(2019). Since many genetic diseases arise from point mutations, this technology has important implications in the study of human health and … putida KT2440. Currently, cytidine deaminase-based base editing system have been extended to Escherichia coli (Nishida et al., 2016), Clostridium beijerinckii (Li et al., 2019), Klebsiella pneumonia (Yu et al., 2018), Corynebacterium glutamicum (Wang et al., 2019), and some Pseudomonas spp. This is in agreement with the previously reported mutation preference of APOBEC1 (Chen et al., 2018). This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The editing differences of Chen’s base editing system (Chen et al., 2018) and ours on the four motifs are possibly because of the different expression types of the APOBEC1-nCas9 complex. pSEVA6BE, pSEVA6BE-NG, pSEVA6BE-YE1 or other derivative plasmids was transformed into Pseudomonas according to a previous electroporation method (Sun et al., 2018). doi: 10.1021/acssynbio.7b00258. Antibiotics were added as the following concentrations: gentamicin (Gm), 50 μg/mL (E. coli), 100 μg/mL (P. putida KT2440, P. entomophila L48, and P. protegens Pf-5) and 30 μg/mL (P. aeruginosa PAO1); kanamycin (Km), 50 μg/mL (E. coli) and 100 μg/mL (P. putida KT2440). Appl. After 66 h cultivation, the shaking flask fermentation showed that the production of PCA in the G136E mutation strain was 264.87 mg/L, which is an increase by 69.01 and 611.17% when compared with that of the AroF-2 unedited strain and the starting strain (Figure 7C). What do I need to know about the customs and importation process for my country? In our study, we not only successfully developed the pSEVA6BE system as a knockout tool for P. putida KT2440, but also expanded the base-editing scope into P. aeruginosa PAO1, P. fluorescens Pf-5 and P. entomophila L48. doi: 10.1016/j.ymben.2018.02.016, Wang, Y., Liu, Y., Li, J., Yang, Y., Ni, X., Cheng, H., et al. BMC Bioinformatics 19:4. doi: 10.1186/s12859-018-2585-4, Jakoèiunas, T., Jensen, M. K., and Keasling, J. D. (2016). doi: 10.1038/nbt1212, Wang, W., Wang, S., Bilal, M., Zhang, X., Zong, Y., and Hu, H. (2018). In recent years, Cas9 assisted genome editing systems have revolutionarily accelerated the development of genetic studies in different organisms (Jakoèiunas et al., 2016), including Pseudomonas spp. After base editing, DNA sequencing (Figure 4A) of PCR products showed that TAG and TAA could be generated with efficiencies of 100% and 80%, respectively. 13, 1–36. Biotechnol. The deletion of hmgA can disable the ring-cleavage reaction of homogentisate, which leads to the accumulation of homogentisate. In our study, sgRNA and APOBEC1-nCas9-UGI were under the control of the PJ23119 constitutive promoter and arabinose-inducible promoter, respectively. All of the plasmids and primers used in this study are listed in Supplementary Tables S2, S3. (2006). Bioeng. The strong environmental adaptability and great metabolic versatility of Pseudomonas spp. Learn more, Please note: Your browser does not fully support some of the features used on Addgene's website. The construction of a cytosine base editor with a narrower editing window provides precision base editing of cytidine-rich sites.

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