CN117467693A - 一种基因组编辑载体及应用 - Google Patents
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Abstract
本公开提供了一种基因组编辑载体及应用。具体的,本公开提供了一种专为乳酸克鲁维酵母优化的CRISPR基因编辑***及应用。特别的,本公开提供了一种乳酸克鲁维酵母CRISPR/Cas9基因组编辑载体及应用。在一个实施方式中,本公开获得了转化效率和同源重组效率均大幅提升的乳酸克鲁维菌基因编辑***,比现有技术提高了转化子数量,并提高了同源重组效率。与此同时,本公开采用的基因组编辑载体,基因组编辑***或基因组编辑方法,均能够良好的用于在细胞中基因功能的研究和/或蛋白生产,特别是酶的生产。
Description
技术领域
本公开属于生物技术领域,其涉及一种基因组编辑载体及应用。具体的,本公开涉及一种专为乳酸克鲁维酵母优化的CRISPR基因编辑***及应用。特别的,本公开涉及一种乳酸克鲁维酵母CRISPR/Cas9基因组编辑载体及应用。
背景技术
乳酸克鲁维酵母(Kluyveromyces lactis)是FDA认定的食品安全级(GRAS)酵母,具有营养要求简单、生长旺盛、生物量大、生长温度适应范围广、分泌蛋白能力强、无内毒素等优点。乳酸克鲁维酵母蛋白表达***培养成分简单、成本低廉、易放大,具备优秀的工业发酵属性,应用于食品药品领域,可实现胞外分泌和胞内合成两种方式的蛋白表达生产,显示出巨大的应用潜力。帝斯曼公司已利用乳酸克鲁维酵母表达***实现了牛凝乳酶的分泌表达和乳糖酶的胞内表达,并成功进入食品市场。然而,为进一步提升该表达***的应用潜力,尚需对乳酸克鲁维酵母进行广泛的遗传工程改造。
微生物的遗传工程改造主要利用同源重组(homology-directed repair,HR)和非同源重组(non-homologous end joining,NHEJ)方式进行。HR的基本过程为,基因组发生双链断裂(double-strand breaks,DSBs)后,供体DNA在断裂位点处与受损基因组序列发生同源重组,实现修复,这一过程不易引入***或缺失突变;NHEJ的基本过程为,断裂DNA两端直接连接,不借助供体DNA介导的同源重组,这一过程容易产生***或缺失突变,造成移码突变。
乳酸克鲁维酵母具有非常活跃的非同源重组修复方式,同源重组修复方式的比例极小。提供的线性修复DNA片段不能有效的整合在目标基因组位点,造成基因组精确编辑等遗传工程改造手段的效率低下。
利用CRISPR/Cas9方法进行基因组编辑,在各种酵母中应用已经非常广泛。具体来说,在gRNA的介导下,Cas9蛋白识别基因组上的PAM及其上游20bp序列,并在PAM上游3bp位置产生双链切口。供体DNA以HR的方式重组***,以达到基因组改造的目的。
但是,现有技术中采用的CRISPR/Cas9方法对于乳酸克鲁维酵母的编辑效率都不高。例如,现有技术文献1利用pUDP025质粒在乳酸克鲁维酵母中可以实现HR效率24%,但该质粒转化效率低下(27个/300ng质粒),较低的同源重组效率也增加了筛选的工作量,无法满足实际应用的要求。与此同时,现有技术文献2公开了一种针对克鲁维酵母优化的CRISPR/Cas9基因编辑***,其中CRISPR/Cas9质粒是基于酿酒酵母2μ质粒构建的,由于其在说明书中(例如说明书第【0008】段)写明将质粒的抗性基因Kan置换为Amp,因此前述质粒在酵母中将失去G418抗性基因表达。然而,现有技术文献2在实施例5中却写明是用G418来进行转化子筛选,这样会造成转化子筛选十分困难,甚至无法进行筛选。
现有技术文献
现有技术文献1:《Genome editing in Kluyveromyces and Ogataea yeastsusing a broad-host-range Cas9/gRNA co-expression plasmid》(PubMed 29438517);
现有技术文献2:CN107574179A
发明内容
发明要解决的问题
基于现有技术中存在的问题,迫切需要一种提高CRISPR编辑***,特别是CRISPR/Cas9编辑***的转化效率,以及编辑效率,使乳酸克鲁维酵母基因组改造更加容易的技术方案。
用于解决问题的方案
本公开涉及如下技术方案。
(1)一种基因组编辑载体,其中,所述基因组编辑载体选自由如下(a)-(f)组成的组中的任意一种:
(b)基因组编辑载体b,其核苷酸序列包含编码如SEQ ID NO:66所示的氨基酸序列的核苷酸序列或如SEQ ID NO:65所示的序列;
(a)基因组编辑载体a,其核苷酸序列包含如SEQ ID NO:45所示的序列和如SEQ IDNO:44所示的序列;
(c)基因组编辑载体c,其核苷酸序列包含如SEQ ID NO:45所示的序列、如SEQ IDNO:44所示的序列和如SEQ ID NO:46所示的序列;
(d)基因组编辑载体d,其核苷酸序列包含如SEQ ID NO:60所示的序列;
(e)基因组编辑载体e,其核苷酸序列包含如SEQ ID NO:61所示的序列;
(f)基因组编辑载体f,其核苷酸序列包含如SEQ ID NO:62所示的序列。
(2)根据(1)所述的基因组编辑载体,其中,所述基因编辑载体选自由如下(a)-(f)组成的组中的任意一种:
(b)基因组编辑载体b,其核苷酸序列包含如SEQ ID NO:46所示的序列或如SEQ IDNO:65所示的序列;
(a)基因组编辑载体a,其核苷酸序列包含如SEQ ID NO:54所示的序列和如SEQ IDNO:55所示的序列;
(c)基因组编辑载体c,其核苷酸序列包含如SEQ ID NO:47所示的序列和如SEQ IDNO:48所示的序列;
(d)基因组编辑载体d,其核苷酸序列包含如SEQ ID NO:60所示的序列、如SEQ IDNO:63所示的序列和如SEQ ID NO:64所示的序列;
(e)基因组编辑载体e,其核苷酸序列包含如SEQ ID NO:61所示的序列、如SEQ IDNO:63所示的序列和如SEQ ID NO:64所示的序列;
(f)基因组编辑载体f,其核苷酸序列包含如SEQ ID NO:62所示的序列、如SEQ IDNO:63所示的序列和如SEQ ID NO:64所示的序列。
(3)根据(2)所述的基因组编辑载体,其中,所述基因编辑载体选自如下(a)-(f)任一项所示的序列或所述基因编辑载体由如下(a)-(f)任一项所示的序列组成:
(b)基因组编辑载体b,其核苷酸序列包含如SEQ ID NO:58所示的序列和如SEQ IDNO:59所示的序列;
(a)基因组编辑载体a,其核苷酸序列包含如SEQ ID NO:54所示的序列、如SEQ IDNO:55所示的序列、如SEQ ID NO:56所示的序列和如SEQ ID NO:57所示的序列;
(c)基因组编辑载体c,其核苷酸序列包含如SEQ ID NO:47所示的序列和如SEQ IDNO:48所示的序列;
(d)基因组编辑载体d,其核苷酸序列包含如SEQ ID NO:60所示的序列、如SEQ IDNO:63所示的序列和如SEQ ID NO:64所示的序列;
(e)基因组编辑载体e,其核苷酸序列包含如SEQ ID NO:61所示的序列、如SEQ IDNO:63所示的序列和如SEQ ID NO:64所示的序列;
(f)基因组编辑载体f,其核苷酸序列包含如SEQ ID NO:62所示的序列、如SEQ IDNO:63所示的序列和如SEQ ID NO:64所示的序列。
(4)一种基因组编辑***,其中,所述***包括如(1)-(3)任一项所述的基因组编辑载体。
(5)一种宿主细胞,其中,所述宿主细胞包含有如(1)-(3)任一项所述的基因组编辑载体或(4)所述的基因组编辑***。
(6)根据(5)所述的宿主细胞,其中,所述宿主细胞为乳酸克鲁维酵母的细胞。
(7)一种基因组编辑方法,其中,所述基因组编辑方法包括利用基因编辑技术对(5)-(6)任一项宿主细胞进行编辑的步骤。
(8)根据(7)所述的方法,其中,所述方法包括将(4)所述的基因组编辑***导入到(5)-(6)任一项所述的宿主细胞中,在所述宿主细胞中的待编辑的基因组位点进行基因组编辑。
(9)根据(7)-(8)任一项所述的方法,其中,所述基因编辑技术为CRISPR技术。
(10)根据(1)-(3)任一项所述的基因组编辑载体,(4)所述的基因组编辑***或如(7)-(9)任一项所述的基因组编辑方法在细胞中基因功能的研究和/或蛋白生产中的应用。
在一种具体的实施方式中,所述蛋白为酶。
(11)一种生产蛋白的方法,其中,所述方法中使用根据(1)-(3)任一项所述的基因组编辑载体,(4)所述的基因组编辑***或如(7)-(9)任一项所述的基因组编辑方法生产所述蛋白。
在一种具体的实施方式中,所述蛋白为酶。
在本公开中,对原始pUDP025质粒进行一系列改造,以乳酸克鲁维酵母菌基因组中ADE2基因作为报告基因,进行缺失突变,最终通过计算成功突变数量来确定最优的基因组编辑方法。
在一个具体的实施方式中,将pUDP025质粒中的gRNA启动子置换为ScSNR52基因启动子,终止子替换为SUP4,构建得到p025-SNR52-SpiCAS-KlADE2质粒。
在一个具体的实施方式中,将pUDP025质粒中SpiCAS置换成HsCAS,构建得到p025-HsCAS-KlADE2质粒。
在一个具体的实施方式中,将pUDP025质粒中gRNA启动子置换为ScSNR52基因启动子,终止子替换为SUP4,同时将SpiCAS置换成HsCAS,构建得到p025-SNR52-HsCAS-KlADE2质粒。
在一个具体的实施方式中,转化的供体DNA序列为线性双链DNA,分别为距离DSB10bp、30bp、120bp、200bp以及KlADE2基因上游1000bp处,通过Gibson Assembly连接的方法获得。
发明的效果
在一个实施方式中,本公开基于乳酸克鲁维来源的自主复制序列、优化密码子的CAS序列、优化编码框的gRNA表达盒来构建CRISPR/Cas9质粒,以ADE2作为报告基因,获得了转化效率和同源重组效率均大幅提升的乳酸克鲁维CRISPR/Cas9基因编辑***。
在一个实施方式中,本公开通过改造,构建了一种可以在乳酸克鲁维酵母中稳定复制和表达的质粒,使CRISPR/Cas9编辑***在乳酸克鲁维酵母中完成高效基因编辑,比现有技术提高了转化子数量,并提高了同源重组效率。
在一个实施方式中,本公开采用的基因组编辑载体,基因组编辑***或基因组编辑方法,均能够良好的用于在细胞中基因功能的研究和/或酶生产,特别是食品用酶的生产。
附图说明
图1示出了p025-SNR52-SpiCAS-KlADE2质粒的示意图。
图2示出了p025-HsCAS-KlADE2质粒的示意图。
图3示出了p025-SNR52-HsCAS-KlADE2质粒的示意图。
图4示出了不同酵母复制起始点的转化效率测试结果。
图5示出了不同来源CAS以及gRNA表达盒序列的转化及编辑效率测试结果
图6和图7分别示出了不同donor DNA存在下的转化效率、编辑效率以及同源重组效率的测试结果。
具体实施方式
定义
当在权利要求和/或说明书中与术语“包含”联用时,词语“一(a)”或“一(an)”可以指“一个”,但也可以指“一个或多个”、“至少一个”以及“一个或多于一个”。
如在权利要求和说明书中所使用的,词语“包含”、“具有”、“包括”或“含有”是指包括在内的或开放式的,并不排除额外的、未引述的元件或方法步骤。
在整个申请文件中,术语“约”表示:一个值包括测定该值所使用的装置或方法的误差的标准偏差。
虽然所公开的内容支持术语“或”的定义仅为替代物以及“和/或”,但除非明确表示仅为替代物或替代物之间相互排斥外,权利要求中的术语“或”是指“和/或”。
如本公开所使用的,术语“多肽”、“肽”和“蛋白质”在本文中互换地使用并且为任意长度的氨基酸聚合物。该聚合物可以是线形或分支的,它可以包含修饰的氨基酸,并且它可以由非氨基酸隔断。该术语也包括已经被修饰(例如,二硫键形成、糖基化、脂质化、乙酰化、磷酸化或任何其他操作,如以标记组分缀合)的氨基酸聚合物。
如本公开所使用的,术语“CRISPR”是指成簇、规律间隔的短回文重复序列(Clustered regularly interspaced short palindromic repeats),其来自微生物的免疫***。
如本公开所使用的,术语“Cas蛋白突变体”包括与野生型Cas蛋白相比核酸内切酶活性丧失或降低的Cas蛋白突变体、同系物或其多肽片段。
如本公开所使用的,术语“野生型Cas蛋白”是指CRISPR-associated蛋白,Cas蛋白与CRISPR序列共同构成CRISPR/Cas***Cas蛋白具有与核酸酶相关的功能结构域,通过识别PAM(protospacer adjacent motif)在特定位置切割靶序列。
如本公开所使用的,所述的“gRNA”又称为guide RNA或向导RNA,并且具有本领域技术人员通常理解的含义。一般而言,向导RNA可以包含同向(direct)重复序列和向导序列(guide sequence),或者基本上由或由同向重复序列和向导序列(在内源性CRISPR***背景下也称为间隔序列(spacer))组成。gRNA在不同的CRISPR***中,依据其所依赖的Cas蛋白的不同,可以包括crRNA和tracrRNA,也可以只含有crRNA。crRNA和tracrRNA可以经过人工改造融合形成single guide RNA(sgRNA)。
如本公开所使用的,原型间隔序列毗邻区(protospacer adjacent motifs,PAM)是指位于Cas蛋白识别的靶序列附近的序列,其可以位于靶序列的3’端(例如,CRISPR/Cpf1***),也可以位于靶序列的5’端(例如,CRISPR/Cas9***)。
如本公开所使用的,术语“靶序列”是指目标核酸中与gRNA互补或至少部分互补的核苷酸序列,在本公开中,“靶序列”与“靶核酸”可以互换地使用。
如本公开所使用的,术语“靶标链”(target strand)是指目标核酸中与gRNA杂交的核苷酸链;术语“非靶标链”(non-target strand)是指目标核酸中与gRNA不发生杂交配对的核苷酸链。
如本公开所使用的,术语“多核苷酸”指由核苷酸组成的聚合物。多核苷酸可以是单独片段的形式,也可以是更大的核苷酸序列结构的一个组成部分,其是从至少在数量或浓度上分离一次的核苷酸序列衍生而来的,能够通过标准分子生物学方法(例如,使用克隆载体)识别、操纵以及恢复序列及其组分核苷酸序列。当一个核苷酸序列通过一个DNA序列(即A、T、G、C)表示时,这也包括一个RNA序列(即A、U、G、C),其中“U”取代“T”。换句话说,“多核苷酸”指从其他核苷酸(单独的片段或整个片段)中去除的核苷酸聚合物,或者可以是一个较大核苷酸结构的组成部分或成分,如表达载体或多顺反子序列。多核苷酸包括DNA、RNA和cDNA序列。“重组多核苷酸”、“重组核酸分子”属于“多核苷酸”中的一种。
如本文所用,术语“杂交”是指使用核酸链通过碱基配对与互补链结合以形成杂交复合物的任何过程来配对互补核酸。
如本公开所使用的,术语“突变体”是指相对于“野生型”,或者“相比较的”多核苷酸或多肽,在一个或多个(例如,若干个)位置处包含改变(即,替换、***和/或缺失的多核苷酸,其中,取代是指用不同的核苷酸置换占用一个位置的核苷酸。缺失是指去除占据某一位置的核苷酸。***是指在邻接并且紧随占据位置的核苷酸之后添加核苷酸。在本公开中,核苷酸的改变也对应为碱基的改变,替换、***或缺失的核苷酸对应为替换、***或缺失的碱基。
如本公开所使用的,术语“突变核苷酸”或“核苷酸突变”,包括“替换、缺失或添加一个或多个核苷酸”。在本公开中,术语“突变”是指核苷酸序列的改变。
如本公开所使用的,术语“载体”指的是DNA构建体,其含有与合适的控制序列可操作地连接的DNA序列,从而在合适的宿主中表达目的基因。
如本公开所使用的,术语“宿主细胞”指已经向其中引入外源多核苷酸的细胞,包括这类细胞的子代。宿主细胞包括“转化体”和“转化的细胞”,这包括原代转化的细胞和从其衍生的子代。
除非另外定义或由背景清楚指示,否则在本公开中的全部技术与科学术语具有如本公开所属领域的普通技术人员通常理解的相同含义。
实施例
本公开的其他目的、特征和优点将从以下详细描述中变得明显。但是,应当理解的是,详细描述和具体实施例(虽然表示本公开的具体实施方式)仅为解释性目的而给出,因为在阅读该详细说明后,在本公开的精神和范围内所作出的各种改变和修饰,对于本领域技术人员来说将变得显而易见。
本实施例中所用到的实验技术与实验方法,如无特殊说明均为常规技术方法,例如下列实施例中未注明具体条件的实验方法,通常按照常规条件如Sambrook等人,分子克隆:实验室手册(New York:Cold Spring Harbor Laboratory Press,1989)中所述的条件,或按照制造厂商所建议的条件。实施例中所使用的材料、试剂等,如无特殊说明,均可通过正规商业渠道获得。
本公开实施例中采用的试剂如下:
全式金生物无缝克隆试剂盒pEASY-Uni Seamless Cloning and Assembly Kit(CU101-02)
DpnI:Fermentas/Thermo Scientific货号:ER1701
v2 NEB货号:R3733S
天根PCR产物纯化试剂盒货号:DP204-03
pUDP025质粒:addgene购买。货号:103874
T4 DNA连接酶NEB货号:M0202V
PUC19质粒:购自淼灵质粒平台,货号P0368
乳酸克鲁维酵母GG799:ASM215156v1
gRNA序列:TTCGTGAACTCCATAACTTT(SEQ ID NO:43)
实施例1:p025-SNR52-SpiCAS-KlADE2质粒的构建
(1)以酿酒酵母基因组(GCA_000146045.2)为模板,采用如下引物(1202-1-1F和1202-1-1R),利用常规PCR方法,对目标片段进行扩增以获得含有ScSNR52启动子的序列作为目标片段1:
1202-1-1F:ACACGCTTTTTCAGTTCGAGTTTATCTTTGAAAAGATAATGTATGATT(SEQ ID NO:1);
1202-1-1R:AAACAAGCACTTGAGGTATTGAAAGATCATTTATCTTTCACTGCGGAG(SEQ ID NO:2);
ScSNR52启动子序列:
tctttgaaaagataatgtatgattatgctttcactcatatttatacagaaacttgatgttttctttcgagtatatacaaggtgattacatgtacgtttgaagtacaactctagattttgtagtgccctcttgggctagcggtaaaggtgcgcattttttcacaccctacaatgttctgttcaaaagattttggtcaaacgctgtagaagtgaaagttggtgcgcatgtttcggcgttcgaaacttctccgcagtgaaagataaatgatc(SEQ ID NO:45)。
含有前述ScSNR52启动子序列的目标片段1序列:
Acacgctttttcagttcgagtttatctttgaaaagataatgtatgattatgctttcactcatatttatacagaaacttgatgttttctttcgagtatatacaaggtgattacatgtacgtttgaagtacaactctagattttgtagtgccctcttgggctagcggtaaaggtgcgcattttttcacaccctacaatgttctgttcaaaagattttggtcaaacgctgtagaagtgaaagttggtgcgcatgtttcggcgttcgaaacttctccgcagtgaaagataaatgatctttcaatacctcaagtgcttgttt(SEQ ID NO:54)
(2)pUDP025质粒为模板,采用如下引物(1202-1-2F和1202-1-2R),利用常规PCR方法,对于目标片段进行扩增,并通过引物加入SUP4终止子序列作为目标片段2:
1202-1-2F:
CTCCGCAGTGAAAGATAAATGATCTTTCAATACCTCAAGTGCTTGTTT(SEQ ID NO:3);
1202-1-2R:
TATCGACAAAGGAAAAGGGGCCTGTAGACATAAAAAACAAAAAAAGCACCACCGACTCGGTGCCACTTTTTCAA(SEQ ID NO:4);
SUP4终止子序列:
tttttttgttttttatgtct(SEQ ID NO:44)。
含有前述SUP4终止子序列的目标片段2:
ctccgcagtgaaagataaatgatctttcaatacctcaagtgcttgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtggtgctttttttgttttttatgtctacaggccccttttcctttgtcgata(SEQ ID NO:55)
(3)以pUDP025为模板,采用如下引物(1202-1-VF1和1202-1-VR1),利用常规PCR方法,对于目标片段进行扩增以获得目标片段3:
1202-1-VF1:gaatgttcaacaatctataatcagtccatagtcaacaagagccctcaa(SEQ IDNO:5);
1202-1-VR1:aatcatacattatcttttcaaagataaactcgaactgaaaaagcgtgt(SEQ IDNO:6)。
目标片段3,其中,前述目标片段中所含有的片段为pUDP025质粒中的片段(SEQ IDNO:56):
