template for the newly inserted repeat. Taken together, our results elucidate fundamental steps in the adaptation process of the CRISPR/Cas system.
URLPMID:4385744 [本文引用: 1]
Clustered regularly interspaced short palindromic repeat (CRISPR) loci and their associated (Cas) proteins provide adaptive immunity against viral infection in prokaryotes. Upon infection, short phage sequences known as spacers integrate between CRISPR repeats and are transcribed into small RNA molecules that guide the Cas9 nuclease to the viral targets (protospacers). Streptococcus pyogenes Cas9 cleavage of the viral genome requires the presence of a 5'-NGG-3' protospacer adjacent motif (PAM) sequence immediately downstream of the viral target. It is not known whether and how viral sequences flanked by the correct PAM are chosen as new spacers. Here we show that Cas9 selects functional spacers by recognizing their PAM during spacer acquisition. The replacement of cas9 with alleles that lack the PAM recognition motif or recognize an NGGNG PAM eliminated or changed PAM specificity during spacer acquisition, respectively. Cas9 associates with other proteins of the acquisition machinery (Cas1, Cas2 and Csn2), presumably to provide PAM-specificity to this process. These results establish a new function for Cas9 in the genesis of prokaryotic immunological memory.
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URLPMID:24766887 [本文引用: 1]
Abstract Clustered regularly interspaced short palindromic repeats (CRISPR), and associated proteins (Cas) comprise the CRISPR-Cas system, which confers adaptive immunity against exogenic elements in many bacteria and most archaea. CRISPR-mediated immunization occurs through the uptake of DNA from invasive genetic elements such as plasmids and viruses, followed by its integration into CRISPR loci. These loci are subsequently transcribed and processed into small interfering RNAs that guide nucleases for specific cleavage of complementary sequences. Conceptually, CRISPR-Cas shares functional features with the mammalian adaptive immune system, while also exhibiting characteristics of Lamarckian evolution. Because immune markers spliced from exogenous agents are integrated iteratively in CRISPR loci, they constitute a genetic record of vaccination events and reflect environmental conditions and changes over time. Cas endonucleases, which can be reprogrammed by small guide RNAs have shown unprecedented potential and flexibility for genome editing and can be repurposed for numerous DNA targeting applications including transcriptional control. Copyright 2014 Elsevier Inc. All rights reserved.
URLPMID:24476820 [本文引用: 1]
Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)-associated enzyme Cas9 is an RNA-guided endonuclease that uses RNA-DNA base-pairing to target foreign DNA in bacteria. Cas9-guide RNA complexes are also effective genome engineering agents in animals and plants. Here we use single-molecule and bulk biochemical experiments to determine how Cas9-RNA interrogates DNA to find specific cleavage sites. We show that both binding and cleavage of DNA by Cas9-RNA require recognition of a short trinucleotide protospacer adjacent motif (PAM). Non-target DNA binding affinity scales with PAM density, and sequences fully complementary to the guide RNA but lacking a nearby PAM are ignored by Cas9-RNA. Competition assays provide evidence that DNA strand separation and RNA-DNA heteroduplex formation initiate at the PAM and proceed directionally towards the distal end of the target sequence. Furthermore, PAM interactions trigger Cas9 catalytic activity. These results reveal how Cas9 uses PAM recognition to quickly identify potential target sites while scanning large DNA molecules, and to regulate scission of double-stranded DNA.
URLPMID:4139937 [本文引用: 1]
Crystal structure of Cas9 in complex with single guide RNA and target DNA elucidates the molecular mechanism of RNA-guided DNA targeting by Cas9.
URLPMID:22745249 [本文引用: 1]
Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
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URLPMID:23287722 [本文引用: 1]
Bacteria and archaea have evolved adaptive immune defenses termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components, is sequence-specific, and upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190k unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.
URLPMID:25307932 [本文引用: 1]
Abstract While the catalog of mammalian transcripts and their expression levels in different cell types and disease states is rapidly expanding, our understanding of transcript function lags behind. We present a robust technology enabling systematic investigation of the cellular consequences of repressing or inducing individual transcripts. We identify rules for specific targeting of transcriptional repressors (CRISPRi), typically achieving 90%-99% knockdown with minimal off-target effects, and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Together they enable modulation of gene expression over a 0908041,000-fold range. Using these rules, we construct genome-scale CRISPRi and CRISPRa libraries, each of which we validate with two pooled screens. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. Screens for sensitivity to a cholera-diphtheria toxin provide broad insights into the mechanisms of pathogen entry, retrotranslocation and toxicity. Our results establish CRISPRi and CRISPRa as powerful tools that provide rich and complementary information for mapping complex pathways. Copyright 0008 2014 Elsevier Inc. All rights reserved.
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URLPMID:26189680 [本文引用: 1]
A protein marker-based, genome-wide CRISPR screen has been developed in primary immune cells to identify genes that control the induction of tumor necrosis factor. Many of the known regulators, as well as dozens of previously unknown candidates, have been identified, individually validated, and classified into three functional modules.
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URLPMID:4089965 [本文引用: 2]
The simplicity of programming the CRISPR (clustered regularly interspaced short palindromic repeats) ssociated nuclease Cas9 to modify specific genomic loci suggests a new way to interrogate gene function on a genome-wide scale. We show that lentiviral delivery of a genome-scale CRISPR-Cas9 knockout (GeCKO) library targeting 18,080 genes with 64,751 unique guide sequences enables both negative and positive selection screening in human cells. First, we used the GeCKO library to identify genes essential for cell viability in cancer and pluripotent stem cells. Next, in a melanoma model, we screened for genes whose loss is involved in resistance to vemurafenib, a therapeutic RAF inhibitor. Our highest-ranking candidates include previously validated genes NF1 and MED12, as well as novel hits NF2, CUL3, TADA2B, and TADA1. We observe a high level of consistency between independent guide RNAs targeting the same gene and a high rate of hit confirmation, demonstrating the promise of genome-scale screening with Cas9.
