Efficient Knocking Out of the Organophosphorus Insecticides Degradation Gene opdB in Cupriavidus nantongensis X1 T via CRISPR/ Cas9 with Red System

X1 is a type strain of the genus , that can degrade eight kinds of organophosphorus insecticides (OPs). Conventional genetic manipulations in species are time-consuming, difficult, and hard to control. The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 ( ) system has emerged as a powerful tool for genome editing applied in prokaryotes and eukaryotes due to its simplicity, efficiency, and accuracy. Here, we combined CRISPR/ with the Red system to perform seamless genetic manipulation in the X1 strain. Two plasmids, pACasN and pDCRH were constructed. The pACasN plasmid contained nuclease and Red recombinase, and the pDCRH plasmid contained the dual single-guide RNA (sgRNA) of organophosphorus hydrolase ( ) in the X1 strain. For gene editing, two plasmids were transferred to the X1 strain and a mutant strain in which genetic recombination had taken place, resulting in the targeted deletion of . The incidence of homologous recombination was over 30%. Biodegradation experiments suggested that the gene was responsible for the catabolism of organophosphorus insecticides. This study was the first to use the CRISPR/ system for gene targeting in the genus , and it furthered our understanding of the process of degradation of organophosphorus insecticides in the X1 strain.