Evol Ecol Res 20: 107-132 (2019) Full PDF if your library subscribes.
Efficient CRISPR-Cas9 editing of major evolutionary loci in sticklebacks
Julia I. Wucherpfennig1, Craig T. Miller2 and David M. Kingsley1,3
1Department of Developmental Biology, Stanford University School of Medicine, Stanford, California, USA, 2Department of Molecular and Cell Biology, University of California-Berkeley, Berkeley, California, USA and 3Howard Hughes Medical Institute, Stanford University School of Medicine, Stanford, California, USA
Correspondence: D.M. Kingsley, Department of Developmental Biology and Howard Hughes Medical Institute, Stanford University School of Medicine, 279 Campus Drive, Stanford, CA 94305-5329, USA. email: firstname.lastname@example.org
Background: Stickleback fish are widely used to study the genetic and ecological basis of phenotypic evolution. Although several major loci have now been identified that contribute to evolutionary differences between wild populations, further study of the phenotypes associated with particular genes and mutations has been limited by the difficulty of generating targeted mutations at precise locations in the stickleback genome.
Approach and aims: We compared different methods of expressing single-guide RNAs (sgRNAs) and Cas9 activity in fertilized stickleback eggs. We used an easily scored pigmentation gene (SLC24A5) to screen for molecular lesions, phenotypic effects, and possible germline transmission of newly induced alleles. We then used the optimized CRISPR methods to target two major evolutionary loci in sticklebacks, KITLG and EDA. We hypothesized that coding region mutations in the KITLG gene would alter body pigmentation and possibly sex determination, and that mutations in the EDA gene would disrupt the formation of most armour plates, fin rays, spines, teeth, and gill rakers.
Results: Targeted deletions were successfully induced at each target locus by co-injecting one-cell stage stickleback embryos with either Cas9 mRNA or Cas9 protein, together with sgRNAs designed to protein-coding exons. Founder animals were typically mosaic for multiple mutations, which they transmitted through the germline at overall rates of 21% to 100%. We found that the copy of KITLG on the X chromosome (KITLGX ) has diverged from the KITLG on the Y chromosome (KITLGY). Predicted loss-of-function mutations in the KITLGX gene dramatically altered pigmentation in both external skin and internal organs, but the same was not true for KITLGY mutations. Predicted loss-of-function mutations in either the KITLGX or KITLGY genes did not lead to sex reversal or prevent fertility. Homozygous loss-of-function mutations in the EDA gene led to complete loss of armour plates, a severe reduction or loss of most soft rays in the dorsal, anal, and caudal fins, and severe reductions in the numbers of teeth and gill rakers. In contrast, long dorsal and pelvic spines remained intact in EDA mutant animals, suggesting that common co-segregation of plate loss and spine reduction in wild populations is unlikely to be due to pleiotropic effects of EDA mutations.
Conclusion: CRISPR-Cas9 approaches can be used to induce germline mutations in key evolutionary loci in sticklebacks. Targeted coding region mutations confirm an important role for KITLG and EDA in skin pigmentation and armour plate reduction, respectively. They also provide new information about the functions of these genes in other body structures.
Keywords: genome editing, stickleback, CRISPR-Cas9, SLC24A5, KITLG, EDA.
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