Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-mediated genome editing can efficiently produce gene-knockout mutants.On the other hand,CRISPR/Cas-derived base edito...Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-mediated genome editing can efficiently produce gene-knockout mutants.On the other hand,CRISPR/Cas-derived base editors offer the ability to induce precise nucleotide substitutions(Komor et al.,2016).Cytidine base editors(CBEs)consist of a cytidine deaminase fused with a Cas9-nickase variant(Cas9n,with a D10A substitu-tion)and can achieve site-specific C-to-T substitution.Similarly,adenine base editors use an adenine deaminase forA-to-G substi-tution.These systems have been used in various organisms(Mishra et al.,2019).However,the Cas9 complex requires target sites containing NGG protospacer adjacent motifs(PAMs),thus restricting selection of potential targets.A number of CBEs have been developed using Cas9 variants(mostly Cas9n),cytidine deaminases(such as rAPOBEC1 and PmCDA1),and uracil glycosylase inhibitor(UGI)domains.These CBEs of the first generation(BE1,rAPOBEC1-dCas9),second generation(BE2,rAPOBEC1-dCas9-UGI),and third generation(BE3,rAPOBEC1-Cas9n-UGI)have moderate editing efficiencies in mammalians(Komor etal.,2016).展开更多
Bacterial blight(BB),caused by Xanthomonas oryzae pathovar oryzae(Xoo),poses a significant threat to rice production,particularly in Asia and West Africa.Breeding resistance against BB in elite rice varieties is cruci...Bacterial blight(BB),caused by Xanthomonas oryzae pathovar oryzae(Xoo),poses a significant threat to rice production,particularly in Asia and West Africa.Breeding resistance against BB in elite rice varieties is crucial to advancing rice breeding program and supporting smallholder farmers.Transcription Activator-Like effectors(TALes)are key virulence factors in Xoo,with some targeting the susceptibility(S)genes such as the sugar transporter SWEET genes in rice.Among these,SWEET14 is an important S gene,with its promoter bound by the TALe TalC which exists across all sequenced African Xoo isolates.In the present study,we utilized CRISPR/Cas9-based cytidine and adenine base editors to alter the effector binding element(EBE)of TalC in the promoter of SWEET14 in rice cultivars Kitaake,IR24,and Zhonghua 11.Mutations with C to T changes in EBE led to reduced SWEET14 induction by TalC-containing Xoo strains,resulting in resistance to African Xoo isolates reliant on TalC for virulence.Conversely,A to G changes retained SWEET14 inducibility and susceptibility to Xoo in edited lines.Importantly,no off-target mutations were detected at predicted sites,and the edited lines exhibited no obvious defects in major agronomic traits in Kitaake.These results underscore the effectiveness of base editing systems for both molecular biology research and crop improvement endeavors.展开更多
基金grants from the Major Program of Guangdong Basic and Applied Research(2019B030302006)the National Natural Science Foundation of China(31921004+1 种基金31971915)the Guangdong special support program of Young Top-Notch Talent in Science and Technology Innovation(2019TQ05N147).
文摘Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)-mediated genome editing can efficiently produce gene-knockout mutants.On the other hand,CRISPR/Cas-derived base editors offer the ability to induce precise nucleotide substitutions(Komor et al.,2016).Cytidine base editors(CBEs)consist of a cytidine deaminase fused with a Cas9-nickase variant(Cas9n,with a D10A substitu-tion)and can achieve site-specific C-to-T substitution.Similarly,adenine base editors use an adenine deaminase forA-to-G substi-tution.These systems have been used in various organisms(Mishra et al.,2019).However,the Cas9 complex requires target sites containing NGG protospacer adjacent motifs(PAMs),thus restricting selection of potential targets.A number of CBEs have been developed using Cas9 variants(mostly Cas9n),cytidine deaminases(such as rAPOBEC1 and PmCDA1),and uracil glycosylase inhibitor(UGI)domains.These CBEs of the first generation(BE1,rAPOBEC1-dCas9),second generation(BE2,rAPOBEC1-dCas9-UGI),and third generation(BE3,rAPOBEC1-Cas9n-UGI)have moderate editing efficiencies in mammalians(Komor etal.,2016).
基金supported by a sub-award to the University of Missouri from the Heinrich Heine University of Dusseldorf funded by the Bill&Melinda Gates Foundation(OPP1155704)(Bing Yang)and the China Scholar Council(Chenhao Li,as a joint Ph.D.student).
文摘Bacterial blight(BB),caused by Xanthomonas oryzae pathovar oryzae(Xoo),poses a significant threat to rice production,particularly in Asia and West Africa.Breeding resistance against BB in elite rice varieties is crucial to advancing rice breeding program and supporting smallholder farmers.Transcription Activator-Like effectors(TALes)are key virulence factors in Xoo,with some targeting the susceptibility(S)genes such as the sugar transporter SWEET genes in rice.Among these,SWEET14 is an important S gene,with its promoter bound by the TALe TalC which exists across all sequenced African Xoo isolates.In the present study,we utilized CRISPR/Cas9-based cytidine and adenine base editors to alter the effector binding element(EBE)of TalC in the promoter of SWEET14 in rice cultivars Kitaake,IR24,and Zhonghua 11.Mutations with C to T changes in EBE led to reduced SWEET14 induction by TalC-containing Xoo strains,resulting in resistance to African Xoo isolates reliant on TalC for virulence.Conversely,A to G changes retained SWEET14 inducibility and susceptibility to Xoo in edited lines.Importantly,no off-target mutations were detected at predicted sites,and the edited lines exhibited no obvious defects in major agronomic traits in Kitaake.These results underscore the effectiveness of base editing systems for both molecular biology research and crop improvement endeavors.
