CRISPR/Cas9-based Editing Of The Acetyl-CoA Carboxylase(ACC1) Gene In Barley

After corn,rice and wheat,barley?Hordeum vulgare L.,2n=2x=14?is the fourth largest cereal crop in the world,and it's the model plant for studying Triticeae crops.Acetyl-CoA carboxylase?ACCase?,encoded by the ACC1 gene,is the key enzyme that catalyzes the first step in fatty acid biosynthesis in the chloroplast.The herbicide developed with ACCase as the target has been widely used to kill grassy weeds among broad-leaved crops.In other studies,an amino acid change?A1992V?in the carboxyltransferase domain of ACCase can make wheat resistant to ACCase inhibitor herbicides.In this study,we used the clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins?CRISPR/Cas9?to edit the barley ACC1 gene.We aimed to create barley lines that are resistant to ACCase inhibitor herbicides and provide guidelines genome editing in barley.The main research results are as follows:1.Based on the wheat ACC1 gene?AF029895?,we assembled ACC1 DNA?MK481069?and cDNA?MK481066?sequences from barley'Tamalpais'and the ACC1 cDNA sequence?MK481065?from barley‘Morex'.We compared the sequence and structure of the ACC1gene in different grass species and summarized the known mutation sites that confer herbicide resistance in grasses.2.According to barley ACC1 gene sequence,four target sites?T1-T4?were selected in the carboxyltransferase domain,specifically around A1992 site;three CRISPR/Cas9 vectors?V1-V3?were constructed.The V1 and V2 vectors each contained one target,and the V3vector contained two targets.3.For gene replacement,three Donor vectors?D1 to D3?were constructed.D1 was a73-bp single-stranded DNA and was designed to repair DNA breaks by homologous recombination.D2 was a double-stranded plasmid,which contained a 465-bp homologous region.D3 was also a double-stranded plasmid,which was a 702-bp homologous fragment and contained the target sites T3 and T4.This vector was desiged to repair DNA break by nonhomologous end-joining.4.The CRISPR/Cas9 and Donor vectors,either V1 plus D1,V2 plus D2,or V3 plus D3,were seperatedly co-transformed into the barley‘Golden Promise'by biolistic bombardment.In total,we treated 2,300 immature embryos,obtained 90 regenerated plants,and recovered84 transgenic plants positive for the hygromycin gene.They represented 20 independent transgenic events,and a 0.87%transformation efficiency.5.We used TILLING to identify the ACC1-edited plants.Among transgenic plants from the V3+D3 combination,eight plants were positive with an edited ACC1 gene,which corresponsed to an editing efficiency of 25%.DNA sequencing revealed that seven plants had three bases deletion at the T3 target,resulting in the deletion of the 1878^th amino acid isoleucine?ACC1:p.Ile1878del?.In one plant,a 26-bp deletion at the T4 site resulted in213-aa truncation in the ACC1 C-terminus?ACC1:p.Ser2099_Lys2311del?.however,no gene substitution occurred in the eight gene-edtied plants.Transgenic plants from the V1 and V2 vectors were negative an edited ACC1 gene.6.Tillers and spikes of the plants with the Ile1878del mutation were studied in the T₁generation.It appeared that the Ile1878del mutation did not affect the fertile tiller number,average tiller height,spike length and spikelet number per spike in the ACC1-edited plants.Meanwhile,herbicide resistance identification was conducted.Unfortunately,the ACC1:p.Ile1878del protein does not confer resistance to the currently tested APP herbicides,including clethodim,haloxyfop,quizalofop-P-ethyl and sethoxydim.