| Rice(Oryza sativa L.),which is widely known as one of the most important crops in the world,also plays an important role in genome structure and function research as a model plant.It’s meaningful to solve the key biology question,genetic improvement of germplasm,and ensure the food security in the processing of pursuing the deeply research of genome structure,related gene function,and expression manipulation in rice.For these purposes,the crucial step is to obtain the reliable mutants with site-specific mutations efficiently.However,because of the extremely limitations of the natural mutants ever discovered and defined,and the powerless performance of exist artificial methods based on MES inducement and T-DNA insertion,which mainly imputed to its long period,huge invest and random mutation,a new method to obtain reliable mutants with site-specific mutations is in urgent needed.Since 1996,the researchers have reported three main site-specific nucleases with DNA cleavage activity named ZFN(zinc finger nuclease),TALEN(transcription activator-like effector nuclease),and CRISPR-Cas9(clustered regularly interspaced short palindromic repeats,CRISPR-CRISPR associated protein 9),which laid the foundation for genome editing technology applied in basic plant functional gene research and directed genetic improvement of germplasm resources.Due to the significant advantages in high editing efficiency,reliability,multiple recognition site,affluent gene resource,strong toolbox expansibility and convenient operation,it has already become the most common-used genome editing system.In recent years,varieties of editing performance of Cas9,Cas12 a,and Cas12 b has been reported,which greatly expanded the range of Cas system in plant gene function research and crop breeding.Until now,the Sp Cas9 nuclease system,separated from Streptococcus pyogenes,and the Lb Cas12 a nuclease system,separated from Lachnospiraceae bacterium were the most widely used CRISPR-Cas systems in plant.However,the Sp Cas9 and Lb Cas12 a system are both facing the same dilemmas such as limited editing sites(the Sp Cas9 has priority on 5’-NGG-3’ PAM sites,and the Lb Cas12 a has priority on 5’-TTTV-3’ PAM sites),the low editing efficiency on non-canonical PAM sites,the incomplete editing toolbox and the intellectual property restrictions on basic genome engineering elements.These problems limited the further application of CRISPR-Cas system on genome functional research and crop breeding.Based on the current situation of CRISPR-Cas’ s system construction,expansion and application in plant,this dissertation dedicates to solving the key bottle-neck problems of CRISPR-Cas9 and CRISPR-Cas12 a system on editing efficiency improvement,editing tool expansion,and new member mining.The main research results shown as below:1.Based on the x Cas9 nuclease that recognizes 5’-NG-3’ PAM sites,we constructed the plant CRISPR-Cas9 system that recognizes non 5’-NGG-3’ PAM sites and evaluated the editing ability.The results showed that,in rice protoplast,the x Cas9 system mainly presented editing efficiency at canonical 5’-NGG-3’ PAM sites(editing efficiencies were from 1.3% to 60.9%),on the contrast rather low efficiency at 5’-NGH-3’ PAM sites(editing efficiencies were from 0.1% to 9.3%).Fortunately,the x Cas9 showed higher specificity than wild type Cas9.We subsequently constructed the single-base editing system based on x Cas9,which achieved the C to T conversion at both 5’-NGG-3’ and 5’-NGH-3’ PAM sites;2.We further constructed the expression system that could also recognize 5’-NG-3’PAM sites based on Sp Cas9-NG 、 Sp Cas9-NGv1 nucleases,following the evaluations of the editing efficiency of these systems.The results inside of rice protoplast indicated that the Sp Cas9-NG and Sp Cas9-NGv1 systems showed editing efficiencies at most 5’-NGN-3’ PAM