gaatgttcaacaatctataatcagtccatagtcaacaagagccctcaaagttgatatcgtttagtgatctccctcgagatgagcggaggatggttgctttttggggagccctaagtggtacgtgcggcgagcgggatgtttccccattaggcaaaggacgaccgggtaacccctcgagaaaaaaaatttttcgccgctaatctggtgttatataaagctccccctgctcctggaatttttttccttgtcaactcacaccggaaatcaaggcatttcattctgagtagttcttaaaaacataatcaacaatggattataaagatgacgatgacaaacctccaaaaaagaagagaaaggtcgataagaaatactcaataggcttagatatcggcacaaatagcgtcggatgggcggtgatcactgatgaatataaggttccgtctaaaaagttcaaggttctgggaaatacagaccgccacagtatcaaaaaaaatcttataggggctcttttatttgacagtggagagacagcggaagcgactcgtctcaaacggacagctcgtagaaggtatacacgtcggaagaatcgtatttgttatctacaggagattttttcaaatgagatggcgaaagtagatgatagtttctttcatcgacttgaagagtcttttttggtggaagaagacaagaagcatgaacgtcatcctatttttggaaatatagtagatgaagttgcttatcatgagaaatatccaactatctatcatctgcgaaaaaaattggtagattctacttataaagcggatttgcgcttaatctatttggccttagcgcatatgattaagtttcgtggtcattttttgattgagggagatttaaatcctgataatagtgatgtggacaaactatttatccagttggtacaaacctacaatcaattatttgaagaaaaccctattaacgcaagtggagtagatgctaaagcgattctttctgcacgattgagtaaatcaagacgattagaaaatctcattgctcagctccccggtgagaagaaaaatggcttatttgggaatctcattgctttgtcattgggtttgacccctaattttaaatcaaattttgatttggcagaagatgctaaattacagctttcaaaagatacttacgatgatgatttagataatttattggcgcaaattggagatcaatatgctgatttgtttttggcagctaagaatttatcagatgctattttactttcagatatcctaagagtaaatactgaaataactaaggctcccctatcagcttcaatgattaaacgctacgatgaacatcatcaagacttgactcttttaaaagctttagttcgacaacaacttccagaaaagtataaagaaatcttttttgatcaatcaaaaaacggatatgcaggttatattgatgggggagctagccaagaagaattttataaatttatcaaaccaattttagaaaaaatggatggtactgaggaattattggtgaaactaaatcgtgaagatttgctgcgcaagcaacggacctttgacaacggctctattacccatcaaattcacttgggtgagctgcatgctattttgagaagacaagaagacttttatccatttttaaaagacaatcgtgagaagattgaaaaaatcttgacttttcgaattccttattatgttggtccattggcgcgtggcaatagtcgttttgcatggatgactcggaagtctgaagaaacaattaccccatggaattttgaagaagttgtcgataaaggtgcttcagctcaatcatttattgaacgcatgacaaactttgataaaaatcttccaaatgaaaaagtactaccaaaacatagtttgctttatgagtattttacggtttataacgaattgacaaaggtcaaatatgttactgaaggaatgcgaaaaccagcatttctttcaggtgaacagaagaaagccattgttgatttactcttcaaaacaaatcgaaaagtaaccgttaagcaattaaaagaagattatttcaaaaaaatagaatgttttgatagtgttgaaatttcaggagttgaagatagatttaatgcttcattaggtacctaccatgatttgctaaaaattattaaagataaagattttttggataatgaagaaaatgaagatatcttagaggatattgttttaacattgaccttatttgaagatagggagatgattgaggaaagacttaaaacatatgctcacctctttgatgataaggtgatgaaacagcttaaacgtcgccgttatactggttggggacgtttgtctcgaaaattgattaatggtattagggataagcaatctggcaaaacaatattagattttttgaaatcagatggttttgccaatcgcaattttatgcagctgatccatgatgatagtttgacatttaaagaagacattcaaaaagcacaagtgtctggacaaggcgatagtttacatgaacatattgcaaatttagctggtagccctgctattaaaaaaggtattttacagactgtaaaagttgttgatgaattggtcaaagtaatggggcggcataagccagaaaatatcgttattgaaatggcacgtgaaaatcagacaactcaaaagggccagaaaaattcgcgagagcgtatgaaacgaatcgaagaaggtatcaaagaattaggaagtcagattcttaaagagcatcctgttgaaaatactcaattgcaaaatgaaaagctctatctctattatctccaaaatggaagagacatgtatgtggaccaagaattagatattaatcgtttaagtgattatgatgtcgatcacattgttccacaaagtttccttaaagacgattcaatagacaataaggtcttaacgcgttctgataaaaatcgtggtaaatcggataacgttccaagtgaagaagtagtcaaaaagatgaaaaactattggagacaacttctaaacgccaagttaatcactcaacgtaagtttgataatttaacgaaagctgaacgtggaggtttgagtgaacttgataaagctggttttatcaaacgccaattggttgaaactcgccaaatcactaagcatgtggcacaaattttggatagtcgcatgaatactaaatacgatgaaaatgataaacttattcgagaggttaaagtgattaccttaaaatctaaattagtttctgacttccgaaaagatttccaattctataaagtacgtgagattaacaattaccatcatgcccatgatgcgtatctaaatgccgtcgttggaactgctttgattaagaaatatccaaaacttgaatcggagtttgtctatggtgattataaagtttatgatgttcgtaaaatgattgctaagtctgagcaagaaataggcaaagcaaccgcaaaatatttcttttactctaatatcatgaacttcttcaaaacagaaattacacttgcaaatggagagattcgcaaacgccctctaatcgaaactaatggggaaactggagaaattgtctgggataaagggcgagattttgccacagtgcgcaaagtattgtccatgccccaagtcaatattgtcaagaaaacagaagtacagacaggcggattctccaaggagtcaattttaccaaaaagaaattcggacaagcttattgctcgtaaaaaagactgggatccaaaaaaatatggtggttttgatagtccaacggtagcttattcagtcctagtggttgctaaggtggaaaaagggaaatcgaagaagttaaaatccgttaaagagttactagggatcacaattatggaaagaagttcctttgaaaaaaatccgattgactttttagaagctaaaggatataaggaagttaaaaaagacttaatcattaaactacctaaatatagtctttttgagttagaaaacggtcgtaaacggatgctggctagtgccggagaattacaaaaaggaaatgagctggctctgccaagcaaatatgtgaattttttatatttagctagtcattatgaaaagttgaagggtagtccagaagataacgaacaaaaacaattgtttgtggagcagcataagcattatttagatgagattattgagcaaatcagtgaattttctaagcgtgttattttagcagatgccaatttagataaagttcttagtgcatataacaaacatagagacaaaccaatacgtgaacaagcagaaaatattattcatttatttacgttgacgaatcttggagctcccgctgcttttaaatattttgatacaacaattgatcgtaaacgatatacgtctacaaaagaagttttagatgccactcttatccatcaatccatcactggtctttatgaaacacgcattgatttgagtcagctaggaggtgaccctccaaaaaagaagagaaaggtctgagcggttttgtataactaaataatattggaaactaaatacgaatacccaaattttttatctaaattttgccgaaagattaaaatctgcagagatatccgaaacaggtaaatggatgtttcaatccctgtagtcagtcaggaacccatattatattacagtattagtcgccgcttaggcacgcctttaattagcaaaatcaaaccttaagtgcatatgccgtataagggaaactcaaagaactggcatcgcaaaaatgaaaacatacgttgaaactacggcaaaggattggtcagatcgcttcatacagggaaagttcggcatcaacatctttggataatatcagaatgagaaagaacagatacgcagtacgttttttggtgagctctttgcacttctttagttctttccatcaatatcagttttttaaacttttaggactaaaagtgatgtttaacttcaaaatgtttaaaattttgttcttcccgacgttcattaagaatactaatacactttaataattattttaatattttgttctaaataatgacttttaattaaaaaagataaaatataaaaacatcataataactcaccagaggttaagaacaaaaaaacaaattagatatctgctaatccaatatagttaaatcaatctttccttggtataatgggtatattacatatatttcaaggaccgacactcctaccaaatatctaaaatttaccatattaacataacatgtatataaacgtcaaatcataatcagcactaacgtctcacggatcgtatatgccgtagcgacaatctaagaactatgcgaggacacgctagggccaaggcaaaaagattccttgattacgtaagggagttagaatcattttgaataaaaaacacgctttttcagttcgagtttatctttgaaaagataatgtatgatt
(4)以pUDP025为模板,采用如下引物(1202-1-VF2和1202-1-VR2),利用常规PCR方法,对于目标片段进行扩增以获得目标片段4:
1202-1-VF2:ttgaaaaagtggcaccgagtcggtggtgctttttttgttttttatgtctacaggccccttttcctttgtcgata(SEQ ID NO:7)
1202-1-VR2:ttgagggctcttgttgactatggactgattatagattgttgaacattc(SEQ IDNO:8)
目标片段4,其中,前述目标片段中所含有的片段为pUDP025质粒中的片段(SEQ IDNO:57):
ttgaaaaagtggcaccgagtcggtggtgctttttttgttttttatgtctacaggccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacaaacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacaagcttacgtctcacggatcgtatatgccgtagcgacaatctaagaactatgcgaggacacgctagcagctgtattcacgtagacggataggtatagccagacatcagcagcatacttcgggaaccgtaggccaccgagcggccgcgattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgggacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgtcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtgcggactatatgtgaaggcatggctatggcacggcagacattccgccagatcatcaataggcacgacatggaggcccagaataccctccttgacagtcttgacgtgcgcagctcaggggcatgatgtgactgtcgcccgtacatttagcccatacatccccatgtataatcatttgcatccatacattttgatggccgcacggcgcgaagcaaaaattacggctcctcgctgcagacctgcgagcagggaaacgctcccctcacagacgcgttgaattgtccccacgccgcgcccctgtagagaaatataaaaggttaggatttgccactgaggttcttctttcatatacttccttttaaaatcttgctaggatacagttctcacatcacatccgaacataaacaaggatccatgggtaaaaagcctgaactcaccgcgacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtgtccgacctgatgcagctctcggagggcgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtttctacaaagatcgttatgtttatcggcactttgcatcggccgcgctcccgattccggaagtgcttgacattggggaatttagcgagagcctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccgaactgcccgctgttctgcaaccggtcgcggaggccatggatgcgatcgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcggtcaatacactacatggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatgagctgatgctttgggccgaggactgccccgaagtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggccgcataacagcggtcattgactggagcgaggcgatgttcggggattcccaatacgaggtcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcagacgcgctacttcgagcggaggcatccggagcttgcaggatcgccgcggctccgggcgtatatgctccgcattggtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagcttgggcgcagggtcgatgcgacgcaatcgtccgatccggagccgggactgtcgggcgtacacaaatcgcccgcagaagcgcggccgtctggaccgatggctgtgtagaagtactcgccgatagtggaaaccgacgccccagcactcgtccgagggcaaaggaataactcgaggacaataaaaagattcttgttttcaagaacttgtcatttgtatagtttttttatattgtagttgttctattttaatcaaatgttagcgtgatttatattttttttcgcctcgacatcatctgcccagatgcgaagttaagtgcgcagaaagtaatatcatgcgtcaatcgtatgtgaatgctggtcgctatactggagctctcgagaaccctcacctttcgagaggacgatgcccgtgtctaaatgattcgaccagcctaagaatgttcaacaatctataatcagtccatagtcaacaagagccctcaa
将上述步骤(1)-(4)中得到的目标片段1、目标片段2、目标片段3和目标片段4分别利用DpnI内切酶进行酶切,37℃消化3hr,后用PCR产物纯化试剂盒对于前述酶切后目标片段分别进行纯化。
将各部分纯化后的产物利用全式金生物无缝克隆试剂盒进行连接,连接各片段的DNA产物的量为0.15pmol。
将前述DNA连接后产物加入到100μl DH5α感受态细胞中,冰上孵育30min,42℃热激45s,后加入1mL LB液体培养基37℃振荡培养1hr,涂布于具有Amp抗性的LB固体培养基上,37℃倒置培养,至单克隆长出。
挑取3个单克隆进行PCR检验并测序确认后,得到最终改造完成质粒,将前述质粒命名为p025-SNR52-SpiCAS-KlADE2。其中,前述质粒的核苷酸序列(从N端至C端)为将目标片段1、目标片段2、目标片段3和目标片段4连接得到。
实施例2:p025-HsCAS-KlADE2质粒的构建
(1)以质粒104910(addgene获得,https://www.addgene.org/104910/)为模板,采用如下引物(1202-2-Ifnew和1202-2-Irnew),利用常规PCR方法,对于目标片段进行扩增以获得含有HsCAS基因片段作为目标片段5:
1202-2-Ifnew:GGCTACggtctcatggacaagaagtactccattg(SEQ ID NO:9);
1202-2-Irnew:GGCTACggtctctcacaccttcctcttcttctt(SEQ ID NO:10);
目标片段5(SEQ ID NO:58):
