Knocking Out Of The Organophosphorus Insecticides Degradation Gene OpdB In Cupriavidus Nantongensis X1~T Via CRISPR/Cas9

Cupriavidus nantongensis X1~T is a type of strain of the genus Cupriavidus.Studies have shown that strain X1~T can degrade eight organophosphorus pesticides(OPs).Conventional methods of genetic manipulation of the genus Cupriavidus are relatively time-consuming and difficult to handle.The gene editing method of third generation,CRISPR/Cas9 system,allows the manipulation of the whole genome of organisms.The successful uses of this genetic tool have been reported in many prokaryotes and eukaryotes.CRISPR/Cas9-based gene editing system has many advantages,such as simplicity of design and operation,high efficiency,accuracy,and widespread use.In this study,we combined the CRISPR/Cas9system with the Red recombination system from lambda phage that could improve homologous recombination efficiency for seamless genetic editing in Cupriavidus nantongensis X1~T.We constructed the two-plasmid system for efficient genetic manipulation of the genome of Cupriavidus nantongensis X1~T,which were named pACasN and pDCRH,respectively.In addition,the pACasN plasmid contained the sequences of Cas9 nuclease and Red recombinase.The pDCRH plasmid contained the dual-single guide RNA(sg RNA)of the target gene of opdB encoding organophosphorus hydrolase(OpdB)and the fragments of the upstream and downstream homologous arms of opdB that promote the occurrence of homologous recombination in the X1~T strain.For gene editing,two plasmids were transferred to the X1~T strain successively.After induction,homologous recombination occurred and the opdB gene was knocked out successfully.The incidence of homologous recombination was calculated over 30%.Biodegradation experiments of the mutant strain of X1~T-ΔopdB showed that the opdB gene is a key gene responsible for the catabolism of organophosphorus insecticides.This study is the first successful application of the CRISPR/Cas9 system for gene targeting in the genus Cupriavidus.It furthered our understanding of the process of degradation of organophosphorus insecticides in the X1~Tstrain.The main results of this study are as follows.Firstly,two plasmids were constructed for knocking out the organophosphorus insecticides degradation gene opdB in Cupriavidus nantongensis X1~T.They were named pACasN and pDCRH,respectively.The pACasN plasmid contained cas9 gene and Lambda Red recombination system.The pDCRH plasmid contained dual sg RNA and homologous recombination repair templates.Secondly,the plasmids of pACasN and pDCRH were successfully transferred into Cupriavidus nantongensis X1~T for the first time.The two plasmids were stably inherited.The transformation efficiency of two plasmids was calculated at the stable growth period.The transformation efficiency was 86.1±21.3 CFU/mg for the pACasN plasmid transferred into the X1~T strain.The transformation efficiency was 7.4±1.9 CFU/mg for the pDCRH plasmid transferred into the X1~T-ΔopdB strain.After two successive transfers of the plasmids into the X1~T strain,the results showed that the transformation efficiency of the pACasN plasmid was 11.6 times higher than the other pDCRH plasmid.Thirdly,the mutant strains of X1~T-ΔopdB were screened on LB plates when reagent induction was performed to express the proteins of Cas9 and Lambda Red recombinant system.The editing efficiency of the X1~Tstrain was calculated over 30%.Fourthly,plasmid curing was performed on the X1~T-ΔopdB strain harboring two plasmids to achieve seam Less genetic editing.Calculated by scribing on the antibiotic resistance plates,the pACasN plasmid was cured with an efficiency of 13/16and the pDCRH plasmid was cured with an efficiency of 4/16.Finally,the biodegradation experiments of the X1~T-ΔopdB strain showed that the opdB gene is a key gene for the catabolism of organophosphorus insecticides in the X1~T strain.