pHNRhCas9NG, single expression cassette-based dual-component dual-transcription unit CRISPR/Cas9 system for plant genome editing.

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, P, 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 100% gene-editing efficiency. Our system offers a novel, scalable tool for plant GEd, advancing CRISPR/Cas9 capabilities.