A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines

Kathryn Rozen-Gagnon,Sasha Novack,Eliana Jacobson,Soon Yi,Charles M Rice

doi:10.1038/s41598-020-80436-5

Abstract

Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~ 700,000 deaths each year. Genetic mutants are valuable tools to interrogate both fundamental vector biology and mosquito host factors important for viral infection. However, very few genetic mutants have been described in mosquitoes in comparison to model organisms. The relative ease of applying CRISPR/Cas9-based gene editing has transformed genome engineering and has rapidly increased the number of available gene mutants in mosquitoes. Yet, in vivo studies may not be practical for screening large sets of mutants or possible for laboratories that lack insectaries. Thus, it would be useful to adapt CRISPR/Cas9 systems to common mosquito cell lines. In this study, we generated and characterized a mosquito optimized, plasmid-based CRISPR/Cas9 system for use in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines. We demonstrated highly efficient editing of the AGO1 locus and isolated U4.4 and Aag2 cell lines with reduced AGO1 expression. Further, we used homology-directed repair to establish knock-in Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1. These experimentally verified plasmids are versatile, cost-effective, and efficiently edit immune competent mosquito cell lines that are widely used in arbovirus studies.

Highlights

Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~ 700,000 deaths each year
To generate mosquito optimized plasmids for efficient clustered regularly interspaced short palindromic repeats (CRISPR)/Cas[9] gene editing in mosquito cells, we obtained a plasmid used in Drosophila, pDCC629
This plasmid relies on two Drosophila promoters to express CRISPR/Cas[9] components: (1) the RNA Pol III U6:96Ab29,47–51 drives transcription of the single guide RNA (sgRNA), and (2) the hsp70Bb47,52 promoter drives expression of the human codon-optimized Streptococcus pyogenes Cas[9]

Summary

Introduction

Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne diseases around the globe, causing ~ 700,000 deaths each year. We updated CRISPR/Cas[9] plasmids that rely on Drosophila promoters[29] with mosquito promoters for use in mosquito cells We applied this system to edit widely utilized, immune-competent Ae. aegypti (Aag2)[40,41,42] and Ae. albopictus (U4.4)[42,43] cell lines. We generated AGO1-edited U4.4 and Aag[2] cell lines with reduced AGO1 protein levels, as well as knockin Aag[2] cell lines that contain a 3xFLAG tag at the N-terminus of endogenous AGO1 These well-characterized and efficient mosquito optimized CRISPR/Cas[9] plasmids will facilitate functional genetic studies in mosquito cell culture systems

Methods

Results

Conclusion