In an a recent experiment published in Gene, Li and Handler (2019) utilized readily available study systems and newly emerging genetic technologies and demonstrated both germline and somatic CRISPR/Cas9 induced mutations in Anastrepha ludens.
The creation of heritable mutations was demonstrated in the transgenic line called As-loxM1, a strain of A. suspense that is dual-marked with two fluorescent protein genes, EGFP and DsRed which are each regulated by the Drosophila melanogaster polyubiquitin constitutive promoter (Handler and Harrell, 2001). Germline mutations directed by a single-guide RNA (sgRNA) targeting the EGFP gene were readily detected in the G1 heterozygotes after backcrossing with wild type, which demonstrated successful mutagenesis of the germline in one generation. A subsequent backcross of G1 heterozygotes with wild type and then intercrossing of G2 heterozygotes resulted in G3 homozygote EGFP knock out . Gene specificity of the sgRNA designed for EGFP was also demonstrated due to the intact function of the adjacent DsRed gene.
In a separate, but equally impactful experiment, Li and Handler demonstrated the creation of CRISPR/Cas9 mediated somatic mutations by targeting a gene which can produce intermediate morphological phenotypes. The gene Atra-2, a homolog of transformer-2 in D. melanogaster, is part of a sex-determination cascade which is required for sexual differentiation in XX females and proper sexual behavior where depressed somatic expression of transformer or transformer-2 results in an intersex female phenotype of genotypically XX females (Baker and Belote, 1983; Gempe and Beye, 2010; Salz, 2011).
Previous studies using RNAi demonstrated its function in A. suspensa (Schetelig et al. 2012). By injecting wild type A. suspensa with Cas9 protein and two sgRNA targeting exons 4 and 6 of Atra-2, the authors created somatic mutations which disrupted Atra-2 expression and the female-specific splicing of downstream sexual differentiation genes doublesex and fruitless. Expression disruption was evident from the intersex phenotypes in the injected G0 which includes dysmorphic genitalia and truncated ovipositors . Though the purpose of injecting two sgRNAs was to create a 774 bp deletion in the Atra-2 gene (the region between the two cut sites) to demonstrate the potential for CRISPR/Cas9 mediated homology driven repair (HDR), only one guide was detected to be efficient in creating a double-stranded break and NHEJ mutations and a 774 bp deletion was not detected. However, this study was successful in demonstrating a rapid evaluation of sgRNA efficiency within one generation and facilitates future studies in optimizing CRISPR/Cas9 mediated HDR in the future.
Gempe, T., & Beye, M. (2011). Function and evolution of sex determination mechanisms, genes and pathways in insects. BioEssays : news and reviews in molecular, cellular and developmental biology, 33(1), 52–60. doi:10.1002/bies.201000043
Handler, A. M., & Harrell, R. A., 2nd. (2001). Transformation of the Caribbean fruit fly, Anastrepha suspensa, with a piggyBac vector marked with polyubiquitin-regulated GFP. Insect Biochem Mol Biol, 31(2), 199-205.
Li, J., & Handler, A. M. (2019). CRISPR/Cas9-mediated gene editing in an exogenous transgene and an endogenous sex determination gene in the Caribbean fruit fly, Anastrepha suspensa. Gene, 691, 160-166. doi:10.1016/j.gene.2018.12.055
Schetelig, M. F., Milano, A., Saccone, G., & Handler, A. M. (2012). Male only progeny in Anastrepha suspensa by RNAi-induced sex reversion of chromosomal females. Insect Biochem Mol Biol, 42(1), 51-57. doi:10.1016/j.ibmb.2011.10.007