GGCTACggtctcatggacaagaagtactccattgggctcgatatcggcacaaacagcgtcggttgggccgtcattacggacgagtacaaggtgccgagcaaaaaattcaaagttctgggcaataccgatcgccacagcataaagaagaacctcattggcgccctcctgttcgactccggggagacggccgaagccacgcggctcaaaagaacagcacggcgcagatatacccgcagaaagaatcggatctgctacctgcaggagatctttagtaatgagatggctaaggtggatgactctttcttccataggctggaggagtcctttttggtggaggaggataaaaagcacgagcgccacccaatctttggcaatatcgtggacgaggtggcgtaccatgaaaagtacccaaccatatatcatctgaggaagaagcttgtagacagtactgataaggctgacttgcggttgatctatctcgcgctggcgcatatgatcaaatttcggggacacttcctcatcgagggggacctgaacccagacaacagcgatgtcgacaaactctttatccaactggttcagacttacaatcagcttttcgaagagaacccgatcaacgcatccggagttgacgccaaagcaatcctgagcgctaggctgtccaaatcccggcggctcgaaaacctcatcgcacagctccctggggagaagaagaacggcctgtttggtaatcttatcgccctgtcactcgggctgacccccaactttaaatctaacttcgacctggccgaagatgccaagcttcaactgagcaaagacacctacgatgatgatctcgacaatctgctggcccagatcggcgaccagtacgcagacctttttttggcggcaaagaacctgtcagacgccattctgctgagtgatattctgcgagtgaacacggagatcaccaaagctccgctgagcgctagtatgatcaagcgctatgatgagcaccaccaagacttgactttgctgaaggcccttgtcagacagcaactgcctgagaagtacaaggaaattttcttcgatcagtctaaaaatggctacgccggatacattgacggcggagcaagccaggaggaattttacaaatttattaagcccatcttggaaaaaatggacggcaccgaggagctgctggtaaagcttaacagagaagatctgttgcgcaaacagcgcactttcgacaatggaagcatcccccaccagattcacctgggcgaactgcacgctatcctcaggcggcaagaggatttctacccctttttgaaagataacagggaaaagattgagaaaatcctcacatttcggataccctactatgtaggccccctcgcccggggaaattccagattcgcgtggatgactcgcaaatcagaagagactatcactccctggaacttcgaggaagtcgtggataagggggcctctgcccagtccttcatcgaaaggatgactaactttgataaaaatctgcctaacgaaaaggtgcttcctaaacactctctgctgtacgagtacttcacagtttataacgagctcaccaaggtcaaatacgtcacagaagggatgagaaagccagcattcctgtctggagagcagaagaaagctatcgtggacctcctcttcaagacgaaccggaaagttaccgtgaaacagctcaaagaggactatttcaaaaagattgaatgtttcgactctgttgaaatcagcggagtggaggatcgcttcaacgcatccctgggaacgtatcacgatctcctgaaaatcattaaagacaaggacttcctggacaatgaggagaacgaggacattcttgaggacattgtcctcacccttacgttgtttgaagatagggagatgattgaagaacgcttgaaaacttacgctcatctcttcgacgacaaagtcatgaaacagctcaagaggcgccgatatacaggatgggggcggctgtcaagaaaactgatcaatgggatccgagacaagcagagtggaaagacaatcctggattttcttaagtccgatggatttgccaaccggaacttcatgcagttgatccatgatgactctctcacctttaaggaggacatccagaaagcacaagtttctggccagggggacagccttcacgagcacatcgctaatcttgcaggtagcccagctatcaaaaagggaatactgcagaccgttaaggtcgtggatgaactcgtcaaagtaatgggaaggcataagcccgagaatatcgttatcgagatggcccgagagaaccaaactacccagaagggacagaagaacagtagggaaaggatgaagaggattgaagagggtataaaagaactggggtcccaaatccttaaggaacacccagttgaaaacacccagcttcagaatgagaagctctacctgtactacctgcagaacggcagggacatgtacgtggatcaggaactggacatcaatcggctctccgactacgacgtggatcatatcgtgccccagtcttttctcaaagatgattctattgataataaagtgttgacaagatccgataaaaatagagggaagagtgataacgtcccctcagaagaagttgtcaagaaaatgaaaaattattggcggcagctgctgaacgccaaactgatcacacaacggaagttcgataatctgactaaggctgaacgaggtggcctgtctgagttggataaagccggcttcatcaaaaggcagcttgttgagacacgccagatcaccaagcacgtggcccaaattctcgattcacgcatgaacaccaagtacgatgaaaatgacaaactgattcgagaggtgaaagttattactctgaagtctaagctcgtctcagatttcagaaaggactttcagttttataaggtgagagagatcaacaattaccaccatgcgcatgatgcctacctgaatgcagtggtaggcactgcacttatcaaaaaatatcccaagcttgaatctgaatttgtttacggagactataaagtgtacgatgttaggaaaatgatcgcaaagtctgagcaggaaataggcaaggccaccgctaagtacttcttttacagcaatattatgaattttttcaagaccgagattacactggccaatggagagattcggaagcgaccacttatcgaaacaaacggagaaacaggagaaatcgtgtgggacaagggtagggatttcgcgacagtccggaaggtcctgtccatgccgcaggtgaacatcgttaaaaagaccgaagtacagaccggaggcttctccaaggaaagtatcctcccgaaaaggaacagcgacaagctgatcgcacgcaaaaaagattgggaccccaagaaatacggcggattcgattctcctacagtcgcttacagtgtactggttgtggccaaagtggagaaagggaagtctaaaaaactcaaaagcgtcaaggaactgctgggcatcacaatcatggagcgatcaagcttcgaaaaaaaccccatcgactttctcgaggcgaaaggatataaagaggtcaaaaaagacctcatcattaagcttcccaagtactctctctttgagcttgaaaacggccggaaacgaatgctcgctagtgcgggcgagctgcagaaaggtaacgagctggcactgccctctaaatacgttaatttcttgtatctggccagccactatgaaaagctcaaagggtcccccgaagataatgagcagaagcagctgttcgtggaacaacacaaacactaccttgatgagatcatcgagcaaataagcgaattctccaaaagagtgatcctcgccgacgctaacctcgataaggtgctttctgcttacaataagcacagggataagcccatcagggagcaggcagaaaacattatccacttgtttactctgaccaacttgggcgcgcctgcagccttcaagtacttcgacaccaccatagacagaaagcggtacacctctacaaaggaggtcctggacgccacactgattcatcagtcaattacggggctctatgaaacaagaatcgacctctctcagctcggtggagacagcagggctgaccccaagaagaagaggaaggtgtgagagaccGTAGCC
其中,目标片段5中的HsCAS序列如下所示(SEQ ID NO:46):
atggacaagaagtactccattgggctcgatatcggcacaaacagcgtcggttgggccgtcattacggacgagtacaaggtgccgagcaaaaaattcaaagttctgggcaataccgatcgccacagcataaagaagaacctcattggcgccctcctgttcgactccggggagacggccgaagccacgcggctcaaaagaacagcacggcgcagatatacccgcagaaagaatcggatctgctacctgcaggagatctttagtaatgagatggctaaggtggatgactctttcttccataggctggaggagtcctttttggtggaggaggataaaaagcacgagcgccacccaatctttggcaatatcgtggacgaggtggcgtaccatgaaaagtacccaaccatatatcatctgaggaagaagcttgtagacagtactgataaggctgacttgcggttgatctatctcgcgctggcgcatatgatcaaatttcggggacacttcctcatcgagggggacctgaacccagacaacagcgatgtcgacaaactctttatccaactggttcagacttacaatcagcttttcgaagagaacccgatcaacgcatccggagttgacgccaaagcaatcctgagcgctaggctgtccaaatcccggcggctcgaaaacctcatcgcacagctccctggggagaagaagaacggcctgtttggtaatcttatcgccctgtcactcgggctgacccccaactttaaatctaacttcgacctggccgaagatgccaagcttcaactgagcaaagacacctacgatgatgatctcgacaatctgctggcccagatcggcgaccagtacgcagacctttttttggcggcaaagaacctgtcagacgccattctgctgagtgatattctgcgagtgaacacggagatcaccaaagctccgctgagcgctagtatgatcaagcgctatgatgagcaccaccaagacttgactttgctgaaggcccttgtcagacagcaactgcctgagaagtacaaggaaattttcttcgatcagtctaaaaatggctacgccggatacattgacggcggagcaagccaggaggaattttacaaatttattaagcccatcttggaaaaaatggacggcaccgaggagctgctggtaaagcttaacagagaagatctgttgcgcaaacagcgcactttcgacaatggaagcatcccccaccagattcacctgggcgaactgcacgctatcctcaggcggcaagaggatttctacccctttttgaaagataacagggaaaagattgagaaaatcctcacatttcggataccctactatgtaggccccctcgcccggggaaattccagattcgcgtggatgactcgcaaatcagaagagactatcactccctggaacttcgaggaagtcgtggataagggggcctctgcccagtccttcatcgaaaggatgactaactttgataaaaatctgcctaacgaaaaggtgcttcctaaacactctctgctgtacgagtacttcacagtttataacgagctcaccaaggtcaaatacgtcacagaagggatgagaaagccagcattcctgtctggagagcagaagaaagctatcgtggacctcctcttcaagacgaaccggaaagttaccgtgaaacagctcaaagaggactatttcaaaaagattgaatgtttcgactctgttgaaatcagcggagtggaggatcgcttcaacgcatccctgggaacgtatcacgatctcctgaaaatcattaaagacaaggacttcctggacaatgaggagaacgaggacattcttgaggacattgtcctcacccttacgttgtttgaagatagggagatgattgaagaacgcttgaaaacttacgctcatctcttcgacgacaaagtcatgaaacagctcaagaggcgccgatatacaggatgggggcggctgtcaagaaaactgatcaatgggatccgagacaagcagagtggaaagacaatcctggattttcttaagtccgatggatttgccaaccggaacttcatgcagttgatccatgatgactctctcacctttaaggaggacatccagaaagcacaagtttctggccagggggacagccttcacgagcacatcgctaatcttgcaggtagcccagctatcaaaaagggaatactgcagaccgttaaggtcgtggatgaactcgtcaaagtaatgggaaggcataagcccgagaatatcgttatcgagatggcccgagagaaccaaactacccagaagggacagaagaacagtagggaaaggatgaagaggattgaagagggtataaaagaactggggtcccaaatccttaaggaacacccagttgaaaacacccagcttcagaatgagaagctctacctgtactacctgcagaacggcagggacatgtacgtggatcaggaactggacatcaatcggctctccgactacgacgtggatcatatcgtgccccagtcttttctcaaagatgattctattgataataaagtgttgacaagatccgataaaaatagagggaagagtgataacgtcccctcagaagaagttgtcaagaaaatgaaaaattattggcggcagctgctgaacgccaaactgatcacacaacggaagttcgataatctgactaaggctgaacgaggtggcctgtctgagttggataaagccggcttcatcaaaaggcagcttgttgagacacgccagatcaccaagcacgtggcccaaattctcgattcacgcatgaacaccaagtacgatgaaaatgacaaactgattcgagaggtgaaagttattactctgaagtctaagctcgtctcagatttcagaaaggactttcagttttataaggtgagagagatcaacaattaccaccatgcgcatgatgcctacctgaatgcagtggtaggcactgcacttatcaaaaaatatcccaagcttgaatctgaatttgtttacggagactataaagtgtacgatgttaggaaaatgatcgcaaagtctgagcaggaaataggcaaggccaccgctaagtacttcttttacagcaatattatgaattttttcaagaccgagattacactggccaatggagagattcggaagcgaccacttatcgaaacaaacggagaaacaggagaaatcgtgtgggacaagggtagggatttcgcgacagtccggaaggtcctgtccatgccgcaggtgaacatcgttaaaaagaccgaagtacagaccggaggcttctccaaggaaagtatcctcccgaaaaggaacagcgacaagctgatcgcacgcaaaaaagattgggaccccaagaaatacggcggattcgattctcctacagtcgcttacagtgtactggttgtggccaaagtggagaaagggaagtctaaaaaactcaaaagcgtcaaggaactgctgggcatcacaatcatggagcgatcaagcttcgaaaaaaaccccatcgactttctcgaggcgaaaggatataaagaggtcaaaaaagacctcatcattaagcttcccaagtactctctctttgagcttgaaaacggccggaaacgaatgctcgctagtgcgggcgagctgcagaaaggtaacgagctggcactgccctctaaatacgttaatttcttgtatctggccagccactatgaaaagctcaaagggtcccccgaagataatgagcagaagcagctgttcgtggaacaacacaaacactaccttgatgagatcatcgagcaaataagcgaattctccaaaagagtgatcctcgccgacgctaacctcgataaggtgctttctgcttacaataagcacagggataagcccatcagggagcaggcagaaaacattatccacttgtttactctgaccaacttgggcgcgcctgcagccttcaagtacttcgacaccaccatagacagaaagcggtacacctctacaaaggaggtcctggacgccacactgattcatcagtcaattacggggctctatgaaacaagaatcgacctctctcagctcggtggagac
前述HsCAS核苷酸序列编码的氨基酸序列如下所示(SEQ ID NO:66):
MDKKYSIGLDIGTNSVGWAVITDEYKVPSKKFKVLGNTDRHSIKKNLIGALLFDSGETAEATRLKRTARRRYTRRKNRICYLQEIFSNEMAKVDDSFFHRLEESFLVEEDKKHERHPIFGNIVDEVAYHEKYPTIYHLRKKLVDSTDKADLRLIYLALAHMIKFRGHFLIEGDLNPDNSDVDKLFIQLVQTYNQLFEENPINASGVDAKAILSARLSKSRRLENLIAQLPGEKKNGLFGNLIALSLGLTPNFKSNFDLAEDAKLQLSKDTYDDDLDNLLAQIGDQYADLFLAAKNLSDAILLSDILRVNTEITKAPLSASMIKRYDEHHQDLTLLKALVRQQLPEKYKEIFFDQSKNGYAGYIDGGASQEEFYKFIKPILEKMDGTEELLVKLNREDLLRKQRTFDNGSIPHQIHLGELHAILRRQEDFYPFLKDNREKIEKILTFRIPYYVGPLARGNSRFAWMTRKSEETITPWNFEEVVDKGASAQSFIERMTNFDKNLPNEKVLPKHSLLYEYFTVYNELTKVKYVTEGMRKPAFLSGEQKKAIVDLLFKTNRKVTVKQLKEDYFKKIECFDSVEISGVEDRFNASLGTYHDLLKIIKDKDFLDNEENEDILEDIVLTLTLFEDREMIEERLKTYAHLFDDKVMKQLKRRRYTGWGRLSRKLINGIRDKQSGKTILDFLKSDGFANRNFMQLIHDDSLTFKEDIQKAQVSGQGDSLHEHIANLAGSPAIKKGILQTVKVVDELVKVMGRHKPENIVIEMARENQTTQKGQKNSRERMKRIEEGIKELGSQILKEHPVENTQLQNEKLYLYYLQNGRDMYVDQELDINRLSDYDVDHIVPQSFLKDDSIDNKVLTRSDKNRGKSDNVPSEEVVKKMKNYWRQLLNAKLITQRKFDNLTKAERGGLSELDKAGFIKRQLVETRQITKHVAQILDSRMNTKYDENDKLIREVKVITLKSKLVSDFRKDFQFYKVREINNYHHAHDAYLNAVVGTALIKKYPKLESEFVYGDYKVYDVRKMIAKSEQEIGKATAKYFFYSNIMNFFKTEITLANGEIRKRPLIETNGETGEIVWDKGRDFATVRKVLSMPQVNIVKKTEVQTGGFSKESILPKRNSDKLIARKKDWDPKKYGGFDSPTVAYSVLVVAKVEKGKSKKLKSVKELLGITIMERSSFEKNPIDFLEAKGYKEVKKDLIIKLPKYSLFELENGRKRMLASAGELQKGNELALPSKYVNFLYLASHYEKLKGSPEDNEQKQLFVEQHKHYLDEIIEQISEFSKRVILADANLDKVLSAYNKHRDKPIREQAENIIHLFTLTNLGAPAAFKYFDTTIDRKRYTSTKEVLDATLIHQSITGLYETRIDLSQLGGD
(2)以质粒pUDP025为模板,采用如下引物(1202-2-Vfnew和1202-2-Vrnew),利用常规PCR方法,对于目标片段进行扩增以获得目标片段6:
1202-2-Vfnew:ggctacggtctcgtgtgagcggttttgtataac(SEQ ID NO:11);
1202-2-Vrnew:GGCTACGGTCTCttccataggtttgtcatcgtcatct(SEQ ID NO:12)。
目标片段6的具体序列如下(SEQ ID NO:59):
