Journal Article

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

Lipzen, Anna, Schmutz, Jeremy, Dong, Oliver Xiaoou, Barry, Kerrie W., Ronald, Pamela C., Duong, Phat Q., Zhang, Nan, Tian, Li, Butler, Corinne, Jain, Rashmi, Martin, Joel A., Li, Yan, Yu, Shu

2020

Overview

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.

• Publisher: Nature Publishing Group UK,Nature Publishing Group,Nature Portfolio
• Subject: 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