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

Joshua Xin de Ang; Katherine Nevard; Rebekah Ireland; Deepak Kumar Purusothaman; Sebald A.N. Verkuijl; Lewis Shackleford; Estela Gonzalez; Michelle A.E. Anderson; Luke Alphey

doi:10.1371/journal.pgen.1010060

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

Joshua Xin de Ang, Katherine Nevard, Rebekah Ireland, Deepak Kumar Purusothaman, Sebald A.N. Verkuijl, Lewis Shackleford, Estela Gonzalez, Michelle A.E. Anderson^*, Luke Alphey^*

^*Corresponding author for this work

Biology

Research output: Contribution to journal › Article › peer-review

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.

Original language	English
Article number	1010060
Number of pages	18
Journal	PLoS Genetics
Volume	18
Issue number	2 February
DOIs	https://doi.org/10.1371/journal.pgen.1010060
Publication status	Published - 18 Feb 2022

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10.1371/journal.pgen.1010060Licence: CC BY

Considerations for homology-based DNA repair in mosquitoes: Impact of sequence heterology and donor template source
© 2022 Ang et al.
Final published version, 1.7 MBLicence: CC BY

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title = "Considerations for homology-based DNA repair in mosquitoes: Impact of sequence heterology and donor template source",

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.",

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AU - Verkuijl, Sebald A.N.

AU - Shackleford, Lewis

AU - Gonzalez, Estela

AU - Anderson, Michelle A.E.

AU - Alphey, Luke

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

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