ggctacggtctcgtgtgagcggttttgtataactaaataatattggaaactaaatacgaatacccaaattttttatctaaattttgccgaaagattaaaatctgcagagatatccgaaacaggtaaatggatgtttcaatccctgtagtcagtcaggaacccatattatattacagtattagtcgccgcttaggcacgcctttaattagcaaaatcaaaccttaagtgcatatgccgtataagggaaactcaaagaactggcatcgcaaaaatgaaaacatacgttgaaactacggcaaaggattggtcagatcgcttcatacagggaaagttcggcatcaacatctttggataatatcagaatgagaaagaacagatacgcagtacgttttttggtgagctctttgcacttctttagttctttccatcaatatcagttttttaaacttttaggactaaaagtgatgtttaacttcaaaatgtttaaaattttgttcttcccgacgttcattaagaatactaatacactttaataattattttaatattttgttctaaataatgacttttaattaaaaaagataaaatataaaaacatcataataactcaccagaggttaagaacaaaaaaacaaattagatatctgctaatccaatatagttaaatcaatctttccttggtataatgggtatattacatatatttcaaggaccgacactcctaccaaatatctaaaatttaccatattaacataacatgtatataaacgtcaaatcataatcagcactaacgtctcacggatcgtatatgccgtagcgacaatctaagaactatgcgaggacacgctagggccaaggcaaaaagattccttgattacgtaagggagttagaatcattttgaataaaaaacacgctttttcagttcgagtttatcattatcaatactgccatttcaaagaatacgtaaataattaatagtagtgattttcctaactttatttagtcaaaaaattagccttttaattctgctgtaacccgtacatgcccaaaatagggggcgggttacacagaatatataacatcgtaggtgtctgggtgaacagtttattcctggcatccactaaatataatggagcccgctttttaagctggcatccagaaaaaaaaagaatcccagcaccaaaatattgttttcttcaccaaccatcagttcataggtccattctcttagcgcaactacagagaacaggggcacaaacaggcaaaaaacgggcacaacctcaatggagtgatgcaacctgcctggagtaaatgatgacacaaggcaattgacccacgcatgtatctatctcattttcttacaccttctattaccttctgctctctctgatttggaaaaagctgaaaaaaaaggttgaaaccagttccctgaaattattcccctacttgactaataagtatataaagacggtaggtattgattgtaattctgtaaatctatttcttaaacttcttaaattctacttttatagttagtcttttttttagttttaaaacaccaagaacttagtttcgaataaacacacataaacaaacaaattgaaactgatgagtccgtgaggacgaaacgagtaagctcgtctttcaatacctcaagtgcttgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtgcttttggccggcatggtcccagcctcctcgctggcgccggctgggcaacatgcttcggcatggcgaatgggacacaggccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacaaacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacaagcttacgtctcacggatcgtatatgccgtagcgacaatctaagaactatgcgaggacacgctagcagctgtattcacgtagacggataggtatagccagacatcagcagcatacttcgggaaccgtaggccaccgagcggccgcgattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgggacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgtcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtgcggactatatgtgaaggcatggctatggcacggcagacattccgccagatcatcaataggcacgacatggaggcccagaataccctccttgacagtcttgacgtgcgcagctcaggggcatgatgtgactgtcgcccgtacatttagcccatacatccccatgtataatcatttgcatccatacattttgatggccgcacggcgcgaagcaaaaattacggctcctcgctgcagacctgcgagcagggaaacgctcccctcacagacgcgttgaattgtccccacgccgcgcccctgtagagaaatataaaaggttaggatttgccactgaggttcttctttcatatacttccttttaaaatcttgctaggatacagttctcacatcacatccgaacataaacaaggatccatgggtaaaaagcctgaactcaccgcgacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtgtccgacctgatgcagctctcggagggcgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtttctacaaagatcgttatgtttatcggcactttgcatcggccgcgctcccgattccggaagtgcttgacattggggaatttagcgagagcctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccgaactgcccgctgttctgcaaccggtcgcggaggccatggatgcgatcgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcggtcaatacactacatggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatgagctgatgctttgggccgaggactgccccgaagtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggccgcataacagcggtcattgactggagcgaggcgatgttcggggattcccaatacgaggtcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcagacgcgctacttcgagcggaggcatccggagcttgcaggatcgccgcggctccgggcgtatatgctccgcattggtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagcttgggcgcagggtcgatgcgacgcaatcgtccgatccggagccgggactgtcgggcgtacacaaatcgcccgcagaagcgcggccgtctggaccgatggctgtgtagaagtactcgccgatagtggaaaccgacgccccagcactcgtccgagggcaaaggaataactcgaggacaataaaaagattcttgttttcaagaacttgtcatttgtatagtttttttatattgtagttgttctattttaatcaaatgttagcgtgatttatattttttttcgcctcgacatcatctgcccagatgcgaagttaagtgcgcagaaagtaatatcatgcgtcaatcgtatgtgaatgctggtcgctatactggagctctcgagaaccctcacctttcgagaggacgatgcccgtgtctaaatgattcgaccagcctaagaatgttcaacaatctataatcagtccatagtcaacaagagccctcaaagttgatatcgtttagtgatctccctcgagatgagcggaggatggttgctttttggggagccctaagtggtacgtgcggcgagcgggatgtttccccattaggcaaaggacgaccgggtaacccctcgagaaaaaaaatttttcgccgctaatctggtgttatataaagctccccctgctcctggaatttttttccttgtcaactcacaccggaaatcaaggcatttcattctgagtagttcttaaaaacataatcaacaatggattataaagatgacgatgacaaacctatggaaGAGACCGTAGCC
其中片段6中的panARS序列如下(SEQ ID NO:65):
TCAACATCTTTGGATAATATCAGAATGAGAAAGAACAGATACGCAGTACGTTTTTTGGTGAGCTCTTTGCACTTCTTTAGTTCTTTCCATCAATATCAGTTttTTAaaCttTTAgGACTAAaAgTGATGTTTAACTTCAAaATgTTTAAAaTTTTGTTCTTCCCGACGTTCATTAAGAATACTAATACACTTTAATAATTAtTTTAATATTTtgTTCTAaATAATGACtTTTAATTAAAAAAGATAAAATATAAAAACATCATAATAACTCACCAGAGGTTAAGAACAAAAAAACAAATTAGATATCTGCTAATCCAATATAGTTAAATCAATCTTTCCTTGGTATAATGGGTATATTACATATATTTCAAGGACCGACACTCCTACCAAATATCTAAAATTTACCATATTAACATAACATGTATATAAACGTCAAATCATAATCAGCACTA
将上述步骤(1)-(2)中得到的目标片段5和目标片段6分别利用DpnI内切酶进行酶切,37℃消化3hr,后用PCR产物纯化试剂盒对于前述酶切后目标片段分别进行纯化。
将各部分纯化后的产物以golden gate方式(https://goldengate.neb.com/)进行连接,将前述各DNA片段以0.15pmol的量加入到10μl反应体系中,另外加入0.5μl T4连接酶,1μl T4连接酶buffer,0.5μl BsaI内切酶,在PCR仪中反应37℃5min,22℃5min为一个循环,30个循环后将前述PCR反应产物加入到100μl DH5α感受态细胞中,冰上孵育30min,42℃热激45s,后加入1mL LB液体培养基37℃振荡培养1hr,涂布于具有Amp+抗性的LB固体培养基上,37℃倒置培养,至单克隆长出。
挑取3个单克隆进行PCR检验并测序确认后,得到最终改造完成质粒,将前述质粒命名为p025-HsCAS-KlADE2。其中,前述质粒的核苷酸序列(从N端至C端)为将目标片段5和目标片段6连接得到。
实施例3:p025-SNR52-HsCAS-KlADE2质粒的构建
(1)以实施例1得到的p025-SNR52-SpiCAS-KlADE2作为模板,采用如下引物(1214-3-1F和crispr-V-R),利用常规PCR方法,对于目标片段进行扩增以获得目标片段7:
1214-3-1F:GGCTACGGTCTCtacgttcattaagaatactaa(SEQ ID NO:13);
crispr-V-R:GGCTACggtctcaacattcttaggctggtcgaatcatttagac(SEQ ID NO:14)。
其中,目标片段7包含实施例1中的目标片段1、目标片段2(SEQ ID NO:47):
GGCTACGGTCTCtacgttcattaagaatactaatacactttaataattattttaatattttgttctaaataatgacttttaattaaaaaagataaaatataaaaacatcataataactcaccagaggttaagaacaaaaaaacaaattagatatctgctaatccaatatagttaaatcaatctttccttggtataatgggtatattacatatatttcaaggaccgacactcctaccaaatatctaaaatttaccatattaacataacatgtatataaacgtcaaatcataatcagcactaacgtctcacggatcgtatatgccgtagcgacaatctaagaactatgcgaggacacgctagggccaaggcaaaaagattccttgattacgtaagggagttagaatcattttgaataaaaaacacgctttttcagttcgagtttatctttgaaaagataatgtatgattatgctttcactcatatttatacagaaacttgatgttttctttcgagtatatacaaggtgattacatgtacgtttgaagtacaactctagattttgtagtgccctcttgggctagcggtaaaggtgcgcattttttcacaccctacaatgttctgttcaaaagattttggtcaaacgctgtagaagtgaaagttggtgcgcatgtttcggcgttcgaaacttctccgcagtgaaagataaatgatctttcaatacctcaagtgcttgttttagagctagaaatagcaagttaaaataaggctagtccgttatcaacttgaaaaagtggcaccgagtcggtggtgctttttttgttttttatgtctacaggccccttttcctttgtcgatatcatgtaattagttatgtcacgcttacattcacgccctccccccacatccgctctaaccgaaaaggaaggagttagacaacctgaagtctaggtccctatttatttttttatagttatgttagtattaagaacgttatttatatttcaaatttttcttttttttctgtacaaacgcgtgtacgcatgtaacattatactgaaaaccttgcttgagaaggttttgggacaagcttacgtctcacggatcgtatatgccgtagcgacaatctaagaactatgcgaggacacgctagcagctgtattcacgtagacggataggtatagccagacatcagcagcatacttcgggaaccgtaggccaccgagcggccgcgattatcaaaaaggatcttcacctagatccttttaaattaaaaatgaagttttaaatcaatctaaagtatatatgagtaaacttggtctgacagttaccaatgcttaatcagtgaggcacctatctcagcgatctgtctatttcgttcatccatagttgcctgactccccgtcgtgtagataactacgatacgggagggcttaccatctggccccagtgctgcaatgataccgcgggacccacgctcaccggctccagatttatcagcaataaaccagccagccggaagggccgagcgcagaagtggtcctgcaactttatccgcctccatccagtctattaattgttgccgggaagctagagtaagtagttcgccagttaatagtttgcgcaacgttgttgccattgctacaggcatcgtggtgtcacgctcgtcgtttggtatggcttcattcagctccggttcccaacgatcaaggcgagttacatgatcccccatgttgtgcaaaaaagcggttagctccttcggtcctccgatcgttgtcagaagtaagttggccgcagtgttatcactcatggttatggcagcactgcataattctcttactgtcatgccatccgtaagatgcttttctgtgactggtgagtactcaaccaagtcattctgagaatagtgtatgcggcgaccgagttgctcttgcccggcgtcaatacgggataataccgcgccacatagcagaactttaaaagtgctcatcattggaaaacgttcttcggggcgaaaactctcaaggatcttaccgctgttgagatccagttcgatgtaacccactcgtgcacccaactgatcttcagcatcttttactttcaccagcgtttctgggtgagcaaaaacaggaaggcaaaatgccgcaaaaaagggaataagggcgacacggaaatgttgaatactcatactcttcctttttcaatattattgaagcatttatcagggttattgtctcatgagcggatacatatttgaatgtatttagaaaaataaacaaataggggttccgcgcacatttccccgaaaagtgccacctgtcatgaccaaaatcccttaacgtgagttttcgttccactgagcgtcagaccccgtagaaaagatcaaaggatcttcttgagatcctttttttctgcgcgtaatctgctgcttgcaaacaaaaaaaccaccgctaccagcggtggtttgtttgccggatcaagagctaccaactctttttccgaaggtaactggcttcagcagagcgcagataccaaatactgttcttctagtgtagccgtagttaggccaccacttcaagaactctgtagcaccgcctacatacctcgctctgctaatcctgttaccagtggctgctgccagtggcgataagtcgtgtcttaccgggttggactcaagacgatagttaccggataaggcgcagcggtcgggctgaacggggggttcgtgcacacagcccagcttggagcgaacgacctacaccgaactgagatacctacagcgtgagctatgagaaagcgccacgcttcccgaagggagaaaggcggacaggtatccggtaagcggcagggtcggaacaggagagcgcacgagggagcttccagggggaaacgcctggtatctttatagtcctgtcgggtttcgccacctctgacttgagcgtcgatttttgtgatgctcgtcaggggggcggagcctatggaaaaacgccagcaacgcggcctttttacggttcctggccttttgctggccttttgctcacatgttctttcctgcgttatcccctgattctgtggataaccgtgcggactatatgtgaaggcatggctatggcacggcagacattccgccagatcatcaataggcacgacatggaggcccagaataccctccttgacagtcttgacgtgcgcagctcaggggcatgatgtgactgtcgcccgtacatttagcccatacatccccatgtataatcatttgcatccatacattttgatggccgcacggcgcgaagcaaaaattacggctcctcgctgcagacctgcgagcagggaaacgctcccctcacagacgcgttgaattgtccccacgccgcgcccctgtagagaaatataaaaggttaggatttgccactgaggttcttctttcatatacttccttttaaaatcttgctaggatacagttctcacatcacatccgaacataaacaaggatccatgggtaaaaagcctgaactcaccgcgacgtctgtcgagaagtttctgatcgaaaagttcgacagcgtgtccgacctgatgcagctctcggagggcgaagaatctcgtgctttcagcttcgatgtaggagggcgtggatatgtcctgcgggtaaatagctgcgccgatggtttctacaaagatcgttatgtttatcggcactttgcatcggccgcgctcccgattccggaagtgcttgacattggggaatttagcgagagcctgacctattgcatctcccgccgtgcacagggtgtcacgttgcaagacctgcctgaaaccgaactgcccgctgttctgcaaccggtcgcggaggccatggatgcgatcgctgcggccgatcttagccagacgagcgggttcggcccattcggaccgcaaggaatcggtcaatacactacatggcgtgatttcatatgcgcgattgctgatccccatgtgtatcactggcaaactgtgatggacgacaccgtcagtgcgtccgtcgcgcaggctctcgatgagctgatgctttgggccgaggactgccccgaagtccggcacctcgtgcacgcggatttcggctccaacaatgtcctgacggacaatggccgcataacagcggtcattgactggagcgaggcgatgttcggggattcccaatacgaggtcgccaacatcttcttctggaggccgtggttggcttgtatggagcagcagacgcgctacttcgagcggaggcatccggagcttgcaggatcgccgcggctccgggcgtatatgctccgcattggtcttgaccaactctatcagagcttggttgacggcaatttcgatgatgcagcttgggcgcagggtcgatgcgacgcaatcgtccgatccggagccgggactgtcgggcgtacacaaatcgcccgcagaagcgcggccgtctggaccgatggctgtgtagaagtactcgccgatagtggaaaccgacgccccagcactcgtccgagggcaaaggaataactcgaggacaataaaaagattcttgttttcaagaacttgtcatttgtatagtttttttatattgtagttgttctattttaatcaaatgttagcgtgatttatattttttttcgcctcgacatcatctgcccagatgcgaagttaagtgcgcagaaagtaatatcatgcgtcaatcgtatgtgaatgctggtcgctatactggagctctcgagaaccctcacctttcgagaggacgatgcccgtgtctaaatgattcgaccagcctaagaatgttgagaccGTAGCC
