Abstract
Two complimentary approaches are widely used to study gene function in zebrafish: induction of genetic mutations, usually using targeted nucleases such as CRISPR/Cas9, and suppression of gene expression, typically using Morpholino oligomers. Neither method is perfect. Morpholinos (MOs) sometimes produce off-target or toxicity-related effects that can be mistaken for true phenotypes. Conversely, genetic mutants can be subject to compensation, or may fail to yield a null phenotype due to leakiness (e.g. use of cryptic splice sites or downstream AUGs). When discrepancy between mutant and morpholino-induced (morphant) phenotypes is observed, experimental validation of such phenotypes becomes very labor intensive. We have developed a simple genetic method to differentiate between genuine morphant phenotypes and those produced due to off-target effects. We speculated that indels within 5′ untranslated regions would be unlikely to have a significant negative effect on gene expression. Mutations induced within a MO target site would result in a Morpholino-refractive allele thus suppressing true MO phenotypes whilst non-specific phenotypes would remain. We tested this hypothesis on one gene with an exclusively zygotic function, tbx5a, and one gene with strong maternal effect, ctnnb2. We found that indels within the Morpholino binding site are indeed able to suppress both zygotic and maternal morphant phenotypes. We also observed that the ability of such indels to suppress morpholino phenotypes does depend on the size and the location of the deletion. Nonetheless, mutating the morpholino binding sites in both maternal and zygotic genes can ascertain the specificity of morphant phenotypes.
Highlights
Introduction of aMO-refractive mutation into mRNA has the potential to exacerbate off-target effects
Since tbx5a mRNA is not contributed maternally, outcross of a parent heterozygous for a potentially MO-refractive mutation would produce a clutch of embryos where half would be genotypically wild type and susceptible to the MO, while the other half would be refractive to the MO
We found that Tbx5a-MO4 was able to almost entirely block translation of mRNAs containing wild type and (− 3) target sites
Summary
Introduction of aMO-refractive mutation into mRNA has the potential to exacerbate off-target effects. Results and discussion Partial suppression of tbx5a morphant phenotypes by the (− 7) mutation in MO target site. Since tbx5a mRNA is not contributed maternally, outcross of a parent heterozygous for a potentially MO-refractive mutation would produce a clutch of embryos where half would be genotypically wild type and susceptible to the MO, while the other half would be refractive to the MO.
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