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

Xin De Ang, J., Nevard, K., Ireland, R., Purusothaman, D.-K. , Verkuijl, S. A. N., Shackleford, L., Gonzalez, E., Anderson, M. A. E. and Alphey, L. (2022) Considerations for homology-based DNA repair in mosquitoes: impact of sequence heterology and donor template source. PLoS Genetics, 18(2), e1010060. (doi: 10.1371/journal.pgen.1010060) (PMID:35180218) (PMCID:PMC8893643)

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.

• Item Type: Articles
• Additional Information: Funding: J.X.D.A., K.N., R.I., S.A.N.V., L.S., E.G., M.A.E.A., and L.A were supported by the Defense Advanced Research Projects Agency (https://www.darpa.mil/program/safe-genes) [N66001-17-2-4054] to Kevin Esvelt at MIT; and the UK Biotechnology and Biological Sciences Research Council (https://bbsrc.ukri.org/) [BBS/E/I/00007033, BBS/E/I/00007038, and BBS/E/I/00007039 to The Pirbright Institute]. DKP’s PhD studentship was funded by The Pirbright Institute.
• Status: Published
• Refereed: Yes
• Glasgow Author(s) Enlighten ID: Purusothaman, Dr Deepak Kumar
• Authors: Xin De Ang, J., Nevard, K., Ireland, R., Purusothaman, D.-K., Verkuijl, S. A. N., Shackleford, L., Gonzalez, E., Anderson, M. A. E., and Alphey, L.
• College/School: College of Medical Veterinary and Life Sciences > School of Infection & Immunity > Centre for Virus Research
• Journal Name: PLoS Genetics
• Publisher: Public Library of Science
• ISSN: 1553-7390
• ISSN (Online): 1553-7404
• Copyright Holders: Copyright © 2022 Ang et al.
• First Published: First published in PLoS Genetics 18(2):e1010060
• Publisher Policy: Reproduced under a Creative Commons license