Development of multiple transgenic CRISPR/Cas9 methods for genome editing in the fall armyworm, Spodoptera frugiperda

The use of CRISPR/Cas9 system in model insects has facilitated functional genomics studies. However, this system has not been applied to many pest insects. Here, we report on the establishment of multiple transgenic CRISPR/Cas9-based genome editing methods in a global agricultural pest, the fall armyworm (FAW), Spodoptera frugiperda . To identify fluorescent proteins suitable for screening for transgenic FAW, nine transgenic lines expressing genes coding for fluorescent proteins under the control of different promoters were produced and evaluated. The enhanced green fluorescent protein and a red fluorescent protein, tdTomato genes driven by the hr5ie1 promoter were found to be suitable for the identification of transgenic FAW. Multiple lines of transgenic FAW expressing Cas9 were generated and microinjection of sgRNAs into the embryos of these lines failed to induce target gene knockout. To overcome this problem, sgRNAs were expressed in FAW using U6-sgRNA and U6-tRNA-sgRNA systems, U6-tRNA-sgRNA system was found to be more efficient than U6-sgRNA system. Expression of Cas9 and sgRNAs in the same transgenic animal or in two separate strains followed by crossing them to bring Cas9 and sgRNA together resulted in an efficient knockout of target genes. The multiple transgenic CRISPR/Cas9-based genome editing methods developed provide invaluable tools for gene editing and functional genomics studies in this global pest and other lepidopteran pests.