Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9
Oliver Xiaoou Dong, Shu Yu, Rashmi Jain, Nan Zhang, Phat Q. Duong, Corinne Butler, Yan Li, Anna Lipzen, Joel Martin, Kerrie Barry, Jeremy Schmutz, Li Tian, Pamela C. Ronald
Abstract
Targeted insertion of transgenes at pre-determined plant genomic safe harbors provides a desirable alternative to insertions at random sites achieved through conventional methods. Most existing cases of targeted gene insertion in plants have either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency with relatively small DNA fragments (<1.8 kb). Here, we report the use of an optimized CRISPR-Cas9-based method to achieve the targeted insertion of a 5.2 kb carotenoid biosynthesis cassette at two genomic safe harbors in rice. We obtain marker-free rice plants with high carotenoid content in the seeds and no detectable penalty in morphology or yield. Whole-genome sequencing reveals the absence of off-target mutations by Cas9 in the engineered plants. These results demonstrate targeted gene insertion of marker-free DNA in rice using CRISPR-Cas9 genome editing, and offer a promising strategy for genetic improvement of rice and other crops.