History of plant genetic mutations ± human influences

Left: Nancy Reichert; Right: Genetic engineering flashback; co-authors of Murai et al. (1983) Science 222:476-482. Row 1 (bottom; L-R): Don Merlo, Carolyn Stock, John Kemp, Tim Hall; rows 2-3 (L-R): Dennis Sutton, Mike Murray, Champa Sengupta-Gopalan, Nancy Reichert, Richard Barker; top row: Jerry Slightom; absent: Norimoto Murai

Generating precise mutations in plant genomes via the CRISPR/Cas system began in 2013. Since that time, the field of genome editing has expanded world-wide and is broadly-focused on numerous plant species. Each major step forward in science is usually rooted in scientific achievements that took place in the past, and genome editing is no exception. This paper is intended to provide a brief history of mutation-based science that led us to this point. It includes information on random (naturally occurring) and induced (human-influenced) mutations, how these assisted in plant domestication and breeding, and the role that plant tissue culture played in generating wanted (and unwanted) mutations. Then, the focus was on 1983 when the field of plant genetic engineering began and changed the way many plant species were genetically enhanced, trait-by-trait. The field, though controversial due to anti-GMO sentiments, helped facilitate genome editing in plants 30 years later; many use(d) Agrobacterium tumefaciens and similar vectors for delivery of relevant CRISPR/Cas DNA into plant cells. This system, and newer derivations, were also based on critical bacterial studies as well as eukaryotic cell/tissue studies. This brief history is not intended to downplay the fantastic genome editing accomplishments achieved by many plant researchers, exemplified in articles that appear in this special issue, but rather to provide a historical lens from which to view it.

Nancy Reichert. History of plant genetic mutations ± human influences. In Vitro Cellular & Developmental Biology-Plant, 57:554-564, 2021.

CRISPR/Cas-mediated genome editing in sorghum – recent progress, challenges, and prospects

Top from left: Aalap Parikh and Eleanor Brant, Bottom from left: Mehmet Cengiz Baloglu and Fredy Altpeter.

Sorghum is a versatile crop with great potential as a sustainable food, feed, and bioenergy source. To mitigate the severely negative impact of climate change and population growth on food and energy security, further elevation of the crops stress tolerance is urgently needed. Genome editing technologies such as CRISPR/Cas have great potential to accelerate functional genomics and crop improvement by supporting targeted modification of almost any crop gene sequence. We describe the recent progress in genome editing of sorghum. In addition, we review remaining challenges and prospects of emerging gene editing technologies for rapid precision breeding of this crop

A. Parikh, E. J. Brant, M. C. Baloglu, and F. Altpeter. CRISPR/Cas-mediated genome editing in sorghum — recent progress, challenges and prospects.In Vitro Cellular and Developmental Biology – Plant. 57,720–730, 2021.

Gene editing in trees and clonal crops: Progress and challenges

Left: Greg Goralogia and Steven Strauss, Department of Forest Ecosystems and Society, Oregon State University (Corvallis, OR); Right: Thomas Redick, Global Environmental Ethics Counsel (GEEC), LLC (Clayton, MO)

As a result of its high efficiency at modifying specific genes, gene editing methods are generating tremendous excitement throughout biology and the agricultural sciences—and trees and clonal  crops are no exception.  However, its application also faces great obstacles, both biological and social.  Biological obstacles include very difficult transformation and regeneration methods for most trees and clonal crop species, especially if the goal is a “clean” product with no transgenic sequences present (to reduce regulatory and market restrictions).  Transgene-free or -mitigated products could be aided by several technologies, including recombinase excision, transient editing, rapid flowering, and viral delivery of gene editing components.  However, none of these are in a “ready to go,” efficient form for the large majority of tree and clonal crops.  Much translational research is needed to develop reliable technologies, but research in this area is very poorly funded by USDA and other relevant federal agencies.  Finally, despite some advancement in science-based regulation in the USA in recent years, notably the USDA SECURE system, very large regulatory barriers persist. These include the challenges of harmonization with other US regulatory agencies (FDA/EPA) and alignment with other countries’ regulatory systems and international conventions—all of which create great uncertainty about pathways to large scale field research and commercial release.  Despite much promise, a great deal of technological and social innovation is needed if gene editing is to find significant applications in most tree and clonal crop species.

