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.
Highlights
The construct used to generate GE1.2, pRHM110, was used to transform immature wheat embryos to determine whether a temperature stress during the callus tissue culture phase could increase the frequency of Cas9-mediated editing
Sequencing revealed no mutations in plants regenerated after either treatment which suggests that increased temperature per se did not drive an increase in activity of Cas9 and editing efficiency in wheat
There has been a lot of research dedicated to the precision of CRISPR/Cas9 targeting in both animals and plants over the past 7 years (Lei et al, 2014; Cram et al, 2019; Labun et al, 2019)
Summary
CRISPR/Cas enabled genome engineering has great potential for improving agricultural productivity, producing more climate and disease resilient crops, increasing nutrient use efficiency or altering quality traits (Wang et al, 2014; Zhou et al, 2015; Morineau et al, 2017; Nekrasov et al, 2017; Sun et al, 2017; Li R. et al, 2018; Li X. et al, 2018; Nakayasu et al, 2018; Okuzaki et al, 2018; Sánchez-León et al, 2018; Zhang et al, 2018; Jouanin et al, 2019a,b). Increased Temperature Improves Wheat Editing differences include: the ability to detect mutations, rate of cutting efficiency, heritability of the detected mutations and conditions under which mutations arise (Lawrenson et al, 2015; Howells et al, 2018; LeBlanc et al, 2018). Targeting a single gene in rice is substantially easier than targeting one in wheat; for every potential rice homolog there could be three homoeologous copies in wheat, or more if there has been a duplication event. This complicates guide design, the strategy for edit identification and reduces the likelihood of identifying a true null knockout in the T0 plant. Each species presents its own set of difficulties which need to be taken into consideration when designing the experiment and setting its targets
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