Tall Festuca is an important cold-season and forage grass, which has been widely used in production all over the world. In summer, the extreme high temperature greatly affects the quality and application value of tall fescue. However, its self-incompatible allohexaploid nature and large genome size (~ 6 gigabases) makes it a challenge for genetic modification and improvement. Consequently, a robust and efficient genome editing tool that can simultaneously target several homoeologous gene copies is crucial to resolve this bottleneck. 

The Molecular Breeding of Turfgrass and Forage Grass Group at the Wuhan Botanical Garden of the Chinese Academy of Sciences used Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 (CRISPR/Cas9) and CRISPR/Cas12a (Cpf1) systems to obtain stable transformation mutants of three homologous alleles, and realized the gene editing of allohexaploid tall festuca. 

The researchers found that the editing efficiency of two sgRNAs (sgrNA1 and sgrNA2) of CRISPR/Cas9 for FaPDS gene were 83.9% and 73.1%, respectively. The editing efficiency of LbCas12a for FaPDS gene was 13.6%-61.5%, which was lower than that of the Cas9 system, but it was more inclined to produce large fragment deletion editing. 

In order to study the heritability of editing site, they selected Cas9 system for gene editing of small molecule heat shock protein FaHSP17.9 in tall fescue, and obtained two mutants of fahsp17.9-3 and fahsp17.9-7, which were related to three homologous allelic frameshift of tall fescue.

Under heat stress, fahsp17.9-3 and fahsp17.9-7 showed obvious heat-sensitive phenotypes compared with the wild type. The turf quality, photosynthetic Fv/Fm, J-I-P electron transfer and chlorophyll level were significantly lower than those of the wild type. The T1 plants were identified by Sanger sequencing, and it was found that the T0 plant editing site could be inherited to T0 plants, and new editing sites could be generated. 

This study entitled "Simultaneous gene editing of three homoeoalleles in self-incompatible allohexaploid grasses" has been recently published in Journal of Integrative Plant Biology and it was supported by the National Natural Science Foundation of China and the Major Science and Technology Special Projects of Shandong Province. 

 The fahsp17.9 mutants induced by CRISPR/Cas9 showed sensitivity to heat stress (Image by WBG)