APPLICATIONS OF TECHNOLOGY:
- Plant genome editing (e.g. targeted gene insertion)
- This invention provides targeted gene insertion at pre-determined genomic safe harbors in plants at a much higher frequency than current methods. This provides a desirable alternative to random transgene insertion obtained through conventional methods.
- Obviation of the need for a selectable marker gene, thus increasing the capacity of the cassette to be inserted. Notably, the absence of a selectable marker gene in the final product would potentially reduce the regulations involved.
- Researchers anticipate that the method can be adapted to stack plant traits conferred by multiple cassettes at a single genetic locus. This would reduce the labor in breeding these complex traits into various plant cultivars.
- Although several cases of targeted gene insertion in plants using CRISPR-Cas have been reported, the efficiencies of targeted insertions of large DNA molecules in plants by CRISPR-Cas are low.
- Most cases of targeted gene insertion in plants reported to date either relied on the presence of a selectable marker gene in the insertion cassette or occurred at low frequency (<2.2%) with relatively small DNA fragments (<1.8 kb).
The advent of genome editing technologies has enabled new avenues for crop improvement. One major application for genome editing is the targeted insertion of genes at pre-determined chromosomal regions known as genomic safe harbors. In the present study, researchers at Berkeley Lab have developed a precise, optimized method to insert marker-free DNA fragments at designated genomic targets in rice (Oryza sativa), a staple crop for more than half of the world’s population. The size of the DNA fragment inserted (5.2 kb) is nearly three times of the largest marker-free DNA fragment gene ever inserted at a designated target in a plant genome.
Specifically, the invention includes the CRISPR-Cas9-mediated targeted insertion of beta-carotene producing genes at a pre-determined genomic safe harbor in the rice genome. The analysis indicates that out of the 55 T0 plants regenerated, eight T0 plants carried a full-length insertion of the carotenoid biosynthesis cassette at the destination site of interest, and three of these eight T0 plants gave rise to viable seed. Ultimately, researchers demonstrated the production of a carotenoid-enriched rice plant that is free of markers which has hitherto not been achieved. Additionally, the resulting plant possesses high carotenoid content in the seeds and has no detectable penalty in morphology or yield.
The abundance of beta-carotene in the rice is particularly significant because the pigment is the precursor to vitamin A, an essential micronutrient for humans. In regions around the world where rice is the major food source and vitamin A deficiency is widespread, the bio-fortified rice lends a valuable socio-economic and positive nutritional impact. Therefore, the CRISPR technology described could be a novel route to address the global prevalence of vitamin A deficiency. Such a technique can potentially be applied to any crop species with an established transformation protocol, offering a promising tool for plant research and for crop genetic improvement.
DEVELOPMENT STAGE: Proven principle
FOR MORE INFORMATION:
Dong, O.X., Yu, S., Jain, R. et al. “Marker-free Carotenoid-enriched Rice Generated through Targeted Gene Insertion using CRISPR-Cas9.” Nature Communications 11, article 1178 (2020).
- Oliver Xiaoou Dong
- Pamela C. Ronald
STATUS: Patent pending.
OPPORTUNITIES: Available for licensing or collaborative research.
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Toaster Software: Optimizing CRISPR Technologies for Genome Editing 2016-056