CN107574179A - 一种为克鲁维酵母优化的CRISPR/Cas9高效基因编辑系统 - Google Patents

一种为克鲁维酵母优化的CRISPR/Cas9高效基因编辑系统 Download PDF

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CN107574179A
CN107574179A CN201610814025.4A CN201610814025A CN107574179A CN 107574179 A CN107574179 A CN 107574179A CN 201610814025 A CN201610814025 A CN 201610814025A CN 107574179 A CN107574179 A CN 107574179A
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kluyveromyces
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郭敏
代田纯
李海洋
于雪
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Kang Code (shanghai) Biological Technology Co Ltd
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Abstract

本发明涉及一种专为克鲁维酵母优化的CRISPR/Cas9安全高效基因编辑系统,属于生物技术领域。现有技术中,酿酒酵母中使用的pCAS质粒上同时具有Cas9基因序列及gRNA元件,能够通过一次转化实现在酿酒酵母中的高效基因组改造,但是在克鲁维酵母中不能稳定复制和表达。本发明向克鲁维酵母细胞中转化Cas9/gRNA融合质粒,质粒靶向克鲁维细胞的内源性DNA序列,并产生双链切口;向克鲁维酵母细胞中转化供体DNA序列,该序列在双链切口处与靶位点产生同源重组,将Tag序列插入目标位点。本发明通过改造,构建了一种克鲁维酵母专用的,可以在克鲁维酵母中稳定复制、表达、并进行基因改造的新的安全、高效CRISPR/Cas9基因编辑系统。

Description

一种为克鲁维酵母优化的CRISPR/Cas9高效基因编辑系统
技术领域
本发明涉及一种专为克鲁维酵母优化的CRISPR/Cas9高效基因编辑系统,属于生物技术领域。
背景技术
微生物基因组改造主要依赖于内源性同源重组修复(endogenous homology-directed repair, HDR)和非同源性末端连接(non- homologous end joining, NHEJ) 两种生物学机制[1]。HDR的基本过程为,基因组发生双链断裂(double-strand breaks,DSBs)后,供体DNA(序列与断裂位点两侧序列同源)在断裂位点处与受损基因组序列发生同源重组,实现修复,这一过程不易引入插入或缺失突变[2]。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,这一过程容易产生插入或缺失突变,引起密码子位移[3]。在正常条件下,HDR引起同源重组的概率很低,但是通过内切酶在基因组上产生切口,并向细胞内转化线性的同源DNA片段,可以极大地增加重组效率。而且将内切酶与具有位点识别功能的元件相结合,能实现高效的、特异性的基因组改造。
CRISPR/Cas (Clustered Regulatory Interspaced Short PalindromicRepeats/CRISPR associated)是广泛存在于细菌和古细菌中的一种生物防御系统[4]。其中经过改造的Ⅱ型系统,CRISPR/Cas9,成为现在广泛使用的基因组改造工具[5]。在guideRNA (gRNA)的介导下,Cas9蛋白识别基因组上protospacer adjacent motif (PAM)及其上游20 bp序列,并在PAM上游3 bp位置产生双链切口。在同时提供供体DNA(donor DNA) 的情况下,被CRISPR/Cas9双链切开的基因可以HDR的方式重组入新的序列,以达到基因改造的目的[6]。在酿酒酵母(Saccharomyces cerevisiae)中,利用CRISPR/Cas9系统进行基因组改造的实例很多,包括基因点突变、基因敲除和基因插入等[7-9]。比如,在现有技术中流行的pCAS质粒上同时具有Cas9基因序列及gRNA元件[10],能够通过一次转化实现在酿酒酵母中的高效基因组改造。克鲁维酵母(Kluyveromyces)是一种子囊孢子酵母,其中的马克斯克鲁维酵母(Kluyveromyces marxianus)和乳酸克鲁维酵母(Kluyveromyceslactis)是工业上广泛使用的酵母。例如乳酸克鲁维酵母是一种能够以乳酸作为其唯一的碳源和能源的酵母。与其他酵母相比,乳酸克鲁维酵母具有许多优点,如超强的分泌能力,良好的大规模发酵特性、食品安全的级别及同时具有蛋白翻译后修饰的能力等,其作为宿主系统表达药用蛋白也已显示出巨大的潜力。但通常酿酒酵母的质粒作为一种2µ质粒(包括pCAS),在克鲁维酵母中不能稳定复制和表达[11]。在目前报道的克鲁维酵母CRISPR/Cas9改造系统中,Cas9基因被直接插入到酵母基因组中,造成Cas9蛋白的持续表达,对克鲁维酵母的工业生产存在一定安全隐患[12],所以构建一个能够在克鲁维酵母中稳定复制和表达的高效安全CRISPR/Cas9系统是生物技术发展必需的,也将是生物医药领域上的一大进步。
发明内容
为了克服现有技术中的缺陷,本发明提供了一种专为克鲁维酵母优化的CRISPR/Cas9高效基因编辑系统,使得CRISPR/Cas9能在克鲁维酵母中稳定复制,安全高效表达,并完成克鲁维酵母基因组的高效编辑。
本发明解决其技术问题所采用的技术方案是:一方面,本发明提供了一种利用优化的CRISPR/Cas9系统对克鲁维酵母基因组进行改造的方法,包括下述步骤:
a)向所述克鲁维酵母细胞中转化Cas9/gRNA融合质粒,该质粒靶向所述细胞的内源性DNA序列,并产生双链切口;
b)向所述克鲁维酵母细胞中转化供体DNA序列,该序列在双链切口处与靶位点产生同源重组,将Tag序列插入目标位点。
在本发明中,所述的克鲁维酵母为乳酸克鲁维酵母,改造的基因为乳酸克鲁维酵母的细胞质苏氨酸氨酰tRNA合成酶Threonyl-tRNAsynthetase(Kl-TRS)基因。先检索Kl-TRS基因序列(http://www.uniprot.org/),确定gRNA序列。
在本发明中,对Kl-TRS基因的改造为在基因末端插入一段长1302 碱基对(bp)的标记Tag序列。
在本发明中,对原始pCAS质粒进行一系列改造,最终使标记Tag可以高效插入克鲁维酵母基因组中,并在克鲁维酵母中稳定遗传和表达。具体改造包括下述步骤:
a)在pCAS质粒中插入pKD1 stabilizing element (SE)序列,构建成质粒pKM-Cas9-SE;
b)将pKM-Cas9-SE质粒中gRNA启动子置换为KlSNR52基因启动子,构建成质粒pKM-Cas9-SE-pKlSNR52;
c)将pKM-Cas9-SE-pKlSNR52质粒中Cas9序列替换为适合克鲁维酵母表达的KLCas9,构建成质粒pKM-KLCas9-SE-pKlSNR52;
d)将pKM-KLCas9-SE-pKlSNR52质粒中抗性基因Kan置换为Amp,完成质粒的最终改造,命名为pKM-CAS1.0;
e)将靶向Kl-TRS基因的gRNA插入pKM-Cas9中,构建成质粒pKM-CAS1.0-TRS1(SEQ ID NO.1)。
在本发明中,转化的供体DNA序列为线性双链DNA,该线性双链供体DNA序列包括插入靶位点的Tag序列以及与靶位点两侧序列同源的序列,具体构建与扩增步骤包括下述步骤:
a)以乳酸克鲁维酵母菌液为模板扩增同源臂序列,将同源臂序列插入pMD18质粒中,构建成中间质粒pKM-TRS-DD1;
b)将Tag序列插入pKM-TRS-DD1质粒两同源臂中间位置,构建成最终质粒pKM-T-DD2;
c)以pKM-TRS-DD2为模板进行PCR扩增,得到线性双链供体DNA(SEQ ID NO.2)。
在本发明中,接着通过LiAc/SS carrier DNA/PEG方法将质粒转化进入克鲁维酵母细胞中,包括下述步骤:
a)将pKM-CAS1.0-TRS1质粒和线性供体DNA 同时转化进入克鲁维酵母细胞中;
b)涂板后挑取单克隆于1 mL液体YPD培养基中振荡培养。
在本发明中,通过PCR扩增检测Tag序列插入靶位点效率,在Tag序列内设计一条引物,在靶基因上位于同源臂外侧的序列设计一条引物,以单克隆菌液为模板进行PCR扩增,电泳检测阳性的为插入成功。
在另一方面,本发明涉及一种CRISPR/Cas9质粒,包含与SEQ ID NO. 1至少80%序列相同的核苷酸。
在另一方面,本发明涉及一种线性双链DNA,包含与SEQ ID NO.2至少80%序列相同的核苷酸。
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普通技术人员通常所理解的相同。本文中述及的所有出版物和其他参考文献都通过引用纳入本文。
本发明的有益效果是: 本发明通过改造,构建了一种在克鲁维酵母中稳定复制和表达的质粒,使得CRISPR/Cas9能在克鲁维酵母中完成稳定、高效、安全的基因编辑的系统。
附图说明
下面结合附图和实施例对本发明进一步说明。
图1是pKM-CAS1.0-TRS1与pCAS质粒转化克鲁维酵母效率对比图,pKM-CAS1.0-TRS1转化克鲁维酵母效率明显高于pCAS质粒的转化效率。
图2是Kl-TRS基因gRNA位置示意图,其中方框中为PAM序列,下划线表示gRNA序列,箭头指示Cas9酶切位点。其中Kl-TRS为乳酸克鲁维酵母细胞质色氨酸氨酰tRNA合成酶(Threonyl-tRNAsynthetase)基因,位于染色体F的567803..570037位点。
图3是pKM-CAS1.0-TRS1质粒图谱。
图4是pKM-TRS-DD2质粒图谱。
图5包括图5a和5b,是基因组插入Tag序列的PCR验证结果图,图5a为琼脂糖凝胶电泳图,其电泳条带大小为1274bp,阳性率达80%以上,图5b为三次重复试验的误差条形图,三次实验结果误差不明显,表明实验结果相对稳定。
具体实施方式
以下结合具体实施例和附图说明对本发明做进一步的说明,但下述的实施例并非用于限定本发明的保护范围。
实施例1—KL-CAS1.0系统改造
pKM-Cas9/gRNA质粒构建
现有技术中,酿酒酵母中使用的pCAS质粒上同时具有Cas9基因序列及gRNA元件,能够通过一次转化实现在酿酒酵母中的高效基因组改造,但是在克鲁维酵母中不能稳定复制和表达,如图1所示。本发明通过改造,构建了一种克鲁维酵母专用的,可以在克鲁维酵母中稳定复制、表达、并进行基因改造的新的安全高效CRISPR/Cas9基因编辑系统,将该基因编辑系统命名为KL-CAS1.0。本发明的克鲁维酵母以乳酸克鲁维酵母为实施例作说明,但不以此为限。
KL-CAS1.0高效pKD1 stabilizing element (SE)元件插入
pKD1是克鲁维酵母转化的常用质粒[13],为了构建一种能在克鲁维酵母中稳定复制并表达的质粒,本发明在pCAS质粒中首先插入pKD1中的SE元件[14]。
pKD1SE元件(SEQ ID NO.3)由上海生工合成(上海,中国),并以引物pKD1SE-F1:GGACGCTCGAAGCCGCGGTGAGCAAAAG和pKD1SE-R1: CCTATGGAAAAACGCCAGCAACGCGGCC进行扩增。以pCAS质粒为模板,以引物pCAS-F1: AGGAACCGTAAAAAGGCCG和pCAS-R1:GGCCTTTTGCTGGCCTTT进行扩增。将pCAS扩增产物8.5 µL与pKD1SE扩增产物8.5 µL混合,加入1 µLDpn I,2 µL10×digestion buffer,37oC温浴3 h。将Dpn I处理后产物10 µL加入100µL DH5α感受态细胞中,冰上放置30 min,42oC热激45 s后,加入1 mL LB液体培养基37oC振荡培养1 h,涂布于Kan抗性LB固体培养,37oC倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养,PCR检测阳性并测序确认后,提取质粒保存,命名为pKM-Cas9-SE。
KL-CAS1.0高效gRNA启动子置换
SNR52基因启动子是一种RNA polymerase III (Pol III)启动子,曾用于酿酒酵母CRISPR系统gRNA转录[7,15]。为了保证pKM-Cas9-SE质粒中gRNA在克鲁维酵母中高效转录,本发明第二步将pKM-Cas9-SE质粒中gRNA启动子置换为乳酸克鲁维酵母中SNR52基因的启动子。
以乳酸克鲁维酵母菌液为模板,以引物pKlSNR52-F1:TTATGCTTAAATGCGTATATGTGTTATGTATTGGTGAACCCAATGGGAAA和引物pKlSNR52-R1:AGCGAGGAGGCTGGGACCATGCCGGCCATCGTTACTTTCTCGGCAGTTCG进行扩增,得到KlSNR52启动子序列(位于染色体F的1157521...1157897位点)。以pKM-Cas9-SE质粒为模板,以引物pKM-Cas9-F1: GATGGCCGGCATGGTCCC和引物pKM-Cas9-R1:TACATAACACATATACGCATTTAAGCATAAACACGCAC进行扩增。将两次扩增产物各8.5 µL,1 µLDpnI,2 µL10×digestion buffer混合,37oC温浴3 h。将Dpn I处理后产物10 µL加入100µLDH5α感受态细胞中,冰上放置30 min,42oC热激45 s后,加入1 mL LB液体培养基37oC振荡培养1 h,涂布于Kan抗性LB固体培养,37oC倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养,PCR检测阳性并测序确认后,提取质粒保存,命名为pKM-Cas9-SE-pKlSNR52。
KL-CAS1.0中乳酸克鲁维酵母专用Cas9的序列置换
为提高Cas9蛋白在乳酸克鲁维酵母中的活性,本发明将原质粒中Streptococcus pyogenes Cas9基因序列置换为优化过的,适合乳酸克鲁维酵母表达的KLCas9
KLCas9序列由上海生工合成,并插入在pUC57质粒中。以pUC57-KLCas9质粒为模板,以pKM-Cas9-F1:TTAATACACGTATTTATTTGTCCAATTACCATGGATAAGAAATACTCTATCGGTTTG和引物pKM-Cas9-R1: AACTTTTCTTTTCTTTTTTGGCCCTCCACCATCACCACCTAATTGAGACAAAT进行扩增,得到KLCas9基因产物。以pKM-Cas9-SE-pKlSNR52质粒为模板,以pKM-Cas9-F2:GGTGGAGGGCCAAAAAAGAAAAG和pKL-Cas9-R2: GGTAATTGGACAAATAAATACGTGT进行扩增。将两次扩增产物各8.5 µL,1 µL Dpn I,2 µL10×digestion buffer混合,37oC温浴3 h。将DpnI处理后产物10 µL加入100µL DH5α感受态细胞中,冰上放置30 min,42oC热激45 s后,加入1 mL LB液体培养基37oC振荡培养1 h,涂布于Kan抗性LB固体培养,37oC倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养,PCR检测阳性并测序确认后,提取质粒保存,命名为pKM-KLCas9-SE-pKlSNR52。
KM-CAS1.0质粒抗性基因置换
因为原质粒与供体DNA中的抗性基因都是Kan,为了便于最终阳性克隆的筛选,本发明将原质粒中Kan基因置换为Amp基因。以pKM-KLCas9-SE-pKlSNR52质粒为模板,以pKM-Cas9-F3: AGACCCCGTAGAAAAGATCAAAGGATCTTCCTGTCGATTCGATACTAACGCC和pKM-Cas9-R3: TTAGAAAAATAAACAAATAGGGGTTCCGCGGCTGGCCGGGTGACCCGGCG进行扩增。以pMD18质粒为模板,以Amp-F1: CGCGGAACCCCTATTTGTTT和Amp-R1: GAAGATCCTTTGATCTTTTCTACGGG进行扩增。将两次扩增产物各8.5 µL,1 µLDpn I,2 µL10×digestion buffer混合,37oC温浴3 h。将Dpn I处理后产物10 µL加入100µL DH5α感受态细胞中,冰上放置30 min,42oC热激45 s后,加入1 mL LB液体培养基37oC振荡培养1 h,涂布于Amp抗性LB固体培养,37oC倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养,PCR检测阳性并测序确认后,得到最终改造完成质粒,命名为pKM-CAS1.0。
2.实施例2—靶基因及gRNA序列确定
2A.乳酸克鲁维酵母靶标基因鉴定
在网站http://www.uniprot.org/进行序列搜索,物种“Kluyveromyceslactis”,关键词“ThreoninetRNAsynthetase”“ThrRS”“TRS”。这里以在此基因尾部插入一段标记DNA为例,其他目标基因或插入位置、序列均可采用类似方法操作。
i.在乳酸克鲁维酵母中存在两种TRS,分别存在于细胞质和线粒体中。检索到蛋白序列以后,在网站https://ihg.gsf.de/ihg/mitoprot.htmL上进行分析,确定检索蛋白存在于细胞质(不含线粒体识别序列,真核同源)还是线粒体(存在线粒体识别序列,原核同源),挑选细胞质TRS基因(http://www.uniprot.org/uniprot/Q6CL41) 作为目标基因,并命名为Kl-TRS
ii.下载基因序列并在网址 http://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE-TYPE=BlastSearch&PROG-DEF=blastn&BLAST-PROG-DEF=megaBlast&BLAST-SPEC=OGP--28985--12363上进行BLAST比对分析,确定基因的染色体定位,并获取基因两端相邻序列信息。
gRNA序列确定
Kl-TRS基因终止密码子附近搜索PAM序列(NGG),选择位于终止密码子上游,且最靠近终止密码子的PAM(位于染色体F的570038...570040位点),并确定Kl-TRSgRNA序列(CTGATAATGTCTTGGCTTAA,位于染色体F的570018...570037位点),如图2所示。
3.实施例3—靶序列pKM-Cas9质粒构建
本发明通过PCR-同源重组方法,将原质粒中的gRNA序列置换为Kl-TRSgRNA序列。具体步骤为:以pKM-CAS1.0质粒为模板,以引物pKM-Cas9-TRS-F1:CTTTCTGATAATGTCTTGGCTTAAGTTTTAGAGCTAGAAATAGCAAG和引物pKM-Cas9-TRS-R1:GCTCTAAAACTTAAGCCAAGACATTATCAGAAAGTCCCATTCGCCAC进行扩增。将扩增产物17 µL,1 µLDpn I,2 µL10×digestion buffer混合,37oC温浴3 h。将Dpn I处理后产物10 µL加入100µL DH5α感受态细胞中,冰上放置30 min,42oC热激45 s后,加入1 mL LB液体培养基37oC振荡培养1 h,涂布于Amp抗性LB固体培养,37oC倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养,PCR检测阳性并测序确认后,提取质粒保存,命名为pKM-CAS1.0-TRS1,如图3所示。
4.实施例4—供体 DNA质粒构建及线性供体DNA扩增
为了便于线性供体DNA的保存及扩增,本发明首先将供体DNA插入到pMD18质粒中,然后通过PCR扩增得到线性供体DNA序列。具体步骤为:以pMD18质粒为模板,以引物pMD18-F1:ATCGTCGACCTGCAGGCATG和引物pMD18-R1: ATCTCTAGAGGATCCCCGGG进行扩增。以乳酸克鲁维酵母菌液为模板,以引物KLLA-T-LF1:GAGCTCGGTACCCGGGGATCCTCTAGAGATTTTAATGTTTAAGGCTCGTGAACGTT和引物KLLA-T-RR1:GCCAAGCTTGCATGCCTGCAGGTCGACGATTTATCTATGTTTATTGGCACACAAGC进行扩增。将两次扩增产物各8.5 µL,1 µLDpn I,2 µL10×digestion buffer混合,37oC温浴3 h。将Dpn I处理后产物10 µL加入100µL DH5α感受态细胞中,冰上放置30 min,42oC热激45 s后,加入1 mL LB液体培养基37oC振荡培养1 h,涂布于Amp抗性LB固体培养,37oC倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养,PCR检测阳性并测序确认后,提取质粒保存,命名为pKM-T-DD1。
以pKM-TRS-DD1为模板,以引物Thr-F1:TTTCATTTGATGCTCGATGAGTTTTTCTAAAGGAATATCCAAACCGATCA和引物Thr-R1:ATTATACCATGTTCCTGTGATACCGGCTTCAGCCAAGACATTATCAGCTC进行扩增。以Tag质粒为模板,以引物Tag-F1: GAAGCCGGTATCACAGGAAC和引物Tag-R1: TTAGAAAAACTCATCGAGCATCAAATG进行扩增。将两次扩增产物各8.5 µL,1 µLDpn I,2 µL10×digestion buffer混合,37oC温浴3h。将Dpn I处理后产物10 µL加入100µL DH5α感受态细胞中,冰上放置30 min,42oC热激45s后,加入1 mL LB液体培养基37oC振荡培养1 h,涂布于Amp抗性LB固体培养,37oC倒置培养至单克隆长出。挑取5个单克隆在LB液体培养基中振荡培养,PCR检测阳性并测序确认后,提取质粒保存,命名为pKM-TRS-DD2,如图4所示。
以pKM-T-DD2质粒为模板,以引物KLLA-T-LF1:GAGCTCGGTACCCGGGGATCCTCTAGAGATTTTAATGTTTAAGGCTCGTGAACGTT和引物KLLA-T-RR1:GCCAAGCTTGCATGCCTGCAGGTCGACGATTTATCTATGTTTATTGGCACACAAGC进行扩增,得到线性供体DNA。
5.实施例5—优化的乳酸克鲁维酵母感受态制备和转化
本发明基于文献[16]中酵母感受态制备及转化方法,经过优化,应用于乳酸克鲁维酵母。
乳酸克鲁维酵母感受态制备
将乳酸克鲁维酵母菌液在YPD固体培养基上划线并挑取单克隆,于25 mL 2×YPD液体培养基中振荡培养过夜,取2 mL菌液于50 mL液体2×YPD培养基中继续振荡培养2-8 h。20oC条件下3000 g离心5 min收集酵母细胞,加入500 µL无菌水重悬,同样条件下离心收集细胞。配制感受态细胞溶液(5% v/v甘油,10% v/v DMSO)并将酵母细胞溶解于500 µL该溶液中。分装50 µL至1.5 mL离心管中,-80oC保存。
乳酸克鲁维酵母感受态转化
将感受态细胞置于37oC融化15-30 s,13000 g离心2 min并去除上清。配制转化缓冲液:PEG 3350 (50% (w/v)) 260 µL,LiAc (1.0 M) 36 µL,carrier DNA (5.0 mg/mL) 20µL,Cas9/gRNA质粒15µL,供体DNA 5 µL,加入无菌水至最终体积360 µL。热激后,13000 g离心30 s去除上清。加入1 mL YPD液体培养基,培养2-3 h,吸取200 µL涂布于固体YPD(200 µg/mL G418)培养基,培养2-3天至单菌落出现。
6.实施例6—乳酸克鲁维酵母基因组插入Tag序列检测
在乳酸克鲁维酵母转化后的平板上挑取10-20个单克隆,置于1 mL YPD(200 µg/mLG418)液体培养基中振荡培养过夜,以菌液为模板,以引物Tag-R2(Tag序列内引物):ACATACGAGCCTTCAGCATTACCAC和引物Thr-F2(供体DNA 5’外侧引物):CACGGTACCAGAATTTACAACACT进行PCR扩增检测,有阳性条带表明Tag序列插入靶位点成功,如图5a所示。同时图5b为三次重复试验的误差条形图,三次实验结果误差不明显,表明实验结果相对稳定。
参考文献
1.Jakočiūnas,T.,M.K.Jensen,and J.D.Keasling,CRISPR/Cas9 advances engineering of microbial cell factories.Metabolic Engineering, 2016. 34: p. 44-59.
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SEQUENCE LISTING
<110> 康码(上海)生物科技有限公司
<120> 一种为克鲁维酵母优化的CRISPR/Cas9高效基因编辑系统
<130>
<160> 3
<170> PatentIn version 3.5
<210> 1
<211> 8956
<212> DNA
<213> 重组质粒
<400> 1
gtatatgtgt tatgtagtat actctttctt caacaattaa atactctcgg tagccaagtt 60
ggtttaaggc gcaagactgt aatttatcac tacgaaatct tgagatcggg cgttcgactc 120
gcccccggga gagatggccg gcatggtccc agcctcctcg ctggcgccgg ctgggcaaca 180
ccttcgggtg gcgaatggga ctttctgata atgtcttggc ttaagtttta gagctagaaa 240
tagcaagtta aaataaggct agtccgttat caacttgaaa aagtggcacc gagtcggtgc 300
tttttttatt ttttgtcact attgttatgt aaaatgccac ctctgacagt atggaacgca 360
aacttctgtc tagtggatat aggtctagag atctgtttag cttgcctcgt ccccgccggg 420
tcacccggcc agccgcggaa cccctatttg tttatttttc taaatacatt caaatatgta 480
tccgctcatg agacaataac cctgataaat gcttcaataa tattgaaaaa ggaagagtat 540
gagtattcaa catttccgtg tcgcccttat tccctttttt gcggcatttt gccttcctgt 600
ttttgctcac ccagaaacgc tggtgaaagt aaaagatgct gaagatcagt tgggtgcacg 660
agtgggttac atcgaactgg atctcaacag cggtaagatc cttgagagtt ttcgccccga 720
agaacgtttt ccaatgatga gcacttttaa agttctgcta tgtggcgcgg tattatcccg 780
tattgacgcc gggcaagagc aactcggtcg ccgcatacac tattctcaga atgacttggt 840
tgagtactca ccagtcacag aaaagcatct tacggatggc atgacagtaa gagaattatg 900
cagtgctgcc ataaccatga gtgataacac tgcggccaac ttacttctga caacgatcgg 960
aggaccgaag gagctaaccg cttttttgca caacatgggg gatcatgtaa ctcgccttga 1020
tcgttgggaa ccggagctga atgaagccat accaaacgac gagcgtgaca ccacgatgcc 1080
tgtagcaatg gcaacaacgt tgcgcaaact attaactggc gaactactta ctctagcttc 1140
ccggcaacaa ttaatagact ggatggaggc ggataaagtt gcaggaccac ttctgcgctc 1200
ggcccttccg gctggctggt ttattgctga taaatctgga gccggtgagc gtgggtctcg 1260
cggtatcatt gcagcactgg ggccagatgg taagccctcc cgtatcgtag ttatctacac 1320
gacggggagt caggcaacta tggatgaacg aaatagacag atcgctgaga taggtgcctc 1380
actgattaag cattggtaac tgtcagacca agtttactca tatatacttt agattgattt 1440
aaaacttcat ttttaattta aaaggatcta ggtgaagatc ctttttgata atctcatgac 1500
caaaatccct taacgtgagt tttcgttcca ctgagcgtca gaccccgtag aaaagatcaa 1560
aggatcttcc tgtcgattcg atactaacgc cgccatccag tgtcgaaaac gagctctcga 1620
gaacccttaa tgtcgacagt cgaacaagaa gcaggcaaag tttagagcac tgcccctccg 1680
cactcaaaaa agaaaaaact aggaggaaaa taaaattctc aaccacacaa acacataaac 1740
acatacaaat acaaatacaa gcttatttac ttgacatcgc gcgatcttcc actattcagc 1800
gccgtccgcc ctctctcgtg ttttttgttt acgcgacaac tatgcgaaat ccggagcaac 1860
gggcaaccgt ttggggaaag accacaccca cgcgcgatcg ccatggcaac gaggtcgcac 1920
acgccccaca cccagacctc cctgcgagcg ggcatgggta caatgtcccc gttgccacag 1980
acaccacttc gtagcacagc gcagagcgta gcgtgttgtt gctgctgaca aaagaaaatt 2040
tttcttagca aagcaaagga ggggaagcac gggcagatag caccgtacca tacccttgga 2100
aactcgaaat gaacgaagca ggaaatgaga gaatgagagt tttgtaggta tatatagcgg 2160
tagtgtttgc gcgttaccat catcttctgg atctatctat tgttcttttc ctcatcactt 2220
tccccttttt cgctcttctt cttgtctttt atttctttct tttttttaat tgttccctcg 2280
attggctatc taccaaagaa tccaaactta atacacgtat ttatttgtcc aattaccatg 2340
gacaagaagt actccattgg gctcgatatc ggcacaaaca gcgtcggttg ggccgtcatt 2400
acggacgagt acaaggtgcc gagcaaaaaa ttcaaagttc tgggcaatac cgatcgccac 2460
agcataaaga agaacctcat tggcgccctc ctgttcgact ccggggagac ggccgaagcc 2520
acgcggctca aaagaacagc acggcgcaga tatacccgca gaaagaatcg gatctgctac 2580
ctgcaggaga tctttagtaa tgagatggct aaggtggatg actctttctt ccataggctg 2640
gaggagtcct ttttggtgga ggaggataaa aagcacgagc gccacccaat ctttggcaat 2700
atcgtggacg aggtggcgta ccatgaaaag tacccaacca tatatcatct gaggaagaag 2760
cttgtagaca gtactgataa ggctgacttg cggttgatct atctcgcgct ggcgcatatg 2820
atcaaatttc ggggacactt cctcatcgag ggggacctga acccagacaa cagcgatgtc 2880
gacaaactct ttatccaact ggttcagact tacaatcagc ttttcgaaga gaacccgatc 2940
aacgcatccg gagttgacgc caaagcaatc ctgagcgcta ggctgtccaa atcccggcgg 3000
ctcgaaaacc tcatcgcaca gctccctggg gagaagaaga acggcctgtt tggtaatctt 3060
atcgccttat ccttaggcct gacccccaac tttaaatcta acttcgacct ggccgaagat 3120
gccaagcttc aactgagcaa agacacctac gatgatgatc tcgacaatct gctggcccag 3180
atcggcgacc agtacgcaga cctttttttg gcggcaaaga acctgtcaga cgccattctg 3240
ctgagtgata ttctgcgagt gaacacggag atcaccaaag ctccgctgag cgctagtatg 3300
atcaagcgct atgatgagca ccaccaagac ttgactttgc tgaaggccct tgtcagacag 3360
caactgcctg agaagtacaa ggaaattttc ttcgatcagt ctaaaaatgg ctacgccgga 3420
tacattgacg gcggagcaag ccaggaggaa ttttacaaat ttattaagcc catcttggaa 3480
aaaatggacg gcaccgagga gctgctggta aagcttaaca gagaagatct gttgcgcaaa 3540
cagcgcactt tcgacaatgg aagcatcccc caccagattc acctgggcga actgcacgct 3600
atcctcaggc ggcaagagga tttctacccc tttttgaaag ataacaggga aaagattgag 3660
aaaatcctca catttcggat accctactat gtaggccccc tcgcccgggg aaattccaga 3720
ttcgcgtgga tgactcgcaa atcagaagag accatcactc cctggaactt cgaggaagtc 3780
gtggataagg gggcctctgc ccagtccttc atcgaaagga tgactaactt tgataaaaat 3840
ctgcctaacg aaaaggtgct tcctaaacac tctctgctgt acgagtactt cacagtttat 3900
aacgagctca ccaaggtcaa atacgtcaca gaagggatga gaaagccagc attcctgtct 3960
ggagagcaga agaaagctat cgtggacctc ctcttcaaga cgaaccggaa agttaccgtg 4020
aaacagctca aagaagacta tttcaaaaag attgaatgtt tcgactctgt tgaaatcagc 4080
ggagtggagg atcgcttcaa cgcatccctg ggaacgtatc acgatctcct gaaaatcatt 4140
aaagacaagg acttcctgga caatgaggag aacgaggaca ttcttgagga cattgtcctc 4200
acccttacgt tgtttgaaga tagggagatg attgaagaac gcttgaaaac ttacgctcat 4260
ctcttcgacg acaaagtcat gaaacagctc aagaggcgcc gatatacagg atgggggcgg 4320
ctgtcaagaa aactgatcaa tgggatccga gataagcagt ctggaaagac aatcctggat 4380
tttcttaagt ccgatggatt tgccaaccgg aacttcatgc agttgatcca tgatgactct 4440
ctcaccttta aggaggacat ccagaaagca caagtttctg gccaggggga cagtcttcac 4500
gagcacatcg ctaatcttgc aggtagccca gctatcaaaa agggaatact gcagaccgtt 4560
aaggtcgtgg atgaactcgt caaagtaatg ggaaggcata agcccgagaa tatcgttatc 4620
gagatggccc gagagaacca aactacccag aagggacaga agaacagtag ggaaaggatg 4680
aagaggattg aagagggtat aaaagaactg gggtcccaaa tccttaagga acacccagtt 4740
gaaaacaccc agcttcagaa tgagaagctc tacctgtact acctgcagaa cggcagggac 4800
atgtacgtgg atcaggaact ggacatcaat cggctctccg actacgacgt ggatcatatc 4860
gtgccccagt cttttctcaa agatgattct attgataata aagtgttgac aagatccgat 4920
aaaaatagag ggaagagtga taacgtcccc tcagaagaag ttgtcaagaa aatgaaaaat 4980
tattggcggc agctgctgaa cgccaaactg atcacacaac ggaagttcga taatctgact 5040
aaggctgaac gaggtggcct gtctgagttg gataaagccg gcttcatcaa aaggcagctt 5100
gttgagacac gccagatcac caagcacgtg gcccaaattc tcgattcacg catgaacacc 5160
aagtacgatg aaaatgacaa actgattcga gaggtgaaag ttattactct gaagtctaag 5220
ctggtctcag atttcagaaa ggactttcag ttttataagg tgagagagat caacaattac 5280
caccatgcgc atgatgccta cctgaatgca gtggtaggca ctgcacttat caaaaaatat 5340
cccaagcttg aatctgaatt tgtttacgga gactataaag tgtacgatgt taggaaaatg 5400
atcgcaaagt ctgagcagga aataggcaag gccaccgcta agtacttctt ttacagcaat 5460
attatgaatt ttttcaagac cgagattaca ctggccaatg gagagattcg gaagcgacca 5520
cttatcgaaa caaacggaga aacaggagaa atcgtgtggg acaagggtag ggatttcgcg 5580
acagtccgga aggtcctgtc catgccgcag gtgaacatcg ttaaaaagac cgaagtacag 5640
accggaggct tctccaagga aagtatcctc ccgaaaagga acagcgacaa gctgatcgca 5700
cgcaaaaaag attgggaccc caagaaatac ggcggattcg attctcctac agtcgcttac 5760
agtgtactgg ttgtggccaa agtggagaaa gggaagtcta aaaaactcaa aagcgtcaag 5820
gaactgctgg gcatcacaat catggagcga tcaagcttcg aaaaaaaccc catcgacttt 5880
ctcgaggcga aaggatataa agaggtcaaa aaagacctca tcattaagct tcccaagtac 5940
tctctctttg agcttgaaaa cggccggaaa cgaatgctcg ctagtgcggg cgagctgcag 6000
aaaggtaacg agctggcact gccctctaaa tacgttaatt tcttgtatct ggccagccac 6060
tatgaaaagc tcaaagggtc tcccgaagat aatgagcaga agcagctgtt cgtggaacaa 6120
cacaaacact accttgatga gatcatcgag caaataagcg aattctccaa aagagtgatc 6180
ctcgccgacg ctaacctcga taaggtgctt tctgcttaca ataagcacag ggataagccc 6240
atcagggagc aggcagaaaa cattatccac ttgtttactc tgaccaactt gggcgcgcct 6300
gcagccttca agtacttcga caccaccata gacagaaagc ggtacacctc tacaaaggag 6360
gtcctggacg ccacactgat tcatcagtca attacggggc tctatgaaac aagaatcgac 6420
ctctctcagc tcggtggaga cggtggaggg ccaaaaaaga aaagaaaagt tgaagatgct 6480
tctgctcatc atcaccatca ccatcaccat taaatccgct ctaaccgaaa aggaaggagt 6540
tagacaacct gaagtctagg tccctattta tttttttata gttatgttag tattaagaac 6600
gttatttata tttcaaattt ttcttttttt tctgtacaga cgcgtgtacg catgtaacat 6660
tatactgaaa accttgcttg agaaggtttt gggacgctcg aagccgcggt gagcaaaagg 6720
ccagcaaaag gccgcggccg ccttatagcc tcagggggtg aaacactcct tctgggctca 6780
agtgactttt tgaaatttca gctgagcctc aggtgagtga aagcgctctc aagactcagg 6840
tgatgtttta taattccggc tgagcctcaa gcgacttttt taaatttcac ttcagcccca 6900
agagagaatt cgcgctgctg agcctcaaaa gcgtaaacca ccccctgagc tcagggggac 6960
tgtttttttg catatttcaa cacgacatgg gcattttggt tattggatgt agaatggttg 7020
aataaacgtg attgtaaaat agagtttgta actacgaata attagttttt gagaagtttg 7080
gtgaatttaa tatttgtatg aggaaagtaa attttaatac ctaaataaac aaaaatatat 7140
ggtgacgtcg gatccaggaa ccgtaaaaag gccgcgttgc tggcgttttt ccataggctc 7200
cgcccccctg acgagcatca caaaaatcga cgctcaagtc agaggtggcg aaacccgaca 7260
ggactataaa gataccaggc gtttccccct ggaagctccc tcgtgcgctc tcctgttccg 7320
accctgccgc ttaccggata cctgtccgcc tttctccctt cgggaagcgt ggcgctttct 7380
catagctcac gctgtaggta tctcagttcg gtgtaggtcg ttcgctccaa gctgggctgt 7440
gtgcacgaac cccccgttca gcccgaccgc tgcgccttat ccggtaacta tcgtcttgag 7500
tccaacccgg taagacacga cttatcgcca ctggcagcag ccactggtaa caggattagc 7560
agagcgaggt atgtaggcgg tgctacagag ttcttgaagt ggtggcctaa ctacggctac 7620
actagaagga cagtatttgg tatctgcgct ctgctgaagc cagttacctt cggaaaaaga 7680
gttggtagct cttgatccgg caaacaaacc accgctggta gcggtggttt ttttgtttgc 7740
aagcagcaga ttacgcgcag aaaaaaagga tctcaagaag atcctttgat cttttctacc 7800
gaagcatctg tgcttcattt tgtagaacaa aaatgcaacg cgagagcgct aatttttcaa 7860
acaaagaatc tgagctgcat ttttacagaa cagaaatgca acgcgaaagc gctattttac 7920
caacgaagaa tctgtgcttc atttttgtaa aacaaaaatg caacgcgaga gcgctaattt 7980
ttcaaacaaa gaatctgagc tgcattttta cagaacagaa atgcaacgcg agagcgctat 8040
tttaccaaca aagaatctat acttcttttt tgttctacaa aaatgcatcc cgagagcgct 8100
atttttctaa caaagcatct tagattactt tttttctcct ttgtgcgctc tataatgcag 8160
tctcttgata actttttgca ctgtaggtcc gttaaggtta gaagaaggct actttggtgt 8220
ctattttctc ttccataaaa aaagcctgac tccacttccc gcgtttactg attactagcg 8280
aagctgcggg tgcatttttt caagataaag gcatccccga ttatattcta taccgatgtg 8340
gattgcgcat actttgtgaa cagaaagtga tagcgttgat gattcttcat tggtcagaaa 8400
attatgaacg gtttcttcta ttttgtctct atatactacg tataggaaat gtttacattt 8460
tcgtattgtt ttcgattcac tctatgaata gttcttacta caattttttt gtctaaagag 8520
taatactaga gataaacata aaaaatgtag aggtcgagtt tagatgcaag ttcaaggagc 8580
gaaaggtgga tgggtaggtt atatagggat atagcacaga gatatatagc aaagagatac 8640
ttttgagcaa tgtttgtgga agcggtattc gcaatatttt agtagctcgt tacagtccgg 8700
tgcgtttttg gttttttgaa agtgcgtctt cagagcgctt ttggttttca aaagcgctct 8760
gaagttccta tactttctag agaataggaa cttcggaata ggaacttcaa agcgtttccg 8820
aaaacgagcg cttccgaaaa tgcaacgcga gctgcgcaca tacagctcac tgttcacgtc 8880
gcacctatat ctgcgtgttg cctgtatata tatatacatg agaagaacgg catagtgcgt 8940
gtttatgctt aaatgc 8956
<210> 2
<211> 3193
<212> DNA
<213> 重组质粒
<400> 2
tttaatgttt aaggctcgtg aacgttctta tagagaattg ccatggagag ttgctgattt 60
cggtgtcatt cacagaaacg aattctccgg tgctttgtct ggtttgactc gtgtaagaag 120
attccaacaa gacgatgctc acattttctg tacccaagac caaatcgaaa atgaaatcgc 180
taacattttc gatttcttaa aatacgttta tggtgtgttc ggtttcgaat tcaagatgga 240
actatccact agaccagaaa aatacgttgg tgaactagag acctggaaca atgcggaatc 300
caaacttgaa actgctttga agaagtgggg tggtaactgg gaattaaatc caggtgatgg 360
tgctttctac ggtccaaaga tcgatatcat gatttccgat gccctaaaga gatggcatca 420
atgtgctacc attcaattgg atttccaact accaaataga tttgaattgg aattcaaaac 480
caaggaattg gaaggtagcg acaattacga aagaccggtc atgattcatc gtgctatctt 540
aggttctgtg gaaagaatga ctgctatctt gacggagcac tttgctggta agtggccatt 600
ctggttatct ccacgtcaaa tccttgtcgt accagtcggt gttaaattcc aagaatatgc 660
tcaagaagtt cgtgacaaga tggttgaagc tggcttctac gcagacgttg atctcaccgg 720
taatacccta caaaagaagg tcaggaacgg tcaaatgttg aaatataact tcattttcat 780
tgttggtgaa caggagatga ctgaaaaatc cgttaatatc agaaacaggg atgttatgga 840
cttacaaggt aagaacgcca ccgttcaagt agacgctgtc attaaccaat tgtccaagtt 900
gaaggaagaa aagagagctg ataatgtctt ggctgaagcc ggtatcacag gaacatggta 960
taatcagctg ggtagcactc tgatcgttac cgctggagca gatggtgctc tgactggcac 1020
ctatgttaca gcacgtggta atgctgaagg ctcgtatgtc ctgacaggtc gctatgattc 1080
tgccccggca actgatggat ctggaacagc gctgggatgg actgttgctt ggaaaaacaa 1140
ctatcgtaat gcccattccg ccaccacatg gagtggtcag tatgttggag gcgccgaagc 1200
tcgtattaat acacagtggc tgctgacaag tggtacaact gaagccaatg cctggaaatc 1260
aaccctggtt ggccatgaca cctttacaaa agtcaaaccg tccgccgctt ctgcgaattc 1320
cgcggggaag tcaacccatc accatcacca ccaccaccac gggtcaggtg caactaactt 1380
ttctctgttg aaacaagctg gagatgtcga agagaatcct ggccccatgg gtaaggaaaa 1440
gactcacgtt tcgaggccgc gattaaattc caacatggat gctgatttat atgggtataa 1500
atgggctcgc gataatgtcg ggcaatcagg tgcgacaatc tatcgattgt atgggaagcc 1560
cgatgcgcca gagttgtttc tgaaacatgg caaaggtagc gttgccaatg atgttacaga 1620
tgagatggtc agactaaact ggctgacgga atttatgcct cttccgacca tcaagcattt 1680
tatccgtact cctgatgatg catggttact caccactgcg atccccggca aaacagcatt 1740
ccaggtatta gaagaatatc ctgattcagg tgaaaatatt gttgatgcgc tggcagtgtt 1800
cctgcgccgg ttgcattcga ttcctgtttg taattgtcct tttaacagcg atcgcgtatt 1860
tcgtctcgct caggcgcaat cacgaatgaa taacggtttg gttgatgcga gtgattttga 1920
tgacgagcgt aatggctggc ctgttgaaca agtctggaaa gaaatgcata agcttttgcc 1980
attctcaccg gattcagtcg tcactcatgg tgatttctca cttgataacc ttatttttga 2040
cgaggggaaa ttaataggtt gtattgatgt tggacgagtc ggaatcgcag accgatacca 2100
ggatcttgcc atcctatgga actgcctcgg tgagttttct ccttcattac agaaacggct 2160
ttttcaaaaa tatggtattg ataatcctga tatgaataaa ttgcagtttc atttgatgct 2220
cgatgagttt ttctaaagga atatccaaac cgatcatata tttgtcaaca gaaattctaa 2280
tttattaacc tttcaacagt tatagtttgt caacactatc tcgtcgtttt aactccctta 2340
ccatgcaagt cgtatattta agctaactat cttttctgtc tcttaactca tgtatattgt 2400
tagatataaa ctaatataaa tttcactaat tatcatcgga aataatatga atgatggacc 2460
tttttaattc gttgtcatag atggaatata gctctaccgg agcaatcaag ccttctgtta 2520
agtgtgattt gttctgttgt catgtatgat gtttgaaaat ttatctcttt ttacaagtac 2580
tctcctttgt tctgcgtgat gcaggataat cctgataaga tttctgcgaa cttcattatt 2640
tataattggt atcgctgtta tcataatcat gttttctaat ttcttcccat gatttaccat 2700
tctttgtgtc tacagcggtg ttctcctttc taattttgtc ccacgatgac agagtgggtt 2760
cctgtgtctc tgtgtcattg aatggatcat aatcatccct cggagtcacc tgagaaggtg 2820
gggtagtttg agagacacca gtctttggca caccttcctt cttatcgtat tgaggtcctg 2880
tatatagccc cattgggtcc ttgttgtcga ataaaactga ggagtttctc acgccagaat 2940
tgccttcctt caatttgtcc attacaagtc tgggatcagg aattcttctt tctggattct 3000
tatatgtcat gtaataatag ttcgcccatc tggtaggcat tcccatgttt aatagcttta 3060
gaatttcgta ctgcttctgt cgactacttt ttggcaaatg cggatcatct tccagtatgt 3120
ctggtttagc agccttaagt ttagatttgt acgccaagga gccaccagct tgtgtgccaa 3180
taaacataga taa 3193
<210> 3
<211> 422
<212> DNA
<213> 人工序列
<400> 3
gcggccgcct tatagcctca gggggtgaaa cactccttct gggctcaagt gactttttga 60
aatttcagct gagcctcagg tgagtgaaag cgctctcaag actcaggtga tgttttataa 120
ttccggctga gcctcaagcg acttttttaa atttcacttc agccccaaga gagaattcgc 180
gctgctgagc ctcaaaagcg taaaccaccc cctgagctca gggggactgt ttttttgcat 240
atttcaacac gacatgggca ttttggttat tggatgtaga atggttgaat aaacgtgatt 300
gtaaaataga gtttgtaact acgaataatt agtttttgag aagtttggtg aatttaatat 360
ttgtatgagg aaagtaaatt ttaataccta aataaacaaa aatatatggt gacgtcggat 420
cc 422

Claims (10)

1.一种利用优化的CRISPR/Cas9系统对克鲁维酵母基因组进行改造的方法,包括下述步骤:
a)向所述克鲁维酵母细胞中转化Cas9/gRNA融合质粒,该质粒靶向所述克鲁维酵母细胞的内源性DNA序列,并产生双链切口;
b)向所述克鲁维酵母细胞中转化供体DNA序列,该序列在双链切口处与靶位点产生同源重组,将Tag序列插入目标位点。
2.如权利要求1所述的方法,其特征在于,所述克鲁维酵母是一种乳酸克鲁维酵母。
3.如权利要求2所述的方法,其特征在于,改造的基因为乳酸克鲁维酵母的细胞质苏氨酸氨酰tRNA合成酶Kl-TRS基因。
4.如权利要求3所述的方法,其特征在于,对Kl-TRS基因的改造为在基因末端插入一段长1302bp的标记Tag序列。
5.如权利要求1所述的方法,其特征在于,对原始CRISPR/Cas9质粒进行的一系列序列改造和系统优化,最终使标记Tag可以高效插入克鲁维酵母基因组中,并在克鲁维酵母中稳定遗传和表达,包括下述步骤:
a)在pCAS质粒中插入pKD1 stabilizing element (SE)序列,构建成质粒pKM-Cas9-SE;
b)将pKM-Cas9-SE质粒中gRNA启动子置换为KlSNR52基因启动子,构建成质粒pKM-Cas9-SE-pKlSNR2;
c)将pKM-Cas9-SE-pKlSNR2质粒中Cas9序列替换为适合克鲁维酵母表达的KLCas9,构建成质粒pKM-KLCas9-SE-pKlSNR52;
d)将pKM-KLCas9-SE-pKlSNR52质粒中抗性基因Kan置换为Amp,完成质粒的最终改造,命名为pKM-CAS1.0;
e)将靶向Kl-TRS基因的gRNA插入pKM-CAS1.0中,构建成质粒pKM-CAS1.0-TRS1。
6.如权利要求1或2所述的方法,其特征在于,转化的供体DNA序列为线性双链DNA,该序列包括插入靶位点的Tag序列以及与靶位点两侧序列同源的序列,包括下述步骤:
a)以乳酸克鲁维酵母菌液为模板扩增同源臂序列,将同源臂序列插入pMD18质粒中,构建成中间质粒pKM-TRS-DD1;
b)将Tag序列插入pKM-T-DD1质粒两同源臂中间位置,构建成最终质粒pKM-TRS-DD2;
c)以pKM-TRS-DD2为模板进行PCR扩增,得到线性双链供体DNA。
7.如权利要求1所述的方法,其特征在于,通过LiAc/SS carrier DNA/PEG方法将质粒转化进入克鲁维酵母细胞中,包括下述步骤:
a)将pKM-CAS1.0-TRS1质粒和线性供体DNA 同时转化进入克鲁维酵母细胞中;
b)涂板后挑取单克隆于1 mL液体YPD培养基中振荡培养。
8.如权利要求1所述的方法,其特征在于,通过PCR扩增检测Tag序列插入靶位点效率,包括下述步骤:在Tag序列内设计一条引物,在靶基因上位于同源臂外侧的序列设计一条引物,以单克隆菌液为模板进行PCR扩增,电泳检测阳性的为插入成功。
9.如权利要求6所述的方法,其特征在于,线性双链供体DNA包含与SEQ IDNO.2至少80%序列相同的核苷酸。
10.一种CRISPR/Cas9质粒,其特征在于,其包含与SEQ ID NO.1至少80%序列相同的核苷酸。
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