Single-base editors,including cytosine base editors(CBEs)and adenine base editors(ABEs),facilitate accurate C·G to T·A and A·T to G·C,respectively,holding promise for the precise modeling and treat...Single-base editors,including cytosine base editors(CBEs)and adenine base editors(ABEs),facilitate accurate C·G to T·A and A·T to G·C,respectively,holding promise for the precise modeling and treatment of human hereditary disorders.Efficient base editing and expanded base conversion range have been achieved in human cells through base editors fusing with Rad51 DNA binding domain(Rad51DBD),such as hyA3A-BE4max.Here,we show that hyA3A-BE4max catalyzes C-to-T substitution in the zebrafish genome and extends editing positions(C_(12)-C_(16))proximal to the protospacer adjacent motif.We develop a codon-optimized counterpart zhyA3A-CBE5,which exhibits substantially high C-to-T conversion with 1.59-to 3.50-fold improvement compared with the original hyA3A-BE4max.With these tools,disease-relevant hereditary mutations can be more efficaciously generated in zebrafish.We introduce human genetic mutation rpl11^(Q42*)and abcc6a^(R1463C) by zhyA3A-CBE5 in zebrafish,mirroring Diamond-Blackfan anemia and Pseudoxanthoma Elasticum,respectively.Our study expands the base editing platform targeting the zebrafish genomic landscape and the application of single-base editors for disease modeling and gene function study.展开更多
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic small vessel disease caused by mutations in the NOTCH3 gene. However, the pathogenesis of CADASIL rem...Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic small vessel disease caused by mutations in the NOTCH3 gene. However, the pathogenesis of CADASIL remains unclear, and patients have limited treatment options. Here, we use human induced pluripotent stem cells (hiPSCs) generated from the peripheral blood mononuclear cells of a patient with CADASIL carrying a heterozygous NOTCH3 mutation (c.1261C>T, p.R421C) to develop a disease model. The correction efficiency of different adenine base editors (ABEs) is tested using the HEK293T-NOTCH3 reporter cell line. ABEmax is selected based on its higher efficiency and minimization of predicted off-target effects. Vascular smooth muscle cells (VSMCs) differentiated from CADASIL hiPSCs show NOTCH3 deposition and abnormal actin cytoskeleton structure, and the abnormalities are recovered in corrected hiPSC-derived VSMCs. Furthermore, CADASIL blood vessel organoids generated for in vivo modeling show altered expression of genes related to disease phenotypes, including the downregulation of cell adhesion, extracellular matrix organization, and vessel development. The dual adeno-associated virus (AAV) split-ABEmax system is applied to the genome editing of vascular organoids with an average editing efficiency of 8.82%. Collectively, we present potential genetic therapeutic strategies for patients with CADASIL using blood vessel organoids and the dual AAV split-ABEmax system.展开更多
The clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated protein(Cas) system has been widely used for genome editing. In this system, the cytosine base editor(CBE) and adenine base edit...The clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated protein(Cas) system has been widely used for genome editing. In this system, the cytosine base editor(CBE) and adenine base editor(ABE) allow generating precise and irreversible base mutations in a programmable manner and have been used in many different types of cells and organisms. However, their applications are limited by low editing efficiency at certain genomic target sites or at specific target cytosine(C) or adenine(A) residues. Using a strategy of combining optimized synergistic core components, we developed a new multiplex super-assembled ABE(sABE) in rice that showed higher base-editing efficiency than previously developed ABEs. We also designed a new type of nuclear localization signal(NLS) comprising a FLAG epitope tag with four copies of a codon-optimized NLS(F4NLS^(r2)) to generate another ABE named F4NLS-sABE. This new NLS increased editing efficiency or edited additional A at several target sites. A new multiplex super-assembled CBE(sCBE) and F4NLS^(r2) involved F4NLS-sCBE were also created using the same strategy. F4NLS-sCBE was proven to be much more efficient than sCBE in rice. These optimized base editors will serve as powerful genome-editing tools for basic research or molecular breeding in rice and will provide a reference for the development of superior editing tools for other plants or animals.展开更多
