A fluorescence-based system for screening CRISPR-Cas13 efficacy and collateral effects

Sudip Dhakal¹, Arjun Challagulla¹, James Wynne² and Kristie Jenkins³

1, Health and Biosecurity, CSIRO, Geelong, VIC; 2, Agriculture and Food, CSIRO, Hobart, TAS; 3, Australian Animal Health Laboratory (AAHL), CSIRO, Geelong, VIC.

With the emergence of various Clustered regularly interspaced palindromic repeats (CRISPR) and Cas systems, editing both DNA and RNA in the various cell models and living organisms have been possible. CRISPR-Cas13 is a class of RNA guided RNA targeting proteins with a high efficiency and specificity. Different Cas13 proteins have shown to cause varying amounts of collateral activity. Hence, it is imperative to assess the collateral activity and on-target action of these Cas13 proteins to predict the aftermath of using these novel class of CRISPR/Cas system.

The aim of this study was to develop a rapid and efficient method to screen target specific and collateral activity of different Cas13 proteins.

In summary, a rapid fluorescence protein based method to compare the efficiency and collateral activity of Cas13 has been developed. In this study, Cas13 variant 3 was the most effective enzyme with more than 95% on-target effect, while it also showed 50% reduction of blue fluorescence as collateral effect.

References

Hu, Y., Chen, Y., Xu, J. et al. Metagenomic discovery of novel CRISPR-Cas13 systems. Cell Discov 8, 107 (2022).

Tong, H., Huang, J., Xiao, Q. et al. High-fidelity Cas13 variants for targeted RNA degradation with minimal collateral effects. Nat Biotechnol 41, 108–119 (2023).