The Preliminary Study Of Intron-splicing Based Single Transcription Unit CRISPR/Cas9 Gene Editor In Tobacco And Cabbage

In recent years,it has become a trend to improve various traits of crops through gene editing technology to meet the development of society and human needs.CRISPR/Cas9 gene editing systems have been rapidly developed because of its simplicity,high efficiency and low cost,and it has provided powerful technical support for rapid breeding and trait improvement of many crops.Currently,increasing the expression level of Cas9 protein as well as sg RNA is used as one of the important ways to improve the editing efficiency of CRISPR/Cas9 gene editing system.CRISPR/Cas9expression cassettes are available in multiple conformations,and Cas9 protein and sg RNA expression can be controlled by the same or different promoters.For example,using a highly expressed constitutive promoter or tissue-specific promoter to express Cas9 protein,and using a species-specific U6 promoter or constitutive promoter to express sg RNA.Among them,placing Cas9 and sg RNA expression under the control of the same promoter to form a single transcription unit（STU）system was shown to be effective in improving gene editing efficiency.In this study,we fused an intron PTG（in PTG）fragment loaded with an sg RNA-released structure to the herbicide（PPT）resistance gene BAR which is based on the previous STU system.An intron splicing-based i STU-CRISPR/Cas9 gene editing system was constructed,and it was proposed to realize the effective improvement of the editing efficiency of the whole gene editing system through the stable expression of BAR gene in the screening process of transgenic plants.Using tobacco（Nicotiana tabacum L.cv.Wisconsin 38）and cabbage（Brassica oleracea var.capitata L.）as subjects,the Nt Sulfur and Nt Dwarf genes in tobacco,the promoter regions of ERF121 and ERF016 genes and the Bo CER1 gene in cabbage were targeted for editing,the results are as follows:1.Functional validation of in PTG fragment-containing intronic fusion BAR gene in tobacco.In this experiment,SL Intron of potato（Solanum tuberosum L.）ST-LS1 gene and MYB Intron of purple cauliflower anthocyanin-related gene MYB were selected for insertion into BAR gene with t RNA-Spacer-g RNA structural fusion as splice intron of in PTG fragment and construction of expression vectors p CA2300-BAR-MYBI and p CA2300-BAR-SLI.They were transformed into wild-type tobacco separately to obtain transgenic tobacco with herbicide resistance.Agarose gel electrophoresis and PCR product sequencing analysis for the c DNA of BAR gene determined that both intron in the fused BAR gene could be spliced correctly and reconstructed the BAR gene function.2.The study of intron splicing-mediated CRISPR/Cas9 gene editor in tobacco single site gene editing.The MYB Intron was used as the splice intron to construct the two-component transcription unit expression system with 35S::Cas9+U6-sg RNA vectors p CABARCas9-U6::Sul/Dw,35S::BAR2ACas9+U6-sg RNAvectors p CABAR2ACas9-U6::Sul/Dw,35S::BAR-Int ^Sul/Dw+35S::Cas9vectors p CABARMYBI^Sul/Dw-Cas9 and single transcription unit expression system with35S::BAR-Int ^Sul/Dw-2A-Cas9 vectors p CABARMYBI^Sul/Dw-2A-Cas9 for a total of 8plant expression vectors.The efficiency of single site editing was tested separately for2 endogenous genes of tobacco,Nt Sulfur and Nt Dwarf,and the results showed that:（1）The i TCTU-CRISPR/Cas9 expression system-based p CABARMYBI^Sul/Dw-Cas9expression vector had low splicing efficiency of BAR gene intron in single site editing of tobacco Nt Sulfur and Nt Dwarf genes,and only 1 unedited positive healing tissue was obtained in the genetic transformation of p CABARMYBI^Sul-Cas9 vector.（2）Both the i STU expression system-based p CABARMYBI^Sul/Dw-2A-Cas9 expression vectors showed higher editing efficiency than the conventional TCTU expression system in tobacco single site gene editing,with knockdown efficiencies of 89.66%for the Nt Sulfur gene and 86.67%for the Nt Dwarf gene.（3）The knockdown efficiency of p CABARMYBI^Sul/Dw-2A-Cas9 expression vector against tobacco Nt Sulfur and Nt Dwarf genes was as high as 96.77%and 100.00%under higher PPT（20 mg/L）screening,i.e.,increasing the herbicide screening concentration can further improve the editing efficiency of the intron splicing-mediated i STU system.3.The study of intron splicing-mediated CRISPR/Cas9 gene editor in multigene,multisite editing of cabbage.Targeting the promoter regions of ERF121 and ERF016,2 genes associated with black rot resistance in cabbage,and 3 loci（Wax1,Wax2,Wax3）of Bo CER1,a gene associated with leaf wax powder phenotype,the intron splicing-based two-component transcription unit 35S::BAR-Int ^ERF/Wax+35S::Cas9 vectors p CABARMYBI^ERF/Wax-Cas9and single transcription unit expression system with 35S::BAR-Int ^ERF/Wax-2A-Cas9vectors p CABARMYBI^ERF/Wax-2A-Cas9 were constructed for a total of 4 plant expression vectors.They were tested for multi-gene,multi-locus editing efficiency in cabbage 159 material,and the results showed that:（1）The p CABARMYBI^ERF/Wax-Cas9expression vector based on the i TCTU-CRISPR/Cas9 expression system was less efficient in the genetic transformation of cabbage,both yielding only 1 resistant healing tissue and a few transgenic positive plants wich is similar to the results seen in the transformation of tobacco.（2）A total of 4 expression vectors were used to obtain 8,2,29 and 55 positive plants during genetic transformation of cabbage,respectively,and no expected mutations were detected in all positive plants at the corresponding target loci with an editing efficiency of 0.00%.The results show that the editing efficiency of the i STU-CRISPR/Cas9 gene editor based on single transcription units monitored by screening marker genes in targeting multi-gene,multi-locus editing needs to be further investigated.