Clustered regularly interspaced short palindromic repeats(CRISPR)—CRISPR-associated protein(Cas)and base editors are fundamental tools in plant genome editing.Cas9 from Streptococcus pyogenes(SpCas9),recognizing an N...Clustered regularly interspaced short palindromic repeats(CRISPR)—CRISPR-associated protein(Cas)and base editors are fundamental tools in plant genome editing.Cas9 from Streptococcus pyogenes(SpCas9),recognizing an NGG protospacer adjacent motif(PAM),is a widely used nuclease for genome editing in living cells.Cas12a nucleases,targeting T-rich PAMs,have also been recently demonstrated in several plant species.Furthermore,multiple Cas9 and Cas12a engineered variants and orthologs,with different PAM recognition sites,editing efficiencies and fidelity,have been explored in plants.These RNA-guided sequence-specific nucleases(SSN)generate double-stranded breaks(DSBs)in DNA,which trigger non-homologous end-joining(NHEJ)repair or homology-directed repair(HDR),resulting in insertion and deletion(indel)mutations or precise gene replacement,respectively.Alternatively,genome editing can be achieved by base editors without introducing DSBs.So far,several base editors have been applied in plants to introduce C-to-T or A-to-G transitions,but they are still undergoing improvement in editing window size,targeting scope,off-target effects in DNA and RNA,product purity and overall activity.Here,we summarize recent progress on the application of Cas nucleases,engineered Cas variants and base editors in plants.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
The CRISPR/Cas9-mediated base editing technology can efficiently generate point mutations in the genome without introducing a double-strand break(DSB)or supplying a DNA donor template for homology-directed repair(HDR)...The CRISPR/Cas9-mediated base editing technology can efficiently generate point mutations in the genome without introducing a double-strand break(DSB)or supplying a DNA donor template for homology-directed repair(HDR).In this study,adenine base editors(ABEs)were used for rapid generation of precise point mutations in two distinct genes,OsWSL5,and OsZEBRA3(Z3),in both rice protoplasts and regenerated plants.The precisely engineered point mutations were stably inherited to subsequent generations.These single nucleotide alterations resulted in single amino acid changes and associated wsl5 and z3 phenotypes as evidenced by white stripe leaf and light green/dark green leaf pattern,respectively.Through selfing and genetic segregation,transgene-free,base edited wsl5 and z3 mutants were obtained in a short period of time.We noticed a novel mutation(V540A)in Z3 locus could also mimic the phenotype of Z3 mutation(S542P).Furthermore,we observed unexpected non-A/G or T/C mutations in the ABE editing window in a few of the edited plants.The ABE vectors and the method from this study could be used to simultaneously generate point mutations in multiple target genes in a single transformation and serve as a useful base editing tool for crop improvement as well as basic studies in plant biology.展开更多
Single-nucleotide variants account for about half of known pathogenic genetic variants in human.Genome editing strategies by reversing pathogenic point mutations with minimum side effects have great therapeutic potent...Single-nucleotide variants account for about half of known pathogenic genetic variants in human.Genome editing strategies by reversing pathogenic point mutations with minimum side effects have great therapeutic potential and are now being actively pursued.The emerge of precise and effcient genome editing strategies such as base editing and prime editing provide powerful tools for nucleotide conversion without inducing double-stranded DNA breaks(DSBs),which have shown great potential for curing genetic disorders.A diverse toolkit of base editors has been devel-oped to improve the editing effciency and accuracy in different context of application.Here,we summarized the evolving of base editors(BEs),their limitations and future perspective of base editing-based therapeutic strategies.展开更多
Mutations in the Rhodopsin(RHO)gene are the main cause of autosomal dominant retinitis pigmentosa(adRP),84%of which are pathogenic gain-of-function point mutations.Treatment strategies for adRP typically involve silen...Mutations in the Rhodopsin(RHO)gene are the main cause of autosomal dominant retinitis pigmentosa(adRP),84%of which are pathogenic gain-of-function point mutations.Treatment strategies for adRP typically involve silencing or ablating the pathogenic allele,while normal RHO protein replacement has no meaningful therapeutic benefit.Here,we present an adenine base editor(ABE)-mediated therapeutic approach for adRP caused by RHO point mutations in vivo.The correctable pathogenic mutations are screened and verified,including T17M,Q344ter,and P347L.Two adRP animal models are created carrying the class 1(Q344ter)and class 2(T17M)mutations,and dual AAV-delivered ABE can effectively repair both mutations in vivo.The early intervention of ABE8e efficiently corrects the Q344ter mutation that causes a severe form of adRP,delays photoreceptor death,and restores retinal function and visual behavior.These results suggest that ABE is a promising alternative to treat RHO mutation-associated adRP.Our work provides an effective spacer-mediated point mutation correction therapy for dominantly inherited ocular disorders.展开更多
