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.

A CRISPR endonuclease gene drive reveals distinct mechanisms of inheritance bias

“…2 a). However, the large distance between the w GDe drive and the M / m locus leads us to believe that this is unlikely, as co-conversion in similar contexts is generally reported to be on the scale of 100s of base pairs 11 , 20 , 47 – 49 . Furthermore, a substantial fraction of conversion tracts have been reported to be unidirectional in A. aegypti 49 .…”

“…However, the large distance between the w GDe drive and the M / m locus leads us to believe that this is unlikely, as co-conversion in similar contexts is generally reported to be on the scale of 100s of base pairs 11 , 20 , 47 – 49 . Furthermore, a substantial fraction of conversion tracts have been reported to be unidirectional in A. aegypti 49 . This suggests that even if large-scale co-conversion was favoured, some repair events should still have caused recombination between w GDe and the sex-determining locus if co-conversion occurred primarily in the other direction relative to the sex-determination locus.…”

Selective targeting of biting females to control mosquito-borne infectious diseases

“…In addition, haplotype-resolved assemblies will reveal the molecular basis underlying the high levels of differentiation between the ~100 Mb M-and m-linked regions of the homomorphic sex chromosomes (e.g., [22]). These results will have a direct impact on identifying m chromosome-specific sequences for sex ratio distortion through m-shredding and increase knock-in efficiency in M-linked sequences by specifically targeting the M-linked haplotypes [94]. Therefore, beyond revealing the pattern of sex-linked sequence differentiation, haplotype-resolved assemblies will enable new ways to study allele-specific expression, long-range allelic interaction, genetic recombination, and epigenetic inheritance (e.g., [95]).…”

CRISPR-Mediated Cassette Exchange (CriMCE): A Method to Introduce and Isolate Precise Marker-Less Edits

“…This focuses molecular identification by PCR and sequencing on individuals carrying the desired edit, therefore reducing the rearing effort required to enrich the frequency of marker-less variants (Supplementary Figure 3). Somewhat surprisingly, rates of HDR-induced editing are relatively high when marked mutations are introduced (Table 1), 23,[28][29][30] but drop substantially when SNPs are directly inserted into a wild-type genomic locus, in Aedes aegypti and An. gambiae (Table 1, Figure 4).…”