CRISPR/Cas9 genome-editing technology has revolutionized plant science
and holds enormous promise for crop improvement.

The exploration of this system received much attention regarding plant
genome editing. Here, by editing the/NtPDS/gene in tobacco, we first
verified that incorporating an OsU3-tRNA promoter combination into the
CRISPR/Cas9 system contributed to the highest editing efficiency, as the
sgRNA expression level was greater than that resulting from the
AtU6-tRNA and AtU6 promoters. Then, we optimized the existing tobacco
CRISPR/Cas9 system, pORE-Cas9, by using the OsU3-tRNA promoter
combination instead of AtU6 and by fusing an AtUb10-Ros1 expression
cassette to the T-DNA to monitor the transgene events.

The new system was named pOREU3TR. As expected, 49 transgene-free and
homozygous gene-edited green plants were effectively screened in the T_1
generation as a result of editing the/NtLHT1/gene in tobacco, and the
plant height and the contents of most free amino acids in the leaves of
the T_2 mutant plants were significantly different from those in the
leaves of WT plants, demonstrating the high efficiency of the new
editing system.

This OsU3-tRNA-sgRNA/AtUb10-Ros1 system provides essential improvements
for increasing the efficiency of plant genome editing.

[www.sciencedirect.com]