Due to the high specificity in targeting DNA and highly convenient programmability, CRISPR-Cas-based antimicrobials applied for eliminating specific strains such as antibiotic-resistant bacteria in the microbiome were gradually developed. However, the generation of escapers makes the elimination efficiency far lower than the acceptable rate (10^-8) recommended by the National Institutes of Health. Here, a systematic study was carried out providing insight into the escaping mechanisms in Escherichia coli, and strategies for reducing the escapers were devised accordingly. We first showed an escape rate of 10^-5-10^-3 in E. coli MG1655 under the editing of pEcCas/pEcgRNA established previously. Detailed analysis of the escapers obtained at ligA site in E. coli MG1655 uncovered that the disruption of cas9 was the main cause of the generation of survivors, notably the frequent insertion of IS5. Hence, the sgRNA was next designed to target the "perpetrator" IS5, and subsequently the killing efficiency was improved 4-fold. Additionally, the escape rate in IS-free E. coli MDS42 was also tested at the ligA site, ∼10-fold decrease compared with MG1655, but the disruption of cas9 was still observed in all survivors manifested in the form of frameshifts or point mutations. Thus, we optimized the tool itself by increasing the copy number of cas9 to retain some cas9 that still has the correct DNA sequence. Fortunately, the escape rates dropped below 10^-8 at 9 of the 16 tested genes. Furthermore, the λ-Red recombination system was added to generate the pEcCas-2.0, and a 100% gene deletion efficiency was achieved at genes cadA, maeB, and gntT in MG1655, whereas those genes were edited with low efficiency previously. Last, the application of pEcCas-2.0 was then expanded to the E. coli B strain BL21(DE3) and W strain ATCC9637. This study reveals the mechanism of E. coli surviving Cas9-mediated death, and a highly efficient editing tool is established based on the mechanism, which will accelerate the further application of CRISPR-Cas.

#	Concept	H-Index	Publications	Citations
1	Engineering	3	3	62
2	Medicine	1	1	11
3	Social Sciences	1	1	16
4	Cell Biology	1	1	11
5	Computer Science	1	1	28

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