Fig. 4

Construction and application of the easy-to-use CRISPR-Cas9 system. a Plasmid maps for the new easy-to-use CRISPR-Cas9 system. b Visual demonstration of efficient LacZ cassette integration via blue-white colony selection. c PCR verification of lacZ integration using a forward primer upstream of the left arm and a reverse primer downstream of the right arm of the chromosome. C-, with parent E. coli cells as the template. d Comparison of the easy-to-use CRISPR-Cas9 system versus previously developed CRISPR-Cas9 systems. The easy-to-use CRISPR-Cas9 system can eliminate plasmids in one single step instead of stepwise elimination. e PCR verification of plasmid elimination for pCAS92 (Upper), pGRNA2 (Middle) and pKD46 (Bottom). f AtzA catalyzes the hydrolytic dechlorination of atrazine to 2-hydroxyatrazine. g Atrazine degradation is indicated by the clear ring on an agar plate supplemented with atrazine. h The diameter of this degradation ring increased with extended incubation time