Base editors(BEs)are a promising tool for precise base conversion in human cells and animals,while the adeno-associated virus(AAV)is the major vector for human gene therapy.However,the size of the DNA cassette require...Base editors(BEs)are a promising tool for precise base conversion in human cells and animals,while the adeno-associated virus(AAV)is the major vector for human gene therapy.However,the size of the DNA cassette required for BE expression exceeds the 4.7 kb packing capacity of the AAV vector,making dual-AAV approaches based on trans-splicing intein necessary.Even with this approach,current split DNA cassettes are still larger than the AAV packing limit,posing a challenge for cellular production of AAV.Moreover,some split strategies yield variable editing results and target coverage.To address these limitations,25 different split sets for BE4max and A3A-BE4max were tested at two target sites respectively,with splitting sites ranging from 493rd to 517th amino acids on the Cas9 peptide.Fortunately,the best Cas9 split site was identified between His511 and Ser512 and the arrangement of the AAV expression cassette was further manipulated to create evenly distributed CBE and ABE intein systems within 4.7 kb.These novel dual-AAV systems,designated 4.6AAV-CBE and 4.7AAV-ABE,were found to have base editing efficiencies similar to wild-type BEs,with a narrower editing window than the current 573 split system.Notably,4.6AAV-CBE yield a higher AAV production titer,up to 2.1-fold in AAV-N and 1.5-fold in AAV-C,compared to the split-573BE system,likely due to the reduction of DNA cassette size within the AAV packaging capacity.Moreover,after packaging and infecting cells with AAV-N and AAV-C at the same volume and number of cells,the multiplicities of infection(MOI)and editing efficiency of 4.6 AAV-CBE were both higher than those of the split-573BE system.This study present advanced dual-AAV systems for ABE and CBE delivery,establishing a basis for safe and efficient BE therapies.展开更多
APOBECs（apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like） are a family of cytidine deaminases that prefer single-stranded nucleic acids as substrates. Besides their physiological functions,APOBEC fam...APOBECs（apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like） are a family of cytidine deaminases that prefer single-stranded nucleic acids as substrates. Besides their physiological functions,APOBEC family members have been found to cause hypermutations of cancer genomes, which could be correlated with cancer development and poor prognosis. Recently, APOBEC family members have been combined with the versatile CRISPR/Cas9 system to perform targeted base editing or induce hypermutagenesis. This combination improved the CRISPR/Cas9-mediated gene editing at single-base precision, greatly enhancing its usefulness. Here, we review the physiological functions and structural characteristics of APOBEC family members and their roles as endogenous mutators that contribute to hypermutations during carcinogenesis. We also review the various iterations of the APOBEC-CRISPR/Cas9 gene-editing tools, pointing out their features and limitations as well as the possibilities for future developments.展开更多
The emerging pests and phytopathogens have reduced the crop yield and quality, which hasthreatened the global food security. Traditional breeding methods, molecular marker-based breedingapproaches and use of genetical...The emerging pests and phytopathogens have reduced the crop yield and quality, which hasthreatened the global food security. Traditional breeding methods, molecular marker-based breedingapproaches and use of genetically modified crops have played a crucial role in strengthening the foodsecurity worldwide. However, their usages in crop improvement have been highly limited due to multiplecaveats. Genome editing tools like transcriptional activator-like effector nucleases and clustered regularlyinterspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 (CRISPR/Cas9) haveeffectively overcome limitations of the conventional breeding methods and are being widely accepted forimprovement of crops. Among the genome editing tools, the CRISPR/Cas9 system has emerged as themost powerful tool of genome editing because of its efficiency, amicability, flexibility, low cost andadaptability. Accumulated evidences indicate that genome editing has great potential in improving thedisease resistance in crop plants. In this review, we offered a brief introduction to the mechanisms of differentgenome editing systems and then discussed recent developments in CRISPR/Cas9 system-based genomeediting towards enhancement of rice disease resistance by different strategies. This review also discussed thepossible applications of recently developed genome editing approaches like CRISPR/Cas12a (formerlyknown as Cpf1) and base editors for enhancement of rice disease resistance.展开更多