Greg S. Goralogia, Thomas P. Redick, Steven H. Strauss. Gene editing in tree and clonal crops: progress and challenges. In Vitro Cellular & Developmental Biology-Plant, 57:683-699, 2021.

Impacts of the regulatory environment for gene editing on delivering beneficial products

Daniel Jenkins, Pairwise Plant Services, Inc. (Durham, NC); Anna Atanassova, BASF Business Coordination Center (Gent, Belgium); Chloe Pavely, Calyxt, Inc (Rosevill, MN); and Raymond Dobert, Bayer Crop Science West St. Louis, MO)

Various genome-editing technologies have been embraced by plant breeders across the world as promising tools for the improvement of different crops to deliver consumer benefits, improve agronomic performance, and increase sustainability. The uptake of genome-editing technologies in plant breeding greatly depends on how governments regulate its use. Some major agricultural production countries have already developed regulatory approaches that enable the application of genome editing for crop improvement, while other governments are in the early stages of formulating policy. Central to the discussion is the principle of “like products should be treated in like ways” and the subsequent utilization of exclusions and exemptions from the scope of GMO regulations for these products. In some countries, the outcomes of genome editing that could also have been achieved through conventional breeding have been defined as not needing GMO regulatory oversight. In this paper, we provide a short overview of plant breeding and the history of plant biotechnology policy development, the different classes of current regulatory systems and their use of exemptions and exclusions for genome-edited plants, and the potential benefits of such approaches as it relates to achieving societal goals.

Daniel Jenkins, Raymond Dobert, Anna Atanassova, Chloe Pavely. Impacts of the regulatory environment for gene editing on delivering beneficial products. In Vitro Cellular & Developmental Biology-Plant, 57:609-626, 2021.

Detection of genome edits in plants – from editing to seed

Left to right: Top: Raymond D. Shillito, Sherry Whitt, Margit Ross & Farhad Ghavami; Bottom: David De Vleesschauwer, Katelijn D’Halluin, Annelies Van Hoecke & Frank Meulewaeter

Genome editing has become a hot topic in recent years, as evidenced by the special issue of IN-VITRO-Plant that was just published.  Meganucleases, Zinc finger nucleases, and TALENS were used to introduced genome edits in the past, and the advent of CRISPR has opened new opportunities for plant science research and to improve plant varieties. In early 2019, we held a discussion at a meeting of the Analytical Excellence in Industry Collaboration about detection of genome edits.  The EURL had just prepared their report on detection of edits obtained by new mutagenesis techniques.  There was at the time only one peer-reviewed article available, and we believed that an examination of the technical aspects of detection and identification of genome edits would be useful. 

The question was and still is, once I have achieved an edit – how do I characterize it, and how can I detect it in breeding populations, and seed and grain?  While detection of large inserts (analogous to traditional GMOs) is technically straightforward, small insertions, deletions, or single base changes are more difficult to detect.  So far, literature has focused on detection in trade; we decided in this publication to focus on the early stages – those experienced during production of the edits. The need for effective tools begins with optimization of the genome editing process.  It continues with selection and characterization of tissue cultures and/or regenerated plants, and throughout the breeding process.   Such tools are also required for ensuring purity of seed production and may be necessary for monitoring genome-edited products in the marketplace.  A wide range of analytical approaches are used, including PCR, digital PCR, and sequencing.  Each one has advantages, and disadvantages.  The biggest challenge is the detection of genome edits present at low levels in large seed, plant, or grain populations. Furthermore, this is complicated by the impossibility to differentiate directed genome edits from conventionally induced and naturally occurring mutations and variation.

