Turning Up the Temperature on CRISPR: Increased Temperature Can Improve the Editing Efficiency of Wheat Using CRISPR/Cas9

Abstract

The application of CRISPR/Cas9 technologies has transformed our ability to target and edit designated regions of a genome. It’s broad adaptability to any organism has led to countless advancements in our understanding of many biological processes. Many current tools are designed for simple plant systems such as diploid species, however, efficient deployment in crop species requires a greater efficiency of editing as these often contain polyploid genomes. Here, we examined the role of temperature to understand if CRISPR/Cas9 editing efficiency can be improved in wheat. The recent finding that plant growth under higher temperatures could increase mutation rates was tested with Cas9 expressed from two different promoters in wheat. Increasing the temperature of the tissue culture or of the seed germination and early growth phase increases the frequency of mutation in wheat when the Cas9 enzyme is driven by the ZmUbi promoter but not OsActin. In contrast, Cas9 expression driven by the OsActin promoter did not increase the mutations detected in either transformed lines or during the transformation process itself. These results demonstrate that CRISPR/Cas9 editing efficiency can be significantly increased in a polyploid cereal species with a simple change in growth conditions to facilitate increased mutations for the creation of homozygous or null knock-outs.

Multiallelic, Targeted Mutagenesis of Magnesium Chelatase With CRISPR/Cas9 Provides a Rapidly Scorable Phenotype in Highly Polyploid Sugarcane

“…The efficiencies of Cas9-and Cas12a-mediated mutagenesis can be elevated by the heat treatment of the callus following the delivery of editing reagents, as previously reported for Arabidopsis, rice, maize, and wheat (LeBlanc et al, 2018;Malzahn et al, 2019;Milner et al, 2020). Therefore, we deployed an MGE-based rapid readout system to compare co-editing efficiencies in response to heat treatment (37 • C) or standard incubation temperature (28 • C) for sugarcane callus.…”

Wheat root systems as a breeding target for climate resilience

“…Recent examples include CRISPR editing of the cotton GhARG gene which resulted in increased lateral root formation and, in barley, knockout mutation of the HvCKX1 gene resulted in greater root length and increased surface area (Wang et al 2017 ; Gasparis et al 2019 ). While no known RSA genes have been modified in wheat to date, and the generation of full gene-edited knockouts in polyploid crops has proven challenging, recent strategies to improve editing in hexaploid wheat make this a realistic prospect in the near future (Milner et al 2020 ). Several countries currently regulate these gene-edited crops as genetically modified events—even if the foreign T-DNA has been removed, leaving just the gene edit.…”

Error‐free recombination in sugarcane mediated by only 30 nucleotides of homology and CRISPR/Cas9 induced DNA breaks or Cre‐recombinase

“…The efficiency of CRISPR/Cas9‐mediated genome editing can be elevated by a heat treatment following gene transfer as recently described for the polyploid crop wheat. [ 6 ] The choice of the sgRNA target site and its position relative to the tandem repeats may also contribute to the HDR efficiency.…”