CN106834320A - Tal效应子介导的dna修饰 - Google Patents
Tal效应子介导的dna修饰 Download PDFInfo
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- CN106834320A CN106834320A CN201610457321.3A CN201610457321A CN106834320A CN 106834320 A CN106834320 A CN 106834320A CN 201610457321 A CN201610457321 A CN 201610457321A CN 106834320 A CN106834320 A CN 106834320A
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Abstract
本发明为TAL效应子介导的DNA修饰,本发明提供涉及基因靶向(例如,用转录激活因子样效应子核酸酶的基因靶向)的材料和方法。
Description
相关申请的交叉参考
本申请要求2009年12月10日提交的美国临时申请序列号61/285,324、2010年6月7日提交的美国临时申请序列号61/352,108和2010年7月22日提交的美国临时申请序列号61/366,685的优先权,所有申请通过引用全文纳入本文。
有关联邦资助的研究的声明
本发明是在国家科学基金会(National Science Foundation)授予的基金号0820831和0504304下由政府资助完成。政府对本发明拥有某些权利。
技术领域
本发明涉及基因靶向的方法,尤其是包括使用转录激活因子样(TAL)效应子序列的方法。
背景技术
通过同源重组修饰染色体的能力(基因靶向)一直是生物学家的奋斗目标。例如,在植物中,基因靶向可有助于了解植物基因功能,为农作物改良提供新的可能。例如,利用基因靶向可进行重排代谢途径所需的遗传手术以产生高价值农作物,包括改变油或糖分布的种子、提高营养品质的食物或对疾病和压力抗性增加的植物。在动物(例如哺乳动物)中,基因靶向可用于治疗疾病。例如,基因靶向可用于对由各种形式突变引起的缺陷基因进行工程校正。这种基因靶向的有效方法难以实现。
发明内容
黄单胞菌属(Xanthomonas)中植物病原菌的TAL效应子通过结合宿主DNA和激活效应子特异的宿主基因,在疾病或引发防御中起重要作用(参见例如,Gu等(2005)Nature435:1122;Yang等(2006)Proc.Natl.Acad.Sci.USA103:10503;Kay等(2007)Science 318:648;Sugio等(2007)Proc.Natl.Acad.Sci.USA 104:10720;和等(2007)Science318:645)。特异性取决于不完善的可变效应子数量,通常是34个氨基酸重复(Schornack等(2006)J.Plant Physiol.163:256)。多态性主要发生在重复位置12和13,本文将其称为重复可变双残基(RVD)。
本发明部分基于TAL效应子的RVD以直接、线性形式对应于其靶位置的核苷酸,一种RVD对应于一种核苷酸,有一些简并性且没有明显的环境依赖性。这个令人意外的发现代表蛋白-DNA识别的新机制,能针对新靶向特异性TAL效应子进行靶位置预测。如本文所述,这些蛋白可作为靶向的嵌合核酸酶用于研究和生物技术,有助于基因组工程改造中的同源重组(例如添加或提高用于植物中生物燃料或生物可再生物的特征)。这些蛋白还用作例如转录因子,且特别用于需要很高水平特异性的治疗应用,例如(非限制示例)针对病原体(如病毒)的治疗。
在一个方面,本发明涉及改良细胞遗传物质的方法,所述方法包括(a)提供含靶DNA序列的细胞;和(b)将转录激活因子样(TAL)效应子-DNA修饰酶导入所述细胞,所述TAL效应子-DNA修饰酶包括(i)修饰双链DNA的DNA修饰酶结构域,和(ii)TAL效应子结构域,包括联合结合靶DNA序列中特异性核苷酸序列的多种TAL效应子重复序列,从而所述TAL效应子-DNA修饰酶修饰细胞或其后代中所述特异性核苷酸序列内或相邻的靶DNA。本方法还可包括将含有与至少部分靶DNA序列同源的序列的核酸提供给细胞,从而在所述靶DNA序列和所述核酸之间产生同源重组。所述细胞可为真核细胞、哺乳动物细胞、植物细胞或原核细胞。所述靶DNA可为染色体DNA。所述导入可包括用编码TAL效应子DNA修饰酶的载体转染细胞、将TAL效应子DNA修饰酶作为蛋白机械注射入细胞、用细菌III型分泌系统将TAL效应子DNA修饰酶作为蛋白递送到细胞,或通过电穿孔将TAL效应子DNA修饰酶作为蛋白导入细胞。所述DNA修饰酶可为内切核酸酶(如II型限制性内切酶如FokI)。
结合所述靶DNA中特异性核苷酸序列的TAL效应子结构域可包括10或更多个DNA结合重复序列,且优选15或更多个DNA结合重复序列。各DNA结合重复序列可包括确定所述靶DNA序列中碱基对识别的重复可变双残基(RVD),其中各DNA结合重复序列负责识别所述靶DNA序列中的一种碱基对,且其中所述RVD包括下述的一种或多种:识别C的HD;识别T的NG;识别A的NI;识别G或A的NN;NS识别A或C或G或T的NS;识别C或T的N*,其中*代表RVD的第二位置中的缺口;识别T的HG;识别T的H*,其中*代表RVD的第二位置中的缺口;识别T的IG;识别G的NK;识别C的HA;识别C的ND;识别C的HI;识别G的HN;识别G的NA;识别G或A的SN;和识别T的YG。各DNA结合重复序列可包括确定所述靶DNA序列中碱基对识别的RVD,其中各DNA结合重复序列负责识别所述靶DNA序列中的一种碱基对,且其中所述RVD包括下述的一种或多种:识别C的HD;识别C的ND;识别C的HI;识别G的HN;识别G的NA;识别G或A的SN;和识别T的YG;和识别G的NK,和下述的一种或多种:识别C的HD;识别T的NG;识别A的NI;识别G或A的NN;NS识别A或C或G或T的NS;识别C或T的N*,其中*代表RVD的第二位置中的缺口;识别T的HG;识别T的H*,其中*代表RVD的第二位置中的缺口;识别T的IG。
在另一方面,本发明涉及生成编码所选核苷酸序列特异性TAL效应子的核酸的方法,所述方法包括:(1)用PspXI线性化初始质粒,所述初始质粒包括编码第一TAL效应子DNA结合重复结构域的核苷酸序列,所述结构域具有特异于所选核苷酸序列的第一核苷酸的重复可变双残基(RVD),其中所述第一TAL效应子DNA结合重复结构域在3’末端具有独特的PspXI位点;(2)将编码一种或多种TAL效应子DNA结合重复结构域的DNA模块连接到所述初始质粒的PspXI位点,所述结构域具有特异于所选核苷酸序列的后续核苷酸的RVD,其中所述DNA模块具有XhoI粘性末端;和(3)重复步骤(1)和(2)直到核酸编码出能结合所选核苷酸序列的TAL效应子。连接后,所述方法还可包括确定PspXI位点中DNA模块的方向。所述方法可包括重复步骤(1)和(2)1-30次。
在另一方面,本发明涉及生成编码转录激活因子样效应子内切核酸酶(TALEN)的核酸的方法,所述方法包括(a)在细胞基因组中鉴定第一核苷酸序列;和(b)合成编码TALEN的核酸,所述核酸包括(i)与所述第一独特核苷酸序列联合结合的多种DNA结合重复序列,和(ii)在所述第一核苷酸序列内或邻近的位置产生双链切割的内切核酸酶,其中各DNA结合重复序列包括确定所述靶DNA中碱基对识别的RVD,其中各DNA结合重复序列负责识别所述靶DNA中的碱基对,其中所述TALEN包括一种或多种下述RVD:识别C的HD;识别T的NG;识别A的NI;识别G或A的NN;NS识别A或C或G或T的NS;识别C或T的N*;识别T的HG;识别T的H*;识别T的IG;识别G的NK;识别C的HA;识别C的ND;识别C的HI;识别G的HN;识别G的NA;识别G或A的SN;和识别T的YG。
TALEN包括以下RVD的一种或多种:识别C的HD;识别C的ND;识别C的HI;识别G的HN;识别G的NA;识别G或A的SN;和识别T的YG;和识别G的NK,和下述的一种或多种:识别C的HD;识别T的NG;识别A的NI;识别G或A的NN;NS识别A或C或G或T的NS;识别C或T的N*;识别T的HG;识别T的H*;识别T的IG。
所述第一核苷酸序列可满足至少一种下述标准:i)最少15碱基长度且为5’-3’方向,T紧接在5’末端位点的前面;ii)在所述第一(5’)位置中没有T或所述第二位置中没有A;iii)在最后的(3’)位置以T结束且接近最后的位置没有G;和iv)碱基组成为0-63%A、11-63%C、0-25%G、2-42%T。
所述方法可包括在所述细胞的基因组中鉴定第一核苷酸序列和第二核苷酸序列,其中所述第一和第二核苷酸序列满足至少一种上述标准且由15-18bp分开。所述内切核酸酶可在所述第一和第二核苷酸序列中产生双链切割。
在另一实施方式中,本发明涉及包括内切核酸酶结构域和靶DNA特异性TAL效应子DNA结合域的TALEN,其中所述DNA结合域包括多种DNA结合重复序列,各重复序列包括确定所述靶DNA中碱基对识别的RVD,其中各DNA结合重复序列负责识别所述靶DNA中的一种碱基对,且其中所述TALEN包括一种或多种下述RVD:识别C的HD;识别T的NG;识别A的NI;识别G或A的NN;NS识别A或C或G或T的NS;识别C或T的N*;识别T的HG;识别T的H*;识别T的IG;识别G的NK;识别C的HA;识别C的ND;识别C的HI;识别G的HN;识别G的NA;识别G或A的SN;和识别T的YG。TALEN可包括以下RVD的一种或多种:识别C的HD;识别C的ND;识别C的HI;识别G的HN;识别G的NA;识别G或A的SN;和识别T的YG;和识别G的NK,和下述的一种或多种:识别C的HD;识别T的NG;识别A的NI;识别G或A的NN;NS识别A或C或G或T的NS;识别C或T的N*;识别T的HG;识别T的H*;识别T的IG。所述内切核酸酶结构域可来自II型限制性内切核酸酶(如FokI)。
在另一方面,本发明涉及包括内切核酸酶结构域和TAL效应子结构域的TALEN,其中所述TALEN的氨基酸序列选自下组:SEQ ID NO:33-SEQ ID NO:55、SEQ ID NO:72、和SEQID NO:73。
本发明还涉及产生动物的方法,所述方法包括:提供包括靶DNA序列的真核细胞,其内需要导入遗传修饰;用TALEN在靶DNA序列内产生双链切割,所述TALEN包括内切核酸酶结构域和结合靶DNA序列的TAL效应子结构域;和从所述细胞或其后代产生动物,其中已发生双链切割。本方法还可包括将含与至少部分靶DNA同源的序列的外源核酸导入细胞,其中所述导入在允许所述外源核酸和细胞或其后代中靶DNA序列发生同源重组的条件下进行;和从已发生同源重组的所述细胞或其后代产生动物。所述动物可以是哺乳动物。所述遗传修饰可包括取代、插入或缺失。
在另一方面,本发明涉及产生植物的方法,所述方法包括提供含靶DNA序列的植物细胞,其内需要导入预选的遗传修饰;用TALEN在靶DNA序列内产生双链切割,所述TALEN包括内切核酸酶结构域和结合靶DNA序列的TAL效应子结构域;和从已发生双链切割的所述细胞或其后代产生植物。本方法还可包括将含与至少部分靶DNA序列同源的序列的外源核酸导入植物细胞,其中所述导入在允许所述外源核酸和细胞或其后代中靶DNA序列发生同源重组的条件下进行;和从已发生同源重组的所述细胞或其后代产生植物。
在另一方面,本发明涉及细胞中靶向遗传重组的方法,所述方法包括将编码靶向所选DNA靶序列的TAL效应子内切核酸酶的核酸导入细胞;在所述细胞中诱导TAL效应子内切核酸酶的表达;和鉴定所选DNA靶序列显示突变的细胞。所述突变可选自下组:遗传物质的缺失、遗传物质的插入以及遗传物质的缺失和插入。所述方法还可包括将供体DNA导入细胞。所述细胞可为昆虫细胞、植物细胞、鱼类细胞或哺乳动物细胞。
在另一方面,本发明涉及产生靶向靶DNA的能力增强的TAL效应子的方法,所述方法包括产生编码TAL效应子的核酸,所述效应子包括具有多种DNA结合重复序列的DNA结合域,其中各重复序列包括确定所述靶DNA中碱基对识别的RVD,其中各DNA结合重复序列负责识别所述靶DNA中的碱基对,其中所述产生包括纳入编码对A、C或G有特异性的第0变异DNA结合重复序列的核酸,因此不再需要所述结合位点-1位置的T。
在另一方面,本发明涉及产生靶向靶DNA的能力增强的TAL效应子的方法,所述方法包括产生编码TAL效应子的核酸,所述效应子包括具有多种DNA结合重复序列的DNA结合域,其中各重复序列包括确定所述靶DNA中碱基对识别的RVD,其中各DNA结合重复序列负责识别所述靶DNA中的碱基对,其中所述产生包括纳入一种或多种含有对G特异性提高的RVD的TAL效应子DNA结合域编码核酸,其中所述RVD选下组:RN、R*、NG、NH、KN、K*、NA、NT、DN、D*、NL、NM、EN、E*、NV、NC、QN、Q*、NR、NP、HN、H*、NK、NY、SN、S*、ND、NW、TN、T*、NE、NF、YN、Y*和NQ,其中*代表RVD的第二位置上的缺口。
本发明还涉及生成选择性识别靶DNA序列中至少一种碱基对的多肽的方法,所述方法包括合成含重复结构域的多肽,其中所述重复结构域包括衍生自转录激活因子样(TAL)效应子的至少一个重复单元,其中所述重复单元包括确定靶DNA序列中碱基对识别的高变区,其中所述重复单元负责所述DNA序列中一种碱基对的识别,且其中所述高变区包括选自下组的元件:(a)识别C/G的HD;(b)识别A/T的NI;(c)识别T/A的NG;(d)识别C/G或A/T或T/A或G/C的NS;(e)识别G/C或A/T的NN;(f)识别T/A的IG;(g)识别C/G的N;(h)识别C/G或T/A的HG;(i)识别T/A的H;和(j)识别G/C的NK。此外,本发明涉及上述方法生产的多肽,和包括本方法所生成多肽的编码序列的DNA。还涉及包括可操作连接上述DNA的启动子的表达盒,和包括所述表达盒的非人宿主细胞。在另一方面,本发明涉及转化的、包括表达盒的非人生物体。
在另一方面,本发明涉及通过多肽选择性识别DNA序列中碱基对的方法,所述方法包括构建含重复结构域的多肽,其中所述重复结构域包括衍生自TAL效应子的至少一个重复单元,其中所述重复单元包括确定DNA序列中碱基对识别的高变区,其中所述重复单元负责识别所述DNA序列中的一种碱基对,且其中所述高变区包括选自下组的元件:(a)识别C/G的HD;(b)识别A/T的NI;(c)识别T/A的NG;(d)识别C/G或A/T或T/A或G/C的NS;(e)识别G/C或A/T的NN;(f)识别T/A的IG;(g)识别C/G的N;(h)识别C/G或T/A的HG;(i)识别T/A的H;和(j)识别G/C的NK。
本发明还涉及调控细胞中靶基因表达的方法,其中提供含多肽的细胞,所述多肽包括重复结构域,其中所述重复结构域包括衍生自TAL效应子的至少一个重复单元,其中所述重复单元包括确定DNA序列中碱基对识别的高变区,其中所述重复单元负责识别所述DNA序列中的一种碱基对,且其中所述高变区包括选自下组的元件:(a)识别C/G的HD;(b)识别A/T的NI;(c)识别T/A的NG;(d)识别C/G或A/T或T/A或G/C的NS;(e)识别G/C或A/T的NN;(f)识别T/A的IG;(g)识别C/G的N;(h)识别C/G或T/A的HG;(i)识别T/A的H;和(j)识别G/C的NK。
在另一方面,本发明涉及包括重复结构域的多肽,其中所述重复结构域包括衍生自TAL效应子的至少一个重复单元,其中所述重复单元包括确定DNA序列中碱基对识别的高变区,其中所述重复单元负责识别所述DNA序列中的一种碱基对,且其中所述高变区包括选自下组的元件:(a)识别C/G的HD;(b)识别A/T的NI;(c)识别T/A的NG;(d)识别C/G或A/T或T/A或G/C的NS;(e)识别G/C或A/T的NN;(f)识别T/A的IG;(g)识别C/G的N;(h)识别C/G或T/A的HG;(i)识别T/A的H;和(j)识别G/C的NK。本发明还涉及包括上述多肽编码序列的DNA。
在另一方面,本发明涉及修饰成包括位于靶DNA序列中碱基对的DNA,从而所述碱基对可被包括重复结构域的多肽特异识别,其中所述重复结构域包括衍生自TAL效应子的至少一种重复单元,其中所述重复单元包括确定DNA序列中碱基对识别的高变区,其中所述重复单元负责识别所述DNA序列中的一种碱基对,且为了通过高变区得到选择性和确定的识别,其中所述碱基对选自下组:(a)由HD识别的C/G;(b)由NI识别的A/T;(c)由NG识别的T/A;(d)由NS识别的C/G或A/T或T/A或G/C;(e)由NN识别的G/C或A/T;(f)由IG识别的T/A;(g)由N识别的C/G或T/A;(h)由HG识别的T/A;(i)由H识别的T/A;和(j)由NK识别的G/C。还涉及包括上述DNA的载体,包括所述DNA的非人宿主细胞和包括所述DNA的转化的非人生物体。
在另一方面,本发明涉及生产包括靶DNA序列的DNA的方法,所述靶DNA序列由含重复结构域的多肽选择性识别,其中所述重复结构域包括衍生自TAL效应子的至少一个重复单元,其中所述重复单元包括确定DNA序列中碱基对识别的高变区,且其中所述重复单元负责识别所述DNA序列中的一种碱基对,所述方法包括合成含能被所述重复单元识别的碱基对的DNA,其中所述碱基对选自下组:(a)由HD识别的C/G;(b)由NI识别的A/T;(c)由NG识别的T/A;(d)由NS识别的C/G或A/T或T/A或G/C;(e)由NN识别的G/C或A/T;(f)由IG识别的T/A;(g)由N识别的C/G或T/A;(h)由HG识别的T/A;(i)由H识别的T/A;和(j)由NK识别的G/C。
在另一方面,本发明涉及修饰植物细胞的遗传物质的方法。本发明法可包括(a)将下述物质导入植物细胞(i)包括经修饰的核苷酸序列的第一重组核酸,其中所述经修饰的靶核苷酸序列根据所述植物细胞中存在的相应靶核苷酸序列,在核苷酸序列中包括一种或多种修饰,且其中所述靶核苷酸序列还包括序列特异性TAL效应子内切核酸酶(TALEN)的识别位点;和(ii)包括编码序列特异性转录激活因子样(TAL)效应子内切核酸酶的核苷酸序列的第二重组核酸;(b)生成含所述植物细胞的植物;(c)分析获自植物或其后代的细胞、种子、或组织在靶核苷酸序列的重组。所述方法还可包括将(iii)包括编码可选标记物的核苷酸序列的第三重组核酸导入植物细胞;和确定所述植物或其后代是否表达所述可选标记物。所述方法还可包括根据所述可选择标记的缺失筛选所述植物或其后代的步骤。编码所述可选择标记的核苷酸序列可以侧接或不侧接与所述植物细胞内源序列(例如第二序列特异性核酸酶的切割位点的序列)相似或相同的序列的一侧或两侧。编码可选择标记物的核苷酸序列可通过序列特异性重组酶的识别位点侧接两侧。所述方法还可包括异交所述植物的步骤,有或没有根据所述可选择标记的缺失筛选异交后代的步骤。所述第一和第二重组核酸可同时导入所述植物细胞。一种或两种所述重组核酸可在导入步骤前线性化。所述第一和第二重组核酸可存在于相同构建体中。
