In rice splice variants that restore the reading frame after frameshifting indel introduction are common, often induced by the indels and sometimes lead to organism-level rescue

The introduction of frameshifting non-3n indels enables the identification of gene-trait associations. However, it has been hypothesised that recovery of the original reading frame owing to usage of non-canonical splice forms could cause rescue. To date there is very little evidence for organism-level rescue by such a mechanism and it is unknown how commonly indels induce, or are otherwise associated with, frame-restoring splice forms. We perform CRISPR/Cas9 editing of randomly selected loci in rice to investigate these issues. We find that the majority of loci have a frame-restoring isoform. Importantly, three quarters of these isoforms are not seen in the absence of the indels, consistent with indels commonly inducing novel isoforms. This is supported by analysis in the context of NMD knockdowns. We consider in detail the two top rescue candidates, in wax deficient anther 1 (wda1) and brittle culm (bc10), finding that organismal-level rescue in both cases is strong but owing to different splice modification routes. More generally, however, as frame-restoring isoforms are low abundance and possibly too disruptive, such rescue we suggest to be the rare exception, not the rule. Nonetheless, assuming that indels commonly induce frame-restoring isoforms, these results emphasize the need to examine RNA level effects of non-3n indels and suggest that multiple non-3n indels in any given gene are advisable to probe a gene's trait associations. Author summaryAs protein coding genes are read in units of three (codons), insertions or deletions (indels) that are not a multiple of three long (non 3n) are expected to be especially harmful. Whether they are is important both for interpreting the results of non-3n indel experiments to probe a gene's functional importance and for diagnostics. Particularly enigmatic are incidences where some non-3n changes in a gene compromise phenotypes while other seemingly comparable ones do not. One explanation for the latter is that a non-3n indel might be rescued via a frame-restoring splice form. Here we examine this hypothesis by inducing non-3n indels in many genes in rice and find that many non-3n indels are associated with a splice form that restores the reading frame. In the majority of these cases the indel appears to induce the potential rescuing splice form. We examine two top hit cases in detail and show functional rescue by splice modification. More generally, the frame-restoring forms are, however, low abundance and probably result in compromised proteins. We conclude then that splice mediated rescue is possible, but probably uncommon. Nonetheless it should not be overlooked in experimental design and interpretation.

Automated summary

The introduction of frameshifting non-3n indels enables the identification of gene-trait associations. However, the recovery of the original reading frame due to non-canonical splice forms is rare, and most frame-restoring isoforms induced by indels are low in abundance and potentially disruptive to proteins, suggesting that such rescue is uncommon. Despite this, it is important to consider the RNA level effects of non-3n indels and explore multiple non-3n indels in any given gene for probing trait associations.