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

Abstract

Targeted insertion of transgenes at predetermined 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.

The importance of thiamine (vitamin B1) in plant health: From crop yield to biofortification

“…Conventional methods of plant genetic modification previously employed cloning genes into the tumor-inducing plasmid of Agrobacterium tumefaciens, exploiting its DNA transmission capabilities to insert novel genes into plant genomes (107). However, the introduction of CRISPR/Cas9 technologies for genetic engineering (108) has enabled targeted insertion of transgenes at specific locations throughout plant genomes (11,13) and without additional insertion of bacterial flanking DNA (109). Recently, these techniques were successful in inserting a carotenoid biosynthesis cassette into rice, which yielded processed rice grains with high carotenoid (vitamin A) content and had no deleterious effects on crop yield or physiology (109).…”

“…However, the introduction of CRISPR/Cas9 technologies for genetic engineering (108) has enabled targeted insertion of transgenes at specific locations throughout plant genomes (11,13) and without additional insertion of bacterial flanking DNA (109). Recently, these techniques were successful in inserting a carotenoid biosynthesis cassette into rice, which yielded processed rice grains with high carotenoid (vitamin A) content and had no deleterious effects on crop yield or physiology (109). Once appropriate genes are identified for thiamine enhancement, a similar approach could be used.…”

Multiplying the efficiency and impact of biofortification through metabolic engineering

“…iron, zinc, vitamin A, and vitamin B9), whilst also taking the aforementioned stability into account. Moreover, with the advent of genome editing technologies, it is now possible to insert a gene cassette at a pre-defined location (safe harbor) in the genome of a crop plant, thereby avoiding adverse effects such as yield losses 62 . On a cautionary note, researchers need vigilance to recognize unintended metabolic consequences of biofortification.…”

Plant science decadal vision 2020–2030: Reimagining the potential of plants for a healthy and sustainable future

“…Two areas with high potential are synergistic with Goal 2: identifying new plant sources for nutritional improvement and increasing the nutritional content of current crops. The methods used to create golden rice represented a scientific tour de force at the time, (Regis, 2019) but present‐day and emerging tools are far more precise (Dong et al., 2020) and can leverage much‐improved knowledge and modeling of metabolic pathways. For example, biochemical engineering has been used to enrich tomatoes for improved health benefits to consumers (Butelli et al., 2008; Zhang et al., 2015).…”