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CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, nonspecific single-stranded deoxyribonuclease (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA endonuclease-targeted CRISPR trans reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics.
URLPMID:23452860 [本文引用: 1]
The authors have developed a CRISPR interference system in which a catalytically dead Cas9 protein can be targeted to a specific genomic site through a complementary small guide RNA, allowing systematic perturbation of gene transcription in bacteria and mammalian cells.
URLPMID:29512652 [本文引用: 3]
Programmable DNA nucleases have provided scientists with the unprecedented ability to probe, regulate, and manipulate the human genome. Zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeat-Cas9 system (CRISPR-Cas9) represent a powerful array of tools that can bind to and cleave a specified DNA... [Show full abstract]
URLPMID:27096362 [本文引用: 1]
Abstract CRISPR-Cas systems that provide defence against mobile genetic elements in bacteria and archaea have evolved a variety of mechanisms to target and cleave RNA or DNA. The well-studied types I, II and III utilize a set of distinct CRISPR-associated (Cas) proteins for production of mature CRISPR RNAs (crRNAs) and interference with invading nucleic acids. In types I and III, Cas6 or Cas5d cleaves precursor crRNA (pre-crRNA) and the mature crRNAs then guide a complex of Cas proteins (Cascade-Cas3, type I; Csm or Cmr, type III) to target and cleave invading DNA or RNA. In type II systems, RNase III cleaves pre-crRNA base-paired with trans-activating crRNA (tracrRNA) in the presence of Cas9 (refs 13, 14). The mature tracrRNA-crRNA duplex then guides Cas9 to cleave target DNA. Here, we demonstrate a novel mechanism in CRISPR-Cas immunity. We show that type V-A Cpf1 from Francisella novicida is a dual-nuclease that is specific to crRNA biogenesis and target DNA interference. Cpf1 cleaves pre-crRNA upstream of a hairpin structure formed within the CRISPR repeats and thereby generates intermediate crRNAs that are processed further, leading to mature crRNAs. After recognition of a 5'-YTN-3' protospacer adjacent motif on the non-target DNA strand and subsequent probing for an eight-nucleotide seed sequence, Cpf1, guided by the single mature repeat-spacer crRNA, introduces double-stranded breaks in the target DNA to generate a 5' overhang. The RNase and DNase activities of Cpf1 require sequence- and structure-specific binding to the hairpin of crRNA repeats. Cpf1 uses distinct active domains for both nuclease reactions and cleaves nucleic acids in the presence of magnesium or calcium. This study uncovers a new family of enzymes with specific dual endoribonuclease and endonuclease activities, and demonstrates that type V-A constitutes the most minimalistic of the CRISPR-Cas systems so far described.
URLPMID:27114038 [本文引用: 1]
Abstract Cpf1 is an RNA-guided endonuclease of a type V CRISPR-Cas system that has been recently harnessed for genome editing. Here, we report the crystal structure of Acidaminococcus sp. Cpf1 (AsCpf1) in complex with the guide RNA and its target DNA at 2.80203 resolution. AsCpf1 adopts a bilobed architecture, with the RNA-DNA heteroduplex bound inside the central channel. The structural comparison of AsCpf1 with Cas9, a type II CRISPR-Cas nuclease, reveals both striking similarity and major differences,02thereby explaining their distinct functionalities. AsCpf1 contains the RuvC domain and a putative novel nuclease domain, which are responsible for cleaving the non-target and target strands, respectively, and for jointly generating staggered DNA double-strand breaks. AsCpf1 recognizes the 5'-TTTN-3' protospacer adjacent motif by base and shape readout mechanisms. Our findings provide mechanistic insights into RNA-guided DNA cleavage by Cpf1 and establish a framework for rational engineering of the CRISPR-Cpf1 toolbox. Copyright 08 2016 Elsevier Inc. All rights reserved.
URL [本文引用: 1]
CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, nonspecific single-stranded deoxyribonuclease (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA endonuclease-targeted CRISPR trans reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics.
URLPMID:29531313 [本文引用: 1]
Develop new tools
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Targeting of multiple genomic loci with Cas9 is limited by the need for multiple or large expression constructs. Here we show that the ability of Cpf1 to process its own CRISPR RNA (crRNA) can be used to simplify multiplexed genome editing. Using a single customized CRISPR array, we edit up to four genes in mammalian cells and three in the mouse brain, simultaneously.
URLPMID:28315752 [本文引用: 1]
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Abstract Targeted and inducible regulation of mammalian gene expression is a broadly important capability. We engineered drug-inducible catalytically inactive Cpf1 nuclease fused to transcriptional activation domains to tune the expression of endogenous genes in human cells. Leveraging the multiplex capability of the Cpf1 platform, we demonstrate both synergistic and combinatorial gene expression in human cells. Our work should enable the development of multiplex gene perturbation library screens for understanding complex cellular phenotypes.
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URLPMID:29567453 [本文引用: 1]
Cereals high in amylose content (AC) and resistant starch (RS) offer potential health benefits. Previous studies using chemical mutagenesis or RNA interference have demonstrated that starch branching enzyme (SBE) plays a major role in determining the fine structure and physical properties of starch. However, it remains a challenge to control starch branching in commercial lines. Here, we use... [Show full abstract]
URLPMID:28448066 [本文引用: 1]
CRISPR-Cas9 systems are bacterial adaptive immune systems that defend against infection by phages. Through the RNA-guided endonuclease activity of Cas9 they degrade double-stranded DNA with a protospacer adjacent motif (PAM) and sequences complementary to the guide RNA. Recently, two anti-CRISPR proteins (AcrIIA2 and AcrIIA4 from Listeria monocytogenes prophages) were identified, both of which inhibit Streptococcus pyogenes Cas9 (SpyCas9) and L. monocytogenes Cas9 activity in bacteria and human cells. However, the mechanism of AcrIIA2- or AcrIIA4-mediated Cas9 inhibition remains unknown. Here we report a crystal structure of SpyCas9 in complex with a single-guide RNA (sgRNA) and AcrIIA4. Our data show that AcrIIA2 and AcrIIA4 interact with SpyCas9 in a sgRNA-dependent manner. The structure reveals that AcrIIA4 inhibits SpyCas9 activity by structurally mimicking the PAM to occupy the PAM-interacting site in the PAM-interacting domain, thereby blocking recognition of double-stranded DNA substrates by SpyCas9. AcrIIA4 further inhibits the endonuclease activity of SpyCas9 by shielding its RuvC active site. Structural comparison reveals that formation of the AcrIIA4-binding site of SpyCas9 is induced by sgRNA binding. Our study reveals the mechanism of SpyCas9 inhibition by AcrIIA4, providing a structural basis for developing ff-switch tools for SpyCas9 to avoid unwanted genome edits within cells and tissues.