(2)以实施例2得到的p025-HsCAS-KlADE2作为模板,采用如下引物(crispr-V-F和1214-3-2R),利用常规PCR方法,对于目标片段进行扩增以获得目标片段8:
crispr-V-F:GGCTACggtctcaatgttcaacaatctataatcagtcc(SEQ ID NO:15);
1214-3-2R:GGCTACGGTCTCtacgtcgggaagaacaaaatt(SEQ ID NO:16)。
其中,目标片段8中包含实施例2中的目标片段5(SEQ ID NO:48):
GGCTACggtctcaatgttcaacaatctataatcagtccatagtcaacaagagccctcaaagttgatatcgtttagtgatctccctcgagatgagcggaggatggttgctttttggggagccctaagtggtacgtgcggcgagcgggatgtttccccattaggcaaaggacgaccgggtaacccctcgagaaaaaaaatttttcgccgctaatctggtgttatataaagctccccctgctcctggaatttttttccttgtcaactcacaccggaaatcaaggcatttcattctgagtagttcttaaaaacataatcaacaatggattataaagatgacgatgacaaacctatggacaagaagtactccattgggctcgatatcggcacaaacagcgtcggttgggccgtcattacggacgagtacaaggtgccgagcaaaaaattcaaagttctgggcaataccgatcgccacagcataaagaagaacctcattggcgccctcctgttcgactccggggagacggccgaagccacgcggctcaaaagaacagcacggcgcagatatacccgcagaaagaatcggatctgctacctgcaggagatctttagtaatgagatggctaaggtggatgactctttcttccataggctggaggagtcctttttggtggaggaggataaaaagcacgagcgccacccaatctttggcaatatcgtggacgaggtggcgtaccatgaaaagtacccaaccatatatcatctgaggaagaagcttgtagacagtactgataaggctgacttgcggttgatctatctcgcgctggcgcatatgatcaaatttcggggacacttcctcatcgagggggacctgaacccagacaacagcgatgtcgacaaactctttatccaactggttcagacttacaatcagcttttcgaagagaacccgatcaacgcatccggagttgacgccaaagcaatcctgagcgctaggctgtccaaatcccggcggctcgaaaacctcatcgcacagctccctggggagaagaagaacggcctgtttggtaatcttatcgccctgtcactcgggctgacccccaactttaaatctaacttcgacctggccgaagatgccaagcttcaactgagcaaagacacctacgatgatgatctcgacaatctgctggcccagatcggcgaccagtacgcagacctttttttggcggcaaagaacctgtcagacgccattctgctgagtgatattctgcgagtgaacacggagatcaccaaagctccgctgagcgctagtatgatcaagcgctatgatgagcaccaccaagacttgactttgctgaaggcccttgtcagacagcaactgcctgagaagtacaaggaaattttcttcgatcagtctaaaaatggctacgccggatacattgacggcggagcaagccaggaggaattttacaaatttattaagcccatcttggaaaaaatggacggcaccgaggagctgctggtaaagcttaacagagaagatctgttgcgcaaacagcgcactttcgacaatggaagcatcccccaccagattcacctgggcgaactgcacgctatcctcaggcggcaagaggatttctacccctttttgaaagataacagggaaaagattgagaaaatcctcacatttcggataccctactatgtaggccccctcgcccggggaaattccagattcgcgtggatgactcgcaaatcagaagagactatcactccctggaacttcgaggaagtcgtggataagggggcctctgcccagtccttcatcgaaaggatgactaactttgataaaaatctgcctaacgaaaaggtgcttcctaaacactctctgctgtacgagtacttcacagtttataacgagctcaccaaggtcaaatacgtcacagaagggatgagaaagccagcattcctgtctggagagcagaagaaagctatcgtggacctcctcttcaagacgaaccggaaagttaccgtgaaacagctcaaagaggactatttcaaaaagattgaatgtttcgactctgttgaaatcagcggagtggaggatcgcttcaacgcatccctgggaacgtatcacgatctcctgaaaatcattaaagacaaggacttcctggacaatgaggagaacgaggacattcttgaggacattgtcctcacccttacgttgtttgaagatagggagatgattgaagaacgcttgaaaacttacgctcatctcttcgacgacaaagtcatgaaacagctcaagaggcgccgatatacaggatgggggcggctgtcaagaaaactgatcaatgggatccgagacaagcagagtggaaagacaatcctggattttcttaagtccgatggatttgccaaccggaacttcatgcagttgatccatgatgactctctcacctttaaggaggacatccagaaagcacaagtttctggccagggggacagccttcacgagcacatcgctaatcttgcaggtagcccagctatcaaaaagggaatactgcagaccgttaaggtcgtggatgaactcgtcaaagtaatgggaaggcataagcccgagaatatcgttatcgagatggcccgagagaaccaaactacccagaagggacagaagaacagtagggaaaggatgaagaggattgaagagggtataaaagaactggggtcccaaatccttaaggaacacccagttgaaaacacccagcttcagaatgagaagctctacctgtactacctgcagaacggcagggacatgtacgtggatcaggaactggacatcaatcggctctccgactacgacgtggatcatatcgtgccccagtcttttctcaaagatgattctattgataataaagtgttgacaagatccgataaaaatagagggaagagtgataacgtcccctcagaagaagttgtcaagaaaatgaaaaattattggcggcagctgctgaacgccaaactgatcacacaacggaagttcgataatctgactaaggctgaacgaggtggcctgtctgagttggataaagccggcttcatcaaaaggcagcttgttgagacacgccagatcaccaagcacgtggcccaaattctcgattcacgcatgaacaccaagtacgatgaaaatgacaaactgattcgagaggtgaaagttattactctgaagtctaagctcgtctcagatttcagaaaggactttcagttttataaggtgagagagatcaacaattaccaccatgcgcatgatgcctacctgaatgcagtggtaggcactgcacttatcaaaaaatatcccaagcttgaatctgaatttgtttacggagactataaagtgtacgatgttaggaaaatgatcgcaaagtctgagcaggaaataggcaaggccaccgctaagtacttcttttacagcaatattatgaattttttcaagaccgagattacactggccaatggagagattcggaagcgaccacttatcgaaacaaacggagaaacaggagaaatcgtgtgggacaagggtagggatttcgcgacagtccggaaggtcctgtccatgccgcaggtgaacatcgttaaaaagaccgaagtacagaccggaggcttctccaaggaaagtatcctcccgaaaaggaacagcgacaagctgatcgcacgcaaaaaagattgggaccccaagaaatacggcggattcgattctcctacagtcgcttacagtgtactggttgtggccaaagtggagaaagggaagtctaaaaaactcaaaagcgtcaaggaactgctgggcatcacaatcatggagcgatcaagcttcgaaaaaaaccccatcgactttctcgaggcgaaaggatataaagaggtcaaaaaagacctcatcattaagcttcccaagtactctctctttgagcttgaaaacggccggaaacgaatgctcgctagtgcgggcgagctgcagaaaggtaacgagctggcactgccctctaaatacgttaatttcttgtatctggccagccactatgaaaagctcaaagggtcccccgaagataatgagcagaagcagctgttcgtggaacaacacaaacactaccttgatgagatcatcgagcaaataagcgaattctccaaaagagtgatcctcgccgacgctaacctcgataaggtgctttctgcttacaataagcacagggataagcccatcagggagcaggcagaaaacattatccacttgtttactctgaccaacttgggcgcgcctgcagccttcaagtacttcgacaccaccatagacagaaagcggtacacctctacaaaggaggtcctggacgccacactgattcatcagtcaattacggggctctatgaaacaagaatcgacctctctcagctcggtggagaccctccaaaaaagaagagaaaggtctgagcggttttgtataactaaataatattggaaactaaatacgaatacccaaattttttatctaaattttgccgaaagattaaaatctgcagagatatccgaaacaggtaaatggatgtttcaatccctgtagtcagtcaggaacccatattatattacagtattagtcgccgcttaggcacgcctttaattagcaaaatcaaaccttaagtgcatatgccgtataagggaaactcaaagaactggcatcgcaaaaatgaaaacatacgttgaaactacggcaaaggattggtcagatcgcttcatacagggaaagttcggcatcaacatctttggataatatcagaatgagaaagaacagatacgcagtacgttttttggtgagctctttgcacttctttagttctttccatcaatatcagttttttaaacttttaggactaaaagtgatgtttaacttcaaaatgtttaaaattttgttcttcccgacgtaGAGACCGTAGCC
将上述步骤(1)-(2)中得到的目标片段7和目标片段8分别利用DpnI内切酶进行酶切,37℃消化3hr,后用PCR产物纯化试剂盒对于前述酶切后目标片段分别进行纯化。
将各部分纯化后的产物以golden gate方式进行连接,将前述各DNA片段以0.15pmol的量加入到10μl反应体系中,另外加入0.5μl T4连接酶,1μl T4连接酶buffer,0.5μl BsaI内切酶,在PCR仪中反应37℃5min,22℃5min为一个循环,30个循环后将前述PCR反应产物加入到100μl DH5α感受态细胞中,冰上孵育30min,42℃热激45s,后加入1mL LB液体培养基37℃振荡培养1hr,涂布于具有Amp+抗性的LB固体培养基上,37℃倒置培养,至单克隆长出。
挑取3个单克隆进行PCR检验并测序确认后,得到最终改造完成质粒,将前述质粒命名为p025-SNR52-HsCAS-KlADE2。其中,前述质粒的核苷酸序列(从N端至C端)为将目标片段7和目标片段8连接得到。
实施例4:p025-ARS12-KlADE2、p025-ARS101-KlADE2和p025-pKD1_ori KlADE2质 粒的制备
(1)以质粒pUDP025为模板,采用如下引物(part1-F和part1-R),利用常规PCR方法,对于目标片段进行扩增以获得目标片段9:
part1-F:GGCTACGGTCTCGGCCAAGGCAAAAAGATTCCTTGAT(SEQ ID NO:17);
part1-R:GGCTACGGTCTCGACACCCTGTGCACGGCGGGAGAT(SEQ ID NO:18);
目标片段9所示的序列如下(SEQ ID NO:63):
GGCTACGGTCTCGGCCAAGGCAAAAAGATTCCTTGATTACGTAAGGGAGTTAGAATCATTTTGAATAAAAAACACGCTTTTTCAGTTCGAGTTTATCATTATCAATACTGCCATTTCAAAGAATACGTAAATAATTAATAGTAGTGATTTTCCTAACTTTATTTAGTCAAAAAATTAGCCTTTTAATTCTGCTGTAACCCGTACATGCCCAAAATAGGGGGCGGGTTACACAGAATATATAACATCGTAGGTGTCTGGGTGAACAGTTTATTCCTGGCATCCACTAAATATAATGGAGCCCGCTTTTTAAGCTGGCATCCAGAAAAAAAAAGAATCCCAGCACCAAAATATTGTTTTCTTCACCAACCATCAGTTCATAGGTCCATTCTCTTAGCGCAACTACAGAGAACAGGGGCACAAACAGGCAAAAAACGGGCACAACCTCAATGGAGTGATGCAACCTGCCTGGAGTAAATGATGACACAAGGCAATTGACCCACGCATGTATCTATCTCATTTTCTTACACCTTCTATTACCTTCTGCTCTCTCTGATTTGGAAAAAGCTGAAAAAAAAGGTTGAAACCAGTTCCCTGAAATTATTCCCCTACTTGACTAATAAGTATATAAAGACGGTAGGTATTGATTGTAATTCTGTAAATCTATTTCTTAAACTTCTTAAATTCTACTTTTATAGTTAGTCTTTTTTTTAGTTTTAAAACACCAAGAACTTAGTTTCGAATAAACACACATAAACAAACAAATTGAAACTGATGAGTCCGTGAGGACGAAACGAGTAAGCTCGTCTTTCAATACCTCAAGTGCTTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTGGCCGGCATGGTCCCAGCCTCCTCGCTGGCGCCGGCTGGGCAACATGCTTCGGCATGGCGAATGGGACACAGGCCCCTTTTCCTTTGTCGATATCATGTAATTAGTTATGTCACGCTTACATTCACGCCCTCCCCCCACATCCGCTCTAACCGAAAAGGAAGGAGTTAGACAACCTGAAGTCTAGGTCCCTATTTATTTTTTTATAGTTATGTTAGTATTAAGAACGTTATTTATATTTCAAATTTTTCTTTTTTTTCTGTACAAACGCGTGTACGCATGTAACATTATACTGAAAACCTTGCTTGAGAAGGTTTTGGGACAAGCTTACGTCTCACGGATCGTATATGCCGTAGCGACAATCTAAGAACTATGCGAGGACACGCTAGCAGCTGTATTCACGTAGACGGATAGGTATAGCCAGACATCAGCAGCATACTTCGGGAACCGTAGGCCACCGAGCGGCCGCGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGGGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGTCATGACCAAAATCCCTTAACGTGAGTTTTCGTTCCACTGAGCGTCAGACCCCGTAGAAAAGATCAAAGGATCTTCTTGAGATCCTTTTTTTCTGCGCGTAATCTGCTGCTTGCAAACAAAAAAACCACCGCTACCAGCGGTGGTTTGTTTGCCGGATCAAGAGCTACCAACTCTTTTTCCGAAGGTAACTGGCTTCAGCAGAGCGCAGATACCAAATACTGTTCTTCTAGTGTAGCCGTAGTTAGGCCACCACTTCAAGAACTCTGTAGCACCGCCTACATACCTCGCTCTGCTAATCCTGTTACCAGTGGCTGCTGCCAGTGGCGATAAGTCGTGTCTTACCGGGTTGGACTCAAGACGATAGTTACCGGATAAGGCGCAGCGGTCGGGCTGAACGGGGGGTTCGTGCACACAGCCCAGCTTGGAGCGAACGACCTACACCGAACTGAGATACCTACAGCGTGAGCTATGAGAAAGCGCCACGCTTCCCGAAGGGAGAAAGGCGGACAGGTATCCGGTAAGCGGCAGGGTCGGAACAGGAGAGCGCACGAGGGAGCTTCCAGGGGGAAACGCCTGGTATCTTTATAGTCCTGTCGGGTTTCGCCACCTCTGACTTGAGCGTCGATTTTTGTGATGCTCGTCAGGGGGGCGGAGCCTATGGAAAAACGCCAGCAACGCGGCCTTTTTACGGTTCCTGGCCTTTTGCTGGCCTTTTGCTCACATGTTCTTTCCTGCGTTATCCCCTGATTCTGTGGATAACCGTGCGGACTATATGTGAAGGCATGGCTATGGCACGGCAGACATTCCGCGCCAGATCATCAATAGGCACGACATGGAGGCCCAGAATACCCTCCTTGACAGTCTTGACGTGCGCAGCTCAGGGGCATGATGTGACTGTCGCCCGTACATTTAGCCCATACATCCCCATGTATAATCATTTGCATCCATACATTTTGATGGCCGCACGGCGCGAAGCAAAAATTACGGCTCCTCGCTGCAGACCTGCGAGCAGGGAAACGCTCCCCTCACAGACGCGTTGAATTGTCCCCACGCCGCGCCCCTGTAGAGAAATATAAAAGGTTAGGATTTGCCACTGAGGTTCTTCTTTCATATACTTCCTTTTAAAATCTTGCTAGGATACAGTTCTCACATCACATCCGAACATAAACAAGGATCCATGGGTAAAAAGCCTGAACTCACCGCGACGTCTGTCGAGAAGTTTCTGATCGAAAAGTTCGACAGCGTGTCCGACCTGATGCAGCTCTCGGAGGGCGAAGAATCTCGTGCTTTCAGCTTCGATGTAGGAGGGCGTGGATATGTCCTGCGGGTAAATAGCTGCGCCGATGGTTTCTACAAAGATCGTTATGTTTATCGGCACTTTGCATCGGCCGCGCTCCCGATTCCGGAAGTGCTTGACATTGGGGAATTTAGCGAGAGCCTGACCTATTGCATCTCCCGCCGTGCACAGGGTGTCGAGACCGTAGCC
(2)以质粒pUDP025为模板,采用如下引物(part2-F和part2-R),利用常规PCR方法,对于目标片段进行扩增以获得目标片段10:
part2-F:GGCTACGGTCTCGGTGTCACGTTGCAAGACCTGCCTGAA(SEQ ID NO:19);
