Are you a food label reader?  If so, you may have noticed some of your favorite snacks bear the phrase “partially produced with genetic engineering.” This makes sense, given that the soy lectin and corn syrup used in many foods is probably isolated from plants genetically modified to be resistant to a powerful herbicide, glyphosate. Genes, originally isolated from bacteria, were inserted into crop plants, conferring glyphosate tolerance to the soybeans, corn, and other crops. Then, federal regulations followed: requiring that human food made with these plants be labeled “partially produced with genetic engineering.”

While these genetically modified plants have been around almost 20 years, new tools for plant biologists have yielded new traits for plants. At the Plant and Animal Genomics Conference held recently in San Diego, a topic of great interest was applications of the CRIPSR/Cas9 system to plants.

One brilliant approach to using CRISPR in plants is to edit the family of genes that confers susceptibility to bacterial blight in rice. Bacterial blight in rice, caused by Xanthomonas oryzae pv. oryzae, is a huge problem in Asia and Africa.

“To understand sensitivity to bacterial blight, it is necessary to first understand the biology of the disease process,” explains Bing Yang, Ph.D., associate professor in genetics, development and cell biology at Iowa State University.

“Bacteria that cause the blight have effector proteins (called TALs; transcription activator-like) that transcriptionally activate a family of genes in rice, referred to as SWEET genes. We strategized that by mutating the promoter region of the SWEET family of genes, the bacterial TAL proteins would no long be able to bind to the promoter. Being unable to bind to the promoter DNA, the bacterial TAL proteins cannot induce expression of the SWEET genes. Hence, TAL proteins could no longer bring about a state of disease susceptibility in rice,” explains Dr. Yang.

“CRISPR experiments can be designed to leave no fingerprint, or exogenous DNA in the plants. From a regulatory standpoint, the USDA should accept rice plants with small deletions or mutations in their genomes as safe for field tests,” concludes Dr. Yang.

Using a similar approach, disease-resistant citrus trees have also been developed. In Florida, the citrus industry faces disease challenges from citrus canker and citrus greening disease caused by two bacteria, Xanthomonas citri and Candidatus Liberibacter asiaticus, respectively.

“Citrus canker is also a big problem,” asserts Nian Wang, Ph.D., associate professor, department of microbiology and cell science, Citrus Research and Education Center, Institute of Food and Agricultural Sciences, University of Florida. “A specific effector protein from the infecting bacteria binds to the promoter region of the canker susceptibility gene CsLOB1 to induce disease symptoms. By utilizing CRISPR techniques, we can target the promoter region or the coding region of the citrus susceptibility gene to mutate it in such a way to prevent binding of bacterial transducers.”

The CRISPR/Cas9 system can be applied in a manner that leave no exogenous DNA in the citrus, which is very beneficial in getting USDA approval.

“Applying the same strategy for citrus greening disease, we have begun research to identify the key virulence factors and their targets,” continues Dr. Wang. “We are mutating the putative targets using the CRISPR technology. We hope to generate citrus trees resistant to citrus greening disease.”

Another talk at the conference was on gene editing in cereals by Ming Luo, Ph.D., of the Commonwealth Scientific and Industrial Research Organisation (CSIRO) in Canberra, Australia. Wheat rust is a huge problem in failure of wheat crops worldwide; finding a solution to the problem would be a milestone in addressing world hunger.

“A pilot study of CRISPR efficacy in rice was successful with a knockout of two closely linked genes. In contrast, the homologous CRISPR experiment in wheat did not lead to any mutations,” declares Dr. Luo. “In contrast, using TALEN in wheat yielded results.

“While CRISPR works in rice and barley, CRISPR editing in wheat has not worked in our hands. We conclude that employing TALENs as a gene-editing tool in wheat is more efficient than CRISPR.”

One drawback to the CRISPR/Cas9 system in plants concerns off-target effects. To assess these effects in plants, whole genome sequencing is the current gold standard.