Raymond D. Shillito, Sherry Whitt, Margit Ross, Farhad Ghavami, David De Vleesschauwer, Katelijn D’Halluin, Annelies Van Hoecke, and Frank Meulewaeter. Detection of genome edits in plants – from editing to seed. In Vitro Cellular & Developmental Biology-Plant, 57:595-608, 2021.

Generating novel plant genetic variation via genome editing to escape the breeding lottery

Left to right: Nathaniel Schleif, Shawn M. Kaeppler,  and Heidi F. Kaeppler

Plant breeding at its core is a game of numbers: breeders try to find the best individuals within a vast sea of genetic possibilities. Any tool which enhances the chances of winning the genetic lottery must be seized upon in order to ensure continued improvement. A promising new tool for plant breeding is genome editing, which has existed for a couple of decades (meganucleases, ZFNs, TALENs) but has become much easier to deploy at scale with the advent of CRISPR/Cas9 technologies. This report reviews how editing has and will continue to revolutionize the breeding process. The largest improvement Cas9-based editing has brought to editing applications is the ease with which it can be designed and deployed to target specific genes and multiple. This targeted approach can be leveraged to domesticate novel species, introduce useful variation into elite lines, and accelerate introgression of wild germplasm. These approaches each rely on having foreknowledge about what genes will be useful for targeting, which limits editing to that which we already know about. However, enhancement of recombination and targeted mutagenesis are each enabled by editing, which broadens opportunities for genome manipulation for crop improvement. While still limited by the efficacy of transformation technologies, larger pool-based approaches are also addressing efficiency hurdles by targeting many more sites at once, which allows for targeting of multiple sequences of know and less known function. The last decade has been highly fruitful in advancing editing-based applications for plant breeding, and it is anticipated that the rate of advancement of plant genome editing technologies and applications will continue into the future.

Nathaniel Schleif, Shawn M. Kaeppler, and Heidi F. Kaeppler. Generating novel plant genetic variation via genome editing to escape the breeding lottery. In Vitro Cellular & Developmental Biology-Plant, 57:627-644, 2021.

Frequent genetic defects in the p16/INK4A tumor suppressor in canine cell models of breast cancer and melanoma

Left to right: Farruk M. Lutful Kabir, Allison Church Bird, R. Curtis Bird

The cyclin-dependent kinase inhibitors (CKIs) belong to a group of key cell cycle proteins that regulate important cancer drug targets such as the cyclin/CDK complexes. Gene defects in the INK4A/B CKI tumor suppressor locus are frequently associated with human and canine cancers. Many of the cancer-associated genetic alterations, known to play roles in mammary tumor development and progression, appear similar in humans and dogs. The objectives of this study were to characterize expression defects in the INK4 genes, and the encoded p16 family proteins, in spontaneous canine primary mammary tumors (CMT) as well as in canine malignant melanoma (CML) cell lines to further develop these models of spontaneous cancers. Gene expression profiles and characterization of p16 protein revealed that the INK4 gene family was expressed differentially and the genes encoding the tumor suppressor p16, p14, and p15 proteins were often identified as defective in CMT and CML cell lines. This altered expression profile for INK4 locus tumor suppressor genes was confirmed by the identification of similar gene defects in primary canine mammary tumor biopsy specimens demonstrating these were not artifacts of culture. They were also comparable to defects found in human breast cancer. These data strongly suggest that defects identified in the INK4 locus in canine cell lines are lesions originating in spontaneous canine cancers and are not the product of selection in culture. The findings further validate canine tumor models for use in developing a clear understanding of the gene defects present and may help identify new therapeutic cancer treatments that restore these tumor suppressor pathways based on precision medicine.

Farruk M. Lutful Kabir, Patricia DeInnocentes, Allison Church Bird, and R. Curtis Bird. Frequent genetic defects in the p16/INK4A tumor suppressor in canine cell models of breast cancer and melanoma. In Vitro Cellular & Developmental Biology-Animal, 57:519-530. 2021.

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