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

Abstract

Aedes (Ae.) aegypti and Ae. albopictus mosquitoes transmit arthropod-borne viruses (arboviruses) 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 knock-down AGO1 cell lines. 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.

Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos

“…A secondary analysis of the flow cytometry data, performed using the variation of the median fluorescence intensity (MFI) of mCherry signal within the GFP + cells, confirmed the relative changes in KO efficiencies ( Supplementary Figure 2b ). These results are in accordance with overall evolutionary distance between the species and U6 promoter sequence average distance and corroborate previous in vitro 10,11,13,14 and in vivo 12,13 results.…”

“…An incompletely addressed challenge for CRISPR genome engineering in mosquitos is the identity of optimal pol III promoters for heterologous expression of sgRNAs [10][11][12][13][14] . We performed side-by-side evaluations of eleven pol III promoters from four mosquito species, as well as a consensus sequence, in each of the three mosquito cell lines.…”

TMEM41B Is a Pan-flavivirus Host Factor

“…Gene disruption was confirmed by high throughput amplicon sequencing and described above. For additional details please see ( Rozen-Gagnon et al., 2020 ).…”

AePUbpromoter length modulates gene expression inAedes aegypti

“…Most tools used today were originally developed in the model insect Drosophila melanogaster. The optimization of these for use in mosquitoes has enabled developments in gene editing tools such as CRISPR/Cas9 (2,3). Promoter fragments for the expression of genes of interest in both cell culture and whole insects play a crucial role in our ability to investigate this mosquito.…”