Development And Application Of Diversified New CRISPR-Cas Genome Editing System In Plants

The effective acquisition of diverse mutant materials with DNA sequence variation of specific functional coding units in plant genome is the material basis for analyzing gene function and the source of further germplasm innovation.However,mutant creation strategies such as spontaneous mutant material collection,radiation or other physical mutagenesis,ethyl methane sulfonate(EMS)or other chemical reagents mutagenesis,and T-DNA random insertion have problems such as time-consuming,labor-intensive,random mutant events,and high screening costs.It is difficult to meet the actual needs of plant gene function basic research and molecular breeding practice.Since 1996,the researchers have reported ZFN(zinc finger nuclease),TALEN(transcription activator-like effectors nuclease),CRISPR-Cas(clustered regularly interspaced short palindromic repeats/CRISPR-associated protein)genome editing techniques.It provides an effective technical support for the efficient,accurate and diverse creation of plant diversity mutants.Among them,the CRISPR-Cas system derived from archaea/bacteria has been widely used in basic research of plant gene function and molecular breeding practice due to its advantages of high activity,wide adaptability,easy construction and low cost.Based on the large number and diverse functional gene cluster resources in archaea/bacteria(even viruses and eukaryotes),researchers have reported CRISPR-Cas genome editing systems based on type II Cas9,type V Cas12 a,type VI Cas13,and type I Cascade,which provides rich resources for the effective application of plant genome editing.Based on Sp Cas9 nuclease isolated from Streptococcus pyogenes and Lb Cas12 a nuclease isolated from Lachnospiraceae bacterium and their derivatives,researchers have realized base insertion/deletion(In Del)editing,base substitution editing,long fragment insertion/deletion/substitution editing,and epigenetic modification regulation editing in microorganisms,animals and plants.It is the most widely used CRISPR-Cas plant genome editing system.However,the existing plant genome editing tools of Sp Cas9 and Lb Cas12 a still have some constraints in practical applications,such as off-target events caused by non-specific DNA double-strand break(DSB),editing sites limited by Cas nuclease specific PAM(protospacer adjacent motif)recognition motif,long Cas9/12 a protein coding sequence affecting delivery,and insufficient development and application of diversified CRISPR gene cluster resources.To varying degrees,the deep application of CRISPR-Cas genome editing system in plant gene function basic research and molecular breeding practice is limited,and it is urgent to further diversify the effective development of CRISPR-Cas plant genome editing tools.In view of the above constraints in the development and application of CRISPR-Cas plant genome editing system,this dissertation focuses on the key scientific issues and core technical bottlenecks of plant genome editing specificity improvement,site expansion,effective development of CRISPR gene cluster resources,and germplasm innovation application.The main research results are as follows:1.Aiming at the problem of genome off-target editing caused by SpCas9 non-specific induction of DSB,by using the D10 A nickase variant(n Cas9)of Sp Cas9 to guide cytidine deaminase to achieve C-to-T base editing in the specific sequence of the target protein-coding gene,the strategy of early termination codon is introduced in the same frame translation to achieve DSB-independent protein-coding gene knockout,thereby reducing the occurrence of off-target editing events.Meanwhile,based on the editing site preference and the specific editing characteristics of the base editor,a guide RNA(sg RNA)design tool,CRISPR-BETS,which can effectively implement stop codon editing was developed.The results of stable transformation of rice showed that the C-to-T editing efficiency of n Sp Cas9(D10A)-Pm CDA1 base editor in T0 regenerated plants was 16.7%-64.0% in the five endogenous sites of rice,and all test sites achieved stop codon editing.Moreover,the detection results of potential off-target sites of rice edited plants showed that no sg RNA-dependent off-target effect was detected.2.Based on the FrCas9 nuclease isolated from Faecalibaculum rodentium,which can recognize the “AT” symmetric PAM motif(5’-NNTA-3’),the plant genome editing tool was developed to realize the effective expansion of plant genome editing sites.The experimental results show that: 1)Based on the plant FrCas9 knockout vector,effective base indel editing can be achieved at the 5’-NNTA-3’ PAM site of rice(instantaneous editing efficiency: 2.7%-53.9%;stable editing efficiency:10.0%-85.7%).The editing events were mainly 1 bp indel in the upstream of PAM.2)Based on the strategy of TREX2 exonuclease fusion,the FrCas9-TREX2 editing vector can effectively achieve 6-30 bp multi-base deletion,which