Li Xin, Wei Ying, Wang Shu-Yan, Wang Shu-Guang, Xia Peng-Fei
School of Environmental Science and Engineering, Shandong University, Qingdao, China.
School of Environmental Science and Engineering, Shandong University, Qingdao, China; Sino-French Research Institute for Ecology and Environment, Shandong University, Qingdao, China; Weihai Research Institute of Industrial Technology, Shandong University, Weihai, China.
J Biol Chem. 2025 Jan;301(1):108113. doi: 10.1016/j.jbc.2024.108113. Epub 2024 Dec 18.
Base editing is preferable for bacterial gene inactivation without generating double-strand breaks, requiring homology recombination, or highly efficient DNA delivery capability. However, the potential of base editing is limited by the adjoined dependence on the editing window and protospacer adjacent motif. Herein, we report an unconstrained base-editing system to enable the inactivation of any genes of interest in bacteria. We employed a dCas9 derivative, dSpRY, and activation-induced cytidine deaminase to build a protospacer adjacent motif-independent base editor. Then, we programmed the base editor to exclude the START codon of a gene of interest instead of introducing premature STOP codons to obtain a universal approach for gene inactivation, namely XSTART, with an overall efficiency approaching 100%. By using XSTART, we successfully manipulated the amino acid metabolisms in Escherichia coli, generating glutamine, arginine, and aspartate auxotrophic strains. While we observed a high frequency of off-target events as a trade-off for increased efficiency, refining the regulatory system of XSTART to limit expression levels reduced off-target events by over 60% without sacrificing efficiency, aligning our results with previously reported levels. Finally, the effectiveness of XSTART was also demonstrated in probiotic E. coli Nissle 1917 and photoautotrophic cyanobacterium Synechococcus elongatus, illustrating its potential in reprogramming diverse bacteria.
碱基编辑对于细菌基因失活而言是更可取的方法，因为它不会产生双链断裂，不需要同源重组，也不需要高效的DNA递送能力。然而，碱基编辑的潜力受到对编辑窗口和原间隔序列相邻基序的附加依赖性的限制。在此，我们报告了一种无限制的碱基编辑系统，可实现对细菌中任何感兴趣基因的失活。我们使用了一种dCas9衍生物dSpRY和激活诱导的胞苷脱氨酶来构建一种不依赖原间隔序列相邻基序的碱基编辑器。然后，我们对碱基编辑器进行编程，使其排除感兴趣基因的起始密码子，而不是引入提前终止密码子，从而获得一种通用的基因失活方法，即XSTART，其总体效率接近100%。通过使用XSTART，我们成功地操纵了大肠杆菌中的氨基酸代谢，产生了谷氨酰胺、精氨酸和天冬氨酸营养缺陷型菌株。虽然我们观察到作为效率提高的代价，脱靶事件的频率较高，但优化XSTART的调控系统以限制表达水平可将脱靶事件减少60%以上，且不牺牲效率，这与先前报道的水平一致。最后，XSTART的有效性也在益生菌大肠杆菌Nissle 1917和光合自养蓝细菌聚球藻中得到了证明，说明了其在对多种细菌进行重编程方面的潜力。