URLPMID:27096363 [本文引用: 1]
The CRISPR-Cas systems, as exemplified by CRISPR-Cas9, are RNA-guided adaptive immune systems used by bacteria and archaea to defend against viral infection. The CRISPR-Cpf1 system, a new class 2 CRISPR-Cas system, mediates robust DNA interference in human cells. Although functionally conserved, Cpf1 and Cas9 differ in many aspects including their guide RNAs and substrate specificity. Here we report the 2.38 crystal structure of the CRISPR RNA (crRNA)-bound Lachnospiraceae bacterium ND2006 Cpf1 (LbCpf1). LbCpf1 has a triangle-shaped architecture with a large positively charged channel at the centre. Recognized by the oligonucleotide-binding domain of LbCpf1, the crRNA adopts a highly distorted conformation stabilized by extensive intramolecular interactions and the (Mg(H2O)6)(2+) ion. The oligonucleotide-binding domain also harbours a looped-out helical domain that is important for LbCpf1 substrate binding. Binding of crRNA or crRNA lacking the guide sequence induces marked conformational changes but no oligomerization of LbCpf1. Our study reveals the crRNA recognition mechanism and provides insight into crRNA-guided substrate binding of LbCpf1, establishing a framework for engineering LbCpf1 to improve its efficiency and specificity for genome editing.
URLPMID:28757251 [本文引用: 1]
Cas13a, a type VI-A CRISPR-Cas RNA-guided RNA ribonuclease, degrades invasive RNAs targeted by CRISPR RNA (crRNA) and has potential applications in RNA technology. To understand how Cas13a is activated to cleave RNA, we have determined the crystal structure of Leptotrichia buccalis (Lbu) Cas13a bound to crRNA and its target RNA, as well as the cryo-EM structure of the LbuCas13a-crRNA complex. The crRNA-target RNA duplex binds in a positively charged central channel of the nuclease (NUC) lobe, and Cas13a protein and crRNA undergo a significant conformational change upon target RNA binding. The guide-target RNA duplex formation triggers HEPN1 domain to move toward HEPN2 domain, activating the HEPN catalytic site of Cas13a protein, which subsequently cleaves both single-stranded target and collateral RNAs in a non-specific manner. These findings reveal how Cas13a of type VI CRISPR-Cas systems defend against RNA phages and set the stage for its development as a tool for RNA manipulation.
URLPMID:28086085 [本文引用: 1]
C2c2, the effector of type VI CRISPR-Cas systems, has two RNase activities ne for cutting its RNA target and the other for processing the CRISPR RNA (crRNA). Here, we report the structures of Leptotrichia shahii C2c2 in its crRNA-free and crRNA-bound states. While C2c2 has a bilobed structure reminiscent of all other Class 2 effectors, it also exhibits different structural characteristics. It contains the REC lobe with a Helical-1 domain and the NUC lobe with two HEPN domains. The two RNase catalytic pockets responsible for cleaving pre-crRNA and target RNA are independently located on Helical-1 and HEPN domains, respectively. crRNA binding induces significant conformational changes that are likely to stabilize crRNA binding and facilitate target RNA recognition. These structures provide important insights into the molecular mechanism of dual RNase activities of C2c2 and establish a framework for its future engineering as a RNA editing tool.
URLPMID:26478180 [本文引用: 1]
Abstract Bacteria acquire memory of viral invaders by incorporating invasive DNA sequence elements into the host CRISPR locus, generating a new spacer within the CRISPR array. We report on the structures of Cas1-Cas2-dual-forked DNA complexes in an effort toward understanding how the protospacer is sampled prior to insertion into the CRISPR locus. Our study reveals a protospacer DNA comprising a 23-bp duplex bracketed by tyrosine residues, together with anchored flanking 3' overhang segments. The PAM-complementary sequence in the 3' overhang is recognized by the Cas1a catalytic subunits in a base-specific manner, and subsequent cleavage at positions 5 nt from the duplex boundary generates a 33-nt DNA intermediate that is incorporated into the CRISPR array via a cut-and-paste mechanism. Upon protospacer binding, Cas1-Cas2 undergoes a significant conformational change, generating a flat surface conducive to proper protospacer recognition. Here, our study provides important structure-based mechanistic insights into PAM-dependent spacer acquisition. Copyright 2015 Elsevier Inc. All rights reserved.
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Gene editing in non-human primates may lead to valuable models for exploring the etiologies and therapeutic strategies of genetically based neurological disorders in humans. However, a monkey model of neurological disorders that closely mimics pathological and behavioral deficits in humans has not yet been successfully generated. Microcephalin 1 (MCPH1) is implicated in the evolution of the human brain, andMCPH1mutation causes microcephaly accompanied by mental retardation. Here we generated a cynomolgus monkey (Macaca fascicularis) carrying biallelicMCPH1mutations using transcription activator-like effector nucleases. The monkey recapitulated most of the important clinical features observed in patients, including marked reductions in head circumference, premature chromosome condensation (PCC), hypoplasia of the corpus callosum and upper limb spasticity. Moreover, overexpression ofMCPH1in mutated dermal fibroblasts rescued the PCC syndrome. This monkey model may help us elucidate the role ofMCPH1in the pathogenesis of human microcephaly and better understand the function of this protein in the evolution of primate brain size.
