Efficient CRISPR-Cas9 editing of major evolutionary loci in sticklebacks.

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 armor 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 organ, 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 armor plates, severe reduction or loss of most soft rays in the dorsal, anal, and caudal fins, and severe reductions in tooth and gill raker number. 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 armor plate reduction, respectively. They also provide new information about the functions of these genes in other body structures.

The genetic basis of novel trait gain in walking fish.

A major goal in biology is to understand how organisms evolve novel traits. Multiple studies have identified genes contributing to regressive evolution, the loss of structures that existed in a recent ancestor. However, fewer examples exist for genes underlying constructive evolution, the gain of novel structures and capabilities in lineages that previously lacked them. Sea robins are fish that have evolved enlarged pectoral fins, six mobile locomotory fin rays (legs) and six novel macroscopic lobes in the central nervous system (CNS) that innervate the corresponding legs. Here, we establish successful husbandry and use a combination of transcriptomics, CRISPR-Cas9 editing, and behavioral assays to identify key transcription factors that are required for leg formation and function in sea robins. We also generate hybrids between two sea robin species with distinct leg morphologies and use allele-specific expression analysis and gene editing to explore the genetic basis of species-specific trait diversity, including a novel sensory gain of function. Collectively, our study establishes sea robins as a new model for studying the genetic basis of novel organ formation, and demonstrates a crucial role for the conserved limb gene tbx3a in the evolution of chemosensory legs in walking fish.

Evolution of stickleback spines through independent cis-regulatory changes at HOXDB.

Understanding the mechanisms leading to new traits or additional features in organisms is a fundamental goal of evolutionary biology. We show that HOXDB regulatory changes have been used repeatedly in different fish genera to alter the length and number of the prominent dorsal spines used to classify stickleback species. In Gasterosteus aculeatus (typically 'three-spine sticklebacks'), a variant HOXDB allele is genetically linked to shortening an existing spine and adding an additional spine. In Apeltes quadracus (typically 'four-spine sticklebacks'), a variant HOXDB allele is associated with lengthening a spine and adding an additional spine in natural populations. The variant alleles alter the same non-coding enhancer region in the HOXDB locus but do so by diverse mechanisms, including single-nucleotide polymorphisms, deletions and transposable element insertions. The independent regulatory changes are linked to anterior expansion or contraction of HOXDB expression. We propose that associated changes in spine lengths and numbers are partial identity transformations in a repeating skeletal series that forms major defensive structures in fish. Our findings support the long-standing hypothesis that natural Hox gene variation underlies key patterning changes in wild populations and illustrate how different mutational mechanisms affecting the same region may produce opposite gene expression changes with similar phenotypic outcomes.

DNA fragility in the parallel evolution of pelvic reduction in stickleback fish.

Evolution generates a remarkable breadth of living forms, but many traits evolve repeatedly, by mechanisms that are still poorly understood. A classic example of repeated evolution is the loss of pelvic hindfins in stickleback fish (Gasterosteus aculeatus). Repeated pelvic loss maps to recurrent deletions of a pelvic enhancer of the Pitx1 gene. Here, we identify molecular features contributing to these recurrent deletions. Pitx1 enhancer sequences form alternative DNA structures in vitro and increase double-strand breaks and deletions in vivo. Enhancer mutability depends on DNA replication direction and is caused by TG-dinucleotide repeats. Modeling shows that elevated mutation rates can influence evolution under demographic conditions relevant for sticklebacks and humans. DNA fragility may thus help explain why the same loci are often used repeatedly during parallel adaptive evolution.

Mutations in RABL3 alter KRAS prenylation and are associated with hereditary pancreatic cancer.

Pancreatic ductal adenocarcinoma is an aggressive cancer with limited treatment options1. Approximately 10% of cases exhibit familial predisposition, but causative genes are not known in most families2. We perform whole-genome sequence analysis in a family with multiple cases of pancreatic ductal adenocarcinoma and identify a germline truncating mutation in the member of the RAS oncogene family-like 3 (RABL3) gene. Heterozygous rabl3 mutant zebrafish show increased susceptibility to cancer formation. Transcriptomic and mass spectrometry approaches implicate RABL3 in RAS pathway regulation and identify an interaction with RAP1GDS1 (SmgGDS), a chaperone regulating prenylation of RAS GTPases3. Indeed, the truncated mutant RABL3 protein accelerates KRAS prenylation and requires RAS proteins to promote cell proliferation. Finally, evidence in patient cohorts with developmental disorders implicates germline RABL3 mutations in RASopathy syndromes. Our studies identify RABL3 mutations as a target for genetic testing in cancer families and uncover a mechanism for dysregulated RAS activity in development and cancer.