Differential expression of genes in pre-blastoderm embryos of oriental fruit fly, Bactrocera dorsalis (Hendel) (Diptera: Tephritidae) microinjected with white locus CRISPR/Cas9 ribo nucleo protein (RNP) complex.

CRISPR/Cas9-based genome editing is currently revolutionizing applications in agriculture, human health, and other domains. Targeted gene editing employing CRISPR/Cas9 is achieved by delivering the various components viz. guide RNA, traCrRNA, and the Cas9 protein in DNA or RNA or RNP format. RNP enables fast, DNA-free editing without genomic integration. While innate immunity to guide RNA is studied in humans and plants, similar research in insects remains unexplored. In the present study, the RNP complex (sgRNA+Cas9) was administered via microinjection of embryos (eggs) in the pre-blastoderm (G0) stage. Transcriptome sequencing was performed using the Illumina Novoseq 6000 platform to understand the prevalence of innate immunity against the delivered guide RNA for the white locus of Bactrocera dorsalis. 33 up- and 67 down-regulated genes were found in the differential gene expression analysis from the RNA-Seq data. The correlation between RT-qPCR and RNA-Seq gene expression levels showed a strong coefficient of determination (R2 = 0.984). The genes related to stress, intron removal, and effector recognition were overexpressed. However, the genes responsible for growth and development were significantly downregulated. Thus, the present study identified the possible reasons that could play a vital role in influencing the editing outcomes employed through the RNP complex.