Development Of Genomic Engineering Tools Based On CRISPR/Cas9 Variants With Expanding Target Scope For Rice Grain Quality Improvement

The CRISPR/Cas9 genome editing system is a powerful and flexible genetic modification tool that can precisely manipulate genetic components,and it has a strong application prospect for the study of gene function and accelerating genetic improvement of crops.Since canonical Streptococcus pyogenes Cas9(SpCas9)recognizes the protospacer adjacent motif(PAM)sequence NGG that limited the targeting scope of genome and base editing,it is of great theoretical and practical significance to develop widely applicable tools for wide-targeting genome editing.On the other hand,the amylose content(AC)of rice grains affects directly the eating taste and food-processing characteristics of rice,which is controlled by the Waxy(Wx)gene(encoding the granule-bound starch synthase I,GBSS I).However,traditional breeding methods cannot effectively generate rice varieties with different AC levels to meet the consumption requirements of different populations and the needs of food industry,so it is significant to breed new rice germplasms with different AC.For this purpose,two strategies were used in this study:first,constructing a series of genome and base editing tools that can target the NG PAM site based on SpCas9 variants including xCas9,Cas9-NG and e Cas9-NG,and comparison of their editing efficiency;second,using different strategies to edit different regions of the Wx gene by SpCas9,including coding sequence(CDS),5’UTR intron splice site(UISS),intron splice site in CDS(ISS),promoter,and putative transcription factor genes regulating Wx expression.By these ways,a serial of new rice mutant lines with moderate and low AC levels were developed.The main results as follows:1.According to the previous reports,the SpCas9 gene was systematically mutated based on our previous SpCas9 vectors to produce the genome editing vectors with the Cas9mutants(xCas9,Cas9-NG and enhance-fidelity e Cas9-NG variants).Using the SpCas9vector as a control,the three new vectors were used to simultaneously edit four target sites containing TGN(N:A,T,C,G)PAM to systematically compare their genome editing efficiency.The results showed that Cas9-NG could stably recognize NG PAM,thus can expand the target range of genome editing,but the editing efficiency decreased to a certain extents compared with SpCas9 in NGG targets.Compared with Cas9-NG,the editing activity of e Cas9-NG decreased to a certain extents.xCas9 showed weak editing activity on NGG target sites,but had low editing activity on other non-NGG targets.Therefore,if there is a selectable PAM(NGG)in the targeted editing site,SpCas9 should be preferentially selected,and Cas9-NG should only be selected when there is no suitable PAM.2.The CBE4 cytosine base editors(Cas9n-CBE,xCas9n-CBE,Cas9n-NG-CBE and e Cas9n-NG-CBE)based on the Cas9 variants(SpCas9,xCas9,Cas9-NG and e Cas9-NG)were constructed,respectively,and their C-T substitution efficiency was compared systematically.The results showed that Cas9n-CBE could edit the canonical NGG PAM;Cas9n-NG-CBE enabled stable editing not only at canonical NGG target sites but also at the non-NGG targets;e Cas9n-NG-CBE seemed to have application potential according to the statistics,but more targets need further testing;xCas9n-CBE did not detect C-T substitution on all targets.The efficiencies of CBE4 base editors tested in this study are not high and some variants showed strong target preferences,so it still needs to be improved.3.The ABE7.10 adenine base editors(Cas9n-ABE,xCas9n-ABE,Cas9n-NG-ABE and e Cas9n-NG-ABE)based on the Cas9 variants(SpCas9,xCas9,Cas9-NG and e Cas9-NG variants)were developed respectively,and their A-G substitution efficiency was compared systematically.The results showed that xCas9n-ABE,Cas9n-NG-ABE and e Cas9n-NG-ABE exhibited low activity on both canonical NGG and non-NGG targets except for Cas9n-ABE that preferred editing on canonical NGG targets,suggesting that these ABE7.10 base editors are inefficient and needs further improvement.4.For genome editing of the CDS of the Wx gene,two CRISPR/Cas9 vectors for single target-site(CT1 or CT2)were constructed to edit the 3rd and 13th exons,respectively,of the CDS of the Wx~a genotype in a indica rice Tian Feng B(TFB)with 25%of AC.The editing events caused frameshift mutations of the gene,thus generate new glutinous rice with about 2～2.5%of AC lines.5.A genome editing vector targeting to the UISS of Wx was constructed and used to edit TFB.Seven mutant lines with different types of base deletions were obtained.Among them,two lines(UISS-#1 and UISS-#6)destroyed the UISS and produced low AC of about10%.Another line(UISS-#2)had deletion of 2 bases near the UISS,and also produced low AC of 9.8%.The levels of AC of other mutants were similar to the TFB.Analysis of the expression levels and splicing products of Wx from endosperm of 15 days after pollination found that the expression of UISS-#1,#2 and#6 all were 10-fold lower than the wild-type TFB,and they also produced multiple non-canonical splicing transcripts.6.Two constructs ISS-T1T2 and ISS-T3T4 were prepared to edit the sequence around ISS of Wx.The results showed that destroying of ISS caused the loss-of-function of Wx to produce glutinous rice.However,deletions of a few bases near the ISS had little effect on the expression of Wx and the level of AC.7.Eight target sites in the promoter region of Wx were designed and four CRISPR constructs(PT1T2,PT3T4,PT5T6 and PT7T8),each for targeting two sites,were prepared.After transformation of TFB,three mutant lines(PT7T8-#4,#5 and#6)with deletions of the fragments containing the putative CAAT box between the T7 and T8 targets produced decreased AC to about 17%.Compared with the wild-type TFB,the expression levels of Wx in these mutant lines were significantly down-regulated.But in other mutants,the levels of AC were similar to that of TFB.The sizes of the deletion fragments in the promoter region were positive corrected with the expression level of Wx.8.The transcription factor genes OsBP-5 and OsEBP-89 that possibly regulate Wx expression were knocked out separately and simultaneously.The results showed that the knockout of OsBP-5 or/and OsEBP-89 had no significant effect on both the expression levels of Wx and subsequently AC in rice.Conclusion:This study focused on the development of a serial of genome editing tool systems based on the SpCas9 variants,and targeted editing different regions of the Wx gene to develop new rice germplasms with moderate or low AC.This study will provide the basis for the selection of genome editing systems and strategies in crop improvement.