URLPMID:28525759 [本文引用: 1]
Subject Code:H09With the support by the National Natural Science Foundation of China,a collaborative study by the research group led by Prof.Chen Yongchang(陈永昌)and Ji Weizhi from the Yunnan Key Laboratory of Primate Biomedicine Research&Institute of Primate Translational Medicine,Kunming University of
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Knock-in mice generated by HMEJ-mediated targeted integration
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Single DNA base pairs are edited in wheat, rice and maize using a Cas9 nickase fusion protein.
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The article offers information on genome modification of crop plants using a CRISPR-Cas system. It states that genome editing technologies using zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can also generate genome modifications. Photographs related to genome editing in rice and wheat using an engineered type II CRISPR-Cas system are also presented.
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Gene-editing technique to treat lung cancer is due to be tested in people in August.
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Several CRISPR-Cas9 orthologues have been used for genome editing. Here, we present the smallest Cas9 orthologue characterized to date, derived fromCampylobacter jejuni(CjCas9), for efficient genome editingin vivo. After determining protospacer-adjacent motif (PAM) sequences and optimizing single-guide RNA (sgRNA) length, we package the CjCas9 gene, its sgRNA sequence, and a marker gene in an all-in-one adeno-associated virus (AAV) vector and produce the resulting virus at a high titer. CjCas9 is highly specific, cleaving only a limited number of sites in the human or mouse genome. CjCas9, delivered via AAV, induces targeted mutations at high frequencies in mouse muscle cells or retinal pigment epithelium (RPE) cells. Furthermore, CjCas9 targeted to theVegfaorHif1agene in RPE cells reduces the size of laser-induced choroidal neovascularization, suggesting thatin vivogenome editing with CjCas9 is a new option for the treatment of age-related macular degeneration. The amount of genetic material that can be packaged in AAV vectors used for genome editing is limited. Here the authors show that the smallest known Cas9 orthologue, cjCas9, can be packaged in a single AAV vector along with sgRNA and a marker gene, and demonstrate efficient gene editing in mice.
URLPMID:4320685 [本文引用: 1]
Abstract CRISPR RNA-guided nucleases (RGNs) are widely used genome-editing reagents, but methods to delineate their genome-wide, off-target cleavage activities have been lacking. Here we describe an approach for global detection of DNA double-stranded breaks (DSBs) introduced by RGNs and potentially other nucleases. This method, called genome-wide, unbiased identification of DSBs enabled by sequencing (GUIDE-seq), relies on capture of double-stranded oligodeoxynucleotides into DSBs. Application of GUIDE-seq to 13 RGNs in two human cell lines revealed wide variability in RGN off-target activities and unappreciated characteristics of off-target sequences. The majority of identified sites were not detected by existing computational methods or chromatin immunoprecipitation sequencing (ChIP-seq). GUIDE-seq also identified RGN-independent genomic breakpoint 'hotspots'. Finally, GUIDE-seq revealed that truncated guide RNAs exhibit substantially reduced RGN-induced, off-target DSBs. Our experiments define the most rigorous framework for genome-wide identification of RGN off-target effects to date and provide a method for evaluating the safety of these nucleases before clinical use.
URLPMID:25664545 [本文引用: 1]
Abstract Although RNA-guided genome editing via the CRISPR-Cas9 system is now widely used in biomedical research, genome-wide target specificities of Cas9 nucleases remain controversial. Here we present Digenome-seq, in vitro Cas9-digested whole-genome sequencing, to profile genome-wide Cas9 off-target effects in human cells. This in vitro digest yields sequence reads with the same 5' ends at cleavage sites that can be computationally identified. We validated off-target sites at which insertions or deletions were induced with frequencies below 0.1%, near the detection limit of targeted deep sequencing. We also showed that Cas9 nucleases can be highly specific, inducing off-target mutations at merely several, rather than thousands of, sites in the entire genome and that Cas9 off-target effects can be avoided by replacing 'promiscuous' single guide RNAs (sgRNAs) with modified sgRNAs. Digenome-seq is a robust, sensitive, unbiased and cost-effective method for profiling genome-wide off-target effects of programmable nucleases including Cas9.
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The RNA-guided CRISPR-Cas9 nuclease from Streptococcus pyogenes (SpCas9) has been widely repurposed for genome editing1–4. High-fidelity (SpCas9-HF1) and enhanced specificity (eSpCas9(1.1)) variants exhibit substantially reduced off-target cleavage in human cells, but the mechanism of target discrimination and the potential to further improve fidelity were unknown5–9. Using single-molecule F02rster resonance energy transfer (smFRET) experiments, we show that both SpCas9-HF1 and eSpCas9(1.1) are trapped in an inactive state10 when bound to mismatched targets. We find that a non-catalytic domain within Cas9, REC3, recognizes target complementarity and governs the HNH nuclease to regulate overall catalytic competence. Exploiting this observation, we designed a new hyper-accurate Cas9 variant (HypaCas9) that demonstrates high genome-wide specificity without compromising on-target activity in human cells. These results offer a more comprehensive model to rationalize and modify the balance between target recognition and nuclease activation for precision genome editing.
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Engineered CRISPR-Cas9 nucleases with altered and improved PAM specificities and their use in genomic engineering, epigenomic engineering, and genome targeting.
