Considerations for homology-based DNA repair in mosquitoes: Impact of sequence heterology and donor template source.

Joshua Xin De Ang,Lewis Shackleford,Luke Alphey,Sebald A N Verkuijl,Estela Gonzalez,Deepak-Kumar Purusothaman,Katherine Nevard,Michelle A E Anderson,Rebekah Ireland,Subba Reddy Palli

doi:10.1371/journal.pgen.1010060

Joshua Xin De Ang, Lewis Shackleford + Show 8 more

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https://doi.org/10.1371/journal.pgen.1010060

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Abstract

The increasing prevalence of insecticide resistance and the ongoing global burden of vector-borne diseases have encouraged new efforts in mosquito control. For Aedes aegypti, the most important arboviral vector, integration rates achieved in Cas9-based knock-ins so far have been rather low, highlighting the need to understand gene conversion patterns and other factors that influence homology-directed repair (HDR) events in this species. In this study, we report the effects of sequence mismatches or donor template forms on integration rates. We found that modest sequence differences between construct homology arms [DNA sequence in the donor template which resembles the region flanking the target cut] and genomic target comprising 1.2% nucleotide dissimilarity (heterology) significantly reduced integration rates. While most integrations (59–88%) from plasmid templates were the result of canonical [on target, perfect repair] HDR events, no canonical events were identified from other donor types (i.e. ssDNA, biotinylated ds/ssDNA). Sequencing of the transgene flanking region in 69 individuals with canonical integrations revealed 60% of conversion tracts to be unidirectional and extend up to 220 bp proximal to the break, though in three individuals bidirectional conversion of up to 725 bp was observed.

Highlights

Aedes aegypti mosquitoes are the primary vector of the viruses that cause dengue [1], chikungunya [2], yellow fever [3] and Zika [4], which account for hundreds of millions of infections each year [5]
To generate a Cas9-based integration, Cas9 and sgRNA are used to cleave a chromosomal locus while a plasmid DNA donor, containing a genetic cargo flanked by sequences homologous to the chromosomal locus, is supplied as a repair template
Due to the homology arms, the broken strands of the chromosome recognise the donor as a repair template and the transgene is copied as a result of homology-directed repair (HDR), thereby integrating the injected template sequence into the genome

Summary

Introduction

Aedes aegypti mosquitoes are the primary vector of the viruses that cause dengue [1], chikungunya [2], yellow fever [3] and Zika [4], which account for hundreds of millions of infections each year [5]. Once a DSB is induced, the cell will either repair the break via non-homologous end-joining (NHEJ) which is error-prone, or via homology-directed repair (HDR), copying the sequences of an uncut homologous chromosome. The use of site-specific knock-ins in Ae. aegypti have included the development of Cas9-based gene drive systems [19,20], driver lines to express transcriptional activators in specific cell types [21–23], and the study of the effects of amino acid changes on protein function [24]. In other organisms, this tool has been used to tag and study endogenous expression of gene products [25]

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