Recently developed CRISPR-mediated base editors,which enable the generation of num erous nucleotide changes in target genomic regions,have been widely adopted for gene correction and generation of crop germ plasms con...Recently developed CRISPR-mediated base editors,which enable the generation of num erous nucleotide changes in target genomic regions,have been widely adopted for gene correction and generation of crop germ plasms containing im portant gain-of-function genetic variations.How ever,to engineer target genes with unknown functional SNPs remains challenging.To address this issue,we present here abase-e diting-mediated gene evolution(BEMGE)m ethod,employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA(sgRNA)library tiling the full-length coding region,for developing novel rice germ plasm swith mutations in any endogenous gene.To this end,OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bom bardment-mediated transform ation.Four different types of amino acid substitutions in the evolved OsALS1,derived from two sites that have never been targeted by natural or human selection during rice dom estication,were identified,conferring varying levels of tolerance to the herbicide bispyribac-sodium.Furtherm ore,the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing w ith the corresponding base editor and sgRNA.Collectively,these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.展开更多
Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease- causing mutations in patients. However, problems such as mosaic...Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease- causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical applicaUon of such approaches. Recently, a base editor （BE） system built on cytidine （C） deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine （T） efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high- fidelity version of base editor 2 （HF2-BE2）, and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.展开更多
Exploiting novel endogenous glyphosate-tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here,through fusions of different effective cytosine and adenine deaminases wi...Exploiting novel endogenous glyphosate-tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here,through fusions of different effective cytosine and adenine deaminases with nCas9-NG, we engineered an effective surrogate two-component composite base editing system, STCBE-2, with improved C-to-T and A-to-G base editing efficiency and expanded the editing window. Furthermore,we targeted a rice endogenous OsEPSPS gene for artificial evolution through STCBE-2-mediated near-saturated mutagenesis. After hygromycin and glyphosate selection, we identified a novel OsEPSPS allele with an Asp-213-Asn(D213N)mutation(OsEPSPS-D213N) in the predicted glyphosate-binding domain, which conferred rice plants reliable glyphosate tolerance and had not been reported or applied in rice breeding. Collectively, we developed a novel dual base editor which will be valuable for artificial evolution of important genes in crops. And the novel glyphosate-tolerant rice germplasm generated in this study will benefit weeds management in rice paddy fields.展开更多
Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the convers...Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T(C>T) or A→G(A>G) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F0 are mosaic, and breeding of F1 heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing(GVBE) to produce point mutant F0 porcine embryos by editing the maternal alleles during the GV to MⅡ transition. Injection of cytosine base editor 3(BE3) mRNA and X-linked Dmdspecific guide RNAs into GVoocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MⅡ oocytes developed into blastocysts after parthenogenetic activation(PA) or in vitro fertilization(IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4%±2.1%. There 40%–78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only C>T mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five(5/19) F0 male embryos are homozygous and three of them were Dmd precise C>T mutation. Nine(9/19) female IVF embryos had two different alleles including a WT and a C>T mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F0 embryos with maternal point mutated alleles in a single step.展开更多
