CRISPR/Cas9-mediated Multiplex Gene Editing In Wheat

Wheat(Triticum aestivum L.)is a major staple food crop globally.The production is an important factor in ensuring food security both in China and the rest part of the world.Breeding for wheat varieties with high yield,good quality,multiple resistance and efficient use of resources has always been a major breeding goal.However,due to the functional redundancy of genes and polyploid nature of wheat,it is very challenging to pyramid several agronomically important traits which are conferred by multigene in an elite variety simultaneously through conventional breeding.In recent years,CRISPR/Cas9 system,as a simple,versatile and efficient genome editing tool,has been widely used in functional genomics research and crop genetic improvement.Establishment of an efficient CRISPR/Cas9 multiplex system in wheat will greatly facilitate deciphering a complex trait controlled by multiple genes,improvement of agriculturally important traits and generation of novel wheat germplasm,thus to accelerate wheat breeding process.In this study,we developed a robust and efficient multiplexing strategy based on CRISPR/Cas9system and an endogenous t RNA processing system.Six genes including Ta Qsd1,Ta NPT1,Ta IPA1,Ta ARE1,Ta SBE?a and Ta SPDT were selected as primary target genes,and multiplex gene editing vectors were constructed for simultaneously editing of two,three,four and five genes,respectively.Then Zhengmai7698 immature embryos were transformed by particle bombardment.The genotype of plants was determined by Hi-Tom and Sanger sequencing of the PCR amplicons using gene specific primers.Furthermore,we investigated the off-target effects of these g RNAs in the edited lines.The copy numbers of transgenes in edited plants in T₀generation were then estimated by dd PCR.Following tissue culture propagation of wheat and segregation,we then carried out genetic analysis of both transgene and edited loci in T₁generation.The results are as follows:1.We successfully established an efficient multiplex gene editing system in wheat.A multiplexing strategy in wheat based on CRISPR/Cas9-mediated gene editing system was successfully established by using an expression cassette,which is driven by a Pol II promoter Actin to express the tandem repeats of t RNA-sg RNA units and is terminated by a Poly A sequence to stabilize the transcripts and a Nos terminator.We demonstrated the simultaneous editing of two,three,four and five genes in this elite wheat variety Zhengmai7698 by multiplex gene editing.The simultaneous editing efficiency of multiple genes can reach as higher as 50%.2.Analysis of potential off-target effects.Among the six target genes,except for the off-target effects in some lines derived from gene combinations containing Ta SPDT and Ta IPA1 genes at the first potential off-target sites,no off-target effects were detected in the rest of tested lines.3.Analysis of transgene copy number.The results showed that the nineteen edited lines contained1 to 9 copies of Cas9 and 1 to 8 copies of hpt II,respectively.No positive correlation was found between the copy number of Cas9 and the editing efficiency.However,in general,higher copy number of Cas9 facilitated the generation of multiple genomic loci edited lines.4.We generated novel transgene-free wheat materials with pyramiding multiple mutations in target genes.Following tissue culture propagation of wheat and segregation,we successfully recovered transgene-free wheat with pyramiding multiple mutations in target genes in one generation within one year.The inheritance analysis of some editing lines indicated that except new mutations were detected in some plants in T₁generation,and the inheritance of the edited loci in most of the lines were in accordance with Mendelian genetics.