CRISPR/Cas9-based Editing of Acetyl-CoA Carboxylase (ACC1) Gene in Barley

Plastid localized acetyl-CoA carboxylase(ACCase;EC 6.4.1.2)is a target for aryloxyphenoxypropionates(APPs)and cyclohexanediones(CHDs),two groups of selective herbicides used in controlling grassy weeds.Wheat and barley are important cereal crops in the grass(Poaceae or Gramineae)family,and thus sensitive for those herbicides.Characterization of this form of ACCase(or ACC1)in wheat and barley is essential if these agents are used in the sustainable agriculture.In this study,it was confirmed that a single ACC1 gene presented on the second chromosome per homologous group in common wheat,wild emmer wheat,goat grass and barley.Using CRISPR/Cas9,the barley ACC1 gene was edited,specifically in the carboxyl transferase(CT)domain that was critical for herbicide responses in grass species.Two new alleles were generated,one with a 3-bp deletion leading to ACC1:p.Ile1878del and one with a 26-bp deletion causing ACC1:p.Ser2099_Lys2311del.Both were recovered as heterozygotes in the T0 generation.All the seven T0 plants harboring the 3-bp deletion grew normally,but the only T0 plant with 26-bp deletion died at the extension stage(Zadoks 32),probably because there was inadequate ACC1 activity when the plant was big.In the T1 generation,the 3-bp deletion(or Ile1878del)did not impact the edited plants in tiller numbers,tiller height,spike length and spikelet numbers,when compared to the wild-type allele in the non-edited segregants.This study demonstrated that CRISPR/Cas9 was practical to generate single amino acid deletions in the ACC1 protein and the Ile1878 deletion did not compromise plant growth.Unfortunately,the ACC1:p.Ile1878del protein did not confer resistance to the currently tested APP herbicides,including clethodim,haloxyfop,quizalofop-Pethyl and sethoxydim.