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Autoimmune diseases are enigmatic and complex, and most been associated with epigenetic changes. Epigenetics describes changes in gene expression related to environmental influences mediated by a variety of effectors that alter the three-dimensional structure of chromatin and facilitate transcription factor or repressor binding. Recent years have witnessed a dramatic change and acceleration in... [Show full abstract]
URLPMID:27096365 [本文引用: 1]
Current genome-editing technologies introduce double-stranded (ds) DNA breaks at a target locus as the first step to gene correction.1,2Although most genetic diseases arise from point mutations, current approaches to point mutation correction are inefficient and typically induce an abundance of random insertions and deletions (indels) at the target locus from the cellular response to dsDNA breaks.1,2Here we report the development of base editing, a new approach to genome editing that enables the direct, irreversible conversion of one target DNA base into another in a programmable manner, without requiring dsDNA backbone cleavage or a donor template. We engineered fusions of CRISPR/Cas9 and a cytidine deaminase enzyme that retain the ability to be programmed with a guide RNA, do not induce dsDNA breaks, and mediate the direct conversion of cytidine to uridine, thereby effecting a C→T (or G→A) substitution. The resulting “base editors” convert cytidines within a window of approximately five nucleotides (nt), and can efficiently correct a variety of point mutations relevant to human disease. In four transformed human and murine cell lines, second- and third-generation base editors that fuse uracil glycosylase inhibitor (UGI), and that use a Cas9 nickase targeting the non-edited strand, manipulate the cellular DNA repair response to favor desired base-editing outcomes, resulting in permanent correction of 6515-75% of total cellular DNA with minimal (typically ≤ 1%) indel formation. Base editing expands the scope and efficiency of genome editing of point mutations.
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Recruiting a hyperactive cytidine deaminase via the guide RNA to dCas9 allows for the introduction of diverse point mutations at the CRISPR target locus to create complex libraries of variants for protein engineering or dissection of protein function.
URLPMID:27898061 [本文引用: 1]
Abstract Combining CRISPR-Cas9 with an enzyme that induces somatic hypermutations allows the rapid generation of diverse variants for gain-of-function screens.
URLPMID:23452860 [本文引用: 1]
The authors have developed a CRISPR interference system in which a catalytically dead Cas9 protein can be targeted to a specific genomic site through a complementary small guide RNA, allowing systematic perturbation of gene transcription in bacteria and mammalian cells.
URLPMID:23849981 [本文引用: 1]
Abstract The genetic interrogation and reprogramming of cells requires methods for robust and precise targeting of genes for expression or repression. The CRISPR-associated catalytically inactive dCas9 protein offers a general platform for RNA-guided DNA targeting. Here, we show that fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in human and yeast cells, with the site of delivery determined solely by a coexpressed short guide (sg)RNA. Coupling of dCas9 to a transcriptional repressor domain can robustly silence expression of multiple endogenous genes. RNA-seq analysis indicates that CRISPR interference (CRISPRi)-mediated transcriptional repression is highly specific. Our results establish that the CRISPR system can be used as a modular and flexible DNA-binding platform for the recruitment of proteins to a target DNA sequence, revealing the potential of CRISPRi as a general tool for the precise regulation of gene expression in eukaryotic cells. Copyright 2013 Elsevier Inc. All rights reserved.
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The CRISPR-Cas adaptive immune system defends microbes against foreign genetic elements via DNA or RNA-DNA interference. We characterize the Class 2 type VI-A CRISPR-Cas effector C2c2 and demonstrate its RNA-guided RNase function. C2c2 from the bacterium Leptotrichia shahii provides interference against RNA phage. In vitro biochemical analysis show that C2c2 is guided by a single crRNA and can be programmed to cleave ssRNA targets carrying complementary protospacers. In bacteria, C2c2 can be programmed to knock down specific mRNAs. Cleavage is mediated by catalytic residues in the two conserved HEPN domains, mutations in which generate catalytically inactive RNA-binding proteins. These results broaden our understanding of CRISPR-Cas systems and suggest that C2c2 can be used to develop new RNA-targeting tools.
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URLPMID:28111461 [本文引用: 1]
Class 2 CRISPR-Cas systems are characterized by effector modules that consist of a single multidomain protein, such as Cas9 or Cpf1. We designed a computational pipeline for the discovery of novel class 2 variants and used it to identify six new CRISPR-Cas subtypes. The diverse properties of these new systems provide potential for the development of versatile tools for genome editing and regulation. In this Analysis article, we present a comprehensive census of class 2 types and class 2 subtypes in complete and draft bacterial and archaeal genomes, outline evolutionary scenarios for the independent origin of different class 2 CRISPR-Cas systems from mobile genetic elements, and propose an amended classification and nomenclature of CRISPR-Cas.
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Abstract Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK ( S pecific H igh Sensitivity E nzymatic R eporter Un LOCK ing) that combines isothermal pre-amplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCKv2: 1) 4-channel single reaction multiplexing using orthogonal CRISPR enzymes; 2) quantitative measurement of input down to 2 aM; 3) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxilary CRISPR-associated enzyme; and 4) lateral flow read-out. SHERLOCKv2 can detect Dengue or Zika virus ssRNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids.
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URLPMID:5394926 [本文引用: 2]
The human genome produces thousands of long noncoding RNAs (lncRNAs)-transcripts >200 nucleotides long that do not encode proteins. Although critical roles in normal biology and disease have been revealed for a subset of lncRNAs, the function of the vast majority remains untested. We developed a CRISPR interference (CRISPRi) platform targeting 16,401 lncRNA loci in seven diverse cell lines, including six transformed cell lines and human induced pluripotent stem cells (iPSCs). Large-scale screening identified 499 lncRNA loci required for robust cellular growth, of which 89% showed growth-modifying function exclusively in one cell type. We further found that lncRNA knockdown can perturb complex transcriptional networks in a cell type-specific manner. These data underscore the functional importance and cell type specificity of many lncRNAs.
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URLPMID:29251726 [本文引用: 1]
Abstract Combinatorial genetic screening using CRISPR-Cas9 is a useful approach to uncover redundant genes and to explore complex gene networks. However, current methods suffer from interference between the single-guide RNAs (sgRNAs) and from limited gene targeting activity. To increase the efficiency of combinatorial screening, we employ orthogonal Cas9 enzymes from Staphylococcus aureus and Streptococcus pyogenes. We used machine learning to establish S. aureus Cas9 sgRNA design rules and paired S. aureus Cas9 with S. pyogenes Cas9 to achieve dual targeting in a high fraction of cells. We also developed a lentiviral vector and cloning strategy to generate high-complexity pooled dual-knockout libraries to identify synthetic lethal and buffering gene pairs across multiple cell types, including MAPK pathway genes and apoptotic genes. Our orthologous approach also enabled a screen combining gene knockouts with transcriptional activation, which revealed genetic interactions with TP53. The "Big Papi" (paired aureus and pyogenes for interactions) approach described here will be widely applicable for the study of combinatorial phenotypes.
