Study On Targeted RNA Screening System In Cotton CRISPR/Cas9 Gene Editing

Cotton is an important economic crop and a source of natural fiber in my country,and cotton genetic improvement is needed to continuously provide better varieties.However,traditional cotton genetic improvement breeding methods cannot meet market demand.The market needs to create new cotton breeding materials more quickly and efficiently.This requires cotton biology as a solid theoretical basis and efficient cotton genetic improvement technology.At present,the whole cotton genome sequencing has been completed,and the research of cotton biology can be fully carried out.The acquisition of cotton mutant materials is an important prerequisite for the development of cotton biology research.The emergence of CRISPR/Cas genome editing technology and its single-base editing derivative technology provides strong technical support for the efficient acquisition of cotton mutant materials and efficient genetic improvement of cotton.Single guide RNA(sgRNA)is one of the important elements of the CRISPR/Cas genome editing technology system and related derivative technologies.However,studies have shown that many sgRNAs cannot work effectively,so multiple candidate sgRNA designs need to be screened to verify their effectiveness.The early verification of the effectiveness of sgRNA used the method of transient transformation of protoplasts or leaves with complete genome editing vectors.These methods are time-consuming and labor-intensive,the success rate is not high,and the reagents consumed are relatively expensive.Especially for cotton with low production efficiency of protoplasts.Therefore,before constructing the cotton CRISPR/Cas genome editing vector,a transient transformation method is needed to quickly verify the effectiveness of the candidate sgRNA.The transient transformation system of model plants is very mature,but the cotton transient transformation system includes protoplast transformation and Agrobacterium injection,which are not very efficient.This research aims to find a simple,efficient,low-cost and effective sgRNA cotton screening technology system.At the same time,it helps to promote the precise molecular breeding of cotton,and provides important technical reserves for the research of cotton functional genomics,which is of great significance.The main results of this study are as follows:(1)Using traditional strategies,the sgRNAs of GhMAPKKK2,GhCBP,and GhAE genes were designed and the corresponding complete genome editing vectors were constructed.After Agrobacterium was transformed,protoplasts were transformed and wild-type upland cotton cotyledons were respectively transformed to transform plants corresponding to the empty vector.Contrast.The genomic DNA of protoplasts and cotyledons obtained by injection of the above-mentioned editing vector and empty-load transformation were extracted respectively,and analyzed and detected by PCR/RE methods,and it was found that all were cut,indicating that the above strategy did not detect gene editing events.(2)The strategy of transforming Cas9 transgenic positive plants with a vector containing only sgRNA.Connect the aforementioned GhU6-5P::MAPKKK2-sgRNA,GhU6-5P::CBP-sgRNA and GhU6-5P::AE-sgRNA vector to the p1300 vector to obtain GhU6-5P::MAPKKK2-sgRNA-1300,GhU6-5P::CBP-sgRNA-1300 and GhU6-5P::AE-sgRNA-1300 vectors were transformed into Agrobacterium,and the true leaves of Cas9 transgenic positive plants were transformed by Agrobacterium injection,and the plants corresponding to the empty vector were transformed as controls.Extract and inject the above-mentioned editing vector and empty-load transformation to obtain true leaf genomic DNA at the injection site.Use detection primers to PCR amplify the above-mentioned genomic DNA and digest the PCR product(GhMAPKKK2-sgRNA uses Nde I,GhCBP-sgRNA uses BcoD I,GhAE-sgRNA Digestion with Mfe I),the results showed that the transformed GhU6-5P::MAPKKK2-sgRNA-1300 vector had residual bands.After recovery,cloning and sequencing showed that the base composition of the restriction endonuclease site upstream of the PAM site appeared different types Mutations,including base substitutions and base deletions.However,the samples transformed with GhU6-5P::CBP-sgRNA-1300 and GhU6-5P::AE-sgRNA-1300 vector vectors showed no residual bands after digestion,indicating that no editing events occurred or editing efficiency was very low.The above results prove that in the transient transformation system,the transformation strategy of transforming Cas9 transgene positive plants with the p1300 editing vector with only guide RNA is more efficient than the co-transformation of sgRNA and Cas9 editing vector.(3)Use the above strategy to verify whether GhMAPKKK2-sgRNA,GhCBP-sgRNA and GhAE-sgRNA are effective.Construction of Gemini virus delivery vectors GhU6-5P-2::MAPKKK2-sgRNA-CLCrVA,GhU6-5P-2::CBP-sgRNA-CLCrVA and GhU6-5P-2::AE-sgRNA-CLCrVA transformed the cotyledons of Cas9-positive plants to verify the effectiveness of this strategy,and transformed plants corresponding to empty vectors as controls.Extract and transform the above-mentioned vector and pCLCrVB mixed vector without injection of new-born true leaf genomic DNA,PCR/RE assay of the above-mentioned genomic DNA with detection primers,and digestion of PCR products(GhMAPKKK2 gene was digested with Nde I,GhCBP gene was digested with BcoD I,GhAE gene was digested with Mfe I).The results showed that the samples transformed with the above vectors had residues After recovery,cloning and sequencing showed that different types of mutations appeared in the base composition of the restriction endonuclease site upstream of the PAM site,including base substitutions,base insertions,and base deletions.However,no gene editing event was detected in samples transformed with GhU6-5P::CBP-sgRNA-1300 and GhU6-5P::AE-sgRNA-1300 vectors.The results indicate that the strategy of using Cas9 transgenic positive plants as transformation recipients can be Efficient and true verification of the effectiveness of sgRNA,eliminating false negative results due to low transformation efficiency,and the strategy of viral vector delivery of sgRNA is more efficient and accurate.The establishment of this sgRNA high-efficiency verification system provides an important technical basis for cotton functional genomics research.(4)Through the effective sgRNAs previously reported:GhPDS-sgRNA,GhCLA1-sgRNA and GhEF1-sgRNA,sgRNA to be detected:GhBsrk1-sgRNA the gemini virus delivery vector GhU6-5P::EF1-sgRNA-CLCrVA,GhU6-5P::PDS-sgRNA-CLCrVA,GhU6-5P::CLA1-sgRNA-CLCrVA and GhU6-5P::Bsrk1-sgRNA-CLCrVA transformed the cotyledons of Cas9-positive plants to verify the effectiveness of this strategy,and transformed the plants corresponding to the empty vector as the control.Extract and transform GhU6-5P::EF1-sgRNA-CLCrVA,GhU6-5P::PDS-sgRNA-CLCrVA,GhU6-5P::CLA1-sgRNA-CLCrVA,GhU6-5P::Bsrk1-sgRNA-CLCrVA and pCLCrVB mixed vector without injection of new-born true leaf genomic DNA,use detection primer pair After PCR/RE assay of the above-mentioned genomic DNA,the PCR product was digested(GhEF 1 gene was digested with Stu I,GhPDS gene was digested with Bfa I,GhCLA1 gene was digested with Bcl I,and GhBsrk1 gene was digested with Nco I).The results showed that the samples transformed with the above-mentioned vectors had residual strips.After recovery,clone sequencing showed that different types of mutations appeared in the base composition of the restriction enzyme site upstream of the PAM site,including base substitutions,base insertions,and base deletions.The above results prove that because the gemini virus can be transferred,this strategy can detect the editing efficiency at the molecular level on the uninjected newborn true leaves.This experiment successfully established the VIGE strategy of viral delivery of sgRNA for targeted editing of cotton genes.The establishment of this sgRNA high-efficiency verification system provides an important technical basis for cotton functional genomics research.