Angela Meccariello, Flavia Krsticevic, Rita Colonna, Giuseppe Del Corsano, Barbara Fasulo, Philippos Aris Papathanos & Nikolai Windbichler

BMC Biology volume 19, Article number: 78 (2021) on line 16 April 2021

Background

Genetic sex ratio distorters are systems aimed at effecting a bias in the reproductive sex ratio of a population and could be applied for the area-wide control of sexually reproducing insects that vector disease or disrupt agricultural production. One example of such a system leading to male bias is X-shredding, an approach that interferes with the transmission of the X-chromosome by inducing multiple DNA double-strand breaks during male meiosis. Endonucleases targeting the X-chromosome and whose activity is restricted to male gametogenesis have recently been pioneered as a means to engineer such traits.

Results

Here, we enabled endogenous CRISPR/Cas9 and CRISPR/Cas12a activity during spermatogenesis of the Mediterranean fruit fly Ceratitis capitata, a worldwide agricultural pest of extensive economic significance. In the absence of a chromosome-level assembly, we analysed long- and short-read genome sequencing data from males and females to identify two clusters of abundant and X-chromosome-specific sequence repeats. When targeted by gRNAs in conjunction with Cas9, cleavage of these repeats yielded a significant and consistent distortion of the sex ratio towards males in independent transgenic strains, while the combination of distinct distorters induced a strong bias (~ 80%).

Conclusion

We provide a first demonstration of CRISPR-based sex distortion towards male bias in a non-model organism, the global pest insect Ceratitis capitata. Although the sex ratio bias reached in our study would require improvement, possibly through the generation and combination of additional transgenic lines, to result in a system with realistic applicability in the field, our results suggest that strains with characteristics suitable for field application can now be developed for a range of medically or agriculturally relevant insect species.

See: https://bmcbiol.biomedcentral.com/articles/10.1186/s12915-021-01010-7

Figure 2:

CRISPR/Cas constructs and activity against white eye in the male germline. a Schematic representation of the transformation constructs. Cas9 or Cas12a coding sequences are under the transcriptional control of the male germline-specific β2-tubulin promoter (pβ2tub). The gRNA target sequences are under the control of the endogenous U6 Pol III promoter (pCcU6), and the constitutively expressed DsRed as a marker of transgenesis is under the control of the Ubiquitin promoter (pUb). Gypsy insulator sequences (gypsy) are indicated adjacent to pβ2tub and pUb. b Wild-type red-eye phenotype of an adult male medfly (left) and white-eye phenotype of a female medfly (right). c The percentage of white-eyed flies obtained by individually crossing hemizygous males expressing Cas9 and the gRNA targeting the white eye gene (Cas9.w) with white-eyed w2∆/w2∆ females (w) is shown in the top panel. As a control, hemizygous females (Cas9.w) were individually crossed with 10 white-eyed w2∆/w2∆ males (w). The middle panel shows the percentage of white-eyed flies obtained by individually crossing hemizygous males expressing Cas12a and the gRNA targeting the white eye gene (Cas12a.w) with white-eyed w2∆/w2∆ females (w), by crossing hemizygous males for Cas12a.m with white-eyed w2∆/w2∆ females (w) or by crossing hemizygous Cas12a.m females with w2∆/w2∆ white-eyed males (w). The bottom panel shows the control crosses between wild-type males with white-eyed females from the w2∆ strain (wt x w) and white-eyed males from the w2∆ strain crossed with wild-type females (w x wt). The numbers indicate the total number of individuals scored (N). d Diagram representation of the autosomal white eye gene of Ceratitis capitata, spanning six exons, and the sequence of the gRNA targeting exon 3 (PAM is shown in yellow). Below, the wild-type sequence is aligned to sequences obtained from F1 white-eyed flies, showing the indels induced by CRISPR/Cas9. The deletion spanning exon 2 of the w2∆ white eye mutant strain is indicated