URLPMID:28933431 [本文引用: 2]
Abstract This corrects the article DOI: 10.1038/nature23451.
URLPMID:25075903 [本文引用: 1]
To the Editor:Genome-wide, targeted loss-of-function pooled screens using the clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated nuclease Cas9 in human and mouse cells provide an alternative screening system to RNA interference (RNAi)1, 2, 3, 4. Previously, we used a genome-scale CRISPR knockout (GeCKO) library to identify loss-of-function mutations conferring vemurafenib resistance in a melanoma model1. However, initial lentiviral delivery systems for CRISPR screening had low viral titer or required a cell line already expressing Cas9, thereby limiting the range of biological systems amenable to screening.We sought to improve both the lentiviral packaging and choice of guide sequences in our original GeCKO library1, where a pooled library of synthesized oligonucleotides was cloned into a lentiviral backbone containing both the Streptococcus pyogenes Cas9 nuclease and the single guide RNA (sgRNA) scaffold. To create a new vector capable of producing higher-titer virus (lentiCRISPRv2), we made several modifications, including removal of one of the nuclear localization signals, human-codon optimization of the remaining nuclear localization signal and P2A bicistronic linker sequences, and repositioning of the U6-driven sgRNA cassette (Fig. 1a). These changes resulted in an approximately tenfold increase in functional viral titer over that of lentiCRISPRv1 (ref. 1; Fig. 1b).
URLPMID:25119044 [本文引用: 1]
The study of cancer genes in mouse models has traditionally relied on genetically-engineered strains made via transgenesis or gene targeting in embryonic stem cells. Here we describe a new method of cancer model generation using the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins) system in vivo in wild-type mice. We used hydrodynamic injection to deliver a CRISPR plasmid DNA expressing Cas9 and single guide RNAs (sgRNAs) to the liver that directly target the tumour suppressor genes Pten (ref. 5) and p53 (also known as TP53 and Trp53) (ref. 6), alone and in combination. CRISPR-mediated Pten mutation led to elevated Akt phosphorylation and lipid accumulation in hepatocytes, phenocopying the effects of deletion of the gene using Cre-LoxP technology. Simultaneous targeting of Pten and p53 induced liver tumours that mimicked those caused by Cre-loxP-mediated deletion of Pten and p53. DNA sequencing of liver and tumour tissue revealed insertion or deletion mutations of the tumour suppressor genes, including bi-allelic mutations of both Pten and p53 in tumours. Furthermore, co-injection of Cas9 plasmids harbouring sgRNAs targeting the -catenin gene and a single-stranded DNA oligonucleotide donor carrying activating point mutations led to the generation of hepatocytes with nuclear localization of -catenin. This study demonstrates the feasibility of direct mutation of tumour suppressor genes and oncogenes in the liver using the CRISPR/Cas system, which presents a new avenue for rapid development of liver cancer models and functional genomics.
URLPMID:24759083 [本文引用: 1]
Genomic rearrangements are frequently observed in cancer cells but have been difficult to generate in a highly specific manner for functional analysis. Here we report the application of CRISPR/Cas technology to successfully generate several types of chromosomal rearrangements implicated as driver events in lung cancer, including the CD74-ROS1 translocation event and the EML4-ALK and KIF5B-RET inversion events. Our results demonstrate that Cas9-induced DNA breaks promote efficient rearrangement between pairs of targeted loci, providing a highly tractable approach for the study of genomic rearrangements.
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URLPMID:27120160 [本文引用: 1]
The bacterial CRISPR/Cas9 system allows sequence-specific gene editing in many organisms and holds promise as a tool to generate models of human diseases, for example, in human pluripotent stem cells. CRISPR/Cas9 introduces targeted double-stranded breaks (DSBs) with high efficiency, which are typically repaired by non-homologous end-joining (NHEJ) resulting in nonspecific insertions, deletions or other mutations (indels). DSBs may also be repaired by homology-directed repair (HDR) using a DNA repair template, such as an introduced single-stranded oligo DNA nucleotide (ssODN), allowing knock-in of specific mutations. Although CRISPR/Cas9 is used extensively to engineer gene knockouts through NHEJ, editing by HDR remains inefficient and can be corrupted by additional indels, preventing its widespread use for modelling genetic disorders through introducing disease-associated mutations. Furthermore, targeted mutational knock-in at single alleles to model diseases caused by heterozygous mutations has not been reported. Here we describe a CRISPR/Cas9-based genome-editing framework that allows selective introduction of mono- and bi-allelic sequence changes with high efficiency and accuracy. We show that HDR accuracy is increased dramatically by incorporating silent CRISPR/Cas-blocking mutations along with pathogenic mutations, and establish a method termed ‘CORRECT’ for scarless genome editing. By characterizing and exploiting a stereotyped inverse relationship between a mutation’s incorporation rate and its distance to the DSB, we achieve predictable control of zygosity. Homozygous introduction requires a guide RNA targeting close to the intended mutation, whereas heterozygous introduction can be accomplished by distance-dependent suboptimal mutation incorporation or by use of mixed repair templates. Using this approach, we generated human induced pluripotent stem cells with heterozygous and homozygous dominant early onset Alzheimer’s disease-causing mutations in amyloid precursor protein (APP) and presenilin 1 (PSEN1) and derived cortical neurons, which displayed genotype-dependent disease-associated phenotypes. Our findings enable efficient introduction of specific sequence changes with CRISPR/Cas9, facilitating study of human disease.
