Studies On High-efficient CRISPR/Cas9 Gene Editing System In Cotton

The acquisition of heritable cotton mutations is essential for identifying gene functions and breeding new cotton varieties,the emergence of CRISPR/Cas9 genome editing technology provides a powerful technical tools for the realization of cotton molecular breeding.At present,the cotton gene editing system has been established and successively upgraded and improved(CRISPR/Cpf1 and single-base editing system).However,to achieve targeted editing of target genes in cotton,genetic transformation is necessary,and the target gene mutants can be obtained through tissue culture.The genetic transformation of cotton is time-consuming and laborious with a long cycle,and limited by the genotype of cotton varieties,only a few cotton varieties can be effectively transformed.In view of the above problems,this research mainly carried out two approaches.The first approach: the cotton pollen was used as a transgene receptor,we constructed a cotton reproductive-specific CRISPR/Cas9 gene editing system,target and knock out the target gene in the pollen,and then used the mutated pollen to obtain cotton mutants through pollination;The second approach: we developed a novel cotton leaf crumple virus(CLCr V)-mediated gene editing system(VIGE).The Cas9 overexpressing(Cas9-OE)cotton was used as the transgene receptor,and exploring the function of Arabidopsis Flowering Locus T(FT)gene in delivering sg RNAs with long-distance transportation into shoot apical meristem(SAM),thus avoiding tissue culture and stably acquiring heritable cotton mutant offspring.It was verified that the above two methods could avoid tissue culture and obtain cotton mutants efficiently.The main results were as follows:1.The tissue-specific of GhPLIM2b and GhMYB24 genes was analyzed with RT-PCR,respectively in cotton roots,pollen,sepals,petals,leaves,and stems.The results showed that GhPLIM2 b gene was specifically expressed in cotton pollen,while GhMYB24 gene was expressed in cotton pollen;Furthermore,the GhPLIM2b(1530 bp)and GhMYB24(1435 bp)promoters were cloned,and the fusion expression vector of GhPLIM2 b P::GUS and GhMYB24P::GUS was successfully constructed.The results of Agrobacterium vacuum infiltration transformation of cotton pollen showed that both GhPLIM2 b and GhMYB24 promoters could drive the expression of GUS in cotton pollen,and cotton pollen was dyed blue,there was no significant difference in transcriptional activity between the two promoters;The GhPLIM2 b and GhMYB24 promoters were used to drive Cas9 gene expression,and editing vectors for targeted editing of cotton GhCLA1,GhGGB and GhERA1 were designed.The results of Agrobacterium vacuum infiltration transformation of cotton pollen showed that both GhMYB24P::Cas9 and GhPLIM2 b P::Cas9 editing vectors could achieve targeted editing of cotton pollen endogenous genes,with an editing efficiency of3.29%-6.45%,and there was no significant difference in editing efficiency between the two editing systems,but there were a few off-target phenomenon.Since most of the transformed pollens had lost their pollination activity,it was impossible to obtain genetically edited progenies by pollination.This study proved the effectiveness of GhPLIM2 b P::Cas9 and GhMYB24P::Cas9 editing systems,and provided an effective gene editing system for rapid obtaining cotton mutants using cotton pollen as transgenic receptor.2.We developed a novel cotton leaf crumple virus(CLCr V)-mediated VIGE system in Arabidopsis.Cas9-OE Arabidopsis was used as a transgene receptor,the CLCr V-mediated VIGE system was used to target Arabidopsis BRI1,GL2,PDS genes,and GUS transgenic have verified the feasibility and efficiency of the system;At the same time,it was proved that the Pro Yao::Cas9 transgenic Arabidopsis,which is highly expressed in meristem can significantly improve the gene editing efficiency of CLCr V-mediated VIGE;Given the deficiency of the VIGE system,Pro Yao::Cas9 and Pro35S::Cas9 Arabidopsis were transformed by fusing 102 bp FT m RNAs with sg RNAs so as to explore the function of Flowering Locus T(FT)gene in delivering sg RNAs into SAM,thus avoiding tissue culture and stably acquiring heritable mutant offspring.The results showed that sg RNAs fused with FT m RNA at the 5' end(FT strategy)effectively enabled gene editing in infected plants and allowed the acquisition of mutations heritable by the next generation,with an efficiency of 4.35-8.79%.In addition,gene-edited offspring by FT-sg RNAs did not contain any components of the CLCr V genome.FT strategy can be used to acquire heritable mutant offspring avoiding tissue culture and stable transformation based on the CLCr V-mediated VIGE system in Arabidopsis,which shows that the strategy has a broad application prospect in crop functional genomics and molecular design breeding.3.The feasibility of CLCrV-mediated VIGE was analyzed in cotton to determine whether the CLCr V could be used to deliver sg RNAs for targeted endogenous genes in cotton.Cas9-OE cotton was used as a transgene receptor,the CLCr V-mediated VIGE system was efficiently achieved target cotton GhPDS and GhCLA1 genes,which proved that CLCr V mediated VIGE system was also suitable for cotton;At the same time,the CLCr V-mediated VIGE system can deliver multiple sg RNAs in cotton simultaneous realizing double mutations of GhPDS and GhCLA1 genes in cotton;In view of the fact that FT-sg RNA strategy can achieve heritable gene-edited offspring in Arabidopsis,it is further verified whether this strategy is also applicable in cotton.The results showed that 102 bp FT m RNA fused to the 5' end of sg RNA can also achieve targeted editing of cotton endogenous genes GhPDS,GhCLA1,GhBsrk1 and GhMAPKKK2;CLCr V-mediated VIGE system can still achieve heritable mutant offspring without relying on FT-sg RNA in cotton.In addition,gene-edited ovule by CLCr V-mediated VIGE did not contain any components of the CLCr V genome.The successful application of the CLCr V-mediated VIGE system in cotton has laid an important technical foundation for the study of cotton biology,the construction of a large-scale cotton gene mutant library,and the realization of precise genetic improvement at the whole genome level.