在另一方面,本发明涉及修饰细胞遗传物质的另一方法。所述方法可包括提供含需要发生同源重组的染色体靶DNA序列的初级细胞;提供包括可切割双链DNA的内切核酸酶结构域的TALEN和包括多种TAL效应子重复序列的TAL效应子结构域,所述重复序列联合结合细胞靶DNA中的特异核苷酸序列;和将所述靶DNA序列接触细胞内的TALEN从而所述TALEN切割细胞靶DNA序列内或相邻核苷酸序列的双链。本方法还可包括提供含有与至少部分靶DNA同源的序列的核酸,从而在所述靶DNA序列和所述核酸之间产生同源重组。所述靶DNA序列可为所述细胞内源序列。所述细胞可为用于体外培养的植物细胞、哺乳动物细胞、鱼类细胞、昆虫细胞或衍生自这些生物体的细胞系,或用于体外培养的直接取自活组织并建立的初级细胞。所述接触可包括用含TALEN编码序列的载体转染细胞并在细胞内表达TALEN蛋白、机械注射TALEN蛋白到细胞中、通过细菌III型分泌系统递送TAL效应子内切核酸酶蛋白到细胞中或通过电穿孔将TALEN蛋白导入细胞。所述内切核酸酶结构域可来自II型限制性内切核酸酶(如FokI)。结合所述靶DNA中特异核苷酸序列的TAL效应子结构域可包括10或更多个DNA结合重复序列,优选15或更多个DNA结合重复序列。所述细胞可来自任何原核或真核生物体。
在另一方面,本发明涉及能在特定位置切割DNA的序列特异性TALEN的设计方法。本方法可包括鉴定与需要导入双链切割的第二核苷酸序列相邻的第一独特内源染色体核苷酸序列;和设计序列特异性TALEN,其包括(a)联合结合所述第一独特内源染色体核苷酸序列的多种DNA结合重复结构域,和(b)在第二核苷酸序列生成双链切割的内切核酸酶。
本发明还涉及包括内切核酸酶结构域和具体DNA序列特异性TAL效应子DNA结合域的TALEN。TALEN还可包括纯化标签。所述内切核酸酶结构域可来自II型限制性内切核酸酶(如FokI)。
在另一方面,本发明涉及生成导入所需核酸的遗传改良动物。本方法可包括提供含内源染色体靶DNA序列的初级细胞,其内需要导入核酸;用TALEN在内源染色体靶DNA序列内产生双链切割,所述TALEN包括内切核酸酶结构域和结合内源染色体靶DNA序列的TAL效应子结构域;在允许外源核酸和内源染色体靶DNA间发生同源重组的条件下将含与至少部分内源染色体靶DNA同源的序列的外源核酸导入所述初级细胞;和从发生同源重组的初级细胞生成动物。所述动物可以是哺乳动物。所述同源的序列可为选自下组的核苷酸序列:同源重组后破坏基因的核苷酸序列、同源重组后替换基因的核苷酸序列、同源重组后在基因中导入点突变的核苷酸序列和同源重组后导入调控位点的核苷酸序列。
在另一方面,本发明涉及生成导入所需核酸的遗传改良植物。本方法可包括提供含内源靶DNA序列的植物细胞,其内需要导入核酸;用TALEN在内源靶DNA序列中产生双链切割,所述TALEN包括内切核酸酶结构域和结合内源靶核苷酸序列的TAL效应子结构域;在允许外源核酸和内源靶DNA间发生同源重组的条件下将含与至少部分内源靶DNA同源的序列的外源核酸导入所述植物细胞;和从已发生同源重组的植物细胞生成植物。
在另一方面,本发明涉及细胞中靶向遗传重组的方法。本方法可包括将编码TALEN的核酸分子导入所述细胞,所述TALEN靶向选择的DNA靶序列;诱导所述细胞中TALEN表达;和鉴定其中所选DNA靶序列显示突变的细胞。所述突变可选自下组:遗传物质的缺失、遗传物质的插入以及遗传物质的缺失和插入。所述方法还可包括将供体DNA导入细胞。所述细胞可为昆虫细胞、植物细胞、鱼类细胞或哺乳动物细胞。
在另一方面,本发明涉及生成编码序列特异性TALEN的核酸的方法,所述方法包括(1)选择包括编码第一TAL效应子DNA结合重复结构域的核苷酸序列的初始质粒,所述结构域具有对所选核苷酸序列的第一核苷酸特异的RVD,其中所述第一TAL效应子DNA结合重复结构域在3’末端具有独特的PspXI位点;(2)用PspXI线性化所述初始质粒;(3)将编码一种或多种TAL效应子DNA结合重复结构域的DNA模块连接到PspXI位点,所述结构域具有特异于所选核苷酸序列后续核苷酸的RVD,其中所述DNA模块具有XhoI粘性末端;和(4)重复步骤(2)和(3)直到核酸编码出能结合所选核苷酸序列的TALEN。在一些情况中,所述方法还可在步骤(3)的连接后包括检查PspXI位点中DNA模块的方向。
除非另外定义,本文使用的所有技术和科学术语的意义与本发明所属领域的普通技术人员通常所理解的相同。虽然与本文所述类似或等同的方法和材料可用来实施本发明,但在下文描述合适的方法和材料。本文中述及的所有出版物、专利申请、专利和其他参考文献都通过引用全文纳入本文。在抵触的情况下,以本说明书(包括定义在内)为准。此外,材料、方法和实施例都仅是说明性,并不构成限制。
在附图和以下描述中详细说明了本发明的一种或多种实施方式。本发明的其他特征、目的和优势通过描述、附图以及权利要求书可显而易见。
附图简要说明
图1A-1D显示TAL效应子-DNA识别密码。图1A是普通TAL效应子的图,显示重复区域(空心框)和代表性的重复序列(SEQ ID NO:1),RVD用下划线标出。图1B显示各种TAL效应子RVD和靶基因启动子序列(SEQ ID NO:2-11)的最佳模式匹配(低熵比对)。星号表示残基13处有缺失。图1C显示B的比对中RVD-核苷酸的关联以及用40个额外水稻黄单胞菌(X.oryzae)TAL效应子扫描所有水稻启动子获得的另10个比对,保留各效应子的最佳比对,感染期间其下游基因活化。图1D显示20个TAL效应子靶位点的侧接核苷酸频率。位置是相对于靶位点的5’末端;N,靶位点长度。用WebLogo生成标记。
图2A和2B提供OsHen1被水稻条斑病菌(Xanthomonas oryzae pv.oryzicola)株系BLS256的Tal1c活化的证据。图2A是半定量RT-PCR结果图,显示用BLS256标记物交换突变体M51、载有空粘粒载体(ev)的M51、载有粘粒pIJF92(含tal1a、tal1b和tal1c)的M51和野生型(WT)株系接种后24小时的水稻叶片中的OsHen1相对转录物丰度,用肌动蛋白基因作参考。图2B是基于通过拯救对M51中单一标记物交换突变做图以及含标记物的XmaI片段的末端测序的示意图。显示粘粒pIJF92中包括的基因组区域、拯救片段的坐标、和BLS256基因组片段的坐标。
图3是参比AvrBs3氨基酸序列(SEQ ID NO:12)。
图4是参比AvrBs3核酸序列(SEQ ID NO:13)。
图5是TAL核酸酶表达载体图。
图6是靶报告质粒图。
图7是TAL核酸酶的示意结构图。TAL DNA结合域的识别位点用大写字母表示,而间隔序列用小写字母表示。
图8是17个半串联重复的AvrBs3识别结构域的氨基酸序列(SEQ ID NO:31)。加框的是位置12和13的高变氨基酸。
图9显示检测TAL有效性的酵母分析方案。
图10绘制了AvrBs3 TAL核酸酶的酵母分析结果。
图11显示单、双、三重AsvBs3重复模块和克隆载体的示意图。
图12A和12B表示大多数TAL效应子中重复区域末端存在的单一代表性TAL效应子重复(图12A)以及代表性截短重复(图12B)。显示核苷酸和编码的氨基酸序列。N代表编码RVD(用“XX”表示)的核苷酸。数字表示氨基酸位置。序列取自tal1c。
图13的示意图描绘tal1c基因,还显示重复区域减为单一、截短重复序列,产生pCS487的过程。M,MscI位置;S,SphI位置。
图14的示意图显示在pCS487的原始截短重复序列末端导入翻译沉默突变以形成PspXI和XhoI位点,产生pCS489。显示原始重复(SEQ ID NO:21)和突变重复(SEQ ID NO:23)中密码子18-21的序列。编码的氨基酸序列(SEQ ID NO:22)没有被突变改变。突变的核苷酸用斜体表示。
图15是卡那霉素抗性质粒pCS488的图,其仅在Gateway进入载体pENTR-D(英杰公司(Invitrogen),加利福尼亚州卡尔斯巴德)中编码tal1c的N和C末端部分,没有所述重复区域。
图16是命名为pCS493的单一重复起始质粒图,其编码具有RVD NI的重复序列。三种名为pCS494、pCS495和pCS496的其他质粒相同,除了其编码的RVD(右边所示)。
图17A显示具有RVD NI的单一重复模块的核苷酸和编码的氨基酸序列。下划线表示5’XhoI相容性粘性末端,MscI位点,和3’PspXI/XhoI相容性粘性末端。粗体显示RVD和编码其的核苷酸。构建与所示相同的3种其他重复模块,除了分别编码HD、NI和NG的RVD编码序列不同。图17B是名为pCS502的单一重复模块质粒图,其含有图17A所示重复编码序列。还生成与pCS502相同的名为pCS503、pCS504和pCS505的质粒,除了其编码的RVD(右边所示)。
图18A显示具有RVD NI的单一重复模块的核苷酸和编码的氨基酸序列,其中核苷酸取代(斜体)阻止连接到PspXI/XhoI位点后5’末端XhoI位点的重建并破坏内部MscI位点。粗体显示RVD和其编码核苷酸。构建与所示相同的3种其他重复模块,除了分别编码HD、NI和NG的RVD编码序列不同。图18B是通过将其他重复模块依次连接到单一重复模块质粒中组装的三种重复模块的示意图。所述第一重复中的MscI位点和3’末端的PspXI位点仍为独特的,且整个模块侧接有两个XhoI位点。
图19是一、二和三重复模块质粒的完整组列表。
图20的流程图显示可用于组装任何重复序列到tal1c“主干”中生成自定义TAL效应子基因的方法步骤。
图21A和21B的示意图显示构建靶向所示核苷酸序列的TAL内切核酸酶中重复模块的组装。在图21A中,质粒pCS519、pCS524、pCS537、pCS551、pCS583和pCS529的重复模块依次添加到起始质粒pCS493的序列中,产生质粒pMAT55、pMAT56、pMAT57、pMAT58、pMAT59和pMAT60。在图21B中,质粒pCS530、pCS533、pCS522和pCS541的重复模块依次添加到质粒pMAT1的序列中,产生质粒pMAT61、pMAT62、pMAT63和pMAT64。
图22A是TAL效应子蛋白的示意图。BamHI片段(由B表示)融合FokI内切核酸酶的催化结构域产生TALEN。N,N-末端;NLS,核定位信号;B,BamHI位点,AD,酸性激活域。图22B描绘了用TAL效应子AvrBs3和PthXo1构建的TALEN的活性。Avr-FokI、AvrBs3 TALEN;Pth-FokI、PthXo1 TALEN、Avr-FokI和Pth-FokI、AvrBs3和PthXo1融合催化失活形式的FokI(Bitinaite等(1998)Proc.Natl.Acad.Sci.USA 95:10570-10575);含有Zif268DNA结合域的锌指核酸酶ZFN(Porteus和Baltimore(2003)Science 300:763)。
图23是参比PthXo1氨基酸序列(SEQ ID NO:16)。
图24是参比PthXo1核酸序列(SEQ ID NO:32)。
图25是pFZ85载体图。
图26显示avrBs3_TALEN的氨基酸序列(SEQ ID NO:33)。
图27显示pthXo1_TALEN的氨基酸序列(SEQ ID NO:34)。
图28A描绘AvrBs3和PthXo1 TALENS在具有不同间隔长度的靶标上的活性。ZFN,Zif268-衍生的锌指核酸酶。图28B描绘异型二聚TALEN的活性。显示含有PthXo1-FokI和AvrBs3-FokI表达载体和具有靶标的质粒的酵母中的活性(Avr-FokI,Pth-FokI),所述靶标由头尾方向被15bp分离的各识别位点组成。还显示单独AvrBs3(Avr-FokI)和PthXo1(Pth-FokI)TALENS以及Zif268(ZFN)在其各自靶标上的活性作为参考。作为阴性对照,对仅具有Avr-FokI、Pth-FokI靶位点质粒的酵母培养物分析LacZ活性(显示为(-))。
图29A的表显示个体自定义TALEN和其各DNA识别序列的RVD序列。图29B描绘自定义TALEN的活性。(-),仅具有靶位点质粒的阴性对照;ZFN,锌指核酸酶阳性对照。
图30显示20个靶向和TAL效应子对的末端核苷酸和RVD频率。
图31是Golden Gate克隆系统的示意图[Engler等(2008)PLoS One 3:e3647;和Engler等(2009)PLoS One 4:e5553]。
图32A和32B显示用本文所述Golden Gate克隆方法组装和克隆自定义TAL效应子重复编码阵列的58个质粒组。Tet,四环素抗性基因,质粒选择标记;spec,壮观霉素抗性基因,质粒选择标记;amp,氨苄青霉素抗性基因,质粒选择标记。
图33显示用图32所示质粒组通过Golden Gate克隆方法组装和克隆自定义TAL效应子重复编码阵列的方法示意图。为了阐明目的,显示任意的重复阵列组装。spec,壮观霉素抗性基因,质粒选择标记;amp,氨苄青霉素抗性基因,质粒选择标记。
图34A-34U显示如本文实施例9所述生成的TALEN的氨基酸序列。图34A,端粒酶-TALEN124;图34B,gridlock-TALEN105;图34C,adh1-TALEN58;图34D,adh1-TALEN63;图34E,adh1-TALEN68;图34F,adh1-TALEN73;图34G,adh1-TALEN89;图34H,gridlock-TALEN106;图34I,adh1-TALEN64;图34J,adh1-TALEN69;图34K,adh1-TALEN74;图34L,tt4-TALEN90;图34M,端粒酶-TALEN121;图34N,端粒酶-TALEN126;图34O,gridlock-TALEN107;图34P,gridlock-TALEN117;图34Q,端粒酶-TALEN131;图34R,端粒酶-TALEN136;图34S,adh1-TALEN60;图34T,tt4-TALEN85;图34U,gridlock-TALEN102。
图35描绘了酵母试验检测的TALEN活性,使用长度增加(9-、10-、12-、13-、15-、16-、17-、或18聚体)的自定义TALEN单体。TALEN靶向拟南芥(Arabidopsis)和斑马鱼基因,如图所示。
图36A显示被两种TALEN对靶向的拟南芥ADH1基因的两种不同DNA靶序列。图36B描绘靶向拟南芥ADH1基因的功能性TALEN对的酵母试验数据。
图37A是用于检测拟南芥原生质体中TALEN诱导突变的限制性内切核酸酶试验的示意图。图37B显示所述限制性内切核酸酶试验中未消化DNA的9种克隆序列。所述克隆中6种具有非同源末端连接(NHEJ)导入的突变。
图38A显示数种系统发生学上不同TAL效应子的第0重复序列,番茄细菌性斑点病菌(Xanthomonas gardneri)的AvrHah1、辣椒细菌性疮痂病菌(X.campestrispv.vesicatoria)的AvrBs3、水稻白叶枯病菌(X.oryzae pv.oryzae)的PthXo1、柑桔溃疡病(X.citri)的PthA和水稻细菌性条斑病菌(X.oryzae pv.oryzicola)的Tal1c。加框表示多态性位置。图38B的示意图显示PthXo1的第0和第1重复。“第0”重复紧接第1重复之前,显示35%的相同性,且具有相似的预测二级结构。第1重复的RVD和第0重复的候选类似残基用下划线表示。*,缺口;H,螺旋;E,延伸。用JPred预测结构(Cole等(2008)Nucl.Acids Res.36:W197-W201)。
图39显示从转染有质粒的人胚胎肾293T细胞分离的总蛋白的western印迹,所述质粒编码所示带V5-标签的TAL效应蛋白AvrBs3、PthXo1和Tal1c,然后用小鼠抗V5抗体进行免疫检测。所示经免疫标记的肌动蛋白作为各泳道等量上样的对照。
图40A显示TALEN HPRT-3254-17的氨基酸序列,图40B显示TALEN HPRT-3286-20r的氨基酸序列。
图41A的示意图显示人染色体HPRT基因中的TALEN靶向位点。显示HPRT-3254-17和HPRT-3286-20r TALEN的结合位点、这些位点间隔中的Bpu10I位点、和扩增所述区域的引物位点。底部坐标给出编码序列的第一核苷酸中碱基对的距离。图41B显示图41A所示区域的PCR扩增产物的Bpu10I消化结果,用来自TALEN处理或未处理细胞的分离基因组DNA作为模板。扩增前用Bpu10I消化基因组DNA。用琼脂糖凝胶电泳分离DNA片段并用溴化乙啶观察。
具体实施方式
本专利申请提供涉及TAL效应子介导的序列特异性DNA识别的材料和方法。如本文所述,TAL效应子的初级氨基酸序列决定了其结合的核苷酸序列。发明人发现TAL效应子氨基酸序列和其DNA靶序列之间的关系是直接的,从而能预测TAL效应子的靶位置,还可定制TAL效应子以结合具体的核苷酸序列。可出于各种目的利用所述预测和定制。在一个实施例中,具体TAL效应子序列可融合内切核酸酶序列,使内切核酸酶靶向特异DNA序列,并随后在靶序列或其附近切割DNA。DNA中的切割(即双链断裂)可显著增加同源重组的频率。因此,联合载有与具体靶DNA序列有高度序列相似性的序列的DNA构建体,TALEN可用于促进复杂基因组中的定点诱变,即高度准确的高效敲除或改变基因功能,或添加基因或其他序列。
因此,本文提供的主题内容包括但不限于,生成遗传改良生物体(包括但不限于植物、真菌、果蝇(Drosophila)、线虫、斑马鱼、小鼠和其他哺乳动物)的材料和方法。该方法可包括例如,用数种重组核酸转染细胞。例如,细胞(如真核细胞)可用含供体核苷酸序列的第一重组核酸构建体和编码TAL核酸酶的第二重组核酸构建体转化,所述供体核苷酸序列包括相对于所述细胞中所发现相应靶核苷酸序列发生的改变。在一些实施方式中,所述细胞还可用编码可选择标记物的第三重组核酸构建体转化。如本文所述,供体核酸构建体的核酸序列可变为纳入转化细胞的基因组中。例如,用本文所述方法产生的植物细胞可生长至产生将已改变供体核苷酸序列纳入到其基因组中的植物。该植物的种子可用于生产具有表型的植物,所述表型例如相对未修饰植物的生长特征改变(如对各种生物和非生物压力的抗性或耐受性增加)、外观改变(如改变的颜色或高度)或组成改变(如碳、氮、油、蛋白、糖(例如糖或淀粉)、氨基酸、脂肪酸或次级代谢物的水平增加或减少)。
