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What are CRISPR/Cas9 and other New Breeding Technologies (NBTs)?

"Anyone who works in any kind of plant engineering is vigorously pursuing these technologies, especially with crops that have complex genomes or that you can’t breed easily.
"Just as ‘traditional’ genetic engineering, gene editing techniques can induce unintended changes in genetic material even if only one or a few base pairs have been altered.

At a Glance

New breeding techniques (NBTs), including CRISPR/Cas9, give scientists the ability to more precisely modify DNA by turning genes genes on or off or editing DNA. They can pinpoint and remove weakness or insert desired traits already found elsewhere in the species. It’s akin to a biological word processing system that allows scientists to cut and paste DNA almost as easily as if they were editing a journal article.

That differs substantially from traditional transgenic approaches, which relies on the insertion of genetic material, usually from foreign species. Because gene editing and other NBTs allow scientists to avoid crossing the so-called “species barrier”—a concern promoted by anti-GMO activists, which led to scare images of Frankenfoods—plants and animals bred with these techniques may face lower regulatory hurdles than traditional transgenic products.

Researchers are working on CRISPR/Cas9-edited versions of commodity crops such as corn, soybeans, canola, rice, and wheat, with new traits like drought resistance and higher yields—both critical features for farmers trying to deal with a changing climate and the fact that the world population is growing faster than our food supply. The technique can also be used to remove allergens in peanuts, or make food more nutritious, all while using genes that naturally occur in the plant. Anti-GMO critics of NBTs refer to CRISPR and other gene editing techniques as “extreme engineering”, and are attempting to have them regulated as transgenic breeding.

Science and Politics

One of the more revolutionary developments in the realm of genetic engineering has been the introduction of several newer techniques with the potential to change how scientists guide the breeding of plants and animals, and address research on humans. They could also radically modify how governments define and regulate genetic engineering on humans and in agriculture.

“This is now the most powerful system we have in biology,” said biochemist David Sabatini. “Any biological process we care about now, we can get the comprehensive set of genes that underlie that process. That was just not possible before.”

Until recently, most genetic engineering relied on transgenic techniques, with modifications made through the addition of genetic material, usually from different species. These modifications led to a range of products, including crops resistant to the herbicide glyphosate and harmful insects. But the regulatory process is arduous, taking 7-13 years and several hundred million dollars, in part because of caution in response to criticism from GMO critics who characterized crops modified using traditional transgenics as “Frankenfoods”.

In fact, the term was coined to encapsulate one of the central concerns of GMO critics: transgenics led to the creation of new crops or animals by moving so-called “foreign genes” from one species to another. “Plants and organisms unable to physically reproduce can become unnaturally intertwined,” reads a typical NGO posting headlined “Frankenfood=Genetically Modified Food”. “A novel gene may be cobbled together from a plant virus, a soil bacterium and a petunia plant, for example — creating a kind of botanical Frankenstein,” reads another.

Comparison of four breeding methodologies. Conventional breeding mainly relies on hybridation between a donor and a recipient line. The progeny is selected for the desired characteristic. To remove unwanted traits from donor plant, the best line of the progeny is obtained by backcrossing with recipient line. Transgenesis is the genetic modification of a recipient line with genes from other species which are sexually incompatible with the recipient plant. Cisgenesis is the genetic modification of a recipient plant transformed with a natural gene for a crossable plant. Genome editing is a type of genetic engineering in which DNA is directly inserted, replaced, or removed from a genome using engineered nucleases, the so called "molecular scissors", to obtain a desirable trait. BCn, backcross n generation; trans-GMO, transgenic-Genetically Modified Organism; cis-GMO, cisgenic-Genetically Modified Organism; GEC, genome-edited crops.
Source: Sanwen Huang/ Chinese Academy of Agricultural Sciences

There are several newer techniques capturing the attention of researchers looking for options that could avoid the regulatory hurdles faced by transgenic crops and animals.

  • Site-Directed Nucleases (SDN) including Zinc finger nuclease technology, CRISPR and TALENs)
  • Oligonucleotide-directed mutagenesis
  • Cisgenesis
  • Intragenesis
  • Grafting
  • Agro-infiltration
  • RNA-dependent DNA methylation
  • Reverse breeding

Gene or Genome Editing is a broad category that includes several techniques allowing scientists to do precise editing, including CRISPR.

Clustered Regulatory Interspaced Short Palindromic Repeats (CRISPR-Cas9), the newest of the gene-editing techniques, has been capturing the most attention. CRISPR offers researchers a relatively inexpensive, easy and quick option to engineer changes. What makes the CRISPR system so special, in part, and so adaptable to the important task of gene-editing, is its relative simplicity. CRISPR is often described as being the equivalent of using a pair of molecular scissors to modify a specific portion of a gene. They can snip away weaknesses or insert strengths already found in the species being developed. The tool allows the researcher to cut out a section of DNA. Then, one of two things happens: The loose ends are essentially glued back together, eliminating the undesired trait or weakness. Or a “repair” with a desired trait is inserted into the void.

CRISPRs do not need to be paired with separate cleaving enzymes as other tools do. They can also easily be matched with tailor-made “guide” RNA (gRNA) sequences designed to lead them to their DNA targets. Tens of thousands of such gRNA sequences have already been created and are available to the research community. CRISPR-Cas9 can also be used to target multiple genes simultaneously, which sets it apart from other gene-editing tools. 

CRISPR editing doesn’t require the use of genetic material from a foreign species. These tools are being used now in a wide range of research projects, including on humans. For example, let’s say you have a history of Huntington’s Disease in your family. As a child, you undergo genetic testing and it turns out that you have a mutation in the HTT gene. This means that when you reach 30-40 years old, you will develop Huntington’s Disease, a neurodegenerative condition with no cure. Huntington’s causes progressive impairment of a person’s physical and mental state.

But a CRISPR-Cas9 treatment could cut out your mutated copy of the HTT gene and replace it with the non-diseased copy of the gene, curing you of Huntington’s Disease. This is, with the advent of the CRISPR-Cas9 system, not a far away dream.

Advances are even further along in the agricultural sector. In 2015, scientists from John Innes Centre and The Sainsbury Laboratory in the UK successfully edited two crops—barley and a relative of broccoli. They were able to demonstrate that subsequent generations were indistinguishable from those bred conventionally. CRISPR has also been shown to work on hornless cattle—it avoids the dehorning practice used by many dairy farmers—and on pigs that don’t need to be castrated or are protected from disease.

The US Department of Agriculture (USDA) determined in April 2016 that it will not regulate a mushroom genetically modified with using CRISPR–Cas9, making it the first CRISPR-edited product to receive a green light from the US government.

Here is a visual representation of how gene silencing and editing with CRISPR works (Science in the News, Harvard)

Gene silencing and editing with CRISPR. (Science in the News, Harvard)

The media is  full of references to NBTs beyond CRISPR. Here is a guide to a few of the more popular terms:

Zinc Finger Nucleases (ZFNs) is the oldest of the gene editing technologies, developed in the 1990s and owned by Sangamo BioSciences. It has been primarily used in research for a variety of human diseases,  including HIV/AIDS and Hemophilia.

Transcription Activator-like Effector Nucleases (TALENs), developed in 2009, offered an easier method of gene editing. Its first reported success came in 2012 when researchers at Iowa State University developed disease resistant rice. The tool also has been used to create pigs that need less food to get fat and Brazilian cattle with larger muscles but more tender meat. TALENS result in the fewest “off-target” effects, including through uncontrolled cellular growth and cancer (when used in humans), which occur when nucleotide sequences identical or similar to the target are cut unintentionally.

Zinc fingers and TALENs made it possible to directly target a particular gene for the first time but they are more time-consuming than CRISPR in their design and construction.

RNA Interference (RNAi): The technology is used to shut off or silence specific genes in a cell by attacking the messenger RNA that carries the instructions for the targeted genetic trait. RNAi has been used in several crops already, including the recently approved Arctic Apple and the Innate Potato, both of which were designed to resist browning.

Scientists also are looking at external applications. Monsanto, for example is working on an RNAi spray to combat weeds that have developed resistance to its Roundup herbicide. The spray would neutralize the resistance in those weeds.

Gene silencing: This is more of a general term used to describe the result of techniques listed above. It can be accomplished through RNAi or through gene editing tools including CRISPR – where it is often referred to as a gene knockout.

Site-directed mutagenesis: This is an in vitro procedure in which a gene is exposed to a primer. The most widely-used methods do not require any modifications or unique strains. It’s a more precise evolution of an older technique known as mutagenesis, in which seeds were exposed to radiation or chemicals. Mutagenesis has been used to develop many foods, including the Ruby and Rio Red grapefruits. In site-directed mutagenesis, researchers target a specific section of DNA, removing some of the randomness from the research.

The Takeaway

Gene editing and other NBTs can offer several advantages over traditional transgenic techniques. Among the most notable is that governments may regulate these products (when developed without foreign DNA) differently than they do traditional transgenics. There has been some movement this direction from the U.S. Department of Agriculture, which has ruled in several instances that it doesn’t need to approve new varieties made without transgenetics. Those products, however, still face scrutiny from the Food and Drug Administration if they are destined for the human food supply.

The accuracy of the new techniques also results in fewer unintended changes. But researchers say there is still the chance of creating so-called off-target effects during the slicing and splicing. With that in mind, GMO critics argue that there’s no reason to view gene-edited crops any differently than transgenic crops.

It’s still not clear whether the US Department of Agriculture has jurisdiction, under current laws, over crops and livestock developed through gene editing that doesn’t involve the insertion of foreign DNA. The agency has concluded that if an edit cannot be distinguished from a natural occurring mutation, it is not a GMO. Still, the United States is currently working on an overhaul of its patchwork method of regulating this fast-moving sector.

The European Union, which heavily restricts many traditional GMOs, is assessing how to regulate plants and animals developed through these techniques. The commission currently defines GMOs as organisms with alterations that can’t occur naturally, but it says it will clarify the issue. In Sweden, authorities concluded that CRISPR-edited plants (as long as they don’t contain foreign DNA) shouldn’t be defined as GMOs under EU legislation.

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