Engineering of a new type of plant base editor for simultaneous adenine transition and transversion within the editing window will greatly expand the scope and potential of base editing in directed evolution and crop ...Engineering of a new type of plant base editor for simultaneous adenine transition and transversion within the editing window will greatly expand the scope and potential of base editing in directed evolution and crop improvement.Here,we isolated a rice endogenous hypoxanthine excision protein,N-methylpurine DNA glycosylase(OsMPG),and engineered two plant A-to-K(K=G or T)base editors,rAKBE01 and rAKBE02,for simultaneous adenine transition and transversion base editing in rice by fusing OsMPG or its mutant mOsMPG to a plant adenine transition base editor,ABE8e.We further coupled either OsMPG or mOsMPG with a transactivation factor VP64 to generate rAKBE03 and rAKBE04,respectively.Testing these four rAKBEs,at five endogenous loci in rice protoplasts,indicated that rAKBE03 and rAKBE04 enabled higher levels of A-to-G base transitions when compared to ABE8e and ABE8e-VP64.Furthermore,whereas rAKBE01 only enabled A-to-C/T editing at one endogenous locus,in comparison with rAKBE02 and rAKBE03,rAKBE04 could significantly improve the A-to-C/T base transversion efficiencies by up to 6.57-and 1.75-fold in the rice protoplasts,respectively.Moreover,although no stable lines with A-to-C transversion were induced by rAKBE01 and rAKBE04,rAKBE04 could enable simultaneous A-to-G and A-to-T transition and transversion base editing,at all the five target loci,with the efficiencies of A-to-G transition and A-to-T transversion editing ranging from 70.97 to 92.31%and 1.67 to 4.84%in rice stable lines,respectively.Together,these rAKBEs enable different portfolios of editing products and,thus,now expands the potential of base editing in diverse application scenario for crop improvement.展开更多
CRISPR-Cas(Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated)has been extensively exploited as a genetic tool for genome editing.The RNA guided Cas nucleases generate DNA doublestrand break(D...CRISPR-Cas(Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated)has been extensively exploited as a genetic tool for genome editing.The RNA guided Cas nucleases generate DNA doublestrand break(DSB),triggering cellular repair systems mainly Non-homologous end-joining(NHEJ,imprecise repair)or Homology-directed repair(HDR,precise repair).However,DSB typically leads to unexpected DNA changes and lethality in some organisms.The establishment of bacteria and plants into major bio-production platforms require efficient and precise editing tools.Hence,in this review,we focus on the non-DSB and template-free genome editing,i.e.,base editing(BE)and prime editing(PE)in bacteria and plants.We first highlight the development of base and prime editors and summarize their studies in bacteria and plants.We then discuss current and future applications of BE/PE in synthetic biology,crop improvement,evolutionary engineering,and metabolic engineering.Lastly,we critically consider the challenges and prospects of BE/PE in PAM specificity,editing efficiency,off-targeting,sequence specification,and editing window.展开更多
CRISPR/Cas9 systems are an established tool in genome engineering.As double strand breaks caused by the standard Cas9-based knock-out techniques can be problematic in some organisms,new systems were developed that can...CRISPR/Cas9 systems are an established tool in genome engineering.As double strand breaks caused by the standard Cas9-based knock-out techniques can be problematic in some organisms,new systems were developed that can efficiently create knock-outs without causing double strand breaks to elegantly sidestep these issues.The recently published CRISPR-BEST base editor system for actinobacteria is built around a C to T or A to G base exchange.These base editing systems however require additional constraints to be considered for designing the sgRNAs.Here,we present an updated version of the interactive CRISPy-web single guide RNA design tool https://crispy.secondarymetabolites.org/that was built to support“classical”CRISPR and now also CRISPRBEST workflows.展开更多
