Henan Engineering Technology Research Center of Green Coating Materials, Yellow River Conservancy Technical Institute, Kaifeng 475004, China; College of Life Sciences, Henan Agricultural University, Key Laboratory of Enzyme Engineering of Agricultural Microbiology, Ministry of Agriculture and Rural Affairs, Zhengzhou 450046, China.
Henan Vocational College of Agriculture, Zhengzhou 451450, China.
Int J Biol Macromol. 2024 Oct;278(Pt 4):135065. doi: 10.1016/j.ijbiomac.2024.135065. Epub 2024 Aug 24.
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.
CRISPR-Cas9 核糖核蛋白 (RNP) 在植物和动物中的基因编辑应用较为常见，但在细菌中的应用尚未见报道。本研究采用 DNA 单链结合蛋白 (SSB) 构建了 SSB/CRISPR-Cas9 RNP 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。将靶向 upp 基因的 RNP 与 SSB 一起导入大肠杆菌、假单胞菌和枯草芽孢杆菌的原生质体中。在含有 5-氟尿嘧啶（5-FU）的平板上获得了转化体，基因编辑效率（相对于原生质体数量的转化体百分比）分别为 9.75%、5.02%和 8.37%，测序分析证实了 100%的非同源重组。当 RNP、SSB 和 100 核苷酸单链寡脱氧核苷酸 (ssODN) 供体导入这些细菌的原生质体时，转化体的编辑效率分别为 45.11%、30.13%和 27.18%，测序证实了 upp 基因的 100%同源重组敲除。此外，将 RNP、SSB 和包含四环素抗性基因 (tetR-dsODN) 的 100 碱基对双链寡脱氧核苷酸 (dsODN) 供体导入 5-FU 平板上，转化体的编辑效率分别为 35.94%、22.46%和 19.08%，测序证实了 upp 基因与 tetR 的 100%同源重组替换。这些结果表明，SSB/CRISPR-Cas9 RNP 系统可以高效、简单、快速地编辑细菌基因组，而无需质粒。本研究首次报道了基于 RNP 的基因编辑在细菌中的应用。