expands the editing feasibility for non-protein coding sequences.3)Through the fusion of A3A/Y130 F,ec Tad A8 e deaminase and FrCas9(E796A)nickase,effective C-to-T(instantaneous editing efficiency: 24.2%-59.5%;stable editing efficiency:35.0%-50.0%),A-to-G base editing(instantaneous editing efficiency:0.23%-39.3%);stable editing efficiency: 20.0%-40.0%);4)FrCas9 had no significant effect on the number of In Dels and SNVs in the whole genome.TREX2-FrCas9 had no significant effect on the number of In Dels,but significantly increased the number of SNVs.FrCas9 and TREX2-FrCas9 had no off-target effect at all sg RNA-dependent off-target sites detected.3.In response to the demand for diversified PAM recognition and miniaturization of Cas proteases,a plant genome editing tool was developed based on type V Cas12i1 and Cas12i2 nucleases isolated from the Lachnospiraceae bacterium,in order to enrich the plant genome editing toolbox.The results showed that: 1)The Cas12i1 nuclease editing vector only showed limited editing activity in rice(instantaneous editing efficiency: 0.02%-0.92%),but after fusing the transcriptional activation domain or inhibition domain,the Cas12i1 nuclease achieved effective directional transcriptional regulation of rice endogenous genes(activation efficiency: 2.1-25.1times;inhibition efficiency: 7.0%-54.0%);2)Cas12i2 nuclease editing vector can achieve effective base deletion/insertion editing in rice(instantaneous editing efficiency: 0.05%-28.5%;stable editing efficiency: 5.0%-30.0%),and Cas12i2 MAX nuclease variant further improved editing efficiency(transient editing efficiency:1.9%-20.9%;stable editing efficiency: 6.25%-68.6%).4.Considering that the Anti-CRISPR(Acr)protein found in the type V Cas12 a nuclease can effectively inhibit the DNA cleavage activity of the corresponding Cas12 a nuclease in bacteria,we tried to introduce the Acr VA1,Acr VA4 and Acr VA5 proteins isolated from Moraxella bovocali into the Lb Cas12 a plant genome editing system,in order to obtain the target site editing material and further reduce the off-target effect caused by non-specific DSB.And realize the valuable application of Acr protein in plant genome editing.The experimental results show that: 1)Acr VA1,Acr VA4 or Acr VA5 were co-expressed in E.coli,respectively.Acr VA1 protein could inhibit the editing activity of Lb Cas12 a and Mb2Cas12 a.2)The genome editing efficiency of Lb Cas12 a and Mb2Cas12 a was significantly inhibited when co-expressing Acr VA1 protein in rice protoplasts and stable transgenic plants,while the inhibitory effect of co-expressing Acr VA5 protein was mild.At the same time,the co-expression of Acr VA1 and Acr VA5 proteins significantly reduced the number of total In Dels and SNVs and the occurrence of cr RNA-dependent off-target events.3)Based on XVE induced expression and tissue-specific expression strategies,Acr VA1 protein was driven respectively,which effectively realized the development and application of induced and tissue-specific controllable editing tools.Based on this,a variety of plant gene expression regulation circuits were developed.5.Based on the development of the above diversified CRISPR-Cas plant genome editing tools,according to the sequence characteristics of key regulatory genes of important agronomic traits in rice and the needs of different editing events,a new germplasm of specific rice endogenous gene editing was created,and the effectiveness and reliability of the editing tools and corresponding editing strategies were evaluated.The experimental results show that: 1)For the Os GW2 gene in rice,a protein-coding gene knockout strategy based on C-to-T base substitution and introduction of early terminator was designed.A new wide-grain rice germplasm with Os GW2 function loss was effectively created under the premise of significantly reducing the off-target editing effect.2)For Os Gn1 a and Os GS3 genes in rice,a FrCas9 plant genome editing vector was constructed to identify the 5’-NNTA-3’ PAM motif editing site,which effectively created new rice germplasms with multiple secondary branches and multiple grains with Os Gn1 a function loss and long grains with Os GS3 function loss.3)For rice micro RNA encoding gene Os MIR156 j,TREX2-FrCas9 editing vector was used to achieve multi-base deletion editing of the functional sequence of Os MIR156 j,which created an effective editing event for the loss of microRNA function in rice.Based on the above results,this dissertation completed the effective construction of an efficient,highly specific and widely adaptable CRISPR-Cas genome editing system for diverse plants,and qualitatively and quantitatively evaluated its editing efficiency and editing characteristics.Based on this,the effective creation of new germplasm materials for important agronomic trait regulatory genes in rice was realized,which provided a theoretical basis and application case for basic research of plant gene function and molecular breeding application practice.