# Systematic evaluation of CrRNA design parameters for optimized Cas13d-mediated RNA targeting in chicken cells

## TL;DR

This study evaluates how to design better crRNAs for the Cas13d system in chicken cells to improve RNA targeting efficiency and reduce off-target effects.

## Contribution

The study provides a systematic framework for optimizing crRNA design parameters for Cas13d in chicken cells.

## Key findings

- Several crRNAs achieved over 95% target knockdown in chicken fibroblast DF1 cells.
- crRNAs tolerate single-nucleotide mismatches but lose activity with 4-8 nucleotide mismatches.
- RfxCas13d and HfCas13d variants show differences in targeting efficiency and collateral activity.

## Abstract

The CRISPR-Cas13 system has emerged as a powerful platform for programmable RNA targeting, offering efficient and sequence-specific silencing of coding and non-coding transcripts. The RNA-targeting capabilities of CRISPR-Cas13 have been harnessed to silence transcripts harbouring pathogenic mutations and combat infectious diseases. However, the molecular basis of on-target and collateral activity are not completely understood, limiting the utility of Cas13 systems. In this study, we delineate the principles for the development of effective crRNAs by targeting DsRed fluorescence reporter and synthetic influenza mRNA in chicken fibroblast DF1 cells. To systematically determine the optimal design for RfxCas13d crRNA, we investigated the minimum length of the crRNA, importance of protospacer flanking sequence, degree of mismatch tolerance, and off target effects. Our data reveal variable knockdown levels between crRNAs, in which several crRNAs achieved over 95% target knockdown. We show that crRNAs exhibit a high degree of tolerance to single-nucleotide mismatches, regardless of their position in the spacer sequence. However, 4-nt mismatches between the spacer and the target significantly reduces targeting efficacy, whereas eight nucleotide mismatches completely abolish the activity of RfxCas13d. Finally, we compared targeting efficiency and collateral activity of two widely used RfxCas13d and HfCas13d variants. Our data extend current understanding of Cas13d-mediated RNA targeting and offer a framework for rational crRNA design to enhance effectiveness in diverse applications, including antiviral strategies.

The online version contains supplementary material available at 10.1007/s10142-025-01776-x.