CRISPR/Cas9-Mediated NHEJ Mutations in Anastrepha suspensa Paves Way for HDR

Shiena Sim, Ph.D., Research Biologist at the USDA ARS Daniel K. Inouye US Pacific Basin Agricultural Research Center, Hilo, Hawaii where she is advancing the Sterile Insect Technique (SIT) and using genomic techniques to answer questions in arthropod biology, ecology, and evolution for the protection of agricultural crops. MORE ABOUT THE AUTHOR

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.

Anastrepha ludens. Jack Dykinga, USDA Agricultural Research Service – This image is Image Number 1322089 at Insect Images, a source for entomological images operated by The Bugwood Network at the University of Georgia and the USDA Forest Service

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).


Simplified illustration on the effect of transformer/transformer-2 in insects on sex phenotype in higher diptera (Gempe and Beye, 2010).

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.

From left to right, wild type A. suspensa male abdomen, wild type A. suspensa female abdomen, and one G0 intersex female abdomens exhibiting dysmorphic genitalia and truncated ovipositors. The numbers represent abdominal segments.  This is a partial reproduction of an image from Li and Handler (2019); see their publication for the complete image.


Baker, B. S., & Belote, J. M. (1983). Sex determination and dosage compensation in Drosophila melanogaster. Annu Rev Genet, 17, 345-393. doi:10.1146/

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

Salz, H. K. (2011). Sex determination in insects: a binary decision based on alternative splicing. Curr Opin Genet Dev, 21(4), 395-400. doi:10.1016/j.gde.2011.03.001

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



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