part2-R:ggctacGGTCTCTGCCGAACTTTCCCTGTATGAAGCG(SEQ ID NO:20)。
目标片段10所示的序列如下(SEQ ID NO:64):
GGCTACGGTCTCGGTGTCACGTTGCAAGACCTGCCTGAAACCGAACTGCCCGCTGTTCTGCAACCGGTCGCGGAGGCCATGGATGCGATCGCTGCGGCCGATCTTAGCCAGACGAGCGGGTTCGGCCCATTCGGACCGCAAGGAATCGGTCAATACACTACATGGCGTGATTTCATATGCGCGATTGCTGATCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACCGTCAGTGCGTCCGTCGCGCAGGCTCTCGATGAGCTGATGCTTTGGGCCGAGGACTGCCCCGAAGTCCGGCACCTCGTGCACGCGGATTTCGGCTCCAACAATGTCCTGACGGACAATGGCCGCATAACAGCGGTCATTGACTGGAGCGAGGCGATGTTCGGGGATTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGGCCGTGGTTGGCTTGTATGGAGCAGCAGACGCGCTACTTCGAGCGGAGGCATCCGGAGCTTGCAGGATCGCCGCGGCTCCGGGCGTATATGCTCCGCATTGGTCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTCGATGATGCAGCTTGGGCGCAGGGTCGATGCGACGCAATCGTCCGATCCGGAGCCGGGACTGTCGGGCGTACACAAATCGCCCGCAGAAGCGCGGCCGTCTGGACCGATGGCTGTGTAGAAGTACTCGCCGATAGTGGAAACCGACGCCCCAGCACTCGTCCGAGGGCAAAGGAATAACTCGAGGACAATAAAAAGATTCTTGTTTTCAAGAACTTGTCATTTGTATAGTTTTTTTATATTGTAGTTGTTCTATTTTAATCAAATGTTAGCGTGATTTATATTTTTTTTCGCCTCGACATCATCTGCCCAGATGCGAAGTTAAGTGCGCAGAAAGTAATATCATGCGTCAATCGTATGTGAATGCTGGTCGCTATACTGGAGCTCTCGAGAACCCTCACCTTTCGAGAGGACGATGCCCGTGTCTAAATGATTCGACCAGCCTAAGAATGTTCAACAATCTATAATCAGTCCATAGTCAACAAGAGCCCTCAAAGTTGATATCGTTTAGTGATCTCCCTCGAGATGAGCGGAGGATGGTTGCTTTTTGGGGAGCCCTAAGTGGTACGTGCGGCGAGCGGGATGTTTCCCCATTAGGCAAAGGACGACCGGGTAACCCCTCGAGAAAAAAAATTTTTCGCCGCTAATCTGGTGTTATATAAAGCTCCCCCTGCTCCTGGAATTTTTTTCCTTGTCAACTCACACCGGAAATCAAGGCATTTCATTCTGAGTAGTTCTTAAAAACATAATCAACAATGGATTATAAAGATGACGATGACAAACCTCCAAAAAAGAAGAGAAAGGTCGATAAGAAATACTCAATAGGCTTAGATATCGGCACAAATAGCGTCGGATGGGCGGTGATCACTGATGAATATAAGGTTCCGTCTAAAAAGTTCAAGGTTCTGGGAAATACAGACCGCCACAGTATCAAAAAAAATCTTATAGGGGCTCTTTTATTTGACAGTGGAGAGACAGCGGAAGCGACTCGTCTCAAACGGACAGCTCGTAGAAGGTATACACGTCGGAAGAATCGTATTTGTTATCTACAGGAGATTTTTTCAAATGAGATGGCGAAAGTAGATGATAGTTTCTTTCATCGACTTGAAGAGTCTTTTTTGGTGGAAGAAGACAAGAAGCATGAACGTCATCCTATTTTTGGAAATATAGTAGATGAAGTTGCTTATCATGAGAAATATCCAACTATCTATCATCTGCGAAAAAAATTGGTAGATTCTACTTATAAAGCGGATTTGCGCTTAATCTATTTGGCCTTAGCGCATATGATTAAGTTTCGTGGTCATTTTTTGATTGAGGGAGATTTAAATCCTGATAATAGTGATGTGGACAAACTATTTATCCAGTTGGTACAAACCTACAATCAATTATTTGAAGAAAACCCTATTAACGCAAGTGGAGTAGATGCTAAAGCGATTCTTTCTGCACGATTGAGTAAATCAAGACGATTAGAAAATCTCATTGCTCAGCTCCCCGGTGAGAAGAAAAATGGCTTATTTGGGAATCTCATTGCTTTGTCATTGGGTTTGACCCCTAATTTTAAATCAAATTTTGATTTGGCAGAAGATGCTAAATTACAGCTTTCAAAAGATACTTACGATGATGATTTAGATAATTTATTGGCGCAAATTGGAGATCAATATGCTGATTTGTTTTTGGCAGCTAAGAATTTATCAGATGCTATTTTACTTTCAGATATCCTAAGAGTAAATACTGAAATAACTAAGGCTCCCCTATCAGCTTCAATGATTAAACGCTACGATGAACATCATCAAGACTTGACTCTTTTAAAAGCTTTAGTTCGACAACAACTTCCAGAAAAGTATAAAGAAATCTTTTTTGATCAATCAAAAAACGGATATGCAGGTTATATTGATGGGGGAGCTAGCCAAGAAGAATTTTATAAATTTATCAAACCAATTTTAGAAAAAATGGATGGTACTGAGGAATTATTGGTGAAACTAAATCGTGAAGATTTGCTGCGCAAGCAACGGACCTTTGACAACGGCTCTATTACCCATCAAATTCACTTGGGTGAGCTGCATGCTATTTTGAGAAGACAAGAAGACTTTTATCCATTTTTAAAAGACAATCGTGAGAAGATTGAAAAAATCTTGACTTTTCGAATTCCTTATTATGTTGGTCCATTGGCGCGTGGCAATAGTCGTTTTGCATGGATGACTCGGAAGTCTGAAGAAACAATTACCCCATGGAATTTTGAAGAAGTTGTCGATAAAGGTGCTTCAGCTCAATCATTTATTGAACGCATGACAAACTTTGATAAAAATCTTCCAAATGAAAAAGTACTACCAAAACATAGTTTGCTTTATGAGTATTTTACGGTTTATAACGAATTGACAAAGGTCAAATATGTTACTGAAGGAATGCGAAAACCAGCATTTCTTTCAGGTGAACAGAAGAAAGCCATTGTTGATTTACTCTTCAAAACAAATCGAAAAGTAACCGTTAAGCAATTAAAAGAAGATTATTTCAAAAAAATAGAATGTTTTGATAGTGTTGAAATTTCAGGAGTTGAAGATAGATTTAATGCTTCATTAGGTACCTACCATGATTTGCTAAAAATTATTAAAGATAAAGATTTTTTGGATAATGAAGAAAATGAAGATATCTTAGAGGATATTGTTTTAACATTGACCTTATTTGAAGATAGGGAGATGATTGAGGAAAGACTTAAAACATATGCTCACCTCTTTGATGATAAGGTGATGAAACAGCTTAAACGTCGCCGTTATACTGGTTGGGGACGTTTGTCTCGAAAATTGATTAATGGTATTAGGGATAAGCAATCTGGCAAAACAATATTAGATTTTTTGAAATCAGATGGTTTTGCCAATCGCAATTTTATGCAGCTGATCCATGATGATAGTTTGACATTTAAAGAAGACATTCAAAAAGCACAAGTGTCTGGACAAGGCGATAGTTTACATGAACATATTGCAAATTTAGCTGGTAGCCCTGCTATTAAAAAAGGTATTTTACAGACTGTAAAAGTTGTTGATGAATTGGTCAAAGTAATGGGGCGGCATAAGCCAGAAAATATCGTTATTGAAATGGCACGTGAAAATCAGACAACTCAAAAGGGCCAGAAAAATTCGCGAGAGCGTATGAAACGAATCGAAGAAGGTATCAAAGAATTAGGAAGTCAGATTCTTAAAGAGCATCCTGTTGAAAATACTCAATTGCAAAATGAAAAGCTCTATCTCTATTATCTCCAAAATGGAAGAGACATGTATGTGGACCAAGAATTAGATATTAATCGTTTAAGTGATTATGATGTCGATCACATTGTTCCACAAAGTTTCCTTAAAGACGATTCAATAGACAATAAGGTCTTAACGCGTTCTGATAAAAATCGTGGTAAATCGGATAACGTTCCAAGTGAAGAAGTAGTCAAAAAGATGAAAAACTATTGGAGACAACTTCTAAACGCCAAGTTAATCACTCAACGTAAGTTTGATAATTTAACGAAAGCTGAACGTGGAGGTTTGAGTGAACTTGATAAAGCTGGTTTTATCAAACGCCAATTGGTTGAAACTCGCCAAATCACTAAGCATGTGGCACAAATTTTGGATAGTCGCATGAATACTAAATACGATGAAAATGATAAACTTATTCGAGAGGTTAAAGTGATTACCTTAAAATCTAAATTAGTTTCTGACTTCCGAAAAGATTTCCAATTCTATAAAGTACGTGAGATTAACAATTACCATCATGCCCATGATGCGTATCTAAATGCCGTCGTTGGAACTGCTTTGATTAAGAAATATCCAAAACTTGAATCGGAGTTTGTCTATGGTGATTATAAAGTTTATGATGTTCGTAAAATGATTGCTAAGTCTGAGCAAGAAATAGGCAAAGCAACCGCAAAATATTTCTTTTACTCTAATATCATGAACTTCTTCAAAACAGAAATTACACTTGCAAATGGAGAGATTCGCAAACGCCCTCTAATCGAAACTAATGGGGAAACTGGAGAAATTGTCTGGGATAAAGGGCGAGATTTTGCCACAGTGCGCAAAGTATTGTCCATGCCCCAAGTCAATATTGTCAAGAAAACAGAAGTACAGACAGGCGGATTCTCCAAGGAGTCAATTTTACCAAAAAGAAATTCGGACAAGCTTATTGCTCGTAAAAAAGACTGGGATCCAAAAAAATATGGTGGTTTTGATAGTCCAACGGTAGCTTATTCAGTCCTAGTGGTTGCTAAGGTGGAAAAAGGGAAATCGAAGAAGTTAAAATCCGTTAAAGAGTTACTAGGGATCACAATTATGGAAAGAAGTTCCTTTGAAAAAAATCCGATTGACTTTTTAGAAGCTAAAGGATATAAGGAAGTTAAAAAAGACTTAATCATTAAACTACCTAAATATAGTCTTTTTGAGTTAGAAAACGGTCGTAAACGGATGCTGGCTAGTGCCGGAGAATTACAAAAAGGAAATGAGCTGGCTCTGCCAAGCAAATATGTGAATTTTTTATATTTAGCTAGTCATTATGAAAAGTTGAAGGGTAGTCCAGAAGATAACGAACAAAAACAATTGTTTGTGGAGCAGCATAAGCATTATTTAGATGAGATTATTGAGCAAATCAGTGAATTTTCTAAGCGTGTTATTTTAGCAGATGCCAATTTAGATAAAGTTCTTAGTGCATATAACAAACATAGAGACAAACCAATACGTGAACAAGCAGAAAATATTATTCATTTATTTACGTTGACGAATCTTGGAGCTCCCGCTGCTTTTAAATATTTTGATACAACAATTGATCGTAAACGATATACGTCTACAAAAGAAGTTTTAGATGCCACTCTTATCCATCAATCCATCACTGGTCTTTATGAAACACGCATTGATTTGAGTCAGCTAGGAGGTGACCCTCCAAAAAAGAAGAGAAAGGTCTGAGCGGTTTTGTATAACTAAATAATATTGGAAACTAAATACGAATACCCAAATTTTTTATCTAAATTTTGCCGAAAGATTAAAATCTGCAGAGATATCCGAAACAGGTAAATGGATGTTTCAATCCCTGTAGTCAGTCAGGAACCCATATTATATTACAGTATTAGTCGCCGCTTAGGCACGCCTTTAATTAGCAAAATCAAACCTTAAGTGCATATGCCGTATAAGGGAAACTCAAAGAACTGGCATCGCAAAAATGAAAACATACGTTGAAACTACGGCAAAGGATTGGTCAGATCGCTTCATACAGGGAAAGTTCGGCAGAGACCgtagcc
将前述(1)-(2)步骤PCR扩增得到的目标片段9、10利用DpnI内切酶进行酶切,37℃消化3hr,后用PCR产物纯化试剂盒对于前述酶切产物进行纯化。
(3)以GG799基因组(即ASM215156v1)为模板,采用如下扩增引物(ARS12-F和ARS12-R)利用常规PCR方法,对于目标片段进行扩增以获得ARS12作为目标片段11:
ARS12-F:ggcatcGGTCTCTCGGCACTCGAGTACTGAAGCATTGGT(SEQ ID NO:21);
ARS12-R:GGCTACGGTCTCTTGGCCGGGGATCCATTATTATCCT(SEQ ID NO:22);
目标片段11(SEQ ID NO:60):
ggctacGGTCTCtcggcaCTCGAGTACTGAAGCATTGGTTGTTTCATCACAGATGAACCAAACTTCTTCAAGAACCATGGTGAAATCCTAATGTCATCTAAAATTCGATCGCATGTTTTGAACGGAAAACTTTTCGAAATATCTCCGTACAAAAGCGTCTTACCATCGTCTACTAAATCTGAAAATGATTCCAACGACTTAGATCTCAGTACTTCTGCTGTTTCCGAAACAGAATCTCTCTGTTCGCCATTCAATTCTAAAGCTGGTTCTTGTGAAGCCATCATCAATTCGAGATTCTATTCTCTGCCAAGCTATCCTCAAATTGTCACAATATACTGTAGCGTGTCAAATGTATCAGAATTGAATCACTGGGAGTATTAGAACGAGCCTAGAAGTGGCAAATAGATATTTTTAATTATGCATTTGCCAAAGGATTGAAGATCTAGTTTTCCTTAAAATTTAATGCAATTACTATTCTGATTCTACTTAATTCTTTTGTTACCCTTTAAAATTTGTCAATAGTGGAAATCTGTAGACAGAACCACAATGAGTGTGTGGTGTACGGATTTATACTCACAGTTTTAAATTTAAACGCATTTATGGCGGTACAAAGGGCATTAAAATGAGTCAAAAAGGGCCTCTATTCAAATTACCTATGCCCTGTTTGATGTAACAGCATTGCTATTATTCCGAAAAGACTGAAACAGCTTTACAACTTGTTTTTCTGAGTACTACGTAAACTATAATTTATAACTCAATGTAACCTATTCAACCTTTGAATGTTGAAATAGGTTCATAAAATCCAGAACTTTAAAACGCAGTCGTTGAGTTAAAAAGAGGAAAAATAGTTCGATCAGTGGCACCGCTTCATAGTGAGCATCTGAAGAATTTTAGTAGTCGTTACTTGAACACCAACAAGCAAAGTAGAAGAGGAACGGTCATCTCTGTCACTGTTTCTATCGTTACTTTTAGAGAATCATCTATGACGGACTTCACAAGCCACTTTCCTACAACGAAATCTGTAAACTGCAGAAGAGAGAAAAACATAATTAAAAAAAAACCAATATAAAAGATGTTGCATGAGGAGTTGAAAAATTTAGGGAGGAAGAGCTTCAGGGTCTAGTCAATATCTTTTCCGAAACTGATCATAGGTAGCTTATAGTGGCGTTTGATCACAGTAAGTGTTAAGATATATACTATCCATGGAATAACAATAATAAGATGGGAAATAAAACGTCTACACTTTCAAAGAATGATTTGACCAGTTTGAAGCAATCTACGTATTTCGATCGCAGAGAGATCCAACAGTGGCACAAAGGGTTTCTACGGGATTGCCCGAACGGCCAGCTAACTAGAGAAGAGTTCATCAAGATATACAAACAATTTTTCCCCTTTGGCTCGCCAGATGAGTTTGCTACGTATGTGTTTAGCGTGTTTGACAAGGATAATAATGGATCCCCggccaaGAGACCGTAGCC
以GG799基因组为模板,采用如下扩增引物(ARS101-F和ARS101-R)利用常规PCR方法,对于目标片段进行扩增以获得ARS101作为目标片段12:
ARS101-F:GGCTACGGTCTCTTGGCCGGGGATCCATTATTATCCT(SEQ ID NO:23);
ARS101R:GGCTACGGTCTCTTGGCCctctgcaggatcagaagtt(SEQ ID NO:24)。
目标片段12(SEQ ID NO:61):
ggctacGGTCTCtcggcaaagcttctacccataacttccatgctcgtattttagcagaaaccagttgatattatgtaagagatcactgagacaaagttgattaagccaataatcgtcgcagtatgcatgttgtaggtaggtacacttttattctatcgtatccttcattgcttatcgatgccggtgttattcttgctatgataggccatcgaatgttaaagggaagatttattgttttgccctttaaagacgaatttggaccgtgaatgtttaaatattcacaaaagagtaataaacatttattttgaccgtgttaataaatttcaataatacacacttggacataaacatattcatacatatagtctttagcttactcagattctgcaacctctccgtctctgccataaaaacctttgtatatagttaggtagccatccccgtcaacagtaccaattatatctcgaatcaaaggatgctgcgttacagaacccttgatcaaaactgtattatcaggtttgtaggcggatcttctaccgccgccagtatgtaagaatttcaaagaatgttcttgtatgtcatcttgatatatttcatcagaatctgcacgttcttcgccctgttgctcgctttcgttgattaaatagctgatatcccaatggtacacatttcccgaagcacctacagtgaggaaacattcgggttccaaggggctgaattggatatcgaccacaggatcgttatctaattggtacaatgtgactatttctgtggcatccttcacttctgtggaatttctagcggcaatggaacgtaaatcccatagtttaataacaccggtcatcagaaccagtaatgaaaagagtatcaaactgtttcgatgcttcaacaacgtttatcgtaccatcatcttcaacttctgatcctgcagagggccaaGAGACCGTAGCC
PCR扩增后,利用PCR产物纯化试剂盒对于前述PCR扩增产物目标片段11、12进行纯化。
(4)pKD1_ori由华大基因全合成,两端添加BsaI后,构建至pKMV商业载体中,命名为质粒pKMV-pKD1_ori。将该质粒使用BsaI酶37℃酶切15min,80℃失活20min。酶切完成后,利用胶回收试剂盒回收大小约为960bp的片段以获得pKD1_ori作为目标片段13。
目标片段13(SEQ ID NO:62):
GATCACAGCGGACGGTGGTGGCATGATGGGGCTTGCGATGCTATGTTTGTTTGTTTTGTGATGATGTATATTATTATTGAAAAACGATATCAGACATTTGTCTGATAATGCTTCATTATCAGACAAATGTCTGATATCGTTTGGAGAAAAAGAAAAGGAAAACAAACTAAATATCTACTATATACCACTGTATTTTATACTAATGACTTTCTACGCCTAGTGTCACCCTCTCGTGTACCCATTGACCCTGTATCGGCGCGTTGCCTCGCGTTCCTGTACCATATATTTTTGTTTATTTAGGTATTAAAATTTACTTTCCTCATACAAATATTAAATTCACCAAACTTCTCAAAAACTAATTATTCGTAGTTACAAACTCTATTTTACAATCACGTTTATTCAACCATTCTACATCCAATAACCAAAATGCCCATGTACCTCTCAGCGAAGTCCAACGGTACTGTCCAATATTCTCATTAAATAGTCTTTCATCTATATATCAGAAGGTAATTATAATTAGAGATTTCGAATCATTACCGTGCCGATTCGCACGCTGCAACGGCATGCATCACTAATGAAAAGCATACGACGCCTGCGTCTGACATGCACTCATTCTGAAGAAGATTCTGGGCGCGTTTCGTTCTCGTTTTCCTCTGTATATTGTACTCTGGTGGACAATTTGAACATAACGTCTTTCACCTCGCCATTCTCAATAATGGGTTCCAATTCTATCCAGGTAGCGGTTAATTGACGGTGCTTAAGCCGTATGCTCACTCTAACGCTACCGTTGTCCAAACAACGGACCCCTTTGTGACGGGTGTAAGACCCATCATGAAGTAAAACATCTCTAACGGTATGGAAAAGAGTGGTACGGTCAAGTTTCCTGGCACGAGTCAATTTTCCCTCTTCG
(5)将(1)-(4)步骤获得的目标片段9、10和11与目标片段9、10和12以及目标片段9、10和13分别以golden gate方式进行连接,将前述DNA产物以0.12pmol的量分别加入到20μl体系中,加入0.5μl T4连接酶,1μl T4连接酶buffer,0.5μl BsaI内切酶在PCR仪中进行连接,反应37℃1h,然后在80℃下反应5min。连接反应结束后,将连接产物加入至100μl DH5α感受态细胞中,冰上孵育30min,42℃热激45s,后加入1mL LB液体培养基37℃振荡培养1hr,涂布于具有Amp+抗性的LB固体培养基上,37℃倒置培养至单克隆长出。
挑取24个单克隆,利用如下检测引物check-ARS-F和check-ARS-R对于前述质粒(含有ARS12的质粒、含有ARS101的质粒以及含有pKD1_ori的质粒)进行检测,并通过测序最终确定。
check-ARS-F:AGTGCATATGCCGTATAAGG(SEQ ID NO:25);
check-ARS-R:GCATGTACGGGTTACAGCAG(SEQ ID NO:26)。
最终获得改造完成质粒,分别命名为p025-ARS12-KlADE2(其中,前述质粒的核苷酸序列(从N端至C端)为将目标片段9、目标片段10和目标片段11连接得到)、p025-ARS101-KlADE2(其中,前述质粒的核苷酸序列(从N端至C端)为将目标片段9、目标片段10和目标片段12连接得到)和p025-pKD1_ori KlADE2(其中,前述质粒的核苷酸序列(从N端至C端)为将目标片段9、目标片段10和目标片段13连接得到)。pUDP025质粒中复制起点panARS,四种质粒用于后续比较各复制起点的转化效率。
实施例5:供体DNA的制备
(1)以乳酸克鲁维GG799基因组(ASM215156v1)为模板,利用常规PCR方法,采用如下引物,扩增距离gRNA为10bp、30bp、120bp、200bp和1000bp的上下游同源臂。
以乳酸克鲁维基因组GG799为模板,利用常规PCR方法,采用如下引物,扩增距离gRNA 10bp的上游同源臂以获得目标片段14:
IF1:cctgcaggtcgactctagaggatccACTTAGCACGTGAAAGTCGTTTTAA(SEQ ID NO:27)
10bp-IR1:TACACGATATAAAGGGTTTTCTGACCTCCTTGGATTTCACGACAAAGTTC(SEQ IDNO:28)
以乳酸克鲁维基因组GG799为模板,利用常规PCR方法,采用如下引物,扩增距离gRNA 10bp的下游同源臂以获得目标片段15:
10bp-IF2:GAACTTTGTCGTGAAATCCAAGGAGGTCAGAAAACCCTTTATATCGTGTA(SEQ IDNO:29)
IR2:cagtgaattcgagctcggtacccggGATTGGGACGTTCTAAACTGCTTAG(SEQ ID NO:30)
以乳酸克鲁维基因组GG799为模板,利用常规PCR方法,采用如下引物,扩增距离gRNA 30bp的上游同源臂以获得目标片段16:
IF1:cctgcaggtcgactctagaggatccACTTAGCACGTGAAAGTCGTTTTAA(SEQ ID NO:27)
30bp-IR1:TACACGATATAAAGGGTTTTCTGACAGTTCCCTCTGCCATCGTAGGCTAA(SEQ IDNO:31)
以乳酸克鲁维基因组GG799为模板,利用常规PCR方法,采用如下引物,扩增距离gRNA 30bp的下游同源臂以获得目标片段17:
30bp-IF2:TTAGCCTACGATGGCAGAGGGAACTGTCAGAAAACCCTTTATATCGTGTA(SEQ IDNO:32)
IR2:cagtgaattcgagctcggtacccggGATTGGGACGTTCTAAACTGCTTAG(SEQ ID NO:30)
以乳酸克鲁维基因组GG799为模板,利用常规PCR方法,采用如下引物,扩增距离gRNA 120bp的上游同源臂以获得目标片段18:
IF1:cctgcaggtcgactctagaggatccACTTAGCACGTGAAAGTCGTTTTAA(SEQ ID NO:27)
120bp-IR1:TACACGATATAAAGGGTTTTCTGACCGCTGTTAACAGCAACGGACTTTGC(SEQ IDNO:33)
以乳酸克鲁维基因组GG799为模板,利用常规PCR方法,采用如下引物,扩增距离gRNA 120bp的下游同源臂以获得目标片段19:
120bp-IF2:GCAAAGTCCGTTGCTGTTAACAGCGGTCAGAAAACCCTTTATATCGTGTA(SEQ IDNO:34)
IR2:cagtgaattcgagctcggtacccggGATTGGGACGTTCTAAACTGCTTAG(SEQ ID NO:30)
以乳酸克鲁维基因组GG799为模板,利用常规PCR方法,采用如下引物,扩增距离gRNA 200bp的上游同源臂以获得目标片段20:
IF1:cctgcaggtcgactctagaggatccACTTAGCACGTGAAAGTCGTTTTAA(SEQ ID NO:27)
200bp-IR1:TACACGATATAAAGGGTTTTCTGACAAAGCGATCGTCTCTGGGTCGGGAT(SEQ IDNO:35)
以乳酸克鲁维基因组GG799为模板,利用常规PCR方法,采用如下引物,扩增距离gRNA 200bp的下游同源臂以获得目标片段21:
200bp-IF2:ATCCCGACCCAGAGACGATCGCTTTGTCAGAAAACCCTTTATATCGTGTA(SEQ IDNO:36)
IR2:cagtgaattcgagctcggtacccggGATTGGGACGTTCTAAACTGCTTAG(SEQ ID NO:30)
以乳酸克鲁维基因组GG799为模板,利用常规PCR方法,采用如下引物,扩增ADE2上游1000bp为上游同源臂,获得目标片段22:
1000bp-IF1:cctgcaggtcgactctagaggatccCAGAGATATAGGGAGGTTTACAGTA(SEQ IDNO:37)
1000bp-IR1:TACACGATATAAAGGGTTTTCTGACGTTGTCTTAGTGAAGAAGGTGAACG(SEQ IDNO:38)
以乳酸克鲁维基因组GG799为模板,利用常规PCR方法,采用如下引物,扩增距离ADE2下游1000bp为下游同源臂,获得目标片段23:
1000bp-IF2:CGTTCACCTTCTTCACTAAGACAACGTCAGAAAACCCTTTATATCGTGTA(SEQ IDNO:39)
IR2:cagtgaattcgagctcggtacccggGATTGGGACGTTCTAAACTGCTTAG(SEQ ID NO:30)
PCR扩增后,利用PCR产物纯化试剂盒对于前述PCR扩增产物目标片段14-23进行纯化。
(2)以pUC19为模板,利用常规PCR方法,采用如下引物,对于目标条带进行扩增以获得目标片段24:
V-F:CTAAGCAGTTTAGAACGTCCCAATCCCGGGTACCGAGCTCGAATTCACTG(SEQ ID NO:40);
V-R:TTAAAACGACTTTCACGTGCTAAGTGGATCCTCTAGAGTCGACCTGCAGG(SEQ ID NO:41)。
以pUC19为模板,利用常规PCR方法,采用如下引物,对于目标条带进行扩增以获得目标片段25
V-F:CTAAGCAGTTTAGAACGTCCCAATCCCGGGTACCGAGCTCGAATTCACTG(SEQ ID NO:40);
1000bp-VR:TACTGTAAACCTCCCTATATCTCTGggatcctctagagtcgacctgcagg(SEQ IDNO:42)
将上述步骤(2)中得到的PCR扩增产物利用DpnI内切酶进行酶切,37℃消化3hr,后用PCR产物纯化试剂盒对于前述酶切后目标片段分别进行纯化。
将上述纯化后的片段24与片段14和15、片段16和17、片段18和19、片段20和21,片段25与片段22和23分别利用全式金生物无缝克隆试剂盒方式进行连接,将上述各片段以0.08pmol的量加入反应体系,同时加入等体积的无缝克隆酶,50℃连接1小时。
将前述连接产物加入至100μl DH5α感受态细胞中进行转化,冰上孵育30min,42℃热激45s,后加入1mL LB液体培养基37℃振荡培养1hr,涂布于具有Amp抗性的LB固体培养基上,37℃倒置培养至单克隆长出。
(3)分别挑选12个单克隆进行PCR检验并测序确认后,得到pUC19-10bp、pUC19-30bp、pUC19-120bp、pUC19-200bp以及pUC19-1000bp质粒。
(4)分别以上述质粒pUC19-10bp、pUC19-30bp、pUC19-120bp、pUC19-200bp为模板,采用如下引物(IF1和IR2),利用常规PCR方法,对于目标片段进行扩增以获得目标片段26、27、28、29:
IF1:CCTGCAGGTCGACTCTAGAGGATCCACTTAGCACGTGAAAGTCGTTTTAA(SEQ ID NO:27);
IR2:CAGTGAATTCGAGCTCGGTACCCGGGATTGGGACGTTCTAAACTGCTTAG(SEQ ID NO:30)。
以上述质粒pUC19-1000bp为模板,采用如下引物(1000bp-IF1和IR-2),利用常规PCR方法,对于目标片段进行扩增以获得目标片段30:
IR2:CAGTGAATTCGAGCTCGGTACCCGGGATTGGGACGTTCTAAACTGCTTAG(SEQ ID NO:30)。
1000bp-IF1:cctgcaggtcgactctagaggatccCAGAGATATAGGGAGGTTTACAGTA(SEQ IDNO:37)
扩增完成后,获得的目标片段26、27、28、29、30。优选的,可以对于前述目标片段通过PCR产物纯化试剂盒进行纯化,以获得对应的供体DNA。
目标片段26对应于10bp供体DNA,其序列如下(SEQ ID NO:49):
cctgcaggtcgactctagaggatccACTTAGCACGTGAAAGTCGTTTTAATATCACTCGAGTGCTGTATGATAATTAGGTTCATTTCTGAACTCACTGGGGGTAATTCTTCGTCGCCATTTATTGTTGAGAAAAAAAGGATGCGCCTTGGGCGGGTAACGATACATATATCTCGGCTACCTTCGGTTATTTTTAAGGAGATTGAGGATTTTCGGCAACCTTCGGTAATACAGCCCACCTTGTAGTACCGACCTTATCCGTGATGTCTGCTTTCCACTTTTGGATGCGGCAAGTCACGGAAATGTGAGGTACTATTAGCCGAATCGCACATGGAGCTGGGTGATGAAGAACGAATTCATTGTCACGTGGATATTTTTGCACGTGACAGCGAGTTTGGTCTTTTTTTTTGACTTGACATGTTTGATGCCTCATCTAGCTTCCAACATTAAAAATTAGATATTACATCAGAGTTGTCTTCTAATTTCAATTTGATGCGAAGAGATGATCATTAAGATACATACATATATATATATATATACTTTCAATCCATATTGGAAGAAGAGAAAGCCTTGTAATCTCATCATATCTTGCCCCATAATTTAAGGAGTTAGTCTGACTGTTTCCGCTTCTCTGGTAGACAGACCATTAAACAGAATATTCCGTTGTCAGAATTGATAACCCTATATAATAACATCACGTTCACCTTCTTCACTAAGACAACATGGATCAAAGAACTGTCGGTATTTTAGGCGGTGGCCAATTGGGCCGTATGATTGTTGAAGCTGCTCATAGACTGAATGTGAAAACTATCATTTTGGATGCAGAAAACTCTCCGGCAAAGCAGATTAATGCTTTGAGCGAACATGTCAATGGTTCCTTTGCCAAACCGGAAGATATCAAGAAAATTGCATCTCTTTGTGATGTCTTAACTGTTGAAATCGAACACGTCGATGTTCCAACTTTGAAAAGGGTTCAACAGGATCACCCTAACTTGAAGATCTATCCCGACCCAGAGACGATCGCTTTGATTCAAGATAAATTTGTTCAAAAGCAATATTTGATTCAGAATAAGATCAGTGTGGCAAAGTCCGTTGCTGTTAACAGCGATGAAGAATCGTTGGCTAAAGTTGGTTTTGAATTTGGTTACCCATTCATGCTTAAATCAAGAACTTTAGCCTACGATGGCAGAGGGAACTTTGTCGTGAAATCCAAGGAGGTCAGAAAACCCTTTATATCGTGTATAATCTTACTACTCCTGTTTCGATCTTTTACATGTCAATAACATAGCCTATTCTTATAACTACTTTAATTGTTAACGCCCCATTTTCTGAATGACGTGGTTCATTTCCCTTTCAAAATGTGAGTCCGATAATTACATTGACATATTGCGTCAATTGATCAACTTTCTGATGTACCAAGTCGCGATCACTTAGGCTTGCAGATACGACCTTAGTGAACTCCGAGAGAAGGATGAATTTTTGCTTCAGGAATGTAGGTTCAGCCACGAGCACAAGCTTTGCTAGTAGTTCTTCATCAGATAAGTTTGAATTGCCGAAATAAAGAGAACCTCTAGCATATGATATTAAACTCTTTGCATTAAATTGAGACTCGTCAAGCGGAATTGATTTGTTGGCATATATCAACGCACATTTCCCATCAAAAGTAACAAACTTTATAAACTTGAACCAGATAATATCTTGAACTATTTTTGGAAGAGCGAACCGCAATAGATTAATAAATATCGGTGGATATCTCATCCATACACATTTCTTCGTATCATTTAGGAAGGAATCATTCTCTTGTACTTTACCATATGATTCTTCTAATTCTTCTGGGTTATCTTCTGATTGTACCGCTGGGTGAATTCTGCTATAATCTTCAGCTGATTTCTGTTCGATATAATCGTTGCAGGAGCGAAAGGAAACGATTGGGGTCTTGTTCAAGCTAGAAGAGTGAGCTGGAATATTCCCAGACGCTAGCTCAATAAGGGTGATAACATCTAGAAGCCGGAACATGCTCTGGAAACAGAGCATGATAGAAAAAGCTTCAAAGGAAGATATCTCAGAATCTACAGCCTCACTTAATTCGGCCACGTCGACAAGTCTTAATTCTGAATCGAATACGTCACATATCAAAATATCCAACTTATTAATATTTTCTCGTAGGTTAATCTTAAGTGTTTGTGACAACGCTAAGCAGTTTAGAACGTCCCAATCccgggtaccgagctcgaattcactg
目标片段27对应于30bp供体DNA,其序列如下(SEQ ID NO:50):
cctgcaggtcgactctagaggatccACTTAGCACGTGAAAGTCGTTTTAATATCACTCGAGTGCTGTATGATAATTAGGTTCATTTCTGAACTCACTGGGGGTAATTCTTCGTCGCCATTTATTGTTGAGAAAAAAAGGATGCGCCTTGGGCGGGTAACGATACATATATCTCGGCTACCTTCGGTTATTTTTAAGGAGATTGAGGATTTTCGGCAACCTTCGGTAATACAGCCCACCTTGTAGTACCGACCTTATCCGTGATGTCTGCTTTCCACTTTTGGATGCGGCAAGTCACGGAAATGTGAGGTACTATTAGCCGAATCGCACATGGAGCTGGGTGATGAAGAACGAATTCATTGTCACGTGGATATTTTTGCACGTGACAGCGAGTTTGGTCTTTTTTTTTGACTTGACATGTTTGATGCCTCATCTAGCTTCCAACATTAAAAATTAGATATTACATCAGAGTTGTCTTCTAATTTCAATTTGATGCGAAGAGATGATCATTAAGATACATACATATATATATATATATACTTTCAATCCATATTGGAAGAAGAGAAAGCCTTGTAATCTCATCATATCTTGCCCCATAATTTAAGGAGTTAGTCTGACTGTTTCCGCTTCTCTGGTAGACAGACCATTAAACAGAATATTCCGTTGTCAGAATTGATAACCCTATATAATAACATCACGTTCACCTTCTTCACTAAGACAACATGGATCAAAGAACTGTCGGTATTTTAGGCGGTGGCCAATTGGGCCGTATGATTGTTGAAGCTGCTCATAGACTGAATGTGAAAACTATCATTTTGGATGCAGAAAACTCTCCGGCAAAGCAGATTAATGCTTTGAGCGAACATGTCAATGGTTCCTTTGCCAAACCGGAAGATATCAAGAAAATTGCATCTCTTTGTGATGTCTTAACTGTTGAAATCGAACACGTCGATGTTCCAACTTTGAAAAGGGTTCAACAGGATCACCCTAACTTGAAGATCTATCCCGACCCAGAGACGATCGCTTTGATTCAAGATAAATTTGTTCAAAAGCAATATTTGATTCAGAATAAGATCAGTGTGGCAAAGTCCGTTGCTGTTAACAGCGATGAAGAATCGTTGGCTAAAGTTGGTTTTGAATTTGGTTACCCATTCATGCTTAAATCAAGAACTTTAGCCTACGATGGCAGAGGGAACTGTCAGAAAACCCTTTATATCGTGTATAATCTTACTACTCCTGTTTCGATCTTTTACATGTCAATAACATAGCCTATTCTTATAACTACTTTAATTGTTAACGCCCCATTTTCTGAATGACGTGGTTCATTTCCCTTTCAAAATGTGAGTCCGATAATTACATTGACATATTGCGTCAATTGATCAACTTTCTGATGTACCAAGTCGCGATCACTTAGGCTTGCAGATACGACCTTAGTGAACTCCGAGAGAAGGATGAATTTTTGCTTCAGGAATGTAGGTTCAGCCACGAGCACAAGCTTTGCTAGTAGTTCTTCATCAGATAAGTTTGAATTGCCGAAATAAAGAGAACCTCTAGCATATGATATTAAACTCTTTGCATTAAATTGAGACTCGTCAAGCGGAATTGATTTGTTGGCATATATCAACGCACATTTCCCATCAAAAGTAACAAACTTTATAAACTTGAACCAGATAATATCTTGAACTATTTTTGGAAGAGCGAACCGCAATAGATTAATAAATATCGGTGGATATCTCATCCATACACATTTCTTCGTATCATTTAGGAAGGAATCATTCTCTTGTACTTTACCATATGATTCTTCTAATTCTTCTGGGTTATCTTCTGATTGTACCGCTGGGTGAATTCTGCTATAATCTTCAGCTGATTTCTGTTCGATATAATCGTTGCAGGAGCGAAAGGAAACGATTGGGGTCTTGTTCAAGCTAGAAGAGTGAGCTGGAATATTCCCAGACGCTAGCTCAATAAGGGTGATAACATCTAGAAGCCGGAACATGCTCTGGAAACAGAGCATGATAGAAAAAGCTTCAAAGGAAGATATCTCAGAATCTACAGCCTCACTTAATTCGGCCACGTCGACAAGTCTTAATTCTGAATCGAATACGTCACATATCAAAATATCCAACTTATTAATATTTTCTCGTAGGTTAATCTTAAGTGTTTGTGACAACGCTAAGCAGTTTAGAACGTCCCAATCccgggtaccgagctcgaattcactg
