Notably, this included circFAM120a, which was the main circRNA hit that the original authors had selected for further characterization in their study.

Given the similarity in guide designs, we wondered if certain published screens also suffered from similar issues (doi.org/10.1038/s415...). By re-analyzing their data + conducting our own RT-qPCR experiments, we confirmed that many of their top hits were likely confounded by off-target effects.

This guide position is problematic because it provides the longest homology between the guide and the off-target linear junction (at the 5’ end of the Cas13 spacer). Despite using a 30 nucleotide spacer, only 22-23 nucleotides of homology are needed for Cas13 activation.

We found that most circRNA-targeting guides did not have an effect on cell growth, especially when compared to the effects of targeting linear mRNAs. Of the circRNA guides that did appear to alter cell growth, we noticed a concerning pattern: nearly all started at the same position along the BSJ.

We were eager to adopt this method ourselves, so we conducted growth screens in the well-studied cell line, K562. Using Cas13d, we targeted 900 of the most highly expressed circRNAs in K562, along with their matched linear isoforms.

CircRNAs are alternatively-spliced isoforms that derive from genes that typically produce linear mRNAs. They consist of the same sequences as their linear counterparts, but are back-spliced to produce closed RNA loops, which are marked by a unique sequence known as the back-splice junction (BSJ).

Came across a poem I wrote in undergrad for extra credit.