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.
Document Details
- Document Type
- Pub Defense Publication
- Publication Date
- Feb 18, 2022
- Source ID
- 10.1371/journal.pgen.1010060
Entities
People
- Deepak-Kumar Purusothaman
- Estela González
- Joshua X D Ang
- Katherine Nevard
- Lewis Shackleford
- Luke Alphey
- Michelle A E Anderson
- Rebekah Ireland
- Sebald An Verkuijl
Organizations
- Biotechnology and Biological Sciences Research Council
- Defense Advanced Research Projects Agency