多核苷酸和多肽
本文提供分离的核酸和多肽。术语“核酸”和“多核苷酸”可互换使用,指RNA和DNA,包括cDNA、基因组DNA、合成(如化学合成的)DNA和含核酸类似物的DNA(或RNA)。多核苷酸可具有任何三维结构。核酸可为双链或单链(即有义链或反义单链)。多核苷酸的非限制性示例包括基因、基因片段、外显子、内含子、信使RNA(mRNA)、转移RNA、核糖体RNA、核酶、cDNA、重组多核苷酸、分支多核苷酸、质粒、载体、任何序列的分离DNA、任何序列的分离RNA、核酸探针和引物、以及核酸类似物。
本发明的多肽(例如TAL效应子-DNA修饰酶作为非限制性示例)可通过编码例如所述多肽的载体导入细胞或用递送载体将多肽本身导入细胞,所述递送载体关联或组合任何细胞透化技术例如声穿孔或电穿孔或这些技术的衍生技术。
如本文所用,涉及核酸时,“分离的”指与基因组如植物基因组中存在的其他核酸分离的核酸,包括正常侧接基因组中核酸的一侧或两侧的核酸。本文所用术语“分离的”涉及核酸时还包括任何非天然产生的序列,因为所述非天然产生的序列未在天然情况下发现且其在天然产生的基因组中没有紧邻序列。
分离的核酸可为例如DNA分子,只要在天然产生基因组中正常发现与该DNA分子直接侧接的核酸序列之一被移除或缺失。因此,分离的核酸包括但不限于,不依赖其他序列作为单独分子(如化学合成的核酸,或PCR或限制性内切核酸酶处理产生的cDNA或基因组DNA片段)存在的DNA分子,以及纳入载体、自主复制质粒、病毒(如拟逆转录病毒、逆转录病毒、慢病毒、腺病毒或疱疹病毒)的DNA,或原核或真核的基因组DNA。此外,分离的核酸可包括重组核酸如作为部分杂交或融合核酸的DNA分子。例如cDNA库或基因组库内的数百个到数百万其他核酸中存在的核酸,或含有基因组DNA限制性酶消化物的凝胶切块不视作分离的核酸。
核酸可通过例如化学合成或聚合酶链式反应(PCR)生产。PCR指扩增靶核酸的程序或技术。PCR可用于从DNA以及RNA中扩增特定序列,包括总基因组DNA或总细胞RNA的序列。各种PCR方法如PCR Primer:A Laboratory Manual(《PCR引物:实验室手册》),Dieffenbach和Dveksler编,冷泉港实验室出版社(Cold Spring Harbor Laboratory Press),1995所述。通常,用感兴趣区域末端或超出其的序列信息来设计寡核苷酸引物,其与待扩增模板相反链的序列相同或相似。还可用各种PCR策略,通过其可将位点特异性核苷酸序列修饰导入模板核酸。
分离的核酸还可通过突变获得。例如,供体核酸序列可用标准技术突变,包括寡核苷酸定点突变和通过PCR的定点突变。参见,Short Protocols in Molecular Biology(《分 子生物学简明实验方案》),第8章,格林出版联合公司(Green Publishing Associates,Inc.)和约翰威利公司(John Wiley and Sons,Inc.),Ausubel等编,1992。
本文所用术语“多肽”指两种或多种亚基氨基酸的化合物,无论是否经过翻译后修饰(如磷酸化或糖基化)。所述亚基用肽键或其他键例如酯键或醚键连接。术语“氨基酸”指天然和/或非天然或合成的氨基酸,包括D/L光学异构体。
涉及多肽时,“分离的”或“纯化的”表示所述多肽一定程度上分离自胞组分,其通常天然存在(如其他多肽、脂质、糖和核酸)。纯化的多肽可在非还原性聚丙烯酰胺凝胶上产生单一主条带。纯化的多肽纯度可至少为约75%(例如纯度至少80%、85%、90%、95%、97%、98%、99%或100%)。纯化的多肽可通过例如从天然来源提取、化学合成或者宿主细胞或转基因植物中的重组生成而获得,且可用例如亲和色谱、免疫沉淀、尺寸排阻色谱和离子交换色谱而纯化。纯化的程度可通过任何合适的方法测量,包括但不限于柱色谱、聚丙烯酰胺凝胶电泳、或高效液相色谱。
重组构建体
本文还提供重组核酸构建体(例如载体)。“载体”是复制子,例如质粒、噬菌体或粘粒,其中插入另一DNA区段以使所插入区段进行复制。通常,载体与合适的控制元件关联时能复制。合适的载体主干包括例如,本领域通常使用的那些如质粒、病毒、人工染色体、BAC、YAC、或PAC。术语“载体”包括克隆和表达载体以及病毒载体和整合载体。“表达载体”是包括一种或多种表达控制序列的载体,且“表达控制序列”是控制和调节另一DNA序列转录和/或翻译的DNA序列。合适的表达载体包括但不限于衍生自例如噬菌体、杆状病毒、烟草花叶病毒、疱疹病毒、细胞巨化病毒、逆转录病毒、牛痘病毒、腺病毒、和腺相关病毒的质粒和病毒载体。大量载体和表达系统可从例如下述公司购得:诺瓦基公司(Novagen)(威斯康星州麦迪逊)、克隆泰克公司(Clontech)(加州帕洛阿尔托)、司查塔基公司(Stratagene)(加利福尼亚州拉由拉市)和英杰/生命技术公司(Invitrogen/Life Technologies)(加利福尼亚州卡尔斯巴德)。
术语“调节区域”、“控制元件”和“表达控制序列”指影响转录或翻译起始和速率,以及转录或多肽产物的稳定性和/或移动性的核酸序列。调控区域包括但不限于,启动子序列、增强子序列、响应元件、蛋白识别位点、诱导型元件、启动子控制元件、蛋白结合序列、5’和3’非翻译区(UTR)、转录起始位点、终止序列、聚腺苷酸化序列、内含子和其他可存在于编码序列中的调节区域,例如分泌信号、核定位序列(NLS)和蛋白酶切割位点。
本文所用的“可操作性连接”表示纳入遗传构建体从而表达控制序列有效控制感兴趣的编码序列表达。当RNA聚合酶能转录编码序列到RNA中,其若为mRNA则可翻译为所述编码序列编码的蛋白时,编码序列“可操作性连接”细胞中的表达控制序列和“在其控制之下”。因此,调控区域可调节例如调控、促进或驱动需要表达修饰靶核酸的植物细胞、植物或植物组织中的转录。
启动子是由DNA分子区域组成的表达控制序列,通常在转录起始位点上游的100核苷酸内(一般接近RNA聚合酶II的起始位点)。启动子参与RNA聚合酶和其他蛋白的识别和结合以起始并调节转录。为了将编码序列置于启动子控制之下,通常需要将所述多肽的翻译阅读框的翻译起始位点置于所述启动子下游的1-约50核苷酸。但是,启动子可置于所述翻译起始位点上游的多至约5000核苷酸处,或翻译起始位点上游的约2000核苷酸处。启动子通常包括至少一个核心(基础)启动子。启动子还可包括至少一种控制元件例如上游元件。所述元件包括上游激活区(UAR)和任选地影响多核苷酸转录的其他DNA序列如合成的上游元件。
选择要包括的启动子取决于数种因素,包括但不限于功效、选择能力、诱导能力、所需的表达水平和细胞或组织特异性。例如,可使用仅或主要在具体组织、器官和细胞类型中分别产生转录的组织-、器官-和细胞-特异性启动子。在一些实施方式中,植物组织特异性启动子可为合适的调节区域,所述组织如茎干、软组织、基本分生组织、维管束、形成层、韧皮部、外皮、芽顶端分生组织、侧芽分生组织、根顶端分生组织、侧根分生组织、叶原基、叶肉或叶表皮。在一些实施方式中,主要特异于种子的启动子(“种子优先启动子”)有用。种子特异性启动子可在种子发育期间促进胚乳和子叶组织中可操作连接核酸的转录。或者,组成型启动子可在整个植物发育期间促进大多数或所有植物组织中可操作连接核酸的转录。其他类型启动子包括但不限于诱导型启动子,例如响应外部刺激如化学剂、发育刺激或环境刺激而产生转录的启动子。
基础启动子是转录起始所需转录复合物组装必要的最小序列。基础启动子通常包括可能位于转录起始位点上游约15-约35核苷酸的“TATA盒”元件。基础启动子还可包括“CCAAT盒”元件(通常为序列CCAAT)和/或GGGCG序列,其可位于转录起始位点上游约40-约200核苷酸,通常为约60-约120核苷酸。
可包括在本文所提供核酸构建体中的启动子非限制性示例包含花椰菜花叶病毒(CaMV)35S转录起始区域、衍生自根癌农杆菌(Agrobacterium tumefaciens)T-DNA的1’或2’启动子、Busk((1997)Plant J.11:1285-1295)所述的玉米叶片特异基因启动子、玉米和其他物种的kn1-相关基因、以及各种植物基因的转录起始区域如玉米泛素1启动子。
5’非翻译区(UTR)转录但不翻译,位于转录起始位点和翻译起始密码子之间,可包括+1核苷酸。3’UTR可位于翻译终止密码子和转录末端之间。UTR可具有特定功能如增加mRNA信使稳定性或翻译衰减。3’UTR的示例包括但不限于聚腺苷酸信号和转录终止序列。编码区域3'末端的聚腺苷酸区还可操作性连接编码序列。所述聚腺苷酸区可衍生自天然基因、多种其他植物基因、或农杆菌(Agrobacterium)T-DNA。
本文提供的载体还可包括例如复制起点和/或支架连接区(SAR)。此外,表达载体可包括设计用于协助操作或检测(例如纯化或定位)所表达多肽的标签序列。标签序列如绿色荧光蛋白(GFP)、谷胱甘肽S转移酶(GST)、聚组氨酸、c-myc、血细胞凝集素或FlagTM标签(柯达公司(Kodak),康涅狄格州纽黑文)序列通常表达为与编码多肽的融合物。所述标签可插入所述多肽内的任何位置,包括羧基或氨基末端。
“递送载体”或“多种递送载体”表示本发明可采用的任何递送载体,用于将本发明需要的试剂/化学品和分子(蛋白或核酸)与细胞接触或递送到细胞内或亚细胞组分内。其包括但不限于脂质体递送载体、病毒递送载体、药物递送载体、化学运载体、聚合物运载体、脂复合体、多聚复合体、树状聚合物、微泡(超声造影剂)、纳米颗粒、乳剂或其他合适的转移载体。这些递送载体可递送分子、化学品、大分子(基因、蛋白)或其他载体如质粒、Diatos公司开发的肽。在这些情况中,递送载体为分子运载体。“递送载体”或“多种递送载体”还指进行转染的递送方法。
术语“载体”或“多种载体”指能够转运其所连接的另一核酸的核酸分子。本发明中的“载体”包括但不限于病毒载体、质粒、RNA载体或线性或环形DNA或RNA分子,其可由染色体、非染色体、半合成或合成核酸组成。载体优选能自主复制(附加型载体)和/或表达其连接的核酸(表达载体)。大量合适的载体为本领域技术人员已知,并可市售获得。
病毒载体包括逆转录病毒、腺病毒、细小病毒(如腺伴随病毒)、冠状病毒、负链RNA病毒如正粘病毒(如流感病毒)、弹状病毒(如狂犬病毒和水泡性口炎病毒)、副粘病毒(如麻疹和仙台)、正链RNA病毒如小RNA病毒和甲型病毒、和双链DNA病毒,包括腺病毒、疱疹病毒(如1和2型单纯性疱疹病毒、EB病毒、巨细胞病毒)、和痘病毒(如牛痘、鸟痘和金丝雀痘)。其他病毒包括例如诺沃克病毒、外衣病毒、黄病毒、呼吸道肠道病毒、乳多空病毒、嗜肝DNA病毒、和肝炎病毒。逆转录病毒的例子包括:鸟白血病肉瘤、哺乳动物C型、B型病毒、D型病毒、HTLV-BLV组、慢病毒、泡沫病毒(Coffin,J.M.,Retroviridae:The viruses and theirreplication(“逆转录病毒:病毒和其复制”),收录于Fundamental Virology(《基础病毒学》)第3版,B.N.Fields,等编,费城的林普科特瑞文出版社(Lippincott-RavenPublishers),1996)。
-“慢病毒载体”表示就基因递送极具前景的基于HIV的慢病毒载体,因为其包装容量相对较大、免疫原性低且能高效稳定转导大范围的不同细胞类型。慢病毒载体通常在三种(包装、包膜和转移)或更多质粒瞬时转染到生产细胞后生成。类似HIV,慢病毒载体通过病毒表面糖蛋白与细胞表面受体的相互作用进入靶细胞。进入后,所述病毒RNA通过病毒逆转录酶复合物介导进行逆转录。逆转录的产物是双链线性病毒DNA,其是受感染细胞DNA中病毒整合的底物。所述慢病毒载体可为“非整合”或“整合”。
-“整合的慢病毒载体(或LV)”指能整合到靶细胞基因组中的非限制性示例载体。
-相反的“非整合慢病毒载体(或NILV)”表示不通过病毒整合酶作用整合靶细胞基因组的有效基因递送载体。
一种优选的载体是附加体,即能在染色体外复制的核酸。载体优选能自主复制和/或表达其连接的核酸。能引导其操作性连接基因表达的载体在本文中称为“表达载体”。本发明的载体包括但不限于YAC(酵母人工染色体)、BAC(细菌人工染色体)、杆状病毒载体、噬菌体、噬菌粒、粘粒、病毒载体、质粒、RNA载体或线性或环形DNA或RNA分子,其可由染色体、非染色体、半合成或合成DNA组成。一般,重组DNA技术中利用的表达载体常为“质粒”的形式,通常指环状双链DNA环,其载体形式不结合染色体。本领域技术人员已知大量合适的载体。载体可含选择标记物,例如:用于真核细胞培养物的新霉素磷酸转移酶、组氨醇脱氢酶、二氢叶酸还原酶、潮霉素磷酸转移酶、单纯疱疹病毒胸苷激酶、腺苷脱氨酶、谷氨酰胺合成酶、和次黄嘌呤鸟嘌呤磷酸核糖基转移酶;用于酿酒酵母(S.cerevisiae)的TRP1;大肠杆菌(E.coli)中的四环素、利福平或氨苄青霉素抗性。所述载体优选为表达载体,其中编码感兴趣多肽的序列置于合适的转录和翻译控制元件控制下以生产或合成所述多肽。因此,所述多核苷酸包括在表达盒中。更具体的,所述载体包括复制起点、可操作连接所述编码多核苷酸的启动子、核糖体结合位点、RNA剪接位点(用基因组DNA时)、聚腺苷酸化位点和转录终止位点。其还可包括增强子或沉默子元件。启动子的选择取决于表达多肽的细胞。合适的启动子包括组织特异性和/或诱导型启动子。诱导型启动子的示例有:由重金属水平增加诱导的真核金属硫蛋白启动子、响应异丙基-β-D-硫代吡喃半乳糖苷(IPTG)而诱导的原核lacZ启动子和温度增加诱导的真核热激蛋白启动子。组织特异性启动子的示例为骨骼肌肌氨酸激酶、前列腺特异抗原(PSA)、α-抗胰蛋白酶、人表面活性蛋白(SP)A和B、β酪蛋白和酸性乳清蛋白基因。
诱导型启动子可由病原体或压力诱导,更优选由诸如冷、热、UV光、或高离子浓度等压力诱导(综述见Potenza等(2004)In vitro Cell Dev Biol 40:1-22)。诱导型启动子可由化学品诱导[综述见Moore等(2006);Padidam(2003);(Wang等(2003);和(Zuo和Chua(2000)]。
递送载体和载体可关联或组合任何细胞透化技术例如声穿孔或电穿孔或这些技术的衍生技术。
应理解重组多核苷酸中可存在多于一个调节区域,如内含子、增强子、上游激活区域和诱导型元件。
重组核酸构建体可包括插入适于转化细胞(如植物细胞或动物细胞)的载体中的多核苷酸序列。重组载体可用例如标准重组DNA技术制备(参见例如,Sambrook等(1989)Molecular Cloning,A Laboratory Manual(《分子克隆,实验室手册》)第2版;纽约州冷泉港的冷泉港实验室公司(Cold Spring Harbor Laboratory,Cold Spring Harbor,N.Y.))。
本文所述重组核酸序列能通过非常规(即随机、非同源、非位点特异)重组纳入细胞的基因组中,或本文所述重组核酸序列适于通过同源重组纳入细胞的基因组中。适于通过同源重组整合的核酸序列侧接与内源靶核苷酸序列相似或相同序列的两侧,这有助于重组核酸在含有内源靶核苷酸序列的基因组中具体位点的整合。适于通过同源重组整合的核酸序列还包括序列特异性核酸酶的识别位点。或者,序列特异性核酸酶的识别位点可位于待转化的细胞基因组中。下述供体核酸序列通常适于通过同源重组整合。
在一些实施方式中,编码可选择标记物的核酸还可适于通过同源重组整合,且因此可侧接与所述植物基因组内的内源序列(如序列特异性核酸酶的切割位点的内源序列)相似或相同的序列两侧。在一些情况中,含选择性标记物编码序列的核酸还可包括序列特异性核酸酶的识别位点。在这些实施方式中,序列特异性核酸酶的识别位点可与供体核酸序列中所含的相同或不同(即可被与供体核酸序列相同的核酸酶识别,或被与供体核酸序列不同的核酸酶识别)。
在一些情况中,重组核酸序列可适于通过位点特异性重组整合到细胞基因组中。本文所用的“位点特异性”重组指核酸序列靶向基因组中具体位点时发生的重组,不通过重组核酸内序列和基因组内序列之间的同源性,而是通过识别特异性核酸序列并催化这些位点之间DNA链相互交换的重组酶作用。因此,位点特异性重组指酶介导的两种确定核苷酸序列的切割和连接。可使用任何合适的位点特异性重组系统,包括例如Cre-lox系统或FLP-FRT系统。在这些实施方式中,除了供体核苷酸序列和核酸酶编码序列以外,还可将编码重组酶的核酸导入细胞,且在一些情况中,将选择标记物序列导入细胞。参见例如,美国专利号4,959,317。
序列特异性内切核酸酶
本文提供序列特异性核酸酶和编码所述序列特异性内切核酸酶的重组核酸。序列特异性内切核酸酶可包括TAL效应子DNA结合域和内切核酸酶结构域。因此,编码该序列特异性内切核酸酶的核酸可包括来自连接核酸酶核苷酸序列的核酸特异性TAL效应子的核苷酸序列。
TAL效应子是植物病原菌蛋白,其通过所述病原体注入植物细胞,其中所述蛋白进入细胞核并作为转录因子启动特定植物基因。TAL效应子的初级氨基酸序列决定其结合的核苷酸序列。因此,可根据TAL效应子预测靶位置,且如本文所述,TAL效应子还可被工程改造并生成以用于结合具体核苷酸序列。
融合TAL效应子编码核酸序列的是编码核酸酶或部分核酸酶的序列,通常为II型限制性内切核酸酶如FokI的非特异性切割结构域(Kim等(1996)Proc.Natl.Acad.Sci.USA93:1156-1160)。其他有用的内切核酸酶可包括例如HhaI、HindIII、NotI、BbvCI、EcoRI、BglI、和AlwI。可利用一些内切核酸酶仅作为二聚物起作用的现象来提高TAL效应子的靶特异性。例如,在一些情况中,各FokI单体可融合识别不同DNA靶序列的TAL效应子序列,且仅当两个识别位点足够接近时所述失活的单体才会在一起产生功能性酶。通过需要DNA结合以活化所述核酸酶,可产生高度位点特异的限制性酶。
本文提供的序列特异性TALEN可识别细胞中存在的预选定靶核苷酸序列内的具体序列。因此,在一些实施方式中,可扫描靶核苷酸序列的核酸酶识别位点,且可根据靶序列选择具体的核酸酶。在其他情况中,TALEN可工程改造成靶向具体的细胞序列。编码所需TALEN的核苷酸序列可插入任何合适的表达载体,且可连接一种或多种表达控制序列。例如,核酸酶编码序列可操作性连接待转化植物物种中引导所述内切核酸酶组成型表达的启动子序列。或者,内切核酸酶编码序列可操作性连接引导条件性表达的启动子序列(例如,在某些营养条件下表达)。例如,花椰菜花叶病毒35S启动子可用于组成型表达。其他组成型启动子包括但不限于胭脂碱合成酶启动子、泛素启动子和肌动蛋白启动子。在一些实施方式中,人工雌激素诱导启动子可用于条件性表达,且高水平的转录可在植物暴露于雌激素时实现。其他可用的条件启动子包括例如,热诱导热激蛋白基因启动子和光调节启动子例如来自编码二磷酸核酮糖羧化酶启动子大亚基的基因。