Base editing,the targeted introduction of point mutations into cellular DNA,holds promise for improving genome-scale functional genome screening to single-nucleotide resolution.Current efforts in prokaryotes,however,r...Base editing,the targeted introduction of point mutations into cellular DNA,holds promise for improving genome-scale functional genome screening to single-nucleotide resolution.Current efforts in prokaryotes,however,remain confined to loss-of-function screens using the premature stop codons-mediated gene inactivation library,which falls far short of fully releasing the potential of base editors.Here,we developed a base editor-mediated functional single nucleotide variant screening pipeline in Escherichia coli.We constructed a library with 31,123 sgRNAs targeting 462 stress response-related genes in E.coli,and screened for adaptive mutations under isobutanol and furfural selective conditions.Guided by the screening results,we successfully identified several known and novel functional mutations.Our pipeline might be expanded to the optimization of other phenotypes or the strain engineering in other microorganisms.展开更多
CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops.The editing capabilities of base editors stri...CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops.The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes.Compared with the welldeveloped adenine base editors(ABEs),cytosine base editors(CBEs)and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice,significantly limiting their application.Here,we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice.We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID*D,and TadA-CDd outperformed TadA-E27R/N46L.A C-to-G base editor(CGBE)engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P.In addition,a dual base editor constructed with a single protein,TadDE,enabled simultaneous,highly efficient C-to-T and A-to-G editing in rice.Collectively,our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice,providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.展开更多
基金supported by grants from Ministry of Science and Technology of the People's Republic of China(2018YFA0801004 and 2018YFA0800103)the National Natural Science Foundation of China(NSFC31530044,NSFC31970780,NSFC82202056).
文摘Single-base editors,including cytosine base editors(CBEs)and adenine base editors(ABEs),facilitate accurate C·G to T·A and A·T to G·C,respectively,holding promise for the precise modeling and treatment of human hereditary disorders.Efficient base editing and expanded base conversion range have been achieved in human cells through base editors fusing with Rad51 DNA binding domain(Rad51DBD),such as hyA3A-BE4max.Here,we show that hyA3A-BE4max catalyzes C-to-T substitution in the zebrafish genome and extends editing positions(C_(12)-C_(16))proximal to the protospacer adjacent motif.We develop a codon-optimized counterpart zhyA3A-CBE5,which exhibits substantially high C-to-T conversion with 1.59-to 3.50-fold improvement compared with the original hyA3A-BE4max.With these tools,disease-relevant hereditary mutations can be more efficaciously generated in zebrafish.We introduce human genetic mutation rpl11^(Q42*)and abcc6a^(R1463C) by zhyA3A-CBE5 in zebrafish,mirroring Diamond-Blackfan anemia and Pseudoxanthoma Elasticum,respectively.Our study expands the base editing platform targeting the zebrafish genomic landscape and the application of single-base editors for disease modeling and gene function study.
基金funded by the National Natural Science Foundation of China(31971365)the Guangdong Basic and Applied Basic Research Foundation(2020B1515120090)the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program(2019BT02Y276).
文摘Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic small vessel disease caused by mutations in the NOTCH3 gene. However, the pathogenesis of CADASIL remains unclear, and patients have limited treatment options. Here, we use human induced pluripotent stem cells (hiPSCs) generated from the peripheral blood mononuclear cells of a patient with CADASIL carrying a heterozygous NOTCH3 mutation (c.1261C>T, p.R421C) to develop a disease model. The correction efficiency of different adenine base editors (ABEs) is tested using the HEK293T-NOTCH3 reporter cell line. ABEmax is selected based on its higher efficiency and minimization of predicted off-target effects. Vascular smooth muscle cells (VSMCs) differentiated from CADASIL hiPSCs show NOTCH3 deposition and abnormal actin cytoskeleton structure, and the abnormalities are recovered in corrected hiPSC-derived VSMCs. Furthermore, CADASIL blood vessel organoids generated for in vivo modeling show altered expression of genes related to disease phenotypes, including the downregulation of cell adhesion, extracellular matrix organization, and vessel development. The dual adeno-associated virus (AAV) split-ABEmax system is applied to the genome editing of vascular organoids with an average editing efficiency of 8.82%. Collectively, we present potential genetic therapeutic strategies for patients with CADASIL using blood vessel organoids and the dual AAV split-ABEmax system.