“Recent work in the model organism Arabidopsis, shows that the CRISPR/Cas9 system correctly targets the desired loci in plant genomes,” states Cara Soyars, University of North Carolina doctoral candidate. “This finding contrasts with off-target CRISPR effects in animals where off-target effects are a serious concern. Extrapolating this to other genera of plants, we postulate that modifications to the Cas9 protein to increase specificity of the binding site is not necessary in plants.”

“Plant genomes have many redundant genes. Hence, to effectively knockout a particular pathway of interest, many genes need to be knocked out,” continues Soyars. “Our lab, the Zachary Nimchuk lab, has developed a system that allows entire families of genes to be targeted in one experiment. While the system is predicted to increase the risk of off-target effects, we have shown with whole genome sequencing that there are very few or no off-target effects in Arabidopsis.

“One of our studies necessitated the targeting of 14 genomic loci at once. Using the multiplexed CRISPR/Cas9 system, we had a 33­–92% success rate. Whole genome sequencing also revealed that chromosomal translocation events are extremely rare after genome manipulation in Arabidopsis via CRISPR/Cas9.

“We really do not know why there is such a lower rate of off-target effects in plants when compared to animals,” clarifies Soyars. “Speculatively, plants use nonhomologous recombination; whereas animals employ homologous recombination when joining double DNA breaks. Perhaps differences in these repair mechanisms explain the difference in off target effects?”

One advantage of the CRISPR/Cas9 system is the applicability across a wide range of organisms. Editing carried out for research purposes does not require the same level of stringency as those for therapeutic applications. However, any plants or animals undergoing genome editing will need to be carefully vetted.

The regulatory body overseeing this is the Animal and Plant Health Inspection Service (APHIS), which is part of the USDA. APHIS released for comment a policy suggesting a path forward. For now, very small changes [like single base insertion or deletions (2–10 base pairs removed)] do not seem to be of much interest to APHIS.

“The ability to make these tiny changes at a very specific place in the genome is the result of using CRISPR/Cas9 technology in plants,” affirms Jeff Wolt, Ph.D., professor of agronomy at Iowa State University. “In the past, genetic additions to plants included either exogenous genes or even some of the machinery to get the modifications incorporated.

“Dr. Bing screened plants to select the edited gene of interest, while selecting against the inclusion of the CRISPR machinery. Dr. Bing confirmed this with lots of sequencing. His letter of inquiry to APHIS posed the question: will these rice plants be subject to regulation? APHIS responded that the material can be used without regulatory oversight.

“Plant researchers are moving forward cautiously, as the all the wonderful technology from previous methods of transgenic manipulation was not fully realized due to public push-back. We need to ensure that what we are doing is well-communicated and transparent,” expounds Dr. Wolt.

“Plant sciences have lagged behind in adopting new technologies for genome editing for a couple of reasons,” he continues. “First, funding levels are generally lower for plant researchers than studies involving animals. Second, the techniques used to change the genome must go through the cell walls of plants; in animals, especially cell lines, it is much simpler to get the components of CRISPR/Cas9 into the cells.”

“Another reason many of the exciting applications of CRISPR in plants are not discussed as often as medical applications,” explains Mark Behlke, M.D, Ph.D., CSO of Integrated DNA Technologies, “is that the development of agricultural applications done by industry is confidential and is not published quickly, or at all. Also, working with crop plant genomes can be more complex than mammalian cells; as these species are often polyploid, which makes manipulation of their genomes more complicated.  Furthermore, plant genomes often have huge repetitive content.

“On the other hand,” Dr. Behlke continues, “advances in CRISPR/Cas9 technology has made genome manipulation accessible for just about any research lab in the world. One method that is especially promising is the use of a DNA-free system to perform genome engineering in plants. In this sort of system, the RNA guide is bound to recombinant Cas9 protein and added directly into cells as a ribonucleoprotein (RNP) complex, with no use of plasmids or other DNA-based expression cassettes.

“A delivery method of coating gold nanoparticles with plasmids and shooting them into whole animals has worked in cattle vaccinations (‘biolistics’). This approach is already being applied to plants, to get the Cas9 RNP complexes into cells through their tough cell walls,” concludes Dr. Behlke.


DeeAnn Visk PhD, is founder and principal writer at DeeAnn Visk Consulting.

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