为了治疗目的,将本发明的TAL效应子DNA修饰酶和药学上可接受的赋形剂以治疗有效量给予。若给予量为生理上显著,则称该组合以“治疗有效量”给予。若试剂使接受者的生理发生可检测的变化,则该试剂为生理上显著。本文中,若试剂的存在使一种或多种靶疾病的症状严重性和损伤或异常的基因组修正降低,则该试剂为生理上显著。包括靶向DNA和/或编码TAL效应子DNA修饰酶的核酸的载体可通过各种方法导入细胞(如注射、直接摄取、弹轰击、脂质体、电穿孔)。TAL效应子DNA修饰酶可用表达载体在细胞中稳定或瞬时表达。真核细胞表达技术为本领域技术人员已知。(参见Current Protocols in Human Genetics(《新编人类遗传学实验方案》):第12章“Vectors For Gene Therapy(基因治疗载体)”和第13章“Delivery Systems for Gene Therapy(基因治疗的递送系统)”)。
本发明的另一方面,所述TAL效应子DNA修饰酶基本无免疫原性,即造成很小或没有不良的免疫反应。可根据本发明使用改善或消除此类有害免疫反应的各种方法。在优选实施方式中,TAL效应子DNA修饰酶基本没有N甲酰甲硫氨酸。避免不需要免疫反应的另一方法是偶联TAL效应子DNA修饰酶和聚乙二醇("PEG")或聚丙二醇("PPG")(平均分子量(MW)优选500-20,000道尔顿)。例如Davis等(US 4,179,337)所述,与PEG或PPG偶联可提供具有抗病毒活性的非免疫原性、生理活性、水溶性TAL效应子DNA修饰酶偶联物。还使用聚乙二醇-聚丙烯醇共聚物的相似方法如Saifer等(US 5,006,333)所述。
供体载体
本文还提供包括供体核苷酸序列的重组核酸。供体核苷酸序列可包括与待转化细胞的基因组内源的预选定靶核苷酸序列相比具有一种或多种修饰(即取代、缺失或插入)的变体序列(本文还称为“修饰的靶核苷酸序列”)。供体核酸内的变体序列通常侧接与细胞内源靶核苷酸序列相似或相同的序列两侧。侧接序列可具有任何合适的长度,且通常至少长50核苷酸(如至少50核苷酸、至少75核苷酸、至少100核苷酸、至少200核苷酸、至少250核苷酸、至少300核苷酸、至少500核苷酸、至少750核苷酸、至少1000核苷酸、约50-约5000核苷酸、约100-2500核苷酸、约100-约1000核苷酸、约100-500核苷酸、约200-约500核苷酸、或约250-400核苷酸)。因此,同源重组可在重组供体核酸构建体和所述变体序列两侧的内源靶标之间发生,从而得到的细胞基因组在来自例如相同基因的内源序列环境中包含所述变体序列。可生成供体核苷酸序列以靶向基因组中任何合适序列。例如在植物中,供体核苷酸序列可靶向脂质生物合成基因、糖生物合成基因、种子贮藏蛋白基因、抗病或抗虫基因、压力耐受基因、耐旱基因、或生成抗营养因子的基因。此外,所述供体核苷酸序列包含本文所述序列特异性核酸酶的识别位点。
选择标记物
本文提供的一些方法包括使用编码可选择或可筛选标记物的第三重组核酸。产生可选择特性的编码多肽的核苷酸序列可纳入含有一种或多种表达控制序列的表达载体中。例如,表达载体可包括编码可选择标记物的序列,其可操作性连接待转化植物细胞中引导组成型表达的启动子序列。合适的可选择标记可包括但不限于,对抗生素如卡那霉素、G418、博来霉素、氨苄青霉素或潮霉素产生抗性的多肽,或除草剂如草丁膦、氯磺隆、或草胺膦。
例如在用于植物的实施方式中,可选择的标记物可产生对除草剂的抗性,除草剂抑制生长点或分生组织,如咪唑并啉酮或磺酰脲。该分类编号中用于突变ALS和AHAS酶的示例性多肽如美国专利号5,767,366和5,928,937所述。美国专利号4,761,373和5,013,659针对耐受各种咪唑并啉酮或磺酰胺除草剂的植物。美国专利号4,975,374涉及含编码突变鄂谷氨酰胺合成酶(GS)基因的植物细胞和植物,其耐受已知能抑制GS的除草剂如草胺膦和甲硫氨酸磺基肟产生的抑制。美国专利号5,162,602公开了对环己二酮和芳氧基苯氧基丙酸除草剂的抑制有抗性的植物。该抗性由改变的乙酰辅酶A羧化酶(ACC酶)产生。
用于抗草甘膦的多肽(以商标名出售)还适用于植物。参见,例如美国专利号4,940,835和4,769,061。美国专利号5,554,798公开了转基因草甘膦抗性玉米植物,其抗性是由改变的5-烯醇式丙酮酰-3-磷酸莽草酸(EPSP)合成酶赋予。该多肽会赋予对草甘膦除草剂组合物的抗性,所述组合物包括但不限于草甘膦盐如三甲基锍盐、异丙胺盐、钠盐、钾盐和铝盐。参见例如美国专利号6,451,735和6,451,732。
对膦化合物如草丁膦铵或草胺膦、和吡啶氧基或苯氧丙酸以及环己酮有抗性的多肽也适用。参见例如,欧洲公开号0 242 246以及美国专利号5,879,903、5,276,268和5,561,236。
其他除草剂包括抑制光合作用的那些,例如三嗪和苄腈(腈水解酶)。参见例如,美国专利号4,810,648。其他除草剂包括2,2-二氯丙酸、烯禾啶、吡氟氯禾灵、咪唑啉酮类除草剂、磺脲类除草剂、三唑嘧啶类除草剂、s-三嗪除草剂和溴苯腈。赋予对原卟啉原氧化酶抗性的除草剂也适用。参见例如,美国专利公开号20010016956和美国专利号6,084,155。
在一些实施方式中,编码可选择标记物的重组核酸序列可适于通过位点特异重组整合到细胞(如植物或动物细胞)基因组中。例如,编码可选择标记物的序列可由重组酶如Cre或FLP的识别序列侧接。在其他实施方式中,编码可选择标记物的重组核酸适于通过同源重组整合到植物基因组中。在该核酸中,编码可选择标记物的序列可侧接有与待引入重组核酸的植物细胞基因组内所发现内源核苷酸序列相似或相同的序列。至少一种内源序列可以是序列特异性核酸酶的切割位点。编码可选择标记的核酸还可含有序列特异核酸酶的识别位点。所述核酸酶可为与靶向供体核苷酸序列的核酸酶相同的序列特异性核酸酶,或为与靶向供体核苷酸序列的核酸酶不同的序列特异性核酸酶。在其他实施方式中,编码可选择标记物的重组核酸适于通过非常规重组整合到植物细胞基因组中。所述核酸通常缺少本文所述适于同源或位点特异重组的核酸内含有的侧接序列和核酸酶识别位点。
方法
本文提供的一种或多种构建体可用于转化细胞和/或DNA修饰酶可导入细胞,从而生成遗传改良的生物体(如植物或动物)。因此,还提供含有本文所述核酸和/或多肽的遗传改良生物体和细胞。在一些实施方式中,转化的细胞具有整合到其基因组中的重组核酸构建体,即可稳定转化。稳定转化的细胞通常在每次细胞分裂保留导入的核酸序列。构建体可以同源方式整合,从而转化细胞内源的核苷酸序列被所述构建体替代,其中所述构建体含有对应于内源序列的序列,但其中含有相对内源序列的一种或多种修饰。应注意虽然含有该经修饰内源序列的植物或动物在本文可称为“遗传改良生物体”(GMO),但修饰的内源序列不视作转基因。构建体还可以非常规方式整合,从而其随机整合到所述转化细胞的基因组中。
或者,细胞可被瞬时转化,从而所述构建体没有整合到其基因组中。例如,含有TALEN编码序列的质粒载体可导入细胞,从而所述TALEN编码序列表达但所述载体未稳定整合到基因组中。瞬时转化的细胞通常在每次细胞分裂中丧失一些或所有导入的核酸构建体,从而足够数量的细胞分裂后在后代细胞中不能检测到所导入的核酸。然而,TALEN编码序列的表达足以实现供体序列和内源靶序列的同源重组。瞬时转化和稳定转化的细胞都可用于本文所述方法。
具体对于遗传改良植物细胞,本文所述方法中使用的细胞可构成部分或所有完整植物。此类植物可以所考虑物种适合的方法生长在培养室、温室或田地中。遗传改良植物可根据具体目的所需进行培育,例如将重组核酸引入其他品系中、将重组核酸转移到其他物种或用于其他理想性状的进一步筛选。或者,对于那些适合这类技术的物种,遗传改良植物可进行无性繁殖。后代包括具体植物或植物品系的子代。所述植物的后代包括F1、F2、F3、F4、F5、F6形成的种子和后代植物,或BC1、BC2、BC3形成的种子和后代植物,或F1BC1、F1BC2、F1BC3形成的种子和后代植物。可培养遗传改良植物产生的种子,然后自交(或异交和自交)获得与核酸构建体同源的种子。
如需要,遗传修饰的细胞(如植物细胞或动物细胞)可悬浮培养,或进行组织或器官培养。为了本文所述方法的目的,可使用固体和/或液体组织培养技术。使用固体培养基时,细胞可直接置于所述培养基上或可置于滤膜上,然后与所述培养基接触。使用液体培养基时,细胞可置于漂浮装置上如接触液体培养基的孔膜。固体培养基通常由液体培养基添加琼脂制得。例如,固体培养基可为含琼脂和适当浓度生长素如2,4-二氯苯氧基乙酸(2,4-D)和适当浓度细胞分裂素如激动素的穆拉什格斯固格(MS)培养基。
细胞可用一种重组核酸构建体或多种(如2、3、4或5)重组核酸构建体转化。若使用多个构建体,其可同时或依次转化。本领域已知各种物种的转化技术。本文所述多肽和/或重组载体可用许多已知方法中的任一种导入宿主基因组中,所述方法包括电穿孔、微注射和生物射弹方法。或者,多肽或载体可与合适的T-DNA侧接区域组合并引入常规的根癌农杆菌(Agrobacterium tumefaciens)宿主载体。本领域熟知所述根癌农杆菌介导的转化技术,包括二元载体的卸装和使用。其他基因转移和转化技术包括通过钙或PEG进行的原生质体转化、电穿孔介导的裸露DNA摄入、脂质体介导的转染、电穿孔、病毒载体介导的转化和微弹轰击(参见例如,美国专利5,538,880、5,204,253、5,591,616和6,329,571)。若植物细胞或组织培养物用作转化的接受组织,植物可用本领域技术人员已知的技术从转化的培养物中再生。
在一些实施方式中,DNA修饰酶(如TALEN)可直接导入细胞。例如,多肽可通过机械注射、用细菌III型分泌系统递送、电穿孔或农杆菌(Agrobacterium)介导的转移来导入细胞。参见例如,Vergunst等(2000)Science 290:979-982对农杆菌VirB/D4转运系统的讨论,和其在介导核蛋白T复合物转移到植物细胞中的用途。
进一步对于植物,本文所述的多核苷酸、载体和多肽可导入许多单子叶和双子叶植物和植物细胞系统,包括双子叶植物如红花、紫花苜蓿、大豆、咖啡、苋菜、油菜籽(高芥子酸和菜籽油)、花生或向日葵、以及单子叶植物如油椰子、甘蔗、香蕉、苏丹草、玉米、小麦、黑麦、大麦、燕麦、水稻、小米、或高粱。裸子植物如杉木和松树也适合。
因此,本文所述方法可用于例如属于下述的双子叶植物:木兰目(Magniolales)、八角目(Illiciales)、樟目(Laurales)、胡椒目(Piperales)、马兜铃目(Aristochiales)、睡莲目(Nymphaeales)、毛茛目(Ranunculales)、罂粟目(Papeverales)、瓶子草科(Sarraceniaceae)、昆栏树目(Trochodendrales)、金缕梅目(Hamamelidales)、杜仲目(Eucomiales)、银毛木目(Leitneriales)、杨梅目(Myricales)、壳斗目(Fagales)、木麻黄目(Casuarinales)、石竹目(Caryophyllales)、肉穗果目(Batales)、蓼目(Polygonales)、白花丹目(Plumbaginales)、五桠果目(Dilleniales)、山茶目(Theales)、锦葵目(Malvales)、荨麻目(Urticales)、玉蕊目(Lecythidales)、菫菜目(Violales)、杨柳目(Salicales)、白花菜目(Capparales)、杜鹃花目(Ericales)、(Diapensales)、柿树目(Ebenales)、报春花目(Primulales)、蔷薇目(Rosales)、豆目(Fabales)、川苔草目(Podostemales)、小二仙草目(Haloragales)、桃金娘目(Myrtales)、山茱萸目(Cornales)、睡莲目(Proteales)、檀香目(Santales)、大花草目(Rafflesiales)、卫矛目(Celastrales)、大戟目(Euphorbiales)、鼠李目(Rhamnales)、无患子目(Sapindales)、胡桃目(Juglandales)、牻牛儿苗目(Geraniales)、遠志目(Polygalales)、伞形目(Umbellales)、龙胆目(Gentianales)、花荵目(Polemoniales)、唇形目(Lamiales)、车前目(Plantaginales)、玄參目(Scrophulariales)、桔梗目(Campanulales)、茜草目(Rubiales)、川续断目(Dipsacales)、和菊目(Asterales)。本文所述方法还用于例如属于下述的单子叶植物:泽泻目(Alismatales)、水鳖目(Hydrocharitales)、茨藻目(Najadales)、霉草目(Triuridales)、鸭跖草目(Commelinales)、谷精草目(Eriocaulales)、帚灯草目(Restionales)、禾本目(Poales)、灯心草目(Juncales)、莎草目(Cyperales)、香蒲目(Typhales)、凤梨目(Bromeliales)、姜目(Zingiberales)、棕榈目(Arecales)、巴拿马草目(Cyclanthales)、露兜树目(Pandanales)、天南星目(Arales)、百合目(Lilliales)、和兰目(Orchidales)、或用于属于裸子植物门(Gymnospermae)的植物例如松杉目(Pinales)、银杏目(Ginkgoales)、苏铁目(Cycadales)和麻黄目(Gnetales)。
本方法在在广泛的植物物种中使用,包括来自双子叶属颠茄属(Atropa)、油丹属(Alseodaphne)、腰果属(Anacardium)、落花生属(Arachis)、琼楠属(Beilschmiedia)、芸苔属(Brassica)、红花属(Carthamus)、木防己属(Cocculus)、巴豆属(Croton)、香瓜属(Cucumis)、柑橘属(Citrus)、西瓜属(Citrullus)、辣椒属(Capsicum)、长春花属(Catharanthus)、椰子属(Cocos)、咖啡属(Coffea)、南瓜属(Cucurbita)、胡萝卜属(Daucus)、杜氏木属(Duguetia)、花菱草属(Eschscholzia)、榕属(Ficus)、草莓属(Fragaria)、海罂粟属(Glaucium)、大豆属(Glycine)、棉属(Gossypium)、向日葵属(Helianthus)、橡胶树属(Hevea)、天仙子属(Hyoscyamus)、(Lactuca)、(Landolphia)、(Linum)、(Litsea)、(Lycopersicon)、羽扇豆属(Lupinus)、木薯属(Manihot)、马郁兰属(Majorana)、苹果属(Malus)、苜蓿属(Medicago)、烟草属(Nicotiana)、木犀榄属(Olea)、银胶菊属(Parthenium)、罂粟属(Papaver)、鳄梨属(Persea)、菜豆属(Phaseolus)、黄连木属(Pistacia)、豌豆属(Pisum)、梨属(Pyrus)、李属(Prunus)、萝卜属(Raphanus)、蓖麻属(Ricinus)、千里光属(Senecio)、风龙属(Sinomenium)、千金藤属(Stephania)、白芥属(Sinapis)、茄属(Solanum)、可可属(Theobroma)、车轴草属(Trifolium)、胡卢巴属(Trigonella)、野豌豆属(Vicia)、蔓长春花属(Vinca)、葡萄属(Vitis)、和(Vigna);单子叶属(Allium)、须芒草属(Andropogon)、剪股颖属(Aragrostis)、天门冬属(Asparagus)、燕麦属(Avena)、狗牙根属(Cynodon)、油棕属(Elaeis)、羊茅属(Festuca)、羊茅黑麦草属(Festulolium)、萱草属(Heterocallis)、大麦属(Hordeum)、青萍属(Lemna)、黑麦草属(Lolium)、芭蕉属(Musa)、稻属(Oryza)、黍属(Panicum)、狼尾草属(Pannesetum)、梯牧草属(Phleum)、早熟禾属(Poa)、黑麦属(Secale)、高粱属(Sorghum)、小麦属(Triticum)、和玉蜀黍属(Zea);或裸子植物属冷杉属(Abies)、杉木属(Cunninghamia)、云杉属(Picea)、松属(Pinus)、和黄杉属(Pseudotsuga)的物种。
可通过根据具体性状或活性例如标记基因或抗生素抗性基因编码的那些来选择或筛选工程改造的细胞从而鉴定和分离转化的细胞、愈伤组织、组织或植物。本领域普通技术人员熟知这类筛选和选择方法。此外,物理和生化方法可用于鉴定转化子。这些包括用于检测多核苷酸的Southern分析或PCR扩增;用于检测RNA转录物的Northern印迹、S1RNA酶保护、引物延伸、或RT-PCR扩增;用于检测多肽和多核苷酸的酶或核酶活性的酶学试验;和检测多肽的蛋白凝胶电泳、Western印迹、免疫沉淀反应和酶联免疫分析。其它技术如原位杂交、酶染色和免疫染色也可用来检测多肽和/或多核苷酸的存在或表达。熟知进行所有参考技术的方法。稳定纳入植物细胞的多核苷酸可用例如标准育种技术导入其他植物。
本发明的内容中,“真核细胞”指下列生物体衍生的和体外培养建立的真菌、酵母、植物或动物细胞或细胞系。更优选地,真菌可为曲霉属(Aspergillus)、青霉属菌(Penicillium)、枝顶孢属(Acremonium)、木霉属(Trichoderma)、葡萄孢属(Chrysoporium)、被孢霉(Mortierella)、克鲁维酵母属(Kluyveromyces)或毕赤酵母属(Pichia)。更优选地,所述真菌可为黑曲霉(Aspergillus niger)、构巢曲霉(Aspergillusnidulans)、米曲霉(Aspergillus oryzae)、土曲霉(Aspergillus terreus)、产黄青霉(Penicillium chrysogenum)、桔青霉(Penicillium citrinum)、产黄头孢霉(Acremoniumchrysogenum)、里氏木霉(Trichoderma reesei)、孢霉(Mortierella alpine)、鲁克文金孢子菌(Chrysosporium lucknowense)、乳酸克鲁维酵母(Kluyveromyces lactis)、巴斯德毕赤酵母(Pichia pastoris)或西弗毕赤酵母(Pichia ciferrii)。
