Development of an Agrobacterium‐delivered CRISPR/Cas9 system for wheat genome editing

Abstract

Summary CRISPR /Cas9 has been widely used for genome editing in many organisms, including important crops like wheat. Despite the tractability in designing CRISPR /Cas9, efficacy in the application of this powerful genome editing tool also depends on DNA delivery methods. In wheat, the biolistics based transformation is the most used method for delivery of the CRISPR /Cas9 complex. Due to the high frequency of gene silencing associated with co‐transferred plasmid backbone and low edit rate in wheat, a large T 0 transgenic plant population are required for recovery of desired mutations, which poses a bottleneck for many genome editing projects. Here, we report an Agrobacterium ‐delivered CRISPR /Cas9 system in wheat, which includes a wheat codon optimized Cas9 driven by a maize ubiquitin gene promoter and a guide RNA cassette driven by wheat U6 promoters in a single binary vector. Using this CRISPR /Cas9 system, we have developed 68 edit mutants for four grain‐regulatory genes, Ta CKX 2‐1 , Ta GLW 7 , Ta GW 2, and Ta GW 8 , in T 0 , T 1 , and T 2 generation plants at an average edit rate of 10% without detecting off‐target mutations in the most Cas9‐active plants. Homozygous mutations can be recovered from a large population in a single generation. Different from most plant species, deletions over 10 bp are the dominant mutation types in wheat. Plants homozygous of 1160‐bp deletion in Ta CKX 2‐D1 significantly increased grain number per spikelet. In conclusion, our Agrobacterium ‐delivered CRISPR /Cas9 system provides an alternative option for wheat genome editing, which requires a small number of transformation events because CRISPR /Cas9 remains active for novel mutations through generations.

Gene editing of the wheat homologs of TONNEAU1‐recruiting motif encoding gene affects grain shape and weight in wheat

“…With the high‐density genetic maps and reference genome sequences available for multiple crops, this strategy of using comparative genomics for understanding the genetic basis of trait control in related crops appeared to be effective and allowed to reduce time and efforts needed to identify causal genes. During the last several years, several genes controlling grain size and weight identified in rice were shown to underlie similar traits in wheat (Zhang et al ., ; Dong et al ., ; Wang et al ., ; Zhang et al ., ). Here, we characterized the wheat TaGW7 gene, the ortholog of the OsGW7 gene in rice.…”

“…In wheat, CRISPR‐Cas9 gene editing was successfully applied to modify such traits of agronomic value as disease resistance (Wang et al ., ), gluten content (Sánchez‐León et al ., ), protein content (Zhang et al ., ), male sterility (Okada et al ., ) and grain size and weight (Wang et al ., ; Zhang et al ., , ). These studies also demonstrated that the editing of either individual copies of duplicated genes on homoeologous chromosomes or all three homoeologous copies of genes is possible (Wang et al ., 2014, 2018a).…”

Genes, mechanisms and novel EST-SSR markers associated with metribuzin tolerance in wheat (Triticum aestivum L.): targets for improving photosynthetic capacity and yield

“…High-throughput identi cation of PAV on a whole-genome level has become possible with the advent of next-generation sequencing (NGS) technologies, at affordable prices [37]. There is a rapidly rising trends in the application of genome editing based crop improvement using CRISPR/Cas genome engineering system [38,39]. The improved understanding of genetic and genomic knowledge of herbicide tolerance will open up the utilities for inducing multiple cleavage events, controlling gene expression, and site speci c transgene insertion.…”

A simple method for spray-on gene editing in planta

“…The structure of the pCas9-GFP plasmid is reported in Zhang et al, 2019 25 , but for ease of understanding a plasmid map is also provided here (Fig. S10).…”