Postharvest Biotechnology

Are fruit and vegetables being genetically engineered?

Recently, two new methods for changing gene expression have been used to develop new cultivars for market that have more pleasing organoleptic properties. These methods, cisgenics and CRISPR-CAS9, can be used to change plant traits without the need for foreign DNA to be present in the plant. They are therefore not transgenic.

What is cisgenics?
Cisgenics describes a method of silencing the genes of an organism so that no foreign DNA is integrated into the plant long-term. How does it differ from transgenic plants? The “cis” in cisgenics means that DNA is from the same species while the “trans” in transgenics indicates the presence of DNA transferred from another species.

How are cisgenics produced? Researchers use one of the plant’s natural defense mechanisms that evolved to protect the cells against viruses, as a means to trick the plant into suppressing the action of its own gene. A portion of the sequence of the target gene (the gene to be silenced), is synthesized in the lab, but designed in a hairpin shape to mimic the ‘look’ of a viral gene. When introduced into the plant, this strange conformation triggers the plant’s defense response, degrading the introduced, and most importantly, the product of the native gene. This process is called RNA interference or RNAi and it creates non-functional genes.

Shown below are two cultivars: Artic® apple and the Innate potato that were produced by RNAi, and were given US government regulatory approval to be marketed to the general public.

A) The ‘non-browning’ Artic® Apple was produced by a Canadian company called Okanagan Specialty Fruits. They used RNAi to suppress a gene responsible for browning called PPO. This significantly reduces the visible discoloration seen when the fruit is bruised or cut as seen in the conventional line.

B) The Innate ‘non-browning’ potato was produced by Simplot, an American company in Idaho. Four genes were silenced by RNAi in this line. One of those genes is PPO. When this gene is suppressed, the tuber will show less browning when cut or bruised. Another gene called INV was also silenced. This allows the Innate potatoes’ tubers to be stored in the cold after harvest without some of the harmful consequences. Most potatoes, including the “chipping” type in the US are stored for months at low temperatures so that they can be sold throughout the year. Cold storage can cause the starch to break down to simple sugars, a process called ‘cold sweetening’. When fried, these sugars react with other tuber components, which can lead to black areas on the chips, which are carcinogenic. Innate potatoes should not produce as much sugars, significantly reducing this problem.

These products are an interesting development because they focused on output or quality traits, and perhaps more importantly, on the postharvest attributes of produce.

What is CRISPR-CAS9 and how can it be used for crop improvement?
This acronym stands for Clustered Regularly Interspaced Short Palindromic Repeats. The name is long and clumsy but what it describes is a system for very easy and directed changes to be made to the genome. The CRISPR technique makes it very simple to ‘surgically’ edit the DNA of any organism, creating very minor changes to its DNA makeup.

The CRISPR system uses Cas9, a bacterial nuclease protein that acts as a molecular scissors. Cas9, along with a carefully designed short ‘guide sequence,’ is inserted into the cell. This guide sequence has to meet certain specifications, but most importantly, a part of it must precisely match the sequence of the gene the researcher wishes to modify. Cas9 will use the guide sequence to find the matching region of the genome, and will make a cut in the DNA at that location. This break in the DNA strand will trigger the cell’s repair system to knit the separated DNA strands back together.

During the repair process, some of the DNA sequence may be removed or additional sequence may be inserted inadvertently at the repair site. These ‘mistakes’ may produce a mutant or damaged gene. To increase their chances of obtaining a defunct gene without leaving a large ‘footprint’ on the genome, researchers will transform many plants with an identical guide sequence along with the Cas9 protein. Each line may be repaired differently, but the researcher will select those with the least amount of change; e.g. 1-5 base pairs or units of DNA modified. Using tomato as an example this could represent a 0.0000000005% change of the genome.

The discovery of CRISPR has been described as one of the greatest revolutions in molecular biology. CRISPR-modified plants have not been regulated as GMOs because of the specificity of the method. CRISPR products, including ‘non-browning’ mushrooms and corn with modified starch, have been given the green light for sale in the US and should be in supermarkets in the near future (Waltz, 2016a, b).

Articles published in 2016 in two of the Nature Publishing Group journals” “Nature” and “Nature Biotechnology” highlighting plant products modified by CRISPR-CAS9 editing that can be directly marketed to consumers.

These products are almost identical in their DNA sequence to the unmodified parent.

In each case, the ‘CRISPRed’ plants were backcrossed to the non-modified parent, followed by selection of offspring, which did not have bacterial DNA, but only the sequence change; i.e., an insertion or deletion of the genome remained. The discovery of CRISPR is significant because the cost of genetically engineering plants and getting them to market is now so low, we should expect to see many crops on the market with better postharvest quality.

References
Adapted from: Beckles, DM (Accepted) ‘Biotechnology and Postharvest Quality.” In: Postharvest Technology of Horticultural Crops. Eds. M.E.Saltveit & J.F. Thompson. University of California, Agriculture and Natural Resources, Oakland, CA 4th Edition

1. Haroldsen et al (2012) California Agriculture 66(2): 62-69
2. http://www.innatepotatoes.com/
3. Waltz, E., 2016a. CRISPR-edited crops free to enter market, skip regulation. Nature  Biotechnology News 34, 582.
4. Waltz, E., 2016b. Gene-edited CRISPR mushroom escapes US regulation. Nature 532, 293.