Considerations for homology-based DNA repair in mosquitoes: Impact of sequence heterology and donor template source.
Journal
PLoS genetics
ISSN: 1553-7404
Titre abrégé: PLoS Genet
Pays: United States
ID NLM: 101239074
Informations de publication
Date de publication:
02 2022
02 2022
Historique:
received:
31
08
2021
accepted:
28
01
2022
revised:
03
03
2022
pubmed:
19
2
2022
medline:
6
4
2022
entrez:
18
2
2022
Statut:
epublish
Résumé
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.
Identifiants
pubmed: 35180218
doi: 10.1371/journal.pgen.1010060
pii: PGENETICS-D-21-01170
pmc: PMC8893643
doi:
Types de publication
Journal Article
Research Support, Non-U.S. Gov't
Langues
eng
Sous-ensembles de citation
IM
Pagination
e1010060Subventions
Organisme : Biotechnology and Biological Sciences Research Council
ID : BBS/E/I/00007033
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BBS/E/I/00007039
Pays : United Kingdom
Organisme : Biotechnology and Biological Sciences Research Council
ID : BBS/E/I/00007038
Pays : United Kingdom
Déclaration de conflit d'intérêts
The authors have declared that no competing interests exist.
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