Establishment Of An Efficient CRISPR/Cas9 Gene Editing System In Agrobacterium Rhizogene-mediated Hairy Root Transformation

CRISPR/Cas9 gene editing technology is widely used in the creation of plant and animal mutants and the study of gene function,and the efficiency of gene editing directly affects the difficulty and efficiency of the research work.Factors affecting gene editing efficiency include the promoter that drives the expression of Cas9 and sg RNA,the gene location of editing target,and the ploidy of editing recipient plants.The promoter that drives Cas9 gene is one of the most studied factors affecting gene editing efficiency.Therefore,it is particularly important to select an appropriate promoter to drive the expression of Cas9 in improving gene editing efficiency.Gene-edited plants were obtained by Agrobacterium tumefaciens-mediated genetic transformation in diploid self-pollinated plants,even if the target of gene editing is heterozygous in plant T1,homozygous plants with gene editing target mutation can be obtained by self-crossing generation.At present,in the study of symbiotic nitrogen fixation of legumes,due to the long cycle or low efficiency of genetic transformation through Agrobacterium tumefaciens,Agrobacterium rhizogene-mediated genetic transformation is generally used to study gene function.When gene editing is carried out through A.rhizogenes-mediated genetic transformation,the phenotypes are analyzed on the resulting transgenic hairy roots,since the corresponding phenotypes can be observed only when the edited genes produce double-spike genotypes for most traits.Therefore,to study the function of genes by gene editing through A.rhizogenes-mediated genetic transformation,gene editing efficiency is the key factor,only high gene editing efficiency and high proportion of double-spike types are conducive to gene function analysis.This study intends to clone promoters strongly expressed in roots to drive the expression of Cas9,and screen promoters with high gene editing efficiency and high double-spike types in roots.Firstly,the promoters with specific or strong expression in roots were cloned,and its high activity was proved by A.rhizogenes-mediated genetic transformation.Then,a gene editing vector of Cas9 driven by the promoter was constructed,and a set of CRISPR/Cas9 gene editing system was established for hairy root editing,and promoters with high gene editing efficiency and high homozygous mutation types were selected in hairy roots.The main achievements are as follows:i.The 2173 bp upstream promoter fragment of Gma-mi R1510b-3P was amplified from soybean to construct the recombinant plasmid driving GUSPlus reporter gene expression.Soybean was transformed by A.rhizogenes-mediated mediated one-step transformation and analyzed by GUS histochemical staining.The results showed that the promoter of Gmami R1510b-3P had high expression activity in soybean roots and nodules.ii.Five recombinant plasmids with different lengths of At GCSpro promoter cloned from Arabidopsis thaliana,from 833 bp to 2411 bp,driving GUSPlus reporter gene expression were constructed.Soybean,tomato,cucumber,Lotus japonicus and polyploid crops including sweet potato,cotton and tobacco were transformed by A.rhizogenes-mediated transformation and analyzed by GUS histochemical staining.The results showed that there was no significant difference in GUS signal activity in roots of At GCSpro2411,At GCSpro1977,At GCSpro1629 and At GCSpro1178.The expression level of GUS in roots of At GCSpro833 was the lowest and there was no GUS expression in root tips.These results indicated that At GCSpro833 had no activity in apical tissues.The At GCSpro promoter can drive the expression of GUSPlus in all transformed dicotyledons,and its expression activity is very high.These results indicate that At GCSpro is highly active in the tissue with active cell division in the root,and At GCSpro promoter is suitable for a wide range of dicotyledons.iii.In order to preliminarily test the editing efficiency of Cas9 driven by different promoters,we first designed a sg RNA targeting soybean gene Rj7 for editing.30 transgenic roots were selected for testing through A.rhizogenes-mediated hairy root transformation.The results showed that,At GCSpro2411,At GCSpro1178,Gma-mi R1510b-3P,2×35Spro and At GCSpro833 drive Cas9 to edit Rj7 and generate homozygous/double-allelic mutation efficiency of 76.7%,70%,73.7%,56.7% and 50%,respectively.This indicates that the editing efficiency of Cas9 driven by At GCSpro2411,At GCSpro1178 and Gma-mi R1510b-3P promoter is higher than that of the widely used 2×35Spro,but the editing efficiency of the truncated At GCSpro833 is lower than that of 2×35Spro,which is not suitable for gene editing.The results showed that the promoters At GCSpro1178,Gma-mi R1510b-3P and 2×35Spro drove Cas9 to simultaneously edit Gm NNL1 and Rfg1 genes,yielding homozygous/double-allelic mutation efficiencies of 6.7%,6.7% and 0%,respectively.In conclusion,At GCSpro is suitable for gene editing in A.rhizogenes-mediated transformation with high editing efficiencyiv.In order to verify whether the At GCSpro promoter has the same high editing efficiency in hairy root transformation in other dicotyledons,the Lj NLP4 and Lj SYMRK gene of Lotusjaponicus and the Sl TRY gene of tomato were edited.The results showed that the homozygous/double-allelic mutation efficiency generated with the At GCSpro promoter was1.7 times and 8.3 times more efficient than 2×35Spro.In conclusion,promoters with high expression in hairy roots were screened out,and a CRISPR/Cas9 gene editing system suitable for root trait gene research was established,which established a good foundation for gene function research and genetic engineering transformation.