CRISPR enables plant biologists to engage grand challenges

New research from UGA plant scientists describe how CRISPR-Cas9 gene editing technology has a significant role to play in developing sustainable agriculture. The study and illustrations are featured on the cover of the August issue of the CRISPR Journal, which details how recent studies in the deployment of genome editing in trees are opening new avenues for sustainable forestry:

The widespread success of CRISPR medicines in the clinic is substantiating the safety and efficacy of genome editing technologies in humans, derisking its deployment in plants. Likewise, the global deregulation of edited crops in several continents, including in Europe, is compelling the EU to contextualize agricultural regulations in a globally interconnected world. Most edited crops that may be imported for food and feed into the EU are indistinguishable from conventional breeding outcomes that could render monitoring and regulations impossible to enforce.

The UGA study, focused on gene editing in poplar trees, reflects the extent to which CRISPR technology has revolutionized biology. The work highlights the challenge of deciphering combinatorial mutations of highly repetitive sequences in tandemly arrayed genes (TAGs). Many genes in eukaryotic genomes arise from tandem duplication (or tandem gene amplification) with highly similar sequences and presumably function. This is known to occur in a species-specific manner and TAGs are thought to play important roles in lineage-specific stress response.  

"It has been very difficult to obtain null mutants by traditional approaches to study their function," said CJ Tsai, Professor and Georgia Research Alliance Eminent Scholar in the Franklin College of Arts and Sciences departments of plant biology and genetics, and the Warnell School of Forestry & Natural Resources. "This is because genetic recombination between closely linked genes is super low meaning a huge population is needed to capture the recombination. CRISPR makes this a lot easier, but the data analysis is way more complicated."

Plant biology doctoral student Yen-Ho Chen was instrumental to the research team, developing the target capture sequencing approach to pinpoint many of the complex mutation outcomes. 

Other co-authors included a former Warnell grad student Shakuntala Sharma, who generated the transgenic CRISPR mutant poplars; postdoctoral researcher W. Patrick Bewg and an REU summer intern Cole R. Gizelbach, who helped with amplicon sequencing; and research scientist Liangjiao Xue, who helped with genome assembly at the TAG locus to improve the sequence accuracy.

"Our approach is simple yet powerful: We used one single gRNA to target a consensus sequence of the tandem genes and achieved efficient knockout," Tsai explained. "The mutation patterns we observed were similar to those reported previously using multiple gRNAs. Genome editing could have off-target effects (editing non-target sites) and the risk of unwanted editing increases with the increased number of gRNA used."

An accompanying commentary article in the same issue further characterizes the work of Tsai's team and the critical role of genome editing strategies to mitigate global climate change. 

Image: Article artwork Brad Gilleland, medical illustrator in the UGA College of Veterinary Medicine.