2023-06-28

Alternative splicing of RNA

Presentation by Claire Harbison

Alternative splicing of RNA causes different isoforms of the same gene.

The idea is to knockdown isoforms and see its effects on phenotypes. How to knowckdown the isoforms?

Cas13 has been shown to target and knockdown exonic regions of RNA. See East-Seletsky et al. and Abudayyah et al.

Cheng et al explored Cas13 guide activity, predicting gRNAs with ML algorithms. Model provided 8 guides across two junctions with singnifcant knockdown of COP1.

  • What is Cas13 guide activity?
    Type VI CRISPR enzymes are RNA-targeting proteins with nuclease activity that enable specific and robust target gene knockdown without altering the genome. Cas13 proteins are guided to their target RNAs by a single CRISPR RNA (crRNA) composed of a direct repeat (DR) stem loop and a spacer sequence (gRNA) that mediates target recognition by RNA–RNA hybridization.

  • What is splice junction?
    In the splicing of RNA, the site of a former intron in a mature mRNA.

  • What is the expression level of COP1?
    Around 50tpm in a regular cell.

Predicted guides span the junctions to see if the guide placement impacts knockdown.

They took the 8 guides, prepare the Cas13 guide RNAs, and added it to the media. Activate Cas13 with doxycycline, Extract cells, extract RNA and perform RT-qPCR. The drug doxycycline activates Cas13 when added, and deactivates when removed from the media. - This is reversible, but there is a time delay of days.

There were 4 guides at common junction, and 4 guides at unique junction. The knockdown was around 30% to 50% in A-375 cell lines.

What other parameters could impact knockdown?

  • Cell type
  • Biological replicates
  • Time of Cas13 activity

Knockdown in stem cells was more promising, where the expression reduced to around 5% to 25%. Guides targeting common junctions generally performed better than guides targeting unique juctions. They all work decently.

In HEKs cells (human embryonic kidney), the knockdown was better than the first experiment and worse than the stem cells.

Cas13 performs similarly across biological replicates in HEKs cells.

For common junction, 24 hours of Cas13 activity is the sweet spot. But for unique junctions, 48 hours of Cas13 activity is the sweet spot. (?) The variability is very minimal, could be a technical noise. One could maybe pick any time duration.

  • Does the prediction of guide RNAs also depend on cell type?
    A predicted guide RNA should work across all cell types. It depends on the secondary structure of the target RNA.