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••••• WHU-China 2023

(1) pRed_cas9_△poxb300 can achieve high genome editing efficiency in E. coli The research article indicated that Cas9 (BBa_K1218011) combined with Lambda-Red recombinases (BBa_K1433005) can achieve high editing efficiency in E. coli MG1655. The author reported 100% genome editing efficiency from randomly selected colonies.[1] Fig. 1 poxb gene knockout with 513 bp deletion: lanes 1,2 and 8 were control group samples, and others were experimental group samples. The Colony PCR product of the edited poxb gene was 1008 bp, and the original was 1521 bp. We conducted ladder experiments on the arabinose induction time to figure out the optimal duration of induced editing. As it is said that the induction should last at least for 6h[1], gradient induction was set up from 6h to 30h. Although double bents always existed, which means that bacteria are not completely edited in this colony (Fig2), 24h was considered as the optimal induction time with minimal double bents and almost 100% editing efficiency (Fig3). Fig. 2 AGE results of genome editing Fig. 3 Results of time gradient induction Interestingly, we found that this system has higher genome editing rates in DH5-alpha than in MG1655. However, as it’s not the main part of our experiments and the lack of time, we didn’t make deeper investigations. (2) pRed_cas9_△poxb300 can achieve plasmid gene editing in E. coli We confirmed that pRed_cas9_△poxb300 can achieve plasmid gene editing. We added an N20s sequence and a batch of gRNAs targeting it into another plasmid. After co-transformation, we induced gene knockout for 24 hours by arabinose. It shows that many of the gRNAs (for example, NO. 11, 13, 14, 15) successfully targeted and deleted the N20s sequence. Fig. 4a The design of plasmid N20s gene knockout Fig. 4b The results of plasmid N20s gene knockout Optimized pCas did not knock out poxb300 gene in E. coli Set the original pCas as control, we transformed the pCasop into E. coli DH5α and induced (fig 5a-d). The bacteria amount of pCasop-containing group is much larger than pCas group, indicating an increase on bacteria viability (fig 5e). The sequencing results prove that there is no targeting deletion in pCasop group (fig 5f). Fig. 5 The design of plasmid N20s gene knockout (a-b) The induced pCas-containing bacteria were spread on plates, after 100-fold and 1,000-fold dilution, respectively. (c-d) The induced pCasop-containing bacteria were spread on plates, after 100-fold and 1,000-fold dilution, respectively. (e) The quantification of bacteria on the corresponding plates. They were evaluated by three individual testers. The bacteria amount of pCasop is strikingly larger than the original pCas. (f) The sequencing results. The genomic knock-out rate of pCas is 70% (N = 10), while pCasop is 0. The pCas-containing strain without induction was set as the positive control. References [1]. Zhao, D., Yuan, S., Xiong, B. et al. Development of a fast and easy method for Escherichia coli genome editing with CRISPR/Cas9. Microb Cell Fact 15, 205 (2016). [2]. Genescript

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