CRISPR-Cas9核糖核蛋白(RNP)在基因编辑中的应用在植物和动物中普遍使用,但其在细菌中的应用尚未见报道。在这项研究中,我们使用DNA单链结合蛋白(SSB)构建SSB/CRISPR-Cas9RNP编辑系统,用于细菌中upp基因的非同源重组和同源重组基因编辑.针对upp基因的RNP,随着SSB,被引入大肠杆菌的原生质体中,假单胞菌,和枯草芽孢杆菌.在含有5-氟尿嘧啶(5-FU)的平板上获得转化体,基因编辑效率(转化体相对于原生质体数量的百分比)为9.75%,5.02%,8.37%,分别,和测序分析证实100%非同源重组。当RNP,SSB,并将100个核苷酸的单链寡脱氧核苷酸(ssODN)供体引入这些细菌的原生质体中,获得的转化体编辑效率为45.11%,30.13%,和27.18%,分别,测序证实了upp基因的100%同源重组敲除。此外,介绍RNP,SSB,和含有四环素抗性基因(tetR-dsODN)的100个碱基对双链寡脱氧核苷酸(dsODN)供体在5-FU平板上产生转化体,编辑效率为35.94%,22.46%,和19.08%,分别,测序证实用tetR100%同源重组替换upp基因。这些结果表明,SSB/CRISPR-Cas9RNP系统可以有效地,简单地说,快速编辑细菌基因组而不需要质粒。这项研究首次报道了基于RNP的基因编辑在细菌中的应用。
The application of CRISPR-Cas9
ribonucleoprotein (RNP) for gene editing is commonly used in plants and animals, but its application in bacteria has not been reported. In this study, we employed DNA single-strand binding protein (SSB) to construct an SSB/CRISPR-Cas9 RNP-editing system for non-homologous recombination and homologous recombination gene editing of the upp gene in bacteria. The RNP targeting the upp gene, along with SSB, was introduced into the protoplasts of Escherichia coli, Pseudomonas, and Bacillus subtilis. Transformants were obtained on plates containing 5-fluorouracil (5-FU) with gene editing efficiencies (percentage of transformants relative to the number of protoplasts) of 9.75 %, 5.02 %, and 8.37 %, respectively, and sequencing analysis confirmed 100 % non-homologous recombination. When RNP, SSB, and a 100-nucleotide single-stranded oligodeoxynucleotide (ssODN) donor were introduced into the protoplasts of these bacteria, transformants were obtained with editing efficiencies of 45.11 %, 30.13 %, and 27.18 %, respectively, and sequencing confirmed 100 % homologous recombination knockout of the upp gene. Additionally, introducing RNP, SSB, and a 100 base-pair double-stranded oligodeoxynucleotide (dsODN) donor containing a tetracycline resistance gene (tetR-dsODN) resulted in transformants on 5-FU plates with editing efficiencies of 35.94 %, 22.46 %, and 19.08 %, respectively, with sequencing confirming 100 % homologous recombination replacement of the upp gene with tetR. These results demonstrate that the SSB/CRISPR-Cas9 RNP system can efficiently, simply, and rapidly edit bacterial genomes without the need for plasmids. This study is the first to report the use of RNP-based gene editing in bacteria.