URLPMID:5526198 [本文引用: 1]
Abstract Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting clustered regularly interspaced short palindromic repeats (CRISPR) effector Cas13a (previously known as C2c2) exhibits a "collateral effect" of promiscuous ribonuclease activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed Specific High-Sensitivity Enzymatic Reporter UnLOCKing (SHERLOCK), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify mutations in cell-free tumor DNA. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage and be readily reconstituted on paper for field applications. Copyright 2017, American Association for the Advancement of Science.
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URLPMID:29326244 [本文引用: 1]
Advances in the understanding of molecular biology of human disease and the development of efficient gene transfer techniques have resulted in practical approaches to human gene therapy, with new techniques being developed at an increasing rate. The first trials have now begun in humans and initial results are positive.
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Chimeric antigen receptors (CARs) are synthetic receptors that reprogram T lymphocytes to target chosen antigens. The targeting of CD19, a cell surface molecule expressed in the vast majority of leukemias and lymphomas, has been successfully translated in the clinic, earning CAR therapy a special distinction in the selection of "cancer immunotherapy" by Science as the breakthrough of the year... [Show full abstract]
URLPMID:27021486 [本文引用: 1]
Abstract The beta-thalassemias are inherited anemias caused by mutations that severely reduce or abolish expression of the beta-globin gene. Like sickle cell disease, a related beta-globin gene disorder, they are ideal candidates for performing a genetic correction in patient hematopoietic stem cells (HSCs). The most advanced approach utilizes complex lentiviral vectors encoding the human -globin gene, as first reported by May et al. in 2000. Considerable progress towards the clinical implementation of this approach has been made in the past five years, based on effective CD34+ cell mobilization and improved lentiviral vector manufacturing. Four trials have been initiated in the US and Europe. Of 16 evaluable subjects, 6 have achieved transfusion independence. One of them developed a durable clonal expansion, which regressed after several years without transformation. While globin lentiviral vectors have so far proven to be safe, this occurrence suggests that powerful insulators with robust enhancer-blocking activity will further enhance this approach. The combined discovery of Bcl11a-mediated -globin gene silencing and advances in gene editing are the foundations for another gene therapy approach, which aims to reactivate fetal hemoglobin (HbF) production. Its clinical translation will hinge on the safety and efficiency of gene targeting in true HSCs and the induction of sufficient levels of HbF to achieve transfusion independence. Altogether, the progress achieved over the past 15 years bodes well for finding a genetic cure for severe globin disorders in the next decade.
URLPMID:4157757 [本文引用: 1]
Abstract We demonstrate CRISPR-Cas9-mediated correction of a Fah mutation in hepatocytes in a mouse model of the human disease hereditary tyrosinemia. Delivery of components of the CRISPR-Cas9 system by hydrodynamic injection resulted in initial expression of the wild-type Fah protein in 0908041/250 liver cells. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype. Our study indicates that CRISPR-Cas9-mediated genome editing is possible in adult animals and has potential for correction of human genetic diseases.
URLPMID:27820943 [本文引用: 1]
Abstract The 0205-haemoglobinopathies, such as sickle cell disease and 0205-thalassaemia, are caused by mutations in the 0205-globin (HBB) gene and affect millions of people worldwide. Ex vivo gene correction in patient-derived haematopoietic stem cells followed by autologous transplantation could be used to cure 0205-haemoglobinopathies. Here we present a CRISPR/Cas9 gene-editing system that combines Cas9 ribonucleoproteins and adeno-associated viral vector delivery of a homologous donor to achieve homologous recombination at the HBB gene in haematopoietic stem cells. Notably, we devise an enrichment model to purify a population of haematopoietic stem and progenitor cells with more than 90% targeted integration. We also show efficient correction of the Glu6Val mutation responsible for sickle cell disease by using patient-derived stem and progenitor cells that, after differentiation into erythrocytes, express adult 0205-globin (HbA) messenger RNA, which confirms intact transcriptional regulation of edited HBB alleles. Collectively, these preclinical studies outline a CRISPR-based methodology for targeting haematopoietic stem cells by homologous recombination at the HBB locus to advance the development of next-generation therapies for 0205-haemoglobinopathies.
URLPMID:4924477 [本文引用: 1]
Abstract Frame-disrupting mutations in the DMD gene, encoding dystrophin, compromise myofiber integrity and drive muscle deterioration in Duchenne muscular dystrophy (DMD). Removing one or more exons from the mutated transcript can produce an in-frame mRNA and a truncated, but still functional, protein. In this study, we developed and tested a direct gene-editing approach to induce exon deletion and recover dystrophin expression in the mdx mouse model of DMD. Delivery by adeno-associated virus (AAV) of clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 endonucleases coupled with paired guide RNAs flanking the mutated Dmd exon23 resulted in excision of intervening DNA and restored the Dmd reading frame in myofibers, cardiomyocytes, and muscle stem cells after local or systemic delivery. AAV-Dmd CRISPR treatment partially recovered muscle functional deficiencies and generated a pool of endogenously corrected myogenic precursors in mdx mouse muscle. Copyright 2016, American Association for the Advancement of Science.
URLPMID:26721684 [本文引用: 1]
Duchenne muscular dystrophy (DMD) is a devastating disease affecting about 1 out of 5000 male births and caused by mutations in the dystrophin gene. Genome editing has the potential to restore expression of a modified dystrophin gene from the native locus to modulate disease progression. In this study, adeno-associated virus was used to deliver the clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system to the mdx mouse model of DMD to remove the mutated exon 23 from the dystrophin gene. This includes local and systemic delivery to adult mice and systemic delivery to neonatal mice. Exon 23 deletion by CRISPR-Cas9 resulted in expression of the modified dystrophin gene, partial recovery of functional dystrophin protein in skeletal myofibers and cardiac muscle, improvement of muscle biochemistry, and significant enhancement of muscle force. This work establishes CRISPR-Cas9-based genome editing as a potential therapy to treat DMD.
