{"title":"Cas9AEY (Cas9-facilitated Homologous Recombination Assembly of non-specific Escherichia coli yeast vector) method of constructing large-sized DNA.","authors":"xiaoshu ma, lei yang, hua ye","doi":"10.1101/2024.09.17.611575","DOIUrl":null,"url":null,"abstract":"Saccharomyces cerevisiae is widely used in DNA assembly due to their efficient homologous recombination [1], but DNA assembly through yeast recombination in vivo usually requires the vector to have the ability to replicate in yeast. The CRISPR-Cas9 system can efficiently edit DNA [2,3], and the system can also be used for DNA editing of plasmids. In this paper, a yeast universal element is selected, which can be inserted into the vector, so that the vector can replicate in yeast cells, and then the intermediate plasmid containing yeast universal element can be obtained by recombination in yeast. At the same time, a pCas-SmR plasmid was designed in this paper. After Donor DNA is added, the CRISPR-Cas9 system can accurately and efficiently knock out the yeast universal element in the intermediate plasmid, remove the pCas-SmR plasmid through sucrose screening, and finally obtain a pure plasmid. Saccharomyces cerevisiae cells are widely used in DNA assembly due to their efficient homologous recombination [1], but DNA assembly through yeast recombination in vivo usually requires the vector to have the ability to replicate in yeast. The CRISPR-Cas9 system can efficiently edit DNA [2,3], and the system can also be used for DNA editing of plasmids. In this paper, a yeast universal element is selected, which can be inserted into the vector, so that the vector has the ability to replicate in yeast cells, and then the intermediate plasmid containing yeast universal element can be obtained by recombination in yeast. At the same time, a pCas-SmR plasmid was designed in this paper. After Donor DNA is added, the CRISPR-Cas9 system can accurately and efficiently knock out the yeast universal element in the intermediate plasmid, remove the pCas-SmR plasmid through sucrose screening, and finally obtain a pure knocked out plasmid.","PeriodicalId":501408,"journal":{"name":"bioRxiv - Synthetic Biology","volume":"52 1","pages":""},"PeriodicalIF":0.0000,"publicationDate":"2024-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"bioRxiv - Synthetic Biology","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1101/2024.09.17.611575","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"","PubModel":"","JCR":"","JCRName":"","Score":null,"Total":0}
引用次数: 0
Abstract
Saccharomyces cerevisiae is widely used in DNA assembly due to their efficient homologous recombination [1], but DNA assembly through yeast recombination in vivo usually requires the vector to have the ability to replicate in yeast. The CRISPR-Cas9 system can efficiently edit DNA [2,3], and the system can also be used for DNA editing of plasmids. In this paper, a yeast universal element is selected, which can be inserted into the vector, so that the vector can replicate in yeast cells, and then the intermediate plasmid containing yeast universal element can be obtained by recombination in yeast. At the same time, a pCas-SmR plasmid was designed in this paper. After Donor DNA is added, the CRISPR-Cas9 system can accurately and efficiently knock out the yeast universal element in the intermediate plasmid, remove the pCas-SmR plasmid through sucrose screening, and finally obtain a pure plasmid. Saccharomyces cerevisiae cells are widely used in DNA assembly due to their efficient homologous recombination [1], but DNA assembly through yeast recombination in vivo usually requires the vector to have the ability to replicate in yeast. The CRISPR-Cas9 system can efficiently edit DNA [2,3], and the system can also be used for DNA editing of plasmids. In this paper, a yeast universal element is selected, which can be inserted into the vector, so that the vector has the ability to replicate in yeast cells, and then the intermediate plasmid containing yeast universal element can be obtained by recombination in yeast. At the same time, a pCas-SmR plasmid was designed in this paper. After Donor DNA is added, the CRISPR-Cas9 system can accurately and efficiently knock out the yeast universal element in the intermediate plasmid, remove the pCas-SmR plasmid through sucrose screening, and finally obtain a pure knocked out plasmid.
Cas9AEY(Cas9-facilitated Homologous Recombination Assembly of non-specific Escherichia coli yeast vector)构建大尺寸 DNA 的方法。
酵母菌因其高效的同源重组而被广泛用于 DNA 组装[1],但在体内通过酵母重组进行 DNA 组装通常需要载体具有在酵母中复制的能力。CRISPR-Cas9 系统可以高效地编辑 DNA [2,3],该系统也可用于质粒的 DNA 编辑。本文选择了一种酵母通用元件,将其插入载体中,使载体可以在酵母细胞中复制,然后通过酵母重组获得含有酵母通用元件的中间质粒。同时,本文还设计了一种 pCas-SmR 质粒。加入Donor DNA后,CRISPR-Cas9系统可以准确有效地敲除中间质粒中的酵母通用元件,并通过蔗糖筛选去除pCas-SmR质粒,最终获得纯质粒。酵母细胞因其高效的同源重组而被广泛应用于DNA组装[1],但在体内通过酵母重组进行DNA组装通常需要载体具有在酵母体内复制的能力。CRISPR-Cas9 系统可以高效地编辑 DNA [2,3],该系统也可用于质粒的 DNA 编辑。本文选择了一种酵母通用元件,将其插入载体中,使载体具有在酵母细胞中复制的能力,然后通过酵母重组获得含有酵母通用元件的中间质粒。同时,本文还设计了一种 pCas-SmR 质粒。加入Donor DNA后,CRISPR-Cas9系统可以准确高效地敲除中间质粒中的酵母通用元件,并通过蔗糖筛选去除pCas-SmR质粒,最终获得纯合的敲除质粒。