基金supported by the Beijing Scholars Program[BSP041]。
文摘The clustered regularly interspaced short palindromic repeats(CRISPR)–CRISPR-associated protein(Cas) system has been widely used for genome editing. In this system, the cytosine base editor(CBE) and adenine base editor(ABE) allow generating precise and irreversible base mutations in a programmable manner and have been used in many different types of cells and organisms. However, their applications are limited by low editing efficiency at certain genomic target sites or at specific target cytosine(C) or adenine(A) residues. Using a strategy of combining optimized synergistic core components, we developed a new multiplex super-assembled ABE(sABE) in rice that showed higher base-editing efficiency than previously developed ABEs. We also designed a new type of nuclear localization signal(NLS) comprising a FLAG epitope tag with four copies of a codon-optimized NLS(F4NLS^(r2)) to generate another ABE named F4NLS-sABE. This new NLS increased editing efficiency or edited additional A at several target sites. A new multiplex super-assembled CBE(sCBE) and F4NLS^(r2) involved F4NLS-sCBE were also created using the same strategy. F4NLS-sCBE was proven to be much more efficient than sCBE in rice. These optimized base editors will serve as powerful genome-editing tools for basic research or molecular breeding in rice and will provide a reference for the development of superior editing tools for other plants or animals.
基金Our plant genome editing research is supported by the National Science Foundation Plant Genome Research Program(IOS-1758745)USDA-NIFA Biotechnology Risk Assessment Research Program(2018-33522-28789)+1 种基金Foundation for Food and Agriculture Research(593603)Syngenta Biotechnology.
文摘Clustered regularly interspaced short palindromic repeats(CRISPR)—CRISPR-associated protein(Cas)and base editors are fundamental tools in plant genome editing.Cas9 from Streptococcus pyogenes(SpCas9),recognizing an NGG protospacer adjacent motif(PAM),is a widely used nuclease for genome editing in living cells.Cas12a nucleases,targeting T-rich PAMs,have also been recently demonstrated in several plant species.Furthermore,multiple Cas9 and Cas12a engineered variants and orthologs,with different PAM recognition sites,editing efficiencies and fidelity,have been explored in plants.These RNA-guided sequence-specific nucleases(SSN)generate double-stranded breaks(DSBs)in DNA,which trigger non-homologous end-joining(NHEJ)repair or homology-directed repair(HDR),resulting in insertion and deletion(indel)mutations or precise gene replacement,respectively.Alternatively,genome editing can be achieved by base editors without introducing DSBs.So far,several base editors have been applied in plants to introduce C-to-T or A-to-G transitions,but they are still undergoing improvement in editing window size,targeting scope,off-target effects in DNA and RNA,product purity and overall activity.Here,we summarize recent progress on the application of Cas nucleases,engineered Cas variants and base editors in plants.
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.
基金This work was supported by National Science Foundation Plant Genome Research Program Grant No.1740874the USDA National Institute of Food and Agriculture and Hatch Appropriations under Project#PEN04659 and Accession#1016432 to Yinong Yang.
文摘The CRISPR/Cas9-mediated base editing technology can efficiently generate point mutations in the genome without introducing a double-strand break(DSB)or supplying a DNA donor template for homology-directed repair(HDR).In this study,adenine base editors(ABEs)were used for rapid generation of precise point mutations in two distinct genes,OsWSL5,and OsZEBRA3(Z3),in both rice protoplasts and regenerated plants.The precisely engineered point mutations were stably inherited to subsequent generations.These single nucleotide alterations resulted in single amino acid changes and associated wsl5 and z3 phