Citation: Hu, N., Tian, H., Li, Y., Li, X., Li, D., Li, L., Wang, S., Zhang, Y., Shi, X., Huang, B., Lu, Q., Wang, T., Pan, X., Tu, L., Dai, D., Zhang, B., Peng, R., & Yan, F. (2025). pHNRhCas9NG, single expression cassette-based dual-component dual-transcription unit CRISPR/Cas9 system for plant genome editing. Trends in Biotechnology. https://doi.org/10.1016/j.tibtech.2025.03.016
Keywords: CRISPR/Cas9, T-DNA insertion, improve genome-editing efficiency, transgenic tomato, DDS, Poly(A), egfp fluorescence
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 genome-editing (GEd) technology has revolutionized plant science, facilitating gene function studies and crop improvement. Despite its success, plant- specific CRISPR/Cas9 systems require further optimization. This study aims to boost plant GEd efficiency by revamping the CRISPR/Cas9 system. We addressed large fragment deletions in T-DNA (transfer DNA) postgenomic insertion by developing a binary expression vector, pHNR, which maintains T-DNA integrity using protective sequences. We discovered an artificial promoter, P35SIC47, effective in tobacco, Arabidopsis, and tomato transformation, and designed a dual-component dual-transcription unit CRISPR/Cas9 system (DDS) with optimal gene expression at a poly(A) length of ~150 base pairs. Enhancing the poly (A) tail length of Cas9 mRNA significantly boosted plant GEd efficiency. We also identified compatible hCas9 versions through transitory expression in tobacco leaves. Utilizing pHNRhCas9NG, we efficiently knocked out ten genes in tomato, achieving almost gene-editing efficiency. Our system offers a novel, scalable tool for plant GEd, advancing CRISPR/Cas9 capabilities.
EGFP Fluorescence Analysis
Histological fluorescence microscopy analysis was conducted to detect the expression of the EGFP gene in the plant material. Tissue samples were collected from different tissue types of transgenic tomato plants, and then examined under a fluorescence microscope for EGFP gene expression. EGFP fluorescence detection of transgenic plants was performed by EGFP fluorescence flashlight (LUYOR-3280RB) from LUYOR.
Photo below: The EGFP fluorescence in transgenic tomatoes (right) were taken using an EGFP fluorescence flashlight LUYOR-3280RB and LUV-520 filter.
Fluorescence Flashlight LUYOR-3280RB
The fluorescence flashlight LUYOR-3280RB is a portable, efficient tool to detect GFP and EGFP expression in plants, live animals, cells, etc. Each fluorescence flashlight is paired with a matching barrier filter for fluorescence photography. Visualize fluorescence and screen transgenic samples in a faster and easier way!
Photo below: GFP/EGFP fluorescence flashlight LUYOR-3280RB