sites(editing efficiencies were from 0.1% to41.7%),but presented a significant decrease at canonical 5’-NGG-3’ PAM sites(editing efficiencies of Sp Cas9-NG system were from 1.6% to 20.3%;editing efficiencies of Sp Cas9-NGv1 system were from 2.3% to 11.9%;editing efficiencies of Sp Cas9 system were from 0.6% to 48.4%).These results were proved by stable transformation: the editing efficiencies of Sp Cas9-NG system were from 6.7% to56.3%,and the editing efficiencies of Sp Cas9-NGv1 were from 3.5% to 46.4%.By fused the Pm CDA1 deaminase domain with Sp Cas9-NG,we achieved the efficient single base editing at 5’-NGH-3’ PAM sites and the editing efficiencies were from30.4 to 45.0%;3.We constructed the Fn Cas12 a system that previously reported with 5’-TTV-3’ PAM sites editing efficiency,and evaluated in rice cells for editing profile.We observed that the Fn Cas12 a system showed editing priority at 5’-TTTV-3’ PAM sites(editing efficiencies were from 10.3% to 37.2%),and this system could also recognize partial5’-TTV-3’ PAM sites(editing efficiencies were from 2.3% to 11.2%).The stable transformation in rice indicated the same results that the best editing PAM site for Fn Cas12 a was 5’-TTTV-3’: the editing efficiencies at 5-TTV-3’ PAM sites were from 0.0% to 57.1%,and the 5’-TTTV-3’ PAM sites were from 87.1% to 89.3%;4.Based on the previously reported As Cas12a-RR(recognize 5’-TYCV-3’ and 5’-CCCC-3’ PAM sites),As Cas12a-RVR(recognize 5’-TATV-3’ PAM sites)variants in mammalian,we constructed the Cas12 a variant system based on Lb Cas12 a and Fn Cas12 a nucleases and tested in rice.In rice protoplast transient expression and stable transformation,the Lb Cas12a-RR system showed higher editing efficiencies(from 0.2% to 41.3% in protoplast and average of 56.2% in stable T0 lines)than Fn Cas12a-RR system(from 0.1% to 5.7% in protoplast and average of 6.7% in stable T0 lines),and the Fn Cas12a-RVR system showed higher editing efficiencies(from 0.1% to 40.9% in protoplast)than Lb Cas12a-RVR system(from 0.1% to 10.2%in protoplast);5.In order to enrich the basic plant CRISPR-Cas expression system,we constructed the i STU(intron-based Single Transcript Unit)system based on intron selfprocessing,and realized the genome editing targeted endogenous gene in rice.In protoplast,the editing efficiencies of i STU-CRISPR/Cas9 system were from 3.8%to 35.2%,and editing efficiencies of i STU-CRISPR/Cas12 a system were from 0.7%to 9.0%.We achieved two sites multiplexed genome editing and long DNA fragment deletion based on i STU-CRISPR/Cas9 system and i STU-CRISPR/Cas12 a system.In rice stable T0 lines,we achieved single site genome editing by i STUCRISPR/Cas9(with average editing efficiency of 48.7%),and i STUCRISPR/Cas12a(with average editing efficiency of 15.3%)system and we also achieved two sites’ multiplexed genome editing by i STU-CRISPR/Cas9 with average editing efficiencies of 36.0%;6.At last,based on data mining,we successfully developed a new CRISPR-Cas9member: Lac Cas9,from Lactobacillus spp.that recognized 5’-NGAAA-3’ PAM sites,as a genome editing tool,which achieved genome editing in rice and wheat.We analyzed its editing profile and constructed multiple editing toolbox.The results are shown below: 1)The Lac Cas9 system shown higher editing efficiencies than Sp Cas9-NG,Sp RY and Lb Cas12 a system at related PAM sites,and the single site editing efficiencies in rice T0 lines was 68.0%,and the multiplexed two sites editing efficiencies was 66.7%;2)By using Lac Cas9 nickase fused Pm CDA1 or ABE8 e deaminase,the Lac Cas9 base editing system could achieved efficient base editing with C to T conversion with a rate of 19.1% to 38.0% in rice T0 lines,and the A to G conversion rate was 14.3%.Based on dead Lac Cas9 fused activation domain and repression domain,we achieved gene expression upregulated and repressed. |
