# Development of an RNA aptamer-assisted CRISPR/Cas9 system for efficiently generating and isolating Cas9-free mutants in plant

## TL;DR

A new CRISPR/Cas9 system using RNA aptamers improves the efficiency of generating and identifying Cas9-free plant mutants.

## Contribution

The 3WJ-4 × Bro/Cas9 system introduces an RNA aptamer as a non-interfering reporter for efficient plant genome editing.

## Key findings

- 3WJ-4 × Bro/Cas9 increased T1 mutation rates by 78.6% compared to the GFP/Cas9 system.
- The system improved sorting efficiency for Cas9-free mutants by 30.2% in the T2 generation.
- It enabled more efficient generation of homozygous double-target mutants in Arabidopsis thaliana.

## Abstract

The CRISPR/Cas9 gene-editing system is a powerful tool in plant genetic engineering; however, screening for Cas9-free edited plants remains complex and time-consuming. To address this limitation, we developed an RNA aptamer-assisted CRISPR/Cas9 system, termed 3WJ-4 × Bro/Cas9. In this system, the engineered RNA aptamer 3WJ-4 × Bro functions as a transcriptional reporter, serving as an alternative to traditional fluorescent proteins and thus avoiding their potential interference with Cas9 activity. Compared to the conventional GFP/Cas9 system, 3WJ-4 × Bro/Cas9 showed more efficient transformation and higher accuracy in fluorescence-based selection of positive T1 transformants, without significantly affecting plant growth. Furthermore, 3WJ-4 × Bro/Cas9 achieved a 78.6% increase in the T1 mutation rate compared to GFP/Cas9, with the homozygous mutation rate reaching 1.78%. In addition, 3WJ-4 × Bro/Cas9 enabled fluorescence-based visual screening in the T2 generation for rapid identification of Cas9-free mutants, improving sorting efficiency by 30.2% over the GFP-based method. Moreover, 3WJ-4 × Bro/Cas9 enabled more efficient generation of homozygous double-target mutants compared to GFP/Cas9. These results demonstrate that the 3WJ-4 × Bro/Cas9 system provides a non-transgenic, efficient, and broadly applicable strategy for plant genome editing and selection.

While gene editing has become an essential tool for plant research and crop improvement, traditional fluorescent protein-assisted CRISPR/Cas9 low accuracy in screening Cas9-free mutants. In this study, we aimed to address the challenge of screening for Cas9-free edited plants in the CRISPR/Cas9 gene editing system. To overcome this limitation, we developed an RNA aptamer-assisted CRISPR/Cas9 system, termed 3WJ-4 × Bro/Cas9, which utilizes an engineered RNA aptamer as a transcriptional reporter for fluorescence-based selection of positive transformants and Cas9-free mutants. Our results show that the 3WJ-4 × Bro/Cas9 system outperforms the conventional GFP/Cas9 system in terms of transformation efficiency, mutation rate, and Cas9-free line identification in Arabidopsis thaliana. This method holds promise to accelerate advances in plant genetics research and support future crop improvement efforts.