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Dong, Oliver Xiaoou; Yu, Shu; Jain, Rashmi; Zhang, Nan; Duong, Phat Q; Butler, Corinne; Li, Yan; Lipzen, Anna; Martin, Joel A; Barry, Kerrie W; Schmutz, Jeremy; Tian, Li; Ronald, Pamela C
Nature communications, 03/2020, Volume: 11, Issue: 1Journal Article
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.
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