本发明中植物可为拟南芥属(Arabidospis)、烟草属(Nicotiana)、番茄属(Solanum)、莴苣(Lactuca)、芸苔属(Brassica)、稻属(Oryza)、天门冬属(Asparagus)、豌豆属(Pisum)、苜蓿属(Medicago)、玉蜀黍属(Zea)、大麦属(Hordeum)、黑麦属(Secale)、小麦属(Triticum)、辣椒属(Capsicum)、香瓜属(Cucumis)、南瓜属(Cucurbita)、西瓜属(Citrullis)、柑橘属(Citrus)、或高粱属(Sorghum)。更优选地,所述植物可为拟南芥(Arabidospis thaliana)、烟草(Nicotiana tabaccum)、番茄(Solanum lycopersicum)、马铃薯(Solanum tuberosum)、茄子(Solanum melongena)、番茄(Solanum esculentum)、莴苣(Lactuca saliva)、欧洲油菜(Brassica napus)、甘蓝菜(Brassica oleracea)、芸苔(Brassica rapa)、光稃稻(Oryza glaberrima)、水稻(Oryza sativa)、石刁柏(Asparagusofficinalis)、豌豆(Pisum sativum)、紫苜蓿(Medicago sativa)、玉米(Zea mays)、大麦(Hordeum vulgare)、黑麦(Secale cereal)、小麦(Triticum aestivum)、硬粒小麦(Triticum durum)、辣椒(Capsicum sativus)、西葫芦(Cucurbita pepo)、西瓜(Citrulluslanatus)、甜瓜(Cucumis melo)、来檬(Citrus aurantifolia)、柚(Citrus maxima)、香橼(Citrus medica)、或柑桔(Citrus reticulata)。
本发明中,动物细胞可为人属(Homo)、家鼠属(Rattus)、小鼠属(Mus)、猪属(Sus)、牛属(Bos)、鱼丹属(Danio)、犬属(Canis)、猫属(Felis)、马属(Equus)、斑鳟属(Salmo)、太平洋鲑属(Oncorhynchus)、原鸡属(Gallus)、火鸡属(Meleagris)、果蝇属(Drosophila)、或广杆属线虫(Caenorhabditis);更优选地,所述动物细胞可为智人(Homo sapiens)、褐家鼠(Rattus norvegicus)、小家鼠(Mus musculus)、野猪(Sus scrofa)、黄牛(Bos taurus)、斑马鱼(Danio rerio)、狼(Canis lupus)、猫(Felis catus)、马(Equus caballus)、虹鳟(Oncorhynchus mykiss)、原鸡(Gallus gallus)、或火鸡(Meleagris gallopavo);所述动物细胞可为非限制示例的大西洋鲑(Salmo salar)、硬骨鱼(Teleost fish)或斑马鱼物种的鱼细胞。本发明所述动物细胞还可为非限制性示例的黑腹果蝇(Drosophilamelanogaster)的昆虫细胞;所述动物细胞还可为非限制性示例的秀丽隐杆线虫(Caenorhabditis elegans)的蠕虫细胞。
本发明中,所述细胞可为用于体外培养的植物细胞、哺乳动物细胞、鱼类细胞、昆虫细胞或衍生自这些生物体的细胞系,或用于体外培养的直接取自活组织并建立的初级细胞。非限制性示例的细胞系可选自下组:CHO-K1细胞、HEK293细胞、Caco2细胞、U2-OS细胞、NIH 3T3细胞、NSO细胞、SP2细胞、CHO-S细胞、DG44细胞、K-562细胞、U-937细胞、MRC5细胞、IMR90细胞、Jurkat细胞、HepG2细胞、HeLa细胞、HT-1080细胞、HCT-116细胞、Hu-h7细胞、Huvec细胞、Molt 4细胞。
所有这些细胞系可用本发明方法修饰以提供细胞系模型用于生产、表达、定量、检测、研究感兴趣的基因或蛋白;这些模型还可用于在各领域如非限制性示例的化学、生物燃料、治疗和农学的研究和生产中筛选感兴趣的生物活性分子。
本发明还提供用TAL效应子内序列特异性DNA结合域的方法以例如改变细胞内的遗传物质、调控基因表达、和在如抗病毒治疗中靶向致病序列。例如,在一些实施方式中,本发明提供修饰细胞遗传物质的方法。在一些实施方式中,本方法包括将含有TAL效应子DNA结合域的多肽,或编码该多肽的核酸导入细胞。所述TAL效应子DNA结合域可融合所有或部分DNA修饰酶(如内切核酸酶)。在一些实施方式中,本方法包括将两种或更多重组核酸导入细胞。第一重组核酸包括含一种或多种修饰(即替换、缺失或插入)的供体核苷酸序列,所述修饰相对于细胞中发现的相应预选定靶核苷酸序列。所述供体核苷酸序列可经历与内源靶核苷酸序列的同源重组,从而所述内源序列或其部分被所述供体序列或其部分取代。靶核苷酸序列通常包括序列特异性TALEN的识别位置。在一些情况中,靶核苷酸序列可包含两种或更多不同TALEN的识别位置(如不同的两种相反的靶序列,从而可使用具有不同DNA序列结合特异性的TALEN)。在该情况中,DNA的切割特异性相比仅使用一种靶序列(或相同靶序列的多拷贝)的情况更高。
第二重组核酸含有编码序列特异性TALEN的核苷酸序列,其结合靶核苷酸序列中的识别位置。在一些情况中,供体核苷酸序列和编码序列特异性核酸酶的核苷酸序列可包含在同一核酸构建体中。或者,所述供体核苷酸序列和TALEN编码序列可包含在单独构建体中,或可生产所述TALEN多肽并直接导入细胞中。
在一些实施方式中,还可使用含有编码选择标记的核苷酸序列的第三重组核酸。所述第二和第三重组核酸可与内源序列重组,从而整合到细胞基因组中。这些重组事件可以是非常规(即随机),或其可通过同源重组或位点特异重组发生。重组核酸可同时或依次转化到细胞中,且可在转化前线性化。
当所述细胞是植物细胞时,本文提供的方法还可包括步骤例如,生成含有所述转化细胞的植物、生成所述植物的后代、选择或筛选表达选择标记(若包括)的植物、生成所选植物的后代、和测试所述植物(如组织、种子、前体细胞或完整植物)或所述植物的后代在靶核苷酸序列上的重组。在一些情况中,所述方法可包括异交所选植物以移除选择标记、和/或根据序列特异性核酸酶的缺失来筛选所选或异交的植物。
在一些实施方式中,本发明提供修饰细胞如原核细胞、动物细胞或植物细胞的遗传物质的方法。本方法可包括在细胞中引入含有经修饰靶核苷酸序列的第一重组核酸,所述靶核苷酸序列相对细胞中存在的对应靶核苷酸序列在其核苷酸序列中包括一种或多种修饰,以及序列特异性TALEN的识别位置,和含有编码序列特异性TALEN的核苷酸序列的第二重组核酸。当细胞是植物细胞时,生成含有所述细胞的植物,并分析获自所述植物(或其后代)的细胞、种子或组织在靶核苷酸序列的重组。所述第一和第二重组核酸可同时或顺序转化到细胞中,且其中之一或二者可在转化前线性化。在一些情况中,所述第一和第二重组核酸可存在于相同构建体中。
在一些情况中,所述方法还可包括将包含编码可选标记物的核苷酸序列的第三重组核酸导入细胞,并确定所述细胞、所述细胞或其后代生成的生物体或其后代是否表达所述可选标记物。所述方法还可包括根据所述可选择标记的缺失来筛选所述细胞、生物体或其后代。编码所述可选择标记的核苷酸可侧接或不侧接第二序列特异性核酸酶切割位点处与所述细胞内源核苷酸序列相似或相同的核苷酸序列两侧或序列特异性重组酶的识别位置两侧。在一些情况中,所述方法还可包括异交所述生物体的步骤。异交的后代可根据选择标记的缺失来筛选。
本发明还提供修饰细胞遗传物质的方法(如植物细胞或动物细胞),包括提供含有靶DNA序列如染色体、线粒体、或叶绿体序列的细胞,其中需要发生同源重组、提供含有DNA修饰酶结构域(如内切核酸酶结构域)和具有多种TAL效应子重复序列的TAL效应子结构域的TALEN,所述TAL效应子重复序列一起结合靶DNA序列内的特异核苷酸序列、提供含有与至少部分靶DNA同源的序列的核酸、和将所述细胞中靶DNA序列接触所述TAL内切核酸酶,从而所述细胞中靶DNA序列内或邻近的核苷酸序列的双链被切割。所述切割可提高所述靶DNA序列处的同源重组频率。所述靶DNA序列可为所述细胞内源序列。本方法可包括将含有所述TAL内切核酸酶编码cDNA的载体导入细胞并在细胞中表达TAL内切核酸酶蛋白。在一些情况中,TAL内切核酸酶蛋白自身可通过例如机械注射、用细菌III型分泌系统递送、电穿孔或农杆菌介导的转移来导入细胞。
本文所述方法可用于各种情况。例如在农业中,本文所述方法用于协助靶位置的同源重组,可用于从植物品系、品种或杂交体中移除先前整合的转基因(例如除草剂抗性转基因)。本文所述方法还可用于修饰内源基因,从而所述基因编码的酶赋予除草剂抗性,例如修饰内源5-烯醇式丙酮酰莽草酸-3-磷酸盐(EPSP)合成酶基因,从而所修饰的酶赋予对草甘膦除草剂的抗性。作为另一示例,本文所述方法用于协助植物或哺乳动物代谢途径(如脂肪酸生物合成)中一种或多种内源基因的调控区域的同源重组,从而所述基因的表达以所需方法修饰。本文所述方法用于协助动物(如大鼠或小鼠)中参与非限制性示例的代谢和内部信号通路的一种或多种感兴趣内源基因中的同源重组,所述基因例如编码细胞表面标记的那些、鉴定为与具体疾病关联的基因和已知负责动物细胞具体表型的任何基因。
本发明还提供用于设计能与具体DNA序列相互作用的序列特异性TAL效应子(例如能在特定位置切割DNA的TALEN)的方法。所述方法可包括鉴定靶核苷酸序列(如内源染色体序列、线粒体DNA序列、或叶绿体DNA序列),其上需要结合有TAL效应子(如邻近第二核苷酸序列的序列,其上需要引入双链切割),和设计含有联合结合靶序列的多种DNA结合重复序列的序列特异性TAL效应子。如本文所述,TAL效应子包括许多不完善的重复,所述重复决定了其与DNA相互作用的特异性。各重复序列结合单个碱基,取决于所述重复序列的残基12和13处的具体双氨基酸序列。因此,通过工程改造TAL效应子内的重复序列(如用标准技术或本文所述的技术),可靶向具体的DNA位置。所述工程改造的TAL效应子可用作例如靶向具体DNA序列的转录因子。图1A显示普通TAL效应子的图,重复区域用空心框表示,代表性重复序列(SEQ ID NO:1)中的RVD用下划线标出。
RVD的示例和其对应靶核苷酸如表1A所示(参见例如PCT公开号WO2010/079430)。
表1A
RVD | 核苷酸 |
HD | C |
NG | T |
NI | A |
NN | G或A |
NS | A或C或G |
N* | C或T |
HG | T |
H* | T |
IG | T |
*表示重复序列中的缺口,对应于RVD第二位置的氨基酸残基的缺失。
其他RVD和其对应靶核苷酸示于表1B。
表1B
例如,当需要序列特异性DNA切割时,可设计含有下述的序列特异性TALEN:(a)一起结合内源染色体核苷酸序列的多种DNA结合重复结构域,和(b)在第二核苷酸序列产生双链切割的内切核酸酶。如本文所述,所述序列特异性DNA切割可用于提高同源重组。TALEN的其他应用包括例如针对病毒的治疗方法。TALEN可工程改造成靶向具体病毒序列,切割病毒DNA并减少或消除毒力。
本文提供的材料和方法可用于以靶向的方法修饰具体基因序列。基因可含有经工程改造TAL效应子可靶向的多种序列。但如本文所述,某些靶序列可被更有效地靶向。例如,如实施例9所示,具有特定特征的序列可被TAL效应子更有效地靶向。因此,本文所述方法可包括鉴定满足具体标准的靶序列。这些包括下述序列:i)最少15碱基长度且为5’-3’方向,T紧接在5’末端位点的前面;ii)在所述第一(5’)位置中没有T或所述第二位置中没有A;iii)在最后的(3’)位置以T结束且接近最后的位置没有G;和iv)碱基组成为0-63%A、11-63%C、0-25%G、和2-42%T。
由于本文所述TALEN通常作为二聚体起作用,本发明所提供方法的一些实施方式可包括在细胞中鉴定第一基因组核苷酸序列和第二基因组核苷酸序列,其中所述第一和第二核苷酸序列满足至少一种上述标准且被15-18bp分开。在一些情况中,TALEN多肽可结合各核苷酸序列,且TALEN所含内切核酸酶可在15-18bp间隔区内切割。
本发明还包括生成已导入所需核酸的遗传改良动物的方法。本方法可包括获得含有内源染色体靶DNA序列的细胞,其内需要导入核酸、将TALEN导入细胞以在内源染色体靶DNA序列内产生双链切割、在允许外源核酸和内源染色体靶DNA序列间发生同源重组的条件下将含与至少部分内源染色体靶DNA同源的序列的外源核酸导入所述细胞、和从已发生同源重组的初级细胞生成动物。所述同源核酸可包括例如同源重组后破坏基因的核苷酸序列、同源重组后替换基因的核苷酸序列、同源重组后在基因中引入点突变的核苷酸序列或同源重组后引入调控位点的核苷酸序列。
本文提供的方法可用于生成已引入所需核酸的遗传改良植物。本方法可包括获得含有内源靶DNA序列的植物细胞,其内需要导入核酸、导入TALEN以在内源靶DNA序列中产生双链切割、在允许外源核酸和内源靶DNA序列间发生同源重组的条件下将含与至少部分内源染色体靶DNA同源的序列的外源核酸导入所述植物细胞、和从已发生同源重组的所述植物细胞生成植物。
与没有进行所述方法的细胞相比,用本文所述TALEN协助的同源重组方法生成的细胞中的DNA被修饰,且含有所述修饰DNA的细胞称为“遗传修饰的”。然而,应注意含有所述细胞的生物体可能不视作用于调节目的的GMO,因为该修饰涉及同源重组且不是转基因的随机整合。因此,用本文所述的TALEN协助方法产生遗传修饰可具有优势,因为例如可避免标准调节程序以及其伴随的时间和成本。
本文所述靶向遗传重组的其他方法可包括将靶向所选DNA靶序列的TALEN编码核酸分子导入细胞(如植物细胞、昆虫细胞、硬骨鱼细胞、或动物细胞)、诱导所述TALEN在细胞中表达、和鉴定所选DNA靶序列显示突变(如遗传物质的缺失、遗传物质的插入、或遗传物质的缺失和插入)的重组细胞。供体DNA也可导入所述细胞。
在一些实施方式中,可使用单体TALEN。本文所述TALEN通常用作二聚体,跨具有间隔区的两部分识别位置,从而两种TAL效应子结构域各融合FokI限制性酶的催化结构域,各所得TALEN的DNA识别位置被间隔序列分开,且各TALEN单体与识别位置的结合使FokI二聚化并在所述间隔区内产生双链断裂(参见例如Moscou和Bogdanove(2009)Science 326:1501)。但还可构建单体TALEN,从而单一TAL效应子融合不需要二聚化作用的核酸酶。例如,一种此类核酸酶是FokI的单链变体,其中所述两种单体表达为单一多肽(Minczuk等(2008)Nucleic Acids Res.36:3926-3938)。其他天然产生或工程改造的单体核酸酶也可用作此目的。用于单体TALEN的DNA识别结构域可衍生自天然产生的TAL效应子。或者,DNA识别结构域可经工程改造以识别具体DNA靶标。工程改造的单链TALEN可能更易于构建和开发,因为其仅需要一种工程改造的DNA识别结构域。
在一些实施方式中,可用两种不同DNA结合域生成二聚体DNA序列特异性核酸酶(如一种TAL效应子结合域和一种来自另一类型分子的结合域)。如上所示,本文所述TALEN通常用作二聚体,跨具有间隔区的两部分识别位置。此核酸酶结构也可用于例如由一种TALEN单体和一种锌指核酸酶单体生成的靶标特异性核酸酶。在所述情况中,TALEN和锌指核酸酶单体的DNA识别位置可被合适长度的间隔区分开。所述两种单体的结合可使FokI二聚化并在所述间隔序列内产生双链断裂。除了锌指外的DNA结合域,如同源域、myb重复或亮氨酸拉链也可融合FokI并用作TALEN单体的伴侣,产生功能性核酸酶。
在一些实施方式中,TAL效应子可用于将其他蛋白结构域(如非核酸酶蛋白结构域)靶向具体核苷酸序列。例如,TAL效应子可连接蛋白结构域,所述结构域不限于:DNA相互作用酶(如甲基酶、拓扑异构酶、整合酶、转座酶或连接酶)、转录激活子或抑制子、或与其他蛋白如组氨酸相互作用或修饰其的蛋白。所述TAL效应子融合物的应用包括例如,产生或修饰表观调节元件,在DNA中产生位点特异性插入、缺失或修复,控制基因表达和修饰染色质结构。
在一些实施方式中,所述靶序列的间隔区可选择或变化以调控TALEN特异性和活性。本文所示TALEN的结果是其用作二聚体,跨具有间隔区的两部分识别位置,表明TALEN可在一定间隔长度范围起作用,且TALEN的活性随着间隔长度而不同。参见例如,下述实施例6。间隔长度的灵活性表明可选择间隔长度以高度特异地靶向具体序列(如基因组中)。此外,不同间隔长度观察到的活性差异表明可选择间隔长度以获得所需TALEN活性水平。
在一些实施方式中,TALEN活性可通过改变DNA结合结内重复序列的数量和组成来调控。如本文实施例7所述,例如基于PthXoI的TALEN显示活性高于基于AvrBs3的TALEN。PthXoI与AvrBs3在其重复序列的数量和RVD组成上均不同。此外,这些蛋白的天然产生DNA识别位置在基于Moscou和Bogdanove(同上)所述TAL效应子DNA密码预测的各识别序列之间的差异性方面不尽相同。此外,数种相同长度(12RVD)但RVD组成不同的自定义TALEN的活性不同,且13RVD自定义TALEN具有比12RVD自定义TALEN更高的活性。因此,不仅TALEN可工程改造成识别感兴趣的DNA序列,而且(1)重复序列的数量可变化以调控活性,(2)可选择不同的结合位点以实现不同的活性水平,和(3)RVD的组成及其与靶位点的拟合(根据所述密码(cipher))可变化以调控TALEN活性。
TALEN是异型二聚体形式时,例如具有包括TAL效应子结构域和FokI核酸酶催化结构域在内的两种不同单体,所述RVD在两种TAL效应子结构域中数量相等,或各结构域可表现不同数量的RVD。例如,若总共22RVD用于结合具体异型二聚TALEN中的DNA,两种TAL效应子结构域中各可发现11个重复;或者,两种TAL效应子结构域之一可发现10个重复,另一种有12个。本发明还涵盖具有作为单体的DNA修饰酶结构域的TALEN。在该情况中,所有RVD可存在于单一TAL效应子结构域中,其融合所述单体酶。在该情况中,为了有效结合,RVD的数量必须与等同二聚TALEN中发现的RVD总数相等。例如,两种不同TAL效应子结构域上并非具有10个重复(如在二聚TALEN的情况中),而是在单一TAL效应子结构域中有20个重复(如在单体TALEN的情况中)。
