Abstract
The CRISPR/Cas9 is being developed as an invaluable system that allows rapid and site-specific genome editing in a wide variety of organisms, including diverse insects. It has been successfully used for gene function annotations of RNAi pathway in insect genomics and will facilitate research on RNAi mechanism. Here, we describe a streamlined method to generate and detect somatic and germline knockout mutations of desired target genes in tephritid pests by injecting mRNA encoding the Cas9 endonuclease and in vitro transcribed single guide RNA (sgRNA) into embryos. Target site selection, sgRNA synthesis, Cas9 synthesis, microinjection, and mutation identification are presented in detail.
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