Junction-targeting designs limit the application of CRISPR-Cas13d in circular RNA perturbation studies

Area of Science:

• Molecular Biology
• Genomics
• RNA Biology

Background:

• Circular RNAs (circRNAs) are formed by backsplicing, with thousands identified but few functionally characterized.
• Distinguishing circRNA effects from linear RNA targets poses a significant challenge in functional studies.
• CRISPR-Cas13 systems offer potential for selective targeting of unique circRNA junctions.

Purpose of the Study:

• To assess the limitations of Cas13d-mediated knockdown for studying circular RNAs.
• To evaluate the efficacy and specificity of single-guide RNAs (sgRNAs) targeting circRNA junctions.
• To determine the functional impact of highly expressed circRNAs on cell proliferation.

Main Methods:

• Performed essentiality screens using CRISPR-Cas13d targeting 900 circRNAs in K562 cells.
• Analyzed off-target knockdown effects on linear RNA isoforms.
• Re-analyzed existing Cas13d screens in other cell types.
• Developed machine learning models to predict Cas13d sgRNA efficacy.
• Assessed circRNA effects on cell proliferation.

Main Results:

• Observed consistent off-target knockdown of linear isoforms by circRNA-targeting sgRNAs.
• Identified similar off-target effects in previously published Cas13d screens.
• Machine learning models predicted low knockdown efficacy for most circRNA-targeting sgRNAs.
• Found 0 out of 346 testable circRNAs had detectable effects on proliferation after accounting for design constraints.

Conclusions:

• CRISPR-Cas13d junction-targeting strategies have significant limitations, including off-target effects and low sgRNA efficacy.
• Current junction-targeting approaches may overestimate the functional importance of circRNAs.
• These findings necessitate careful consideration of design constraints for future circRNA perturbation studies.