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URLPMID:29258297 [本文引用: 1]
Abstract Although genetic factors contribute to almost half of all cases of deafness, treatment options for genetic deafness are limited. We developed a genome-editing approach to target a dominantly inherited form of genetic deafness. Here we show that cationic lipid-mediated in vivo delivery of Cas9-guide RNA complexes can ameliorate hearing loss in a mouse model of human genetic deafness. We designed and validated, both in vitro and in primary fibroblasts, genome editing agents that preferentially disrupt the dominant deafness-associated allele in the Tmc1 (transmembrane channel-like gene family 1) Beethoven (Bth) mouse model, even though the mutant Tmc1 Bth allele differs from the wild-type allele at only a single base pair. Injection of Cas9-guide RNA-lipid complexes targeting the Tmc1 Bth allele into the cochlea of neonatal Tmc1 Bth/+ mice substantially reduced progressive hearing loss. We observed higher hair cell survival rates and lower auditory brainstem response thresholds in injected ears than in uninjected ears or ears injected with control complexes that targeted an unrelated gene. Enhanced acoustic startle responses were observed among injected compared to uninjected Tmc1 Bth/+ mice. These findings suggest that protein-RNA complex delivery of target gene-disrupting agents in vivo is a potential strategy for the treatment of some types of autosomal-dominant hearing loss.
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URLPMID:27618611 [本文引用: 1]
Abstract Sequence-specific nucleases have been exploited to create targeted gene knockouts in various plants(1), but replacing a fragment and even obtaining gene insertions at specific loci in plant genomes remain a serious challenge. Here, we report efficient intron-mediated site-specific gene replacement and insertion approaches that generate mutations using the non-homologous end joining (NHEJ) pathway using the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) system. Using a pair of single guide RNAs (sgRNAs) targeting adjacent introns and a donor DNA template including the same pair of sgRNA sites, we achieved gene replacements in the rice endogenous gene 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) at a frequency of 2.0%. We also obtained targeted gene insertions at a frequency of 2.2% using a sgRNA targeting one intron and a donor DNA template including the same sgRNA site. Rice plants harbouring the OsEPSPS gene with the intended substitutions were glyphosate-resistant. Furthermore, the site-specific gene replacements and insertions were faithfully transmitted to the next generation. These newly developed approaches can be generally used to replace targeted gene fragments and to insert exogenous DNA sequences into specific genomic sites in rice and other plants.
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URLPMID:3945887 [本文引用: 1]
One-step generation of knockout pigs by zygote injection of CRISPR/Cas systemCell Research advance online publication, January 31 2014. doi:10.1038/cr.2014.11Authors: Tang Hai, Fei ...
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URLPMID:4154755 [本文引用: 1]
Abstract The CRISPR/Cas9 system has been adapted as an efficient genome editing tool in laboratory animals such as mice, rats, zebrafish and pigs. Here, we report that CRISPR/Cas9 mediated approach can efficiently induce monoallelic and biallelic gene knockout in goat primary fibroblasts. Four genes were disrupted simultaneously in goat fibroblasts by CRISPR/Cas9-mediated genome editing. The single-gene knockout fibroblasts were successfully used for somatic cell nuclear transfer (SCNT) and resulted in live-born goats harboring biallelic mutations. The CRISPR/Cas9 system represents a highly effective and facile platform for targeted editing of large animal genomes, which can be broadly applied to both biomedical and agricultural applications.
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URLPMID:26564781 [本文引用: 1]
Abstract Genetically modified pigs are increasingly used for biomedical and agricultural applications. The efficient CRISPR/Cas9 gene editing system holds great promise for the generation of gene-targeting pigs without selection marker genes. In this study, we aimed to disrupt the porcine myostatin (MSTN) gene, which functions as a negative regulator of muscle growth. The transfection efficiency of porcine fetal fibroblasts (PFFs) was improved to facilitate the targeting of Cas9/gRNA. We also demonstrated that Cas9/gRNA can induce non-homologous end-joining (NHEJ), long fragment deletions/inversions and homology-directed repair (HDR) at the MSTN locus of PFFs. Single-cell MSTN knockout colonies were used to generate cloned pigs via somatic cell nuclear transfer (SCNT), which resulted in 8 marker-gene-free cloned pigs with biallelic mutations. Some of the piglets showed obvious intermuscular grooves and enlarged tongues, which are characteristic of the double muscling (DM) phenotype. The protein level of MSTN was decreased in the mutant cloned pigs compared with the wild-type controls, and the mRNA levels of MSTN and related signaling pathway factors were also analyzed. Finally, we carefully assessed off-target mutations in the cloned pigs. The gene editing platform used in this study can efficiently generate genetically modified pigs with biological safety.
URLPMID:26641533 [本文引用: 1]
Porcine reproductive and respiratory syndrome (PRRS) is an economically devastating viral disease causing heavy losses to the swine industry worldwide. Many studies have shown that transient delivery of small interfering RNA (siRNA) or adenovirus-mediated RNA interfere (RNAi) could potentially inhibit porcine reproductive and respiratory syndrome virus (PRRSV) replication in vivo and in vitro. Here, we applied RNAi to produce transgenic (TG) pigs that constitutively expressed PRRSV-specific siRNA derived from small hairpin RNA (shRNA). First, we evaluated siRNA expression in the founding and F1 generation pigs and confirmed stable transmission. Then, we detected the expression of IFN-β and protein kinase R (PKR) and found no difference among TG, non-transgenic (NTG), and wild-type pigs. Lastly, the F1 generation pigs, including TG and NTG piglets, were challenged with 3×1066·67 TCID6368 of JXA1, a highly pathogenic porcine reproductive and respiratory syndrome virus (HP-PRRSV). Our results showed that the in vivo siRNA expression substantially reduced the serum HP-PRRSV titers and increased survival time by 3 days when TG pigs were compared with the NTG controls. These data suggested that RNAi-based genetic modification might be used to breed viral-resistant livestock with stable siRNA expression with no complications of siRNA toxicity.
