Highly efficient transgene-free plant genome editing in tobacco using an optimized CRISPR/Cas9 system, pOREU3TR

Abstract Background: CRISPR-Cas9 Genome-editing technology has revolutionized the plant science and hold enormous promise in crop improvement. When performing the gene editing system for crops, it is crucial to eliminate of the Cas9/sgRNA T-DNA cassette in the T1 generation. Results: Here, we firstly validated that incorporating an OsU3-tRNA promoter combination in the CRISPR/Cas9 system contributed to the highest mutagenesis efficiency that increased sgRNA expression levels over the AtU6-tRNA and AtU6 promoters by editing the NtPDS gene (Ntab0595110) in tobacco. Then we optimized the existing tobacco CRISPR/Cas9 system by using the OsU3-tRNA promoter combination instead of AtU6, and fusing an AtUb10-Ros1 expression cassette in T-DNA for monitoring the transgene events. The new vector was named as pOREU3TR. As expected, 52 transgene-free and homozygous gene-edited green plants were effectively screened at T1 generation by editing the NtLHT1 gene (Ntab0818090) in tobacco, and the contents of most free amino acids in the T2 mutants ntlht1 leaves were detected significantly different from those in the wild type leaves, demonstrating the highly efficient of the system.Conclusions: This OsU3-tRNA-sgRNA/AtUb10-Ros1 system provides essential improvements to increase the efficiency of plant genome editing.