目标片段28对应于120bp供体DNA,其序列如下(SEQ ID NO:51):
cctgcaggtcgactctagaggatccACTTAGCACGTGAAAGTCGTTTTAATATCACTCGAGTGCTGTATGATAATTAGGTTCATTTCTGAACTCACTGGGGGTAATTCTTCGTCGCCATTTATTGTTGAGAAAAAAAGGATGCGCCTTGGGCGGGTAACGATACATATATCTCGGCTACCTTCGGTTATTTTTAAGGAGATTGAGGATTTTCGGCAACCTTCGGTAATACAGCCCACCTTGTAGTACCGACCTTATCCGTGATGTCTGCTTTCCACTTTTGGATGCGGCAAGTCACGGAAATGTGAGGTACTATTAGCCGAATCGCACATGGAGCTGGGTGATGAAGAACGAATTCATTGTCACGTGGATATTTTTGCACGTGACAGCGAGTTTGGTCTTTTTTTTTGACTTGACATGTTTGATGCCTCATCTAGCTTCCAACATTAAAAATTAGATATTACATCAGAGTTGTCTTCTAATTTCAATTTGATGCGAAGAGATGATCATTAAGATACATACATATATATATATATATACTTTCAATCCATATTGGAAGAAGAGAAAGCCTTGTAATCTCATCATATCTTGCCCCATAATTTAAGGAGTTAGTCTGACTGTTTCCGCTTCTCTGGTAGACAGACCATTAAACAGAATATTCCGTTGTCAGAATTGATAACCCTATATAATAACATCACGTTCACCTTCTTCACTAAGACAACATGGATCAAAGAACTGTCGGTATTTTAGGCGGTGGCCAATTGGGCCGTATGATTGTTGAAGCTGCTCATAGACTGAATGTGAAAACTATCATTTTGGATGCAGAAAACTCTCCGGCAAAGCAGATTAATGCTTTGAGCGAACATGTCAATGGTTCCTTTGCCAAACCGGAAGATATCAAGAAAATTGCATCTCTTTGTGATGTCTTAACTGTTGAAATCGAACACGTCGATGTTCCAACTTTGAAAAGGGTTCAACAGGATCACCCTAACTTGAAGATCTATCCCGACCCAGAGACGATCGCTTTGATTCAAGATAAATTTGTTCAAAAGCAATATTTGATTCAGAATAAGATCAGTGTGGCAAAGTCCGTTGCTGTTAACAGCGGTCAGAAAACCCTTTATATCGTGTATAATCTTACTACTCCTGTTTCGATCTTTTACATGTCAATAACATAGCCTATTCTTATAACTACTTTAATTGTTAACGCCCCATTTTCTGAATGACGTGGTTCATTTCCCTTTCAAAATGTGAGTCCGATAATTACATTGACATATTGCGTCAATTGATCAACTTTCTGATGTACCAAGTCGCGATCACTTAGGCTTGCAGATACGACCTTAGTGAACTCCGAGAGAAGGATGAATTTTTGCTTCAGGAATGTAGGTTCAGCCACGAGCACAAGCTTTGCTAGTAGTTCTTCATCAGATAAGTTTGAATTGCCGAAATAAAGAGAACCTCTAGCATATGATATTAAACTCTTTGCATTAAATTGAGACTCGTCAAGCGGAATTGATTTGTTGGCATATATCAACGCACATTTCCCATCAAAAGTAACAAACTTTATAAACTTGAACCAGATAATATCTTGAACTATTTTTGGAAGAGCGAACCGCAATAGATTAATAAATATCGGTGGATATCTCATCCATACACATTTCTTCGTATCATTTAGGAAGGAATCATTCTCTTGTACTTTACCATATGATTCTTCTAATTCTTCTGGGTTATCTTCTGATTGTACCGCTGGGTGAATTCTGCTATAATCTTCAGCTGATTTCTGTTCGATATAATCGTTGCAGGAGCGAAAGGAAACGATTGGGGTCTTGTTCAAGCTAGAAGAGTGAGCTGGAATATTCCCAGACGCTAGCTCAATAAGGGTGATAACATCTAGAAGCCGGAACATGCTCTGGAAACAGAGCATGATAGAAAAAGCTTCAAAGGAAGATATCTCAGAATCTACAGCCTCACTTAATTCGGCCACGTCGACAAGTCTTAATTCTGAATCGAATACGTCACATATCAAAATATCCAACTTATTAATATTTTCTCGTAGGTTAATCTTAAGTGTTTGTGACAACGCTAAGCAGTTTAGAACGTCCCAATCccgggtaccgagctcgaattcactg
目标片段29对应于200bp供体DNA,其序列如下(SEQ ID NO:52):
cctgcaggtcgactctagaggatccACTTAGCACGTGAAAGTCGTTTTAATATCACTCGAGTGCTGTATGATAATTAGGTTCATTTCTGAACTCACTGGGGGTAATTCTTCGTCGCCATTTATTGTTGAGAAAAAAAGGATGCGCCTTGGGCGGGTAACGATACATATATCTCGGCTACCTTCGGTTATTTTTAAGGAGATTGAGGATTTTCGGCAACCTTCGGTAATACAGCCCACCTTGTAGTACCGACCTTATCCGTGATGTCTGCTTTCCACTTTTGGATGCGGCAAGTCACGGAAATGTGAGGTACTATTAGCCGAATCGCACATGGAGCTGGGTGATGAAGAACGAATTCATTGTCACGTGGATATTTTTGCACGTGACAGCGAGTTTGGTCTTTTTTTTTGACTTGACATGTTTGATGCCTCATCTAGCTTCCAACATTAAAAATTAGATATTACATCAGAGTTGTCTTCTAATTTCAATTTGATGCGAAGAGATGATCATTAAGATACATACATATATATATATATATACTTTCAATCCATATTGGAAGAAGAGAAAGCCTTGTAATCTCATCATATCTTGCCCCATAATTTAAGGAGTTAGTCTGACTGTTTCCGCTTCTCTGGTAGACAGACCATTAAACAGAATATTCCGTTGTCAGAATTGATAACCCTATATAATAACATCACGTTCACCTTCTTCACTAAGACAACATGGATCAAAGAACTGTCGGTATTTTAGGCGGTGGCCAATTGGGCCGTATGATTGTTGAAGCTGCTCATAGACTGAATGTGAAAACTATCATTTTGGATGCAGAAAACTCTCCGGCAAAGCAGATTAATGCTTTGAGCGAACATGTCAATGGTTCCTTTGCCAAACCGGAAGATATCAAGAAAATTGCATCTCTTTGTGATGTCTTAACTGTTGAAATCGAACACGTCGATGTTCCAACTTTGAAAAGGGTTCAACAGGATCACCCTAACTTGAAGATCTATCCCGACCCAGAGACGATCGCTTTGTCAGAAAACCCTTTATATCGTGTATAATCTTACTACTCCTGTTTCGATCTTTTACATGTCAATAACATAGCCTATTCTTATAACTACTTTAATTGTTAACGCCCCATTTTCTGAATGACGTGGTTCATTTCCCTTTCAAAATGTGAGTCCGATAATTACATTGACATATTGCGTCAATTGATCAACTTTCTGATGTACCAAGTCGCGATCACTTAGGCTTGCAGATACGACCTTAGTGAACTCCGAGAGAAGGATGAATTTTTGCTTCAGGAATGTAGGTTCAGCCACGAGCACAAGCTTTGCTAGTAGTTCTTCATCAGATAAGTTTGAATTGCCGAAATAAAGAGAACCTCTAGCATATGATATTAAACTCTTTGCATTAAATTGAGACTCGTCAAGCGGAATTGATTTGTTGGCATATATCAACGCACATTTCCCATCAAAAGTAACAAACTTTATAAACTTGAACCAGATAATATCTTGAACTATTTTTGGAAGAGCGAACCGCAATAGATTAATAAATATCGGTGGATATCTCATCCATACACATTTCTTCGTATCATTTAGGAAGGAATCATTCTCTTGTACTTTACCATATGATTCTTCTAATTCTTCTGGGTTATCTTCTGATTGTACCGCTGGGTGAATTCTGCTATAATCTTCAGCTGATTTCTGTTCGATATAATCGTTGCAGGAGCGAAAGGAAACGATTGGGGTCTTGTTCAAGCTAGAAGAGTGAGCTGGAATATTCCCAGACGCTAGCTCAATAAGGGTGATAACATCTAGAAGCCGGAACATGCTCTGGAAACAGAGCATGATAGAAAAAGCTTCAAAGGAAGATATCTCAGAATCTACAGCCTCACTTAATTCGGCCACGTCGACAAGTCTTAATTCTGAATCGAATACGTCACATATCAAAATATCCAACTTATTAATATTTTCTCGTAGGTTAATCTTAAGTGTTTGTGACAACGCTAAGCAGTTTAGAACGTCCCAATCccgggtaccgagctcgaattcactg
目标片段30对应于1000bp供体DNA,其序列如下(SEQ ID NO:53):
cctgcaggtcgactctagaggatccCAGAGATATAGGGAGGTTTACAGTAGGCAAATACGGAATAATGCAGTCATATTAAGCATTTCTTGCTTGAGTTTGGGTTACTTAATTTGACACAATAACCGAACTTTTTCTCTCTGACATTTCTTAAGGCTTTTTAACCATAAAAAAGAAATAGTATTTGGCAAAAGTAGTATGAATAAAGATAATGTCGAAAAATAGTGTTTCATTTCAAGTGGTAGATAGATTTCTTTGCCGTTAAATTGTGGATAAAGTTCTAACTTGGGATGTACTTTGTTTAGTGATATCATCGCATGTTAGCTAGTCACACTTAGCACGTGAAAGTCGTTTTAATATCACTCGAGTGCTGTATGATAATTAGGTTCATTTCTGAACTCACTGGGGGTAATTCTTCGTCGCCATTTATTGTTGAGAAAAAAAGGATGCGCCTTGGGCGGGTAACGATACATATATCTCGGCTACCTTCGGTTATTTTTAAGGAGATTGAGGATTTTCGGCAACCTTCGGTAATACAGCCCACCTTGTAGTACCGACCTTATCCGTGATGTCTGCTTTCCACTTTTGGATGCGGCAAGTCACGGAAATGTGAGGTACTATTAGCCGAATCGCACATGGAGCTGGGTGATGAAGAACGAATTCATTGTCACGTGGATATTTTTGCACGTGACAGCGAGTTTGGTCTTTTTTTTTGACTTGACATGTTTGATGCCTCATCTAGCTTCCAACATTAAAAATTAGATATTACATCAGAGTTGTCTTCTAATTTCAATTTGATGCGAAGAGATGATCATTAAGATACATACATATATATATATATATACTTTCAATCCATATTGGAAGAAGAGAAAGCCTTGTAATCTCATCATATCTTGCCCCATAATTTAAGGAGTTAGTCTGACTGTTTCCGCTTCTCTGGTAGACAGACCATTAAACAGAATATTCCGTTGTCAGAATTGATAACCCTATATAATAACATCACGTTCACCTTCTTCACTAAGACAACGTCAGAAAACCCTTTATATCGTGTATAATCTTACTACTCCTGTTTCGATCTTTTACATGTCAATAACATAGCCTATTCTTATAACTACTTTAATTGTTAACGCCCCATTTTCTGAATGACGTGGTTCATTTCCCTTTCAAAATGTGAGTCCGATAATTACATTGACATATTGCGTCAATTGATCAACTTTCTGATGTACCAAGTCGCGATCACTTAGGCTTGCAGATACGACCTTAGTGAACTCCGAGAGAAGGATGAATTTTTGCTTCAGGAATGTAGGTTCAGCCACGAGCACAAGCTTTGCTAGTAGTTCTTCATCAGATAAGTTTGAATTGCCGAAATAAAGAGAACCTCTAGCATATGATATTAAACTCTTTGCATTAAATTGAGACTCGTCAAGCGGAATTGATTTGTTGGCATATATCAACGCACATTTCCCATCAAAAGTAACAAACTTTATAAACTTGAACCAGATAATATCTTGAACTATTTTTGGAAGAGCGAACCGCAATAGATTAATAAATATCGGTGGATATCTCATCCATACACATTTCTTCGTATCATTTAGGAAGGAATCATTCTCTTGTACTTTACCATATGATTCTTCTAATTCTTCTGGGTTATCTTCTGATTGTACCGCTGGGTGAATTCTGCTATAATCTTCAGCTGATTTCTGTTCGATATAATCGTTGCAGGAGCGAAAGGAAACGATTGGGGTCTTGTTCAAGCTAGAAGAGTGAGCTGGAATATTCCCAGACGCTAGCTCAATAAGGGTGATAACATCTAGAAGCCGGAACATGCTCTGGAAACAGAGCATGATAGAAAAAGCTTCAAAGGAAGATATCTCAGAATCTACAGCCTCACTTAATTCGGCCACGTCGACAAGTCTTAATTCTGAATCGAATACGTCACATATCAAAATATCCAACTTATTAATATTTTCTCGTAGGTTAATCTTAAGTGTTTGTGACAACGCTAAGCAGTTTAGAACGTCCCAATCccgggtaccgagctcgaattcactg
实施例6:乳酸克鲁维酵母细胞基因编辑效率检测
首先测试酵母复制起始点:
分别将质粒p025-pKD1_ori-KlADE2、p025-ARS101-KlADE2、p025-ARS12-KlADE2以及pUDP025(复制起点为panARS)电转化至乳酸克鲁维酵母细胞中。
实验结果如图4所示。其中每μg pUDP025可获得15个转化子,并且基因编辑效率为33%,其余复制起点编辑效率均为0,因此,panARS为最佳复制起点。
进一步的,测试不同来源CAS以及gRNA表达盒序列:
分别取质粒p025-SNR52-HsCAS-KlADE2、p025-HsCAS-KlADE2、p025-SNR52-SpiCAS-KlADE2各1μg电转化入乳酸克鲁维酵母细胞中(以pUDP025质粒作为对照),需稀释涂平板计数。
实验结果如图5所示。转化前述质粒获得的转化子均可达2*104个以上,并且编辑效率最高的可达71%。其中p025-HsCAS-KlADE2质粒的编辑效率最高,且每μg DNA转化子数量可达22400个。对于不同的基因和基因组位点,编辑效率通常有显著差别,因此在保持相似的编辑效率的基础上,获得更多的转化子数量的基因编辑质粒将更有优势。
综合以上结果,p025-SNR52-HsCAS-KlADE2质粒、p025-HsCAS-KlADE2质粒、p025-SNR52-SpiCAS-KlADE2质粒均可以进行基因编辑,其中,p025-HsCAS-KlADE2质粒为最优基因编辑质粒。
进一步的,测试添加不同供体DNA后的基因编辑效率及同源重组效率:
通过电转化将实施案例5中制备的各供体DNA(1μg)和p025-HsCAS-KlADE2质粒(1μg)转化进入乳酸克鲁维酵母细胞中。
实验结果如图6和图7所示。根据图6,通过对比发现当加入不同供体DNA后,转化效率和非同源重组编辑效率均差距不大,距离ADE2 10bp(前文为距离ADE2)时获得的转化子最多,为3.71*104CFU/μg DNA;距离双链断裂(DSB)10bp(前文为距离ADE2)时编辑效率最高,为93.6%。根据图7,当供体DNA序列距离双链断裂(DSB)10bp时同源重组效率最高,随着距离变远同源重组的效率会降低。
本公开的上述实施例仅是为清楚地说明本公开所作的举例,而并非是对本公开的实施方式的限定。对于所属领域的普通技术人员来说,在上述说明的基础上还可以做出其它不同形式的变化或变动。这里无需也无法对所有的实施方式予以穷举。凡在本公开的精神和原则之内所作的任何修改、等同替换和改进等,均应包含在本公开权利要求的保护范围之内。
Claims (11)
1.一种基因组编辑载体,其中,所述基因组编辑载体选自由如下(a)-(f)组成的组中的任意一种:
(b)基因组编辑载体b,其核苷酸序列包含编码如SEQ ID NO:66所示的氨基酸序列的核苷酸序列或如SEQ ID NO:65所示的序列;
(a)基因组编辑载体a,其核苷酸序列包含如SEQ ID NO:45所示的序列和如SEQ ID NO:44所示的序列;
(c)基因组编辑载体c,其核苷酸序列包含如SEQ ID NO:45所示的序列、如SEQ ID NO:44所示的序列和如SEQ ID NO:46所示的序列;
(d)基因组编辑载体d,其核苷酸序列包含如SEQ ID NO:60所示的序列;
(e)基因组编辑载体e,其核苷酸序列包含如SEQ ID NO:61所示的序列;
(f)基因组编辑载体f,其核苷酸序列包含如SEQ ID NO:62所示的序列。
2.根据权利要求1所述的基因组编辑载体,其中,所述基因编辑载体选自由如下(a)-(f)组成的组中的任意一种:
(b)基因组编辑载体b,其核苷酸序列包含如SEQ ID NO:46所示的序列或如SEQ ID NO:65所示的序列;
(a)基因组编辑载体a,其核苷酸序列包含如SEQ ID NO:54所示的序列和如SEQ ID NO:55所示的序列;
(c)基因组编辑载体c,其核苷酸序列包含如SEQ ID NO:47所示的序列和如SEQ ID NO:48所示的序列;
(d)基因组编辑载体d,其核苷酸序列包含如SEQ ID NO:60所示的序列、如SEQ ID NO:63所示的序列和如SEQ ID NO:64所示的序列;
(e)基因组编辑载体e,其核苷酸序列包含如SEQ ID NO:61所示的序列、如SEQ ID NO:63所示的序列和如SEQ ID NO:64所示的序列;
(f)基因组编辑载体f,其核苷酸序列包含如SEQ ID NO:62所示的序列、如SEQ ID NO:63所示的序列和如SEQ ID NO:64所示的序列。
3.根据权利要求1所述的基因组编辑载体,其中,所述基因编辑载体选自如下(a)-(f)任一项所示的序列或所述基因编辑载体由如下(a)-(f)任一项所示的序列组成:
(b)基因组编辑载体b,其核苷酸序列包含如SEQ ID NO:58所示的序列和如SEQ ID NO:59所示的序列;
(a)基因组编辑载体a,其核苷酸序列包含如SEQ ID NO:54所示的序列、如SEQ ID NO:55所示的序列、如SEQ ID NO:56所示的序列和如SEQ ID NO:57所示的序列;
(c)基因组编辑载体c,其核苷酸序列包含如SEQ ID NO:47所示的序列和如SEQ ID NO:48所示的序列;
(d)基因组编辑载体d,其核苷酸序列包含如SEQ ID NO:60所示的序列、如SEQ ID NO:63所示的序列和如SEQ ID NO:64所示的序列;
(e)基因组编辑载体e,其核苷酸序列包含如SEQ ID NO:61所示的序列、如SEQ ID NO:63所示的序列和如SEQ ID NO:64所示的序列;
(f)基因组编辑载体f,其核苷酸序列包含如SEQ ID NO:62所示的序列、如SEQ ID NO:63所示的序列和如SEQ ID NO:64所示的序列。
4.一种基因组编辑***,其中,所述***包括如权利要求1-3任一项所述的基因组编辑载体。
5.一种宿主细胞,其中,所述宿主细胞包含有如权利要求1-3任一项所述的基因组编辑载体或权利要求4所述的基因组编辑***。
6.根据权利要求5所述的宿主细胞,其中,所述宿主细胞为乳酸克鲁维酵母的细胞。
7.一种基因组编辑方法,其中,所述基因组编辑方法包括利用基因编辑技术对权利要求5-6任一项宿主细胞进行编辑的步骤。
8.根据权利要求7所述的方法,其中,所述方法包括将权利要求4所述的基因组编辑***导入到权利要求5-6任一项所述的宿主细胞中,在所述宿主细胞中的待编辑的基因组位点进行基因组编辑。
9.根据权利要求7-8任一项所述的方法,其中,所述基因编辑技术为CRISPR技术。
10.根据权利要求1-3任一项所述的基因组编辑载体,权利要求4所述的基因组编辑***或如权利要求7-9任一项所述的基因组编辑方法在细胞中基因功能的研究和/或蛋白生产中的应用;优选的,所述蛋白为酶。
11.一种生产蛋白的方法,其中,所述方法中使用根据权利要求1-3任一项所述的基因组编辑载体,权利要求4所述的基因组编辑***或如权利要求7-9任一项所述的基因组编辑方法生产所述蛋白;优选的,所述蛋白为酶。
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