本发明的另一方面,二聚体或单体TALEN内重复的总数量至少为14。本发明的另一方面,二聚体或单体TALEN内重复的总数量至少为20。本发明的另一方面,二聚体或单体TALEN内重复的总数量至少为24。本发明的另一方面,二聚体或单体TALEN内重复的总数量至少为30。
本专利申请还提供生成具有增强的靶向靶DNA能力的TAL效应蛋白的方法。所述方法可包括例如生成编码TAL效应子的核酸,其具有含多种DNA结合重复序列的DNA结合域,各重复序列包含确定所述靶DNA中碱基对识别的RVD,其中各DNA识别重复序列负责识别所述靶DNA中的一种碱基对。如以下实施例12所述,在结合位点的-1位置放宽对T的需求可提高经工程改造TAL效应蛋白的靶向能力。因此,生成编码核酸的TAL效应子可包括纳入对A、C或G特异的编码第0变异DNA结合重复序列的核酸,从而消除所述结合位点-1位置对T的需求。
此外,本文提供生成具有增强的靶向靶DNA能力的TAL效应子的方法。所述方法可包括生成编码TAL效应子的核酸,其包括含多种DNA结合重复序列的DNA结合域,各重复序列包含确定所述靶DNA中碱基对识别的RVD。如以下实施例12所述,NN(识别G的最常见RVD)的特异性通常表现较弱且可随环境变化,但某些RVD可对G提高特异性。因此,本文提供的方法可包括使用对G可能具有更强特异性的替代RVD。例如,可使用选自下组的一种或多种RVD:RN、R*、NG、NH、KN、K*、NA、NT、DN、D*、NL、NM、EN、E*、NV、NC、QN、Q*、NR、NP、HN、H*、NK、NY、SN、S*、ND、NW、TN、T*、NE、NF、YN、Y*、和NQ,其中星号表示RVD的第二位置上的缺口。
制品生产
本发明还提供生产的制品,例如编码TALEN的核酸分子、TALEN多肽、含有该核酸分子或多肽的组合物、或TAL内切核酸酶工程改造的细胞系。所述物质可用作例如研究工具或治疗性应用。
在一些实施方式中,制品可包括用本发明所述方法生成的植物的种子。可用本领域已知的方法调整所述种子并用本领域熟知的包装材料包装以制备制品。种子包装可具有标记,例如标签或固定在包装材料上的标记、打印在包装材料上的标记或插入包装内的标记。所述标记可表明所述包装内含有的种子可生产遗传改良植物的农作物,且可描述遗传修饰所改变的相对未修饰植物的性状。
其他定义
-本文用单字母密码命名多肽序列中的氨基酸残基或亚基,其中例如Q表示Gln或谷氨酰胺残基、R表示Arg或精氨酸残基且D表示Asp或天冬氨酸残基。
-氨基酸替换表示一种氨基酸残基被另一种取代,例如在肽序列中用谷氨酰胺残基取代精氨酸残基是一种氨基酸取代。
-核苷酸命名如下:单字母密码用于命名核苷酸碱基:a是腺嘌呤、t是胸腺嘧啶、c是胞嘧啶、g是鸟嘌呤。对于简并核苷酸,r代表g或a(嘌呤核苷酸),k代表g或t,s代表g或c,w代表a或t,m代表a或c,y代表t或c(嘧啶核苷酸),d代表g、a或t,v代表g、a或c,b代表g、t或c,h代表a、t或c,和n代表g、a、t或c。
-术语“DNA修饰酶”指能修饰细胞遗传物质的任何蛋白,而不论DNA修饰的水平(切割、共价相互作用、水介导的相互作用...)。DNA相互作用蛋白(如甲基化酶、拓扑异构酶、整合酶、转座酶或连接酶)、转录激活子或抑制子、其他蛋白如组氨酸和核酸酶应包括在“DNA修饰酶”的含义中。包括在TAL效应子-DNA修饰酶中时,所述DNA修饰酶指DNA修饰酶结构域。
-术语“核酸酶”意在包括外切核酸酶和内切核酸酶。
-术语“内切核酸酶”指能催化DNA或RNA分子内(优选DNA分子)核酸之间键水解(切割)的任何野生型或变体酶。内切核酸酶的非限制性示例包括II型限制性内切核酸酶例如FokI、HhaI、HindIII、NotI、BbvCI、EcoRI、BglI、和AlwI。通常具有约12-45碱基对(bp),更优选14-45bp长度的多核苷酸识别位点时,内切核酸酶还包括稀有切割内切核酸酶(rare-cutting endonucleases)。稀有切割内切核酸酶通过在确定的基因座诱导DNA双链断裂(DSB)来显著增加HR(Rouet,Smih等1994;Rouet,Smih等1994;Choulika,Perrin等1995;Pingoud和Silva 2007)。稀有切割内切核酸酶可为例如归巢性内切核酸酶(Paques和Duchateau 2007)、工程改造的锌指结构域和限制性酶如FokI的催化结构域融合产生的嵌合锌指核酸酶(Porteus和Carroll 2005)或化学内切核酸酶(Eisenschmidt,Lanio等2005;Arimondo,Thomas等2006;Simon,Cannata等2008)。化学内切核酸酶中,化学或肽切割物与核酸的多聚物或识别特定靶序列的另一DNA偶联,从而将切割活性靶向特定序列。化学内切核酸酶还涵盖合成核酸酶如邻二氮菲、DNA切割分子和三链形成寡核苷酸(TFO)的偶联物,已知用于结合特定DNA序列(Kalish和Glazer 2005)。所述化学内切核酸酶包括在本发明术语“内切核酸酶”内。所述内切核酸酶的示例包括I-Sce I、I-Chu I、I-Cre I、I-Csm I、PI-Sce I、PI-Tli I、PI-Mtu I、I-Ceu I、I-Sce II、I-Sce III、HO、PI-Civ I、PI-Ctr I、PI-Aae I、PI-Bsu I、PI-Dha I、PI-Dra I、PI-Mav I、PI-Mch I、PI-Mfu I、PI-Mfl I、PI-MgaI、PI-Mgo I、PI-Min I、PI-Mka I、PI-Mle I、PI-Mma I、PI-Msh I、PI-Msm I、PI-Mth I、PI-Mtu I、PI-Mxe I、PI-Npu I、PI-Pfu I、PI-Rma I、PI-Spb I、PI-Ssp I、PI-Fac I、PI-MjaI、PI-Pho I、PI-Tag I、PI-Thy I、PI-Tko I、PI-Tsp I、I-MsoI。
本发明的内切核酸酶可为部分转录激活因子样(TAL)效应物内切核酸酶(TALEN)。
-“TALEN”指包括转录激活因子样(TAL)效应子结合域和内切核酸酶结构域的蛋白,这两种结构域的融合产生“单体TALEN”。一些单体TALEN本身可具有功能,其他需要与另一单体TALEN二聚化。两种单体TALEN相同时所述二聚化可产生同二聚TALEN,或单体TALEN不同时可产生异二聚TALEN。例如RVD数量不同时,和/或至少一种RVD的内容物(即氨基酸序列)不同时,两种单体TALEN不同。“TAL效应子-DNA修饰酶”指包括转录激活因子样效应子结合域和DNA修饰酶结构域的蛋白。
“变体”指“变体蛋白”,即天然不存在的蛋白,其通过遗传工程改造或随机突变获得,即工程改造的蛋白。该变体蛋白可例如通过用不同氨基酸替换野生型、天然产生蛋白的氨基酸序列中至少一个残基。可通过例如定点突变和/或随机突变引入所述替换。
“细胞”或“多个细胞”指任何原核或真核活细胞、衍生自这些生物体用于体外培养的细胞系、动物或植物来源的初级细胞。
“初级细胞”或“多个初级细胞”指直接从活组织(即活体材料)取得并建立用于体外生长的细胞,与连续致瘤或人工永生细胞系相比,其经历非常少的群体加倍并因此更能代表其衍生组织的主要功能组分和特征。因此这些细胞代表其所指体内状态的更具价值模型。
-"同源"指与另一序列具有足够相同性的序列,从而引起序列间的同源重组,更具体地有至少95%相同性、优选97%相同性和更优选99%。
-"相同性"指两种核酸分子或多肽间的序列相同性。可通过比较各序列中的位置来确定相同性,所述序列可进行比对用于比较目的。比较序列的位置被相同碱基占据时,所述分子在该位置相同。核酸或氨基酸序列之间的相似或相同性程度随着核酸序列共有位置的相同或匹配核苷酸数量而变化。各种比对算法和/或程序可用于计算两种序列之间的相同性,包括FASTA或BLAST,其可作为部分GCG序列分析软件包(威斯康星大学,威斯康星州麦迪逊)而获得,并使用例如默认设置。
-“突变”指多核苷酸(cDNA、基因)或多肽序列中一种或多种核苷酸/氨基酸的替换、缺失、插入。所述突变可影响基因或其调节序列的编码序列。其还可影响基因组序列的结构或所编码mRNA的结构/稳定性。
-“基因”表示基本遗传单元,由沿着染色体线性排列的DNA区段组成,其编码具体蛋白或蛋白区段。基因通常包括启动子、5'非翻译区、一种或多种编码序列(外显子)、任选的内含子、3'非翻译区。基因还可包括终止子、增强子和/或沉默子。
-术语“感兴趣的基因”指编码已知或推定基因产物的任何核苷酸序列。
-本文所用的术语“基因座”是(如基因的)DNA序列在染色体上的具体物理位置。术语“基因座”通常指靶序列在染色体上的具体物理位置。
-“融合蛋白”指本领域熟知过程的结果,所述过程包括连接原始编码分离蛋白的两种或更多基因,所述“融合蛋白”的翻译产生具有衍生自各原始蛋白的功能特征的单一多肽。
-“催化结构域”指含有酶激活位点的所述酶的蛋白结构域或模块;激活位点指所述酶催化底物的部分。酶,而非其催化结构域,根据其催化的反应来分类和命名。酶委员会编号(EC编号)是基于所催化化学反应的酶编号分类方案(互联网址chem.qmul.ac.uk/iubmb/enzyme/)。本发明范围中,任何催化结构域都可用作TAL效应子结构域的伴侣并与之融合以生成嵌合融合蛋白产生TAL效应子-DNA修饰酶。此类催化结构域的非限制性示例可为MmeI、EsaSSII、CstMI、NucA、EndA大肠杆菌(Escherichia coli)、NucM、EndA肺炎链球菌(Streptococcus pneumonia)、SNase金黄色葡萄球菌(Staphylococcus aureus)、SNase猪葡萄球菌(Staphylococcus hyicus)、SNase弗氏志贺菌(shigella flexneri)、枯草杆菌(Bacillus subtilis)yncB、脱氧核糖核酸内切酶I肠细菌噬菌体T7、牛EndoG、ttSmr DNA错配修复蛋白mutS、Metnase的切割结构域。
除非另有说明,本发明的实施将采用细胞生物学、细胞培养、分子生物学、转基因生物学、微生物学、重组DNA和免疫学的常规技术,它们均在本领域技术范围内。这些技术在文献中已有充分描述。参见例如,Current Protocols in Molecular Biology(《新编分子 生物学实验指南》)(Ausubel,2000,约翰威利公司(Wiley and son Inc,),美国国会图书馆);Molecular Cloning:A Laboratory Manual(《分子克隆:实验室手册》),第三版,(Sambrook等,2001,纽约冷泉港:冷泉港实验室出版);Oligonucleotide Synthesis(《寡核 苷酸合成》)(M.J.Gait编,1984);美国专利号4,683,195;Nucleic Acid Hybridization (《核酸杂交》)(Harries和Higgins编.1984);Transcription and Translation(《转录和翻 译》)(Hames和Higgins编1984);Culture of Animal Cells(《动物细胞培养》)(Freshney,ARL有限公司(Alan R.Liss,Inc.),1987);Immobilized Cells and Enzymes(《免疫细胞和 酶》)(IRL出版社(IRL Press),1986);Perbal,A Practical Guide to Molecular Cloning (《分子克隆实践指南))(1984);Methods in Enzymology(《酶学方法》)系列(Abelson和Simon主编,纽约学术出版社公司(Academic Press,Inc.)),特定是154和155卷(Wu等编)和185卷,"Gene Expression Technology"(基因表达技术)(Goeddel编);Gene Transfer Vectors For Mammalian Cells(《哺乳动物细胞的基因转移载体》)(Miller和Calos编,1987,冷泉港实验室);Immunochemical Methods in Cell and Molecular Biology(《细胞 和分子生物学的免疫化学方法》)(Mayer和Walker编,伦敦的学术出版社,1987);Handbook of Experimental Immunology(《实验免疫学手册》),I-IV卷(Weir和Blackwell编,1986);和Manipulating the Mouse Embryo(《小鼠胚胎操作》),(冷泉港实验室出版社,纽约冷泉港,1986)。
本发明的以上描述提供了制作和使用其的方法和过程,从而本领域任何技术人员能制作和使用,这能具体用于所附权利要求的主题内容,其组成原始描述的部分。
如上所用,短语“选自下组”、“选自”等包括具体材料的混合物。
本文描述数字限制或范围时包括端点。数字限制或范围内的值和子区间也好像明确写出那样具体包括在内。
提供以上描述使本领域技术人员能制作或使用本发明,并在具体应用和其要求的环境中提供。优选实施方式的各种改良对本领域技术人员显而易见,且本文定义的基本原则可应用于其他实施方式和应用而不背离本发明的精神和方法。因此,本发明并不旨在限于所示实施方式中,而应按照与本文所公开原理和特征一致的最广泛的范围。
总体描述本发明后,还可通过参考某些具体实施例获得进一步的理解,本文提供的所述实施例仅用于说明,本发明在下述实施例中进一步描述,除非另有说明,其不限制权利要求中所述的本发明范围。
实施例
实施例1–确定TAL效应子-DNA识别的密码
为了测定RVD和TAL靶位点中连续核苷酸之间是否有一对一线性对应,用TAL效应子RVD序列扫描10种TAL效应子的各已知靶基因的预测启动子区域(即紧接所示翻译起始位点前面的1,000bp)以用于比对,所述比对使RVD核苷酸关联中的熵最小化(任意)。下式用于定量熵,其中R是效应子的RVD组、D是4种核苷酸(A、C、G、T)组,且fi,j代表观察到的第i位RVD关联第j位核苷酸的频率:
各启动子中存在多种低熵位点。然而对于效应子AvrBs3,仅一种对54bp upa20启动子片段作图,所述片段先前鉴定为活化的充分必要条件,且其与AvrBs3直接激活基因常见的UPA盒一致(Kay等,同上)。对于效应子PthXo1和AvrXa27,也各仅有一种位点覆盖其靶标Os8N3和Xa27的激活和未激活等位基因之间的多态性。这三种位点的比对中RVD核苷酸关联性一致,所以基于该关联选择剩余的比对,精确产生每TAL效应子-靶配对的一个位点(图1B和表2)。各位点前面有T(图1D)。
为了评估RVD-核苷酸关联产生的特异性,先基于10种最小熵TAL效应子-靶位点比对中观察到的所有RVD-核苷酸关联频率来生成权矩阵(图1B)。然后用所述权矩阵扫描粳稻日本晴(Oryza sativa spp.japonica cv.Nipponbare)(Osa1,6.0版,rice.plantbiology.msu.edu)中各非冗余基因模型的启动子区域(翻译起始位点前的1000bp)与水稻病原体水稻条斑病菌(AvrXa27、PthXo1、PthXo6、PthXo7和Tal1c)的5种TAL效应子的最佳匹配。对于AvrXa27,包括Xa27的上游序列(GenBank登录号AY986492)。该上游序列在日本晴中不存在。观察到的关联频率权重为90%,且剩下的10%平均分配给所有可能的关联频率。用权矩阵评分(y轴)排列比对,标定为图1B中衍生自RVD-核苷酸关联频率的频率评分的负log。因此,评分越低,匹配越好。对于PthXo1、PthXo6、PthXo7和Tal1c,实验鉴定的靶基因是最佳或近似最佳的匹配。较好的匹配前面没有T,在用于鉴定靶标的微整列上没有表现,或缺少内含子和EST证据。扫描反向互补启动子序列没有产生优于已知靶标正向位点的评分比对。此结果不意味着TAL效应子结合正链,但表明其在相对正链的正向方向上起作用。第5效应子AvrXa27的已知靶标是抗病基因Xa27(Gu等,同上)。该匹配较差的排名(5,368)反映出校准的、或新近的和次佳的宿主适应性。较好的评分位点可能包括被AvrXa27靶向的基因以用于发病机理。
再使用权矩阵,通过用40种其他水稻黄单胞菌(X.oryzae)TAL效应子扫描所有水稻启动子和基于公开微整列数据(PLEXdb.org,登录号OS3)保留其下游基因在感染期间被活化的最佳比对来获得10种其他比对(表3)。如同初始组,T先于各位点,且没有反向链位点评分更佳。总共20个比对的RVD-核苷酸关联频率示于图1C。其构成非常简单的密码。
20TAL效应子核苷酸比对的扩展组中RVD-核苷酸频率用于生成新的权矩阵,并在Python v2.5(www.python.org)中编写计算机脚本。该脚本可用于扫描任何DNA序列集合,寻找具体TAL效应子的匹配,其具有用户可定义的权重因子以用于观察与未观察到的RVD-核苷酸关联。参见Moscou和Bogdanove(同上)。
密码有一些简并性。强关联可代表锚定,其构成主要的结合亲和性,弱关联提供灵活性量度。或者,可涉及邻近效应。通过下述过程研究后者的可能性:测定根据任一边RVD调整的各RVD的核苷酸关联频率并将它们与总观察频率作比较-换句话说是通过根据左或右的邻近RVD分选RVD-核苷酸配对并比较各配对的相对频率,从而用所述配对的总频率分选。邻近分选的RVD-核苷酸关联的频率未显著偏离总观察频率,表明所述关联是环境独立性的。
侧接所述20个靶位点的序列除了-1位的T之外没有显示保守核苷酸,但其倾向于在位点后富含C且整体G很少(图1D)。除了少数例外,位点开始于标注的转录起始点下游60bp内,且没有位点靠近所述转录起始点87bp内(表2和表3)。确定RVD/核苷酸关联的其他规则描述于实施例4和5。
基于这些结果,可进行基因组中TAL效应子靶标的预测和靶标的从头构建。预测位点的能力会加快疾病中重要宿主基因的鉴定。构建靶标的能力在设计响应保守或多种TAL效应子的持久抗性基因中具有潜力。如本文所述,还可以定制用于任意基因活化的TAL效应子或靶向用于DNA修饰的融合蛋白。
表2
用于实验鉴定TAL效应子-靶标配对的预测靶位点特征
RVD,重复可变双残基;TcS,标注的转录起始位点;TlS,转录起始位点。位置是相对于靶位点的5’末端。
1Gu等,同上
2Kay等(2007)Science 318:648
3 等(2007)Science 318:645
4Herbers等(1992)Nature 356:172
5 等(2009)Plant Physiol.
6Schornack等(2008)New Phytologist 179:546
7Yang等(2006)Proc.Natl.Acad.Sci.USA 103:10503
8Sugio等(2007)Proc.Natl.Acad.Sci.USA
表3
感染期间激活的水稻中水稻条斑病菌TAL效应子候选靶标
RVD,重复可变双残基;r,扫描的58,918个基因模型的排名,基于RVD权矩阵评分;TcS,标注的转录起始位点;n.p.,不存在;TlS,转录起始位点。位置是相对于靶位点的5’末端。q值用于比对以跨接种后多至96小时中5个时间点进行模拟,重复4次;给出的改变倍数是在96小时时(PLEXdb,登录号OS3)。
实施例2–TALEN能在酵母中起作用
质粒构建:TAL效应子AvrBs3的蛋白编码序列通过用BamHI消化质粒获得。主要编码所述重复结构域的DNA区段用SphI切割。AvrBs3的氨基酸序列可在GENBANK登录号P14727和SEQ ID NO:12(图3)中找到,核苷酸序列在登录号X16130和SEQ ID NO:13(图4)中。在图4中,BamHI和SphI位点以粗体和下划线显示。AvrBs3 BamHI和SphI片段克隆到核酸酶表达载体pDW1789_TAL(图.5)中,邻近编码FokI核酸酶结构域的序列。为了将AvrBs3靶位点克隆到靶报告质粒中,合成含有反向排列的两种AvrBs3识别位点和其之间18bp间隔序列的两种互补DNA寡聚物,其在5’和3’末端分别具有BglII和SpeI突出。制备其他具有识别位点的报告质粒,间隔长度为6、9、12和15bp。退火的DNA寡聚物克隆到报告质粒pCP5中(图6),其用BglII和SpeI消化。
酵母试验:所述靶报告质粒转化到酵母株系YPH499(MATα株系)中,并在缺少色氨酸的合成完整培养基(SC-W)上筛选转化体。TALEN表达载体转化到YPH500(MATα株系)中;并将转化体接种于缺乏组氨酸的SC培养基(SC-H)上。载有靶报告质粒的酵母克隆和载有TALEN表达质粒的克隆分别在30℃的液体SC-W和SC-H培养基中培养过夜。培养基调节到相同的OD600,且各200μl混合到200μl YPD培养基中。所述混合物在30℃培养4小时以使两种类型的酵母株系接合。沉淀混合的培养物并在30℃的5ml SC-W-H培养基中重悬过夜或直到OD600达到0.5-1的范围。收获所述细胞并如(Townsend等(2009)Nature459:442-445)所述进行定量β-半乳糖苷酶试验。
结果:TAL-FokI融合蛋白是由TAL DNA识别结构域和非特异FokI DNA切割结构域组成的位点特异性核酸酶。TAL DNA识别结构域可工程改造成结合不同DNA序列。如本文实施例1所述,已解密一类新的DNA结合域,即TAL效应子的DNA识别特异性。具体地,TAL效应子的DNA结合域包含各种数量的串联、34氨基酸重复,其可识别并结合特定DNA序列。除了所述重复的位置12和13的两个相邻高度可变残基之外,所述重复的氨基酸序列保守。这些位置一起指定DNA结合位点的个体核苷酸,一个重复对应一个核苷酸。TALEN的结构见图7所示。TALEN起二聚体作用,各单体由工程改造的TAL DNA识别重复序列融合FokI内切核酸酶的非特异性切割结构域而构成。DNA识别重复序列可工程改造成结合感兴趣基因组内的靶DNA序列。TAL核酸酶单体结合被间隔序列分开的两种DNA半位点之一。所述间隔使FokI单体二聚化并在所述半位点之间的间隔序列中产生双链DNA断裂(DSB)。
为了开发TAL效应子DNA识别结构域的潜力,进行实验测定天然TAL效应子融合FokI核酸酶结构域时能否作为核酸酶起作用。通过使用TAL核酸酶表达构建体和靶报告构建体,进行基于酵母的试验。如图5所示,核酸酶表达构建体的主干含有酵母TEF1启动子控制下的FokI核酸酶结构域和N末端核酸酶定位信号(NLS)。数种限制性位点定位在FokI核酸酶结构域和NLS基序之间以有利于各种TAL效应子的克隆。如图6所示,靶报告构建体具有被破坏的lacZ报告基因,其含有125bp编码序列重复。所述重复侧接URA3基因和被TAL DNA结合域识别的靶序列(由两种半位点和间隔序列构成)。若TALEN在靶位点结合并生成DNA双链断裂(DSB),该断裂在酵母中主要由重复的lacZ序列之间的同源重组通过单链退火来修复(Haber(1995)Bioessays17:609)。重组能重建功能性lacZ基因并缺失URA3(赋予5-氟乳清酸抗性)。TALEN的相对切割活性通过测定lacZ酶活性来测量。
在这些研究中,具有如SEQ ID NO:31(图8)所示中央核酸酶重复区域的天然TAL效应子AvrBs3克隆到核酸酶表达载体中,并将具有18bp间隔序列的AvrBs3靶位点(反向排列的两个结合位点)克隆到靶报告载体中。用图9和上述的方案进行酵母试验。结果显示转化有AvrBs3核酸酶质粒和所述靶报告质粒的酵母细胞的lacZ活性比仅含有所述靶报告质粒的对照酵母细胞显著更高(高15.8倍)(图10)。仅用主要编码重复结构域的SphI片段制备的核酸酶融合物没有观察到活性。这表明除了DNA结合域之外的序列为TALEN活性所需。间隔长度为6和9bp的报告质粒也未能显示活性,表明两个结合位点之间的间隔对FokI的二聚化很关键。这些数据表明AvrBs3 TAL核酸酶可作为位点特异性核酸酶起作用,切割酵母中其关联靶序列。
实施例3-用于自定义TALEN的TAL效应子重复序列的模块化组装
对应于四种个体TAL效应子重复序列中每一个的102个碱基对的互补寡核苷酸,各指定不同核苷酸,将其合成、退火并单独或以所有排列中的2和3种重复序列组合克隆到高拷贝细菌克隆载体中,用标准限制性消化和连接技术产生4种单一重复模块、16种双重复模块、和64种三重复模块(如图11所示)。通过将合适的模块顺序引入Gateway-ready高拷贝细菌克隆载体中来组装所需的TAL效应子编码序列,所述载体包括tal1c基因的截短形式,其缺少中央重复区域,除了最终的特征性半重复。例如,可通过顺序引入5个3重模块和1个双模块到截短的tal1c载体中来组装18重复TAL效应子编码序列。
实施例4–用于模块化组装TAL效应子重复序列的系统
开发用于生成自定义TAL效应子编码基因的质粒和方法。TAL效应子的功能特异性如本文所述通过重复序列中的RVD测定;重复序列和蛋白中别处的其他多态性很少且对功能特异性不重要。因此,通过用含有所需RVD的重复序列替代任意TAL效应子基因的重复区域来生成自定义TAL效应子基因。RVD之外的重复序列匹配共有序列(见下)。编码TAL效应子重复序列的DNA片段顺序组装到编码一种、两种、或三种重复的模块中,且所述模块克隆到原始重复被移除的TAL效应子基因中。除了最后的(半)重复,各编码重复的序列为LTPAQVVAIASXXGGKQALETVQRLLPVLCQDHG(SEQ ID NO:18;图12A)。最后(半)重复的序列为LTPAQVVAIASXXGGKQALES(SEQ ID NO:20;图12B)。在两种序列中,“XX”表示RVD的位置。模块重复中所用的RVD为NI、HD、NN、和NG,其分别特异性结合A、C、G和T。下述实验中,移除了重复序列的水稻细菌性条斑病菌(Xanthomonas oryzae pv.oryzicola)株系BLS256的tal1c基因用作构建自定义TAL效应子基因的“主干”。
本文所述方法包括5部分:(1)生成单重复起始质粒;(2)生成单重复模块质粒;(3)生成多重复模块;(4)生成一、二和三重复模块质粒的完整组;和(5)自定义TAL效应子编码序列的组装。
为了生成单重复起始质粒,用MscI消化tal1c基因并重新连接以移除除了第一重复的第一部分和最后截短型重复的最后部分以外的完整重复区域,产生的质粒称为pCS487(图13)。得到的基因编码RVD NI且和大多数TAL效应子基因类似,含有侧接重复区域的两个SphI位点。所述基因不含XhoI位点。
然后,将翻译性沉默突变引入pCS487,产生独特的PspXI位点,其在密码子19和20的中间涵盖独特的XhoI位点。图14描述突变,显示密码子18-21的原始和改变的核苷酸序列(分别为SEQ ID NO:21和SEQ ID NO:23),二者都编码氨基酸序列ALES(SEQ ID NO:22)。得到的质粒称为pCS489。
进一步突变,生成具有RVD HD、NN、和NG三种其他构建体,产生的质粒分别称为pCS490、pCS491、和pCS492。涵盖经修饰重复区域的SphI片段从pCS489、pCS490、pCS491和pCS492中转移到卡那霉素抗性质粒pCS488(图15),其在Gateway进入载体pENTR-D(英杰公司(Invitrogen),加利福尼亚州卡尔斯巴德)中仅编码tal1c的N和C末端部分,而没有所述重复区域。所述转移分别产生单重复起始质粒pCS493(图16)、pCS494、pCS495、和pCS496。截短重复中的PspXI/XhoI位点在这些质粒中仍保持独特。pCS488中的TAL效应子基因和其各衍生物前面有Shine-Dalgarno和Kozak序列,分别用于在原核和真核生物中有效翻译。
然后构建单重复模块质粒。就四种选定RVD(NI、HD、NN、和NG)中的每一个生成一种质粒。各质粒具有连接到PspXI位点后重建XhoI而非PspXI位点的5’相容性粘性末端,和重建XhoI和PspXI位点的3’相容性粘性末端。通过将具有突出端的退火的合成、互补寡核苷酸(图17A)克隆到pBluescript SK-的PspXI/XhoI位点中生成所述质粒,分别产生质粒pCS502(图17B)、pCS503、pCS504、和pCS505。各质粒能在独特的重建PspXI位点处的单重复模块的3’末端引入其他重复,或可用重建的XhoI位点切割所述重复模块。
生成各用于NI、HD、NN和NG的其他单重复模块。各具有连接到PspXI位点后不重建PspXI或XhoI位点的5’相容性粘性末端,重建XhoI和PspXI位点的3’相容性粘性末端,和破坏内部MscI位点的翻译性沉默核苷酸替换(图18A)。通过退火具有突出端的合成、互补寡核苷酸生成这些模块。将这些其他单重复模块中任一种连接到单重复模块质粒(pCS502,pCS503,pCS504,或pCS505)的独特的PspXI/XhoI位点中,没有在5’连接处产生新的XhoI位点,但恢复了独特的3’PspXI/XhoI位点,从而得到的质粒可通过PspXI切割而线性化以引入更多的其他重复。反复该过程可产生含有多个重复的模块(图18B)。此外,各完整多重复模块可用XhoI切割。由于其他单重复模块中的MscI位点被破坏,初始重复中的MscI位点保持独特,且用于在后续亚克隆所述多重复模块后检查方向。
其他单重复模块反复克隆到单重复模块质粒中,与所述单重复模块质粒一起生成所有可能的一、二和三重复模块的完整组,总计84种质粒,称为pCS502-pCS585(图19)。以相同方法生成含有多于三重复(如4、5、6、7、8、9、10或多于10重复)的模块。
然后设计方法以将任何重复序列组装到tal1c“主干”中,生成自定义TAL效应子基因。所述方法包括下述步骤,其还示于图20:
(1)选择具有第一需要重复的单重复起始质粒(pCS493、pCS494、pCS495、或pCS495,分别编码RVD NI、HD、NN、或NG);
(2)用PspXI线性化所述质粒;
(3)用XhoI从合适的模块质粒(pCS502-pCS585)上分离用于后续重复的模块;
(4)连接;
(5)通过用MscI消化来检查方向并用基于载体的引物从3’末端确认序列;和
(6)重复步骤2-5直到组装了所有重复。
实施例5–用于TALEN模块组装的质粒库
本文所述的TALEN重复序列组装(如用图20所示的步骤)产生许多所含重复数量增加的中间质粒。各自储存这些质粒,从而生成用于TALEN模块组装的质粒库(pMAT)。例如,图21A和21B显示构建靶向所示核苷酸序列的TAL内切核酸酶中的重复模块组装。在图21A中,质粒pCS519、pCS524、pCS537、pCS551、pCS583和pCS529的重复模块依次添加到起始质粒pCS493的序列中,产生质粒pMAT55、pMAT56、pMAT57、pMAT58、pMAT59和pMAT60。在图21B中,质粒pCS530、pCS533、pCS522和pCS541的重复模块依次添加到质粒pMAT1的序列中,产生质粒pMAT61、pMAT62、pMAT63和pMAT64。
实施例6–生成并测试自定义TALEN
用实施例4和5所述的系统,TAL DNA识别结构域用于产生识别和切割具体DNA靶标的TALEN(图22A)。为了评估TALEN功能,改良酵母试验,其中LacZ活性作为DNA切割的指示物(Townsend等,同上)。在本试验中,靶质粒和TALEN表达质粒通过接合一起引入相同细胞。所述靶质粒具有含125-bp编码序列重复的lacZ报告基因。所述重复侧接由给定TALEN识别的靶位点。靶位点发生双链DNA断裂时,通过重复序列之间的单链退火对其进行修复,产生功能性的lacZ基因,其表达可用标准β-半乳糖苷酶试验测量产生可量化的读数(图22A)。已证明所述试验能良好预测ZFN通过NHEJ产生染色体突变的能力或刺激高等真核生物中用于基因编辑的同源重组的能力(Townsend等,同上;和Zhang等(2010)Proc.Natl.Acad.Sci.USA107:12028-12033)。
使用两种良好表征的TAL效应子–辣椒病原体辣椒细菌性疮痂病菌(Xanthomonascampestris pv.vesicatoria)的AvrBs3和水稻病原体水稻白叶枯病菌的PthXo1(Bonas等(1989)Mol.Gen.Genet.218:127-136;和Yang等(2006)Proc.Natl.Acad.Sci.USA 103:10503-10508)。AvrBs3的氨基酸序列可在GENBANK登录号P14727和SEQ ID NO:12(图3)中找到,核酸序列在登录号X16130和SEQ ID NO:13(图4)中。PthXo1的氨基酸序列可在GENBANK登录号ACD58243和SEQ ID NO:16(图23)中找到,核酸序列在登录号CP000967,基因ID6305128和SEQ ID NO:32(图24)中。由于起始密码子的错误注释,GENBANK登录号ACD58243下的PthXo1氨基酸序列在N末端截短。完整序列示于图23。
AvrBs3和PthXo1的重复结构域在保守的SphI片段内完整编码(图4和24)。TAL效应子编码基因还具有BamHI限制性片段,其涵盖重复结构域的编码序列以及前287个氨基酸和后231个氨基酸(图4和24;还参见图22A)。BamHI片段中缺失TAL效应子转录激活结构域。SphI片段和BamHI片段都融合核酸酶表达载体pFZ85中存在的编码FokI的DNA片段(图25)。FokI核酸酶和AvrBs3和PthXo1编码的BamHI片段之间的融合蛋白示于图26和27,SEQ IDNOS:33和34。
FokI单体必须二聚化才能切割,但两种DNA识别位置之间的合适间隔长度尚不清楚。对于其中锌指列与FokI被4-7个氨基酸接头分隔的ZFN,两种识别位点之间的典型间隔为5-7bp(Handel等(2009)Mol.Ther.17:104-111)。因此,例如,235氨基酸将本文所用的BamHI TALEN构建体中的重复结构域与FokI分开,对BamHI和SphI构建体可使用各种间隔长度(6、9、12、15和18bp)。作为阳性对照,使用具有DNA结合域的明确锌指核酸酶,所述结构域衍生自小鼠转录因子Zif268(Porteus和Baltimore(2003)Science 300:763)。作为阴性对照,TAL效应子结构域融合催化灭活的FokI变体或针对非关联DNA靶标测试。
200μl过夜培养物中含TALEN表达或靶质粒的单倍体细胞类型在30℃的YPD培养基中接合。4小时后,用5ml选择培养基替代YPD培养基并于30℃孵育过夜。裂解接合培养物,添加ONPG底物,并用96-孔板读数计读取吸光度(Townsend等,同上)。β-半乳糖苷酶水平作为底物切割速率的函数计算。具有15bp间隔分开两种识别位点的靶报告构建体获得的结果示于图22B。主要编码重复列的源自SphI片段的所有核酸酶表达载体未显示活性,表明需要重复列以外的氨基酸序列来发挥功能(图22B)。然而对衍生自BamHI片段的AvrBs3和PthXo1TALEN均观察到很强的活性(图22B)。PthXo1 TALEN的活性类似ZFN阳性对照。所述活性需要功能性FokI结构域且对给定TALEN识别的DNA靶标特异。
还进行实验测试TAL效应子结合位点间的各种距离(12-30bp的11种长度变体),从而鉴定能使FokI最有效二聚化的间隔长度(图28A)。两种酶显示两种最佳的间隔长度-一种为15bp且另一种为21bp(AvrBs3)或24bp(PthXo1)。对于PthXo1,在13bp和更长的所有测试间隔长度中观察到活性。但是AvrBs3的一些间隔长度没有显示活性,表明间隔长度对某些TALEN至关重要。
上述实验测试了同二聚TALEN的活性,其在所述间隔区的任一侧结合反向放置的两种相同识别序列。因为该回文结构位点不可能在基因组靶标天然发生,所以进行实验测试TALEN能否作为异二聚体起作用。AvrBs3和PthXo1的识别位点以头尾方向置于15bp间隔区任一边。测量单独AvrBs3和PthXo1 TALENS以及Zif268对其各自靶标的活性作为对照。作为阴性对照,分析仅具有就异二聚位点而言的靶位点质粒的酵母培养物中的LacZ活性。异二聚TALEN所得的活性近似于所述两种同二聚化酶观察到的活性均值(图28B)。
为了测试重复结构域能否将靶TALEN组装到任意染色体序列中,选择先前针对带ZFN的突变而靶向的两种基因–拟南芥的ADH1和斑马鱼的gridlock(Foley等(2009)PLoSOne 4:e4348;和Zhang等,同上)。在编码区域搜索前面有5’T并与Moscou和Bogdanove(同上)所鉴定TAL效应子结合位点的核苷酸组成相似的12-13bp序列。ADH1和gridlock中,平均每7-9bp产生该位点。ADH1中选择四个12bp位点(染色体基因序列的360、408、928和975位置),gridlock中选择1个13bp位点(染色体基因序列的2356位置;图29A)。用天然TAL效应子中最丰富的RVD(NI用于A、HD用于C、NN用于G、和NG用于T)构建TAL效应子重复结构域以识别这些靶标。为了构建自定义TALEN,含这些RVD的重复序列单独合成并组装到一、二、或三重复的模块中,如实施例4和5所示。这些模块依次连接到原始重复序列已被移除的tal1c基因衍生物中(Moscou和Bogdanove,同上),且这些工程改造的TAL效应子中的BamHI片段融合编码pFZ85中FokI催化结构域的序列(图25)。产生靶向拟南芥的ADH1和斑马鱼gridlock基因的5种自定义TALEN。
得到的自定义TALEN作为同二聚TALEN(即,相同DNA结合位点以反向在16-18bp间隔区任一侧重复)在酵母试验中测试,尽管注意到需要构建异二聚TALEN以引导在天然产生的DNA靶标上切割。基于下一邻近(和反向)候选位点的3’末端与15bp最近的距离选择间隔长度。16bp间隔用于ADH1-360-12、ADH1-408-12r,且18bp间隔用于ADH1-928-12、ADH1-975-12r和gridlock-2356-13r。如上所述进行酵母试验。
就ADH1-360-12和gridlock-2356-13r TALEN观察到强烈核酸酶活性(图29B)。ADH1-928-12TALEN具有中度活性,但其显著高于阴性对照。产生阳性结果的各TALEN的核酸酶活性特异于关联靶标。这些结果表明新的功能性TALEN可通过组装自定义重复结构域产生。
实施例7–天然产生的靶标和TAL效应子对在核苷酸和RVD组成中显示总体和位置
偏好
评价Moscou和Bogdanove(同上)分析的20对靶标和TAL效应子对核苷酸或RVD频率的总体组成偏好和位置效应。观察到位点(正链)一般富含A和C,很少含G。A的平均百分比为31±16%(1标准偏差)。C的平均百分比为37±13%。G的平均百分比为9±8%,T的平均百分比为22±10%。由于所述比对长度不同,位置影响的分析限于各末端的5个位置。令人惊奇的是,位置1和3处的靶序列相较T对A有明显的偏好,位置N和可能的2处偏好T。G在位置N-1特别稀少。该偏好由效应子中匹配的RVD反映,位置1和3上最常见NI,位置1上没有NG,位置N上几乎都是NG,且位置N-1很少有NN(图30)。
实施例8–快速组装和克隆自定义TAL效应子重复列的方法和试剂
Golden Gate克隆方法[Engler等(2008),同上;和Engler等(2009),同上]利用IIS型限制性内切核酸酶(如BsaI)在其识别位点之外切割的能力来产生自定义突出端用于同时顺序连接多种DNA片段。用此方法,数种DNA片段可以特定顺序融合成列并在单一反应中克隆到所需的目标载体中(图31)。
基于Golden Gate系统开发了组装自定义TAL效应子重复编码列的方法和试剂。将BsaI位点置于TAL效应子重复编码序列任一侧时,切割可释放侧接4-bp突出端的重复片段。由于切割位点并非序列特异性,通过交错可使重复克隆具有顺序、互补的突出端(粘性末端),使多重复列顺序组装。
生成58个质粒的库(图32A和32B)以同时将多至10个重复单元组装到“亚列”中,然后同时将一、二、或三个这些亚列和最终的截短重复一起组装到完整的自定义列中。合成4片段的10个交错组,组中各片段编码具有4种最常见RVD,HD、NG、NI和NN之一的不同重复模块,将这些组克隆到载有四环素抗性基因的载体中,共计40种质粒。合成编码20个氨基酸的末端截短TAL效应子重复的4种其他片段,各片段编码不同的四种最常见RVD之一,将所述片段克隆到载有壮观霉素抗性基因的不同载体中,产生另外4种质粒,称为“最终重复质粒”(图32A)。交错组中的所有片段侧接载体中的BsaI位点,从而用BsaI的切割释放具有不同粘性末端的片段,以按照合适的顺序组装;即重复模块1的片段3’末端处的突出仅与重复模块2的片段5’末端处的突出互补,重复模块2的片段3’末端处的突出仅与重复模块3的片段5’末端处的突出互补,等等。最后重复质粒的片段侧接不同IIS型限制性内切核酸酶Esp3I的位点。如下所述构建14种其他质粒作为目标载体接受组装的亚列。
构建第一目标载体质粒pFUS_A以接受具有10重复的第一亚列,将其组装到具有21或更少重复的最终列中(计数最终截短的重复)。构建pFUS_A从而BsaI切割在一侧产生与第一重复模块的5’末端突出互补的的突出端,并在另一侧产生与第10重复模块的3’末端突出互补的突出端。为了接受具有10或更少重复的第二亚列用以组装到最终列中,构建目标载体质粒pFUS_B1、pFUS_B2、pFUS_B3、pFUS_B4、pFUS_B5、pFUS_B6、pFUS_B7、pFUS_B8、pFUS_B9、和pFUS_B10,其在用BsaI切割时产生与所述第一重复模块的5’末端和对应编号位置的重复模块的3’末端的突出分别互补的突出端(例如用于亚列3’末端的pFUS_B6突出端匹配用于位置6的4种重复模块片段的突出端)。克隆到pFUS_A和pFUS_B系列质粒中的列侧接有载体中的Esp3I位点且用Esp3I消化时释放具有独特互补突出端的列,所述突出端使其能与最终截短重复片段一起顺序连接到目标载体pTAL中,其编码缺失重复区域的TALEN。构建pTAL从而用Esp3I切割可使重复列以正确位置和正确方向插入,这是通过一个末端的突出端与第一个10重复亚列的5’末端突出端互补且另一末端的突出端与最后截短重复片段的3’末端突出端互补(图33)。
构建最后的两种目标载体质粒pFUS_A30A和pFUS_A30B用于接受第一和第二个10重复亚列,其组装到具有22-31重复的最后列中。构建pFUS_A30A和pFUS_A30B,从而用Esp3I消化会释放具有合适互补突出端的列,所述列可与pFUS_B载体的第三列和最终重复质粒(用Esp3I消化产生相似的释放)的最后截短重复片段一起顺序连接到pTAL中(图32)。
所有目标载体具有克隆到IIS型限制性内切核酸酶中的LacZ基因,可进行重组子的蓝白筛选。除了载有氨苄青霉素抗性的基因pTAL,所有目标载体载有壮观霉素抗性基因。
为了用这些试剂快速构建自定义TAL效应子重复列,建立下述方法。在第一步中,用于具有10或更少重复的必需亚列的适当单独RVD模块质粒与适当的目标载体在一个管中一起混合。加入T4DNA连接酶和BsaI内切核酸酶,反应在PCR仪中孵育10个循环:37℃5分钟和16℃10分钟,分别为两种酶的最佳温度。然后用PLASMID-SAFETM核酸酶处理所述反应混合物用以水解所有线性dsDNA片段,从而阻止较短的不完整列通过体内重组克隆,然后所述混合物用于化学转化感受态大肠杆菌(E.coli)细胞。分离得到的重组质粒并确认正确的构建体。然后在第二步中,第一步中确认的质粒与合适的最终重复质粒和pTAL一起混合,并如第一步所述进行消化和连接反应循环。最后,将反应产物导入大肠杆菌,分离全长、最后列构建体并加以确认。单人可在一周时间内完成所述操作方案。
用本实施例的方法和试剂制备表4A中TALENS 85、102和117的表达构建体以及以下实施例14所述TALENS HPRT-3254-17和HPRT-3286-20r的表达构建体。
用侧接所述重复区域的保守SphI限制性内切核酸酶位点,能容易地将克隆到pTAL中的重复列亚克隆到其他TAL效应子基因环境中。
实施例9–自定义TALEN数据显示出对“规则”的最初支持以及RVD数量和活性之间
的关联
实施例6描述对TALEN DNA结合域进行工程改造的实验,从而其能识别独特的DNA序列。所述这些自定义TALEN识别拟南芥ADH1和斑马鱼gridlock基因中的位点。其他自定义TAL效应子DNA结合域工程改造成不仅识别这些基因中的位点,还识别拟南芥TT4基因和斑马鱼端粒酶(telomerase)中的位点(Foley等,同上;和Zhang等,同上)。这些自定义TALEN用实施例3、4和8中所述的方法制备。在工程改造所述自定义TALEN中,观察到的组成和位置偏好采用为设计原则或“规则”。首先,对编码区域的序列进行搜索,搜索的序列前面有5’T且至少长15bp并且具有与上述均值一致的核苷酸组成。具体地,仅选择那些0-63%A、11-63%C、0-25%G、和2-42%T的位点。所述位点平均每7-9bp产生。然后选择与上述观察到的位置偏好相符的位点。此组中,各基因中的2对结合位置鉴定为长15-19bp且被15-18bp分隔,从而结合工程改造的TALEN可使FokI二聚化。模块组装方法(实施例3和4)生成部分长度的构建体。
总计21种中间和全长TALEN设计成靶向16种核苷酸序列,各具有9个重复或更长的列。这些TALEN的氨基酸序列在图34A-34U中提供(SEQ ID NO:35-55)。用实施例2和6所述的酵母试验测试这21种TALEN切割DNA的能力。分析数据见图35并总结于表4A。
一些中间、部分长度的TALEN对应于破坏了核苷酸组成和末端T规则的靶标。表4A显示长度,与这两个规则的一致性,以及相对于各TALEN的ZFN268的活性。结果显示了总体趋势:RVD列的长度增加会提高所得TALEN活性。这表明DNA靶标在体内识别前需要的最少数量RVD。此外,与规则的一致性看来很重要。6种没有可检测活性的TALEN中,有两种违反了靶标组成规则,两种末端不是NG,另一种破坏了两个规则(有一种符合两个规则)。活性低于ZFN268的25%的8种TALEN中有3种违反了规则之一,且活性为ZFN268的25-50%的4种TALEN之一的RVD序列末端不是NG。注意到活性为ZFN268的50%或更高的TALEN符合所有规则,且对于相同长度的TALEN,破坏规则者的活性通常低于符合规则的列。即使对于不破坏规则的中间体,相应的全长TALEN与涉及长度的总体趋势一致时具有更高的活性(表4A和图35)。相同靶标上TALEN长度差异造成的间隔长度变化可引起所述观察,但可耐受一定范围的间隔长度(Christian等,同上)。
数据中可见一些明显的复杂性。例如,符合规则的相同长度TALEN之间的活性不同,一些短列具有适度高活性,一些符合规则的长列只有很少或没有活性(表4B)。但是,得到的结果支持结论:1)通常更多重复数量产生更高的活性,和2)与组成和位置偏好规则的一致性对活性很重要。因此,得出下述设计原则。
·TAL效应子结合位点设计为最少15碱基长度且方向为5’到3’,其5’末端前紧接T。
·位点的第一(5’)位置可没有T或第二位置没有A。
·位点必须以T结尾(3’),且在接近最后的位置可不具有G。
·位点的碱基组成必须落入特定范围(均值±两个标准偏差):A 0-63%,C 11-63%,G 0-25%,和T 2-42%。
表4A
酵母试验中测试的TALEN的活性、与原则的一致性、和长度
1测试的靶序列由对应核苷酸序列的反向重复组成,其中HD、NG、NI和NN分别对应C、T、A、和G,由16-18bp的间隔序列分开。
表4B
表4A节选,活性水平分选
实施例10–异二聚TALEN对在酵母试验中切割其预期的天然产生靶序列
实施例2、6和9的数据证明自定义TALEN可工程改造成识别新的靶DNA序列。用识别同二聚靶位点的单独TALEN单体收集自定义TALEN的酵母活性数据。即,TALEN的靶序列在15-18bp间隔任一侧反向重复。然而内源染色体序列的切割通常需要两种不同自定义TALEN识别间隔任一侧的两种不同序列。如实施例6所示,用酵母试验中对应的嵌合靶位点证明AvrBs3和PthXo1 TALENS一同具有该能力。我们测试了两种不同自定义TALEN能否识别和切割天然产生的DNA序列。利用实施例2所述的酵母试验,分析设计用于切割拟南芥ADH1基因中两种不同靶序列的自定义TALEN在这些靶标上的活性。靶位点的DNA序列和对应的TALEN示于图36A。TALEN的氨基酸序列示于图34。酵母试验中β-半乳糖苷酶活性绘制在图36B中。TALEN对其天然产生的靶序列的活性显著高于阴性对照,表明TALEN可工程改造成识别并切割内源靶DNA序列。
实施例11–TALEN切割拟南芥中的天然基因并通过不精确的非同源末端连接引入
突变
对设计成识别拟南芥ADH1基因中靶序列的活性TALEN对之一进行测试以测定其能否结合、切割和突变染色体DNA。包含该对(pTALENs 69和74)的单独ADH1TALEN中各自克隆到植物表达载体pFZ14中,将TALEN置于组成型35S启动子的控制之下(Zhang等,同上)。然后将得到的构建体通过电穿孔导入拟南芥原生质体。48小时后,分离基因组DNA并用Tth111l消化。Tth111l切割位点位于两种TALEN识别位点之间的间隔序列中(图37A)。预期TALEN对染色体DNA的切割可通过不精确的非同源末端连接(NHEJ)引入突变,这会使Tth111l无法切割。然后用PCR扩增涵盖TALEN识别位点的375bp片段。再用Tth111l消化PCR产物以移除未被TALEN介导的NHEJ修饰的大多数剩余基因组DNA。然后消化产物进行琼脂糖凝胶电泳。观察到未切割的PCR产物,且这类未切割的PCR产物诊断内源靶序列处的核酸酶活性(本情况中为TALEN活性)(Zhang等,同上)。通过DNA测序来克隆并分析未切割的DNA。测序9种独立克隆显示6种带有NHEJ引入的突变(图37B)。因此,TALENS切割内源染色体基因座并引入DNA双链断裂和突变。
实施例12–提高靶向能力
在TAL效应子DNA密码的核心处,四种最常见的RVD基于关联频率对4种核苷酸各具有明显的一对一特异性。HD、NG和NI显著,但NN不太显著(图1C)。NN与G的联合最频繁,频率和A几乎一样,有时与C或T关联。对于13RVD序列中四个位置随机组装的TAL效应子和NN,在人工靶标的所有对应位置具有G会产生最佳活性(Boch等(2009)Science 326:1509-1512)。A降低但不消除活性,C和T消除可检测的活性。仅当24RVD效应子PthXo1的结合位点的第一位置处(为NN)C、T或A取代G时观察到显著的活性降低(Romer等(2010)New Phytol.187:1048-1057)。然而,这与下述观察相反:短许多的AvrHah1(14RVD)起始于与A配对的NN,且23RVD效应子PthXo6在位置4-6具有各配对A的一排三种NN,然而这两种蛋白都有高活性(参见Schornack等(2008)New Phytol.179:546-556;和Romer等,上述)。因此NN对G的特异性看来普遍较弱且可根据环境变化。
直接先于TAL效应子靶位点的胸腺嘧啶所观察到的不变性为数种效应子所必需[Boch等,同上;Romer等,同上;和Romer等(2009)Plant Physiol.150:1697-1712]。直接先于TAL效应子中重复区域的高度保守氨基酸序列(图38A),在氨基酸序列和预测的二级结构上与所述重复共有显著的相似性(图38B和Bodganove等(2010)Curr.Opin.Plant Biol.13:394-401)。假定该序列(称为“第0”重复)是结合位点的-1位置需要T的基础,且RVD类似位置的该残基(图38B)指定所述核苷酸。
基于这些发现,假定通过纳入对G高度特异的重复和通过放宽-1处对T的要求,工程改造的TAL效应蛋白的靶向能力可提高。进行实验以测试新的稀有RVD,其对G的特异性强于NN所示,并用常见RVD替换第0重复中的RVD类似残基。
对G具有强特异性的新型稀有RVD:上面公开的模块(参见例如实施例4)使用四种具体RVD(NI、HD、NN、和NG)以特定结合四种核苷酸残基(分别为A、C、G、和T)。含有其他RVD的重复也可能有用,且相较NI、HD、NN和NG可能对四种碱基具有更高的特异性和/或亲和性。为了改良对G的特异性,构建编码新型稀有RVD的数种重复序列。稀有RVD NK、HN和NA与G关联,表明N可能和所述残基之一或另一种同样重要(图1C)。因此,构建编码具有表5所示RVD的重复的广泛衍生物组。左列列出位置12具有极性氨基酸(R、K、D、E、Q、H、S、T、或Y)和位置13具有N的RVD。右列列出RVD的第一位置具有N以及第二位置具有17种其他氨基酸(G、L、V、R、K、D、E、Q、H、T、M、C、P、Y、W、或F)任一种的组合。为了在没有N时能产生更高特异性,还将重复序列制备为位置12具有极性氨基酸(R、K、D、E、Q、H、S、T、或Y)和位置13具有缺口(*)(中列)。
测试新的人工RVD在用于TAL效应子转录激活活性的基于定量报告基因的试验中的功能,如上述烟草中基于GUS或双荧光素酶报告基因的农杆菌介导的瞬时表达试验,或酿酒酵母中基于lacZ报告基因的TALEN试验(参见例如实施例2)。将含有RVD的待测试重复模块纳入到具有可检测和亚饱和水平活性的TAL效应子或TALEN中,在DNA靶标组上测试得到的蛋白的活性差异,所述DNA靶标组在对应位置具有所有四种核苷酸的整合排列。具体地,PthXo1变体作为起始在植物原位和酵母试验中活性最小并响应三种已添加重复处的错配,含有新型稀有重复(同义同子性的三种)中每一个的TALEN针对在对应位置中各具有G的靶标进行体内测试。对于显示活性提高的任一种,用在那些位置改变为其他核苷酸的靶标重复所述试验以确保特异性。
表5.待测试的RVDa
aN*、NG和NS nt的关联频率已知。星号表示对应RVD中第2位置的缺口(即共有重复序列的第13位置)。
用于放宽T在-1位置特异性的第0重复中RVD类似位置的常见RVD取代:第0重复和重复共有序列的二级结构预测和比对表明第0重复中KR*(星号表示缺口)占据的位置与RVD类似,因此其为-1处指定T的残基。用HD,NG,NI和NN取代KR并单独取代R*的PthXo1变体构建在上述Tal1c“主干”构建体中。在植物原位和酵母试验中使用-1位置具有对应核苷酸即分别为C、T、A和G的靶标将这些变体的活性与野生型效应子作比较。构建PthXo1其他变体,其在共有重复序列的位置11处具有残基S,取代第0重复的位置11处的K。还构建具有该取代以及共有重复序列位置16处的残基K取代第0重复位置15处V的其他变体(表6)。可包括用于TAL效应子活性的邻近TATA盒。此外,PthXo1用于本实验,因为不同于AvrBs3(-1处的T似乎是TATA盒的一部分),最接近PthXo1结合位点的TATA盒位于下游46bp处且不会受-1处的修饰干扰。
若上述修饰没有引起对G的靶向增强或靶向前面有除了T之外的核苷酸的序列的能力增强,则测试人工RVD综合组对G的特异性,并就第0重复测试常见RVD以外的取代。
实施例13-新预测的核苷酸特异性RVD
观察到当表1A和1B所列RVD被RVD中的第2氨基酸残基(即总重复的第13)分组时,氨基酸和RVD指定的核苷酸之间有几乎完美的关联,不考虑RVD第1位置的核苷酸(表7)。因此,以缺口(由星号表示)结尾的RVD指定C或T,或者T;D结尾的RVD指定C;G结尾的RVD指定T;且N结尾的RVD指定G或A,或者G。还观察到RVD的位置1处的氨基酸为H、I、N、S或Y。这些观察表明RVD特异性由第2位置中的残基确定,不取决于第1位置的残基是H、I、N、S、或Y。因此,就组合了第2位置观察到的残基与第1位置的残基H、I、S、N或Y的数种新(即尚未观察的)RVD预测特异性。因此,预测I*、S*和Y*指定C或T,或者T;预测SD和YD指定C;预测SG指定T;并预测IN和YN指定G或A或者G。而且,尽管第2位置仅一种K情况,但基于观察到的NK特异性,预测HK、IK、SK和YK指定G。
如实施例2和11所述测试这些新RVD并就定量TAL效应子和TALEN活性试验中的功能和特异性与现有RVD作比较。
表71根据第2残基的RVD分组和顺序
第1残基 | 第2残基 | 核苷酸 |
N | * | C或T |
H | * | T |
H | A | C |
N | A | G |
H | D | C |
N | D | C |
H | G | T |
I | G | T |
N | G | T |
Y | G | T |
N | I | A |
H | I | C |
N | K | G |
H | N | G |
S | N | G或A |
N | N | |
N | S |
1星号表示缺口。具有类似特异性的RVD组以粗线加框。
实施例14–自定义TALEN切割动物细胞内源基因并通过不精确的非同源末端连接
引入突变
为了测试TALEN是否能用于动物细胞的靶向突变,先测试人胚胎肾(HEK)293T细胞中TAL效应子AvrBs3、PthXo1和Tal1c的表达。从AvrBs3、PthXo1和Tal1c编码基因中移除终止密码子并将基因亚克隆到哺乳动物表达载体pcDNA3.2/V5-DEST中(英杰公司(Invitrogen),加利福尼亚州卡尔斯巴德),所述基因与所述载体下游编码蛋白免疫检测所用V5表位的序列同框。pcDNA3.2/V5-DEST将TAL效应子基因置于组成型人巨细胞病毒(CMV)启动子的控制下。用Lipofectamine 2000(英杰公司)将得到的质粒单独转染到HEK 293T细胞中,24小时后,从各转染批次的细胞中分离总蛋白并用小鼠抗V5抗体进行聚丙烯酰胺凝胶电泳、Western印迹和免疫标记。使用SuperSignal Weat Pico化学发光试剂盒(赛默飞世尔公司(ThermoScientific,Inc.)),通过山羊抗小鼠抗体-辣根过氧化物酶偶联物检测标记的蛋白。用肌动蛋白免疫标记和检测确认等量加样。各TAL效应蛋白均可检测地表达且没有明显的降解(图39)。
然后,如实施例9所述设计一对TALEN以靶向内源人HPRT基因中的序列,即HPRT-3254-17和HPRT-3286-20r(图40A和图40B)。用实施例8所述的基于Golden Gate克隆的方法和试剂构建编码HPRT-3254-17的质粒pTALEN141和编码HPRT-3286-20r的质粒pTALEN142。然后TALEN基因亚克隆到哺乳动物表达载体pCDNA3.1(-)中(英杰公司),这将它们置于组成型CMV启动子的控制下,产生质粒pTALEN141M和pTALEN 142M。然后用TALEN141M和pTALEN142M一起转染HEK 293T细胞,并用pCDNA3.1(-)单独转染作为阴性对照。72小时后,分离基因组DNA并用限制性内切核酸酶Bpu10I消化。Bpu10I位点存在于分离HPRT中HPRT-3254-17和HPRT-3286-20r结合位点的间隔区内(图41A)。Bpu10I消化后,用PCR从经TALEN处理样品和对照样品中扩增跨TALEN靶位点的244bp片段。从两种样品中扩增到预期片段,表明Bpu10I对基因组DNA的消化不完全。然而用Bpu10I后续消化PCR产物可完全切割扩增自对照样品的产物,但来自TALEN处理样品的产物切割不完全(图41B)。经TALEN处理样品中存在抗切割的PCR产物证明内源Bpu10I位点体内突变,这是HPRT中指定靶标处TALEN介导的双链断裂的非同源末端连接不完全修复的结果。因此,TALEN可用于哺乳动物细胞中的靶向突变。
其他实施方式
应理解虽然本发明已结合其详述进行描绘,但以上描述意在说明而不是限制本发明的范围,该范围由所附权利要求的范围限定。其他方面、优势和修改在以下权利要求的范围内。
Claims (13)
1.一种编码序列特异性内切核酸酶的重组核酸表达载体,包括连接来自核酸酶的核苷酸序列的序列特异性TAL效应子的核苷酸序列,其可操作性连接至启动子序列。
2.如权利要求1所述的重组核酸表达载体,其中所述核酸酶是HhaI、HindIII、NotI、BbvCI、EcoRI、BglI、和AlwI或II型内切核酸酶例如FokI。
3.如权利要求1所述的重组核酸表达载体,其中所述序列特异性TAL效应子的核苷酸序列编码在位置12和13处具有高度可变残基的许多串联重复,其可识别并结合特异DNA序列。
4.如权利要求3所述的重组核酸表达载体,其中所述重复是34氨基酸重复,选自AvrBs3的中央区域(SEQ ID NO.3)。
5.如权利要求1-4中任一项所述的重组核酸表达载体,其中所述序列特异性TAL效应子的核苷酸序列通过将合适的重复顺序引入Gateway-ready高拷贝细菌克隆载体中来组装,所述载体包括tal基因的截短形式,其缺少中央重复区域,除了最终的特征性半重复。
6.如权利要求1-5中任一项所述的重组核酸表达载体,其中所述载体为质粒形式。
7.生产序列特异性内切核酸酶mRNA或蛋白质的方法,包括转录或翻译权利要求1-6中任一项所述的重组核酸的步骤。
8.权利要求7所述的方法获得的序列特异性内切核酸酶mRNA。
9.一种治疗组合物,其包含权利要求1-6中任一项所述的重组核酸或权利要求7所述的mRNA。
10.权利要求9所述的治疗组合物,其用于治疗病毒疾病。
11.一种在细胞中靶向遗传重组的方法,所述方法包括:
(a)将编码靶向所选DNA靶序列的TAL效应子内切核酸酶的权利要求1-6中任一项所述的核酸或权利要求8所述的mRNA引入分离的细胞;
(b)诱导所述TAL效应子内切核酸酶在细胞中表达;和
(c)鉴定所选DNA靶序列显示突变的细胞。
12.如权利要求11所述的方法,其中步骤c)中的所述突变选自下组:遗传物质的缺失、遗传物质的插入、或遗传物质的缺失和插入。
13.如权利要求11或12所述的方法,其中所述细胞是昆虫细胞、植物细胞、鱼细胞或哺乳动物细胞。
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