Gene Editing Without Foreign DNA

Scientists perform plant-genome modifications on crops without using plasmids.

By | February 1, 2016

PLASMID-FREE EDITING: To mutate a specific gene of interest in a plant (lettuce shown here), scientists first grow protoplasts—plant cells lacking a cell wall (1). Preassembled CRISPR complexes, including a tailor-made stretch of guide RNA and the nuclease Cas9, are introduced into the protoplasts (2). The complex homes in on the target gene and cuts the DNA at a locus specified by the guide RNA (3). Protoplasts are then grown in clumps called calli (4), which are regenerated into a mature, genetically modified specimen (5).
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© GEORGE RETSECK; WIKIMEDIA COMMONS
The ongoing quest to increase the yield of crops and produce varieties resistant to disease, drought, and pests has been aided by the development of gene-editing technologies. These days, probably the most commonly used gene-editing approach in labs is the CRISPR/Cas9 system, in which a guide RNA—specially designed to match part of the sequence of a target gene—positions the Cas9 nuclease at that gene, enabling it to chop the DNA.

To date, researchers have been using DNA plasmids, both naked and inside infecting bacteria, to transfer Cas9 and guide RNAs into plant tissues and cells. However, says geneticist Jen Sheen of Harvard Medical School, this approach runs the risk of creating additional mutations—either from the integration of the plasmid itself into the plant genome, or from the persistence of the encoded gene-editing factors, which can “continue to make mutations.”

Sunghwa Choe of Seoul National University and colleagues have therefore devised a technique that avoids the use of plasmids altogether. They preassemble the Cas9 protein and guide RNA complex in vitro and then mix the complex with polyethylene glycol, which allows direct transfer by endocytosis into protoplasts—plant cells that have had their cell walls removed.

The edited protoplasts can then be cultured into small clumps of plant tissue called calli, from which a mature plant can be regenerated. Choe and colleagues have created genetically modified lettuce plants using this approach and have also edited genes in the protoplasts of three other species.

Avoiding the use of plasmids should not only prevent any additional unwanted DNA damage, but might also allow the genetically modified plants to skip regulatory oversight, explains Choe. (Nature Biotechnol, 33:1162-64, 2015)

 

PLANT GENOME EDITING APPROACH METHOD OF TRANSFER OFF-TARGET MUTATIONS EDITING EFFICIENCY (MUTATION FREQUENCY) SUITABILITY FOR SEXUAL AND ASEXUAL CROPS
Agrobacterium tumefaciens–mediated  transfer of plasmid (T-DNA) encoding
Cas9 and guide RNA
A. tumefaciens bacteria carrying the plasmid infect plant tissue. Possible: plasmid DNA
integrates randomly into
plant genome. Also, persistence
of Cas9 and guide RNA genes may lead to additional
off-target edits.
Approximately 2 percent (Nature Biotechol, 31:691-93, 2013) Sexually reproducing crops, such as wheat, rice, and maize, can be crossbred to remove the T-DNA.Asexual crops, such as grapes, potatoes, and bananas, will retain
the T-DNA.
Transfection of preassembled Cas9/guide RNA complex Assembled particles are transferred into protoplasts using solvents. None detected. Once complex degrades, there’s no chance of unwanted edits. Up to 46 percent Suitable for sexual and asexual crops alike

 

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