Marker-free carotenoid-enriched rice generated through targeted gene insertion using CRISPR-Cas9

• Published in: Nature communications Vol. 11; no. 1; pp. 1178 - 10
• Main Authors: 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.
• Format: Journal Article
• Language: English
• Published: London Nature Publishing Group UK 04.03.2020; Nature Publishing Group; Nature Portfolio
• Subjects: 13/106; 38/22; 38/23; 38/77; 45/29; 631/449/1659; 631/449/447/2311; 631/449/711; 631/61/318; BASIC BIOLOGICAL SCIENCES; Biosynthesis; Biosynthetic Pathways - genetics; Carotenoids; Carotenoids - analysis; Carotenoids - metabolism; Cereal crops; CRISPR; CRISPR-Cas Systems - genetics; Deoxyribonucleic acid; DNA; DNA, Plant - genetics; Gene Editing - methods; Gene Knock-In Techniques - methods; Gene sequencing; Genome editing; Genome, Plant - genetics; Genomes; Genomics; Humanities and Social Sciences; Insertion; Inserts; Markers; Morphology; multidisciplinary; Mutation; Oryza - genetics; Oryza - metabolism; Plant Breeding - methods; Plants, Genetically Modified; Rice; Science; Science (multidisciplinary); Seeds; Seeds - chemistry; Transgenes; Transgenic plants; Whole Genome Sequencing
• ISSN: 2041-1723; 2041-1723
• DOI: 10.1038/s41467-020-14981-y

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. Existing examples of targeted gene insertion in plants either rely on a selectable marker gene or result in short DNA inserts. Here, the authors use an optimized CRISPR-Cas9 method to insert a 5.2 kb carotenoid biosynthesis cassette into genomic safe harbors in rice, and obtain marker-free lines with high carotenoid content.