Evaluation of Factors Affecting In Planta Gene Editing Efficiency in Wheat (Triticum aestivum L.)

Abstract

Gene editing in polyploid crops still suffers from low efficiency, and further improvement is needed for its routine implementation in the modern breeding practice. Here we examined factors that affect the CRISPR/Cas9-mediated gene editing efficiency in allohexaploid wheat plants. We selected three guide RNAs (gRNAs) and evaluated the potential of using heat shock at the seedlings stage to increase editing efficiency in transgenic plants. Only one out of three gRNAs demonstrated significantly increased editing efficiency following heat shock treatment. We also examined the expression of DNA repair and replication gene orthologues in response to heat shock in wheat leaves. Misregulation of the chromatin remodelers following the heat shock treatment could potentially be involved in the increase of editing efficiency in wheat. Overall, the editing efficiency of gRNAs observed in our study correlated with predictive scores from the gRNA design tools. The editing rate of the top-ranked gRNAs could potentially be increased using heat treatment of the transgenic plants.

Diversity of transgene integration and gene-editing events in wheat (Triticum aestivum L.) transgenic plants generated using Agrobacterium-mediated transformation

“…The gRNA#10 seeding sequence targeting three sub-genomic copies of TaGW2 was cloned into the JD633 plasmid carrying GRF–GIF and Cas9/gRNA expression cassettes. The CAPS assay for three sub-genomes targeted by gRNA#10 was performed as described previously ( Xu et al, 2022 ) using Sma I restriction digestion. PC-D, digested WT control (cv.…”

“…Previously, others and we have demonstrated that biolisticmediated transformation can effectively generate edited wheat plants (Li et al, 2021;Xu et al, 2022). Here, we tested the efficiency of GE with the single-guide RNA (gRNA#10) targeting six copies of the wheat grain width and weight2 gene (TaGW2) (Wang et al, 2018).…”

“…On the other hand, if the mutations act synergistically, a combination of a few gRNAs targeting different genes in a single plant could be beneficial for the fast recovery of the mutants with a desired phenotype. We successfully recovered T 0 plants with GE events at three sub-genomes as determined using CAPS and probe-based qPCR assays ( Figure 4 , Supplementary Figure S3 ) for the previously tested gRNA#10 ( Xu et al, 2022 ). The mutagenized loci were faithfully propagated into the following generation ( Supplementary Figure S4 ).…”

“…The editing efficiency at the target sites was assessed using either the cleaved amplified polymorphic sequence (CAPS) assay, as described previously ( Xu et al, 2022 ), or the qPCR method. In the CAPS assay, the gRNA#10 target regions were amplified using sub-genome-specific or universal primers ( Supplementary Table S6 ) and digested with Sma I.…”

Advances in Genome Editing for Sustainable Agriculture

“…The work by Xu et al 7 addresses the low genome-editing efficiency in polyploid crops. By examining factors that affect the CRISPR/Cas9-mediated gene editing efficiency in allohexaploid wheat plants, the authors found that heat shock treatment could significantly increase editing efficiency.…”