Volume 29, Issue 6 p. 569-577
Original Article

Germline mutagenesis of Nasonia vitripennis through ovarian delivery of CRISPR-Cas9 ribonucleoprotein

D. Chaverra-Rodriguez

D. Chaverra-Rodriguez

Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, CA, USA

Equal contributions.

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E. Dalla Benetta

E. Dalla Benetta

Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, CA, USA

W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, CA, USA

Equal contributions.

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C. C. Heu

C. C. Heu

Department of Entomology, The Pennsylvania State University, University Park, PA, USA

The Center for Infectious Disease Dynamics, The Pennsylvania State University, University Park, PA, USA

The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, USA

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J. L. Rasgon

J. L. Rasgon

Department of Entomology, The Pennsylvania State University, University Park, PA, USA

The Center for Infectious Disease Dynamics, The Pennsylvania State University, University Park, PA, USA

The Huck Institutes of the Life Sciences, The Pennsylvania State University, University Park, PA, USA

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P. M. Ferree

P. M. Ferree

W. M. Keck Science Department, Claremont McKenna, Pitzer, and Scripps Colleges, Claremont, CA, USA

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O. S. Akbari

Corresponding Author

O. S. Akbari

Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, CA, USA

Correspondence: Omar S. Akbari, Division of Biological Sciences, Section of Cell and Developmental Biology, University of California, San Diego, La Jolla, CA 92093, USA. Tel.: +1 858 246 0640; e-mail: [email protected]

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First published: 26 July 2020
Citations: 32

Abstract

CRISPR/Cas9 gene editing is a powerful technology to study the genetics of rising model organisms, such as the jewel wasp Nasonia vitripennis. However, current methods involving embryonic microinjection of CRISPR reagents are challenging. Delivery of Cas9 ribonucleoprotein into female ovaries is an alternative that has only been explored in a small handful of insects, such as mosquitoes, whiteflies and beetles. Here, we developed a simple protocol for germline gene editing by injecting Cas9 ribonucleoprotein in adult N. vitripennis females using either ReMOT control (Receptor-Mediated Ovary Transduction of Cargo) or BAPC (Branched Amphiphilic Peptide Capsules) as ovary delivery methods. For ReMOT Control we used the Drosophila melanogaster-derived peptide ‘P2C’ fused to EGFP to visualize the ovary delivery, and fused to Cas9 protein for gene editing of the cinnabar gene using saponin as an endosomal escape reagent. For BAPC we optimized the concentrations of protein, sgRNA and the transfection reagent. We demonstrate delivery of protein cargo such as EGFP and Cas9 into developing oocytes via P2C peptide and BAPC. Additionally, somatic and germline gene editing were demonstrated. This approach will greatly facilitate CRISPR-applied genetic manipulation in this and other rising model organisms.

Data availability statement

All data are described in the text and/or included in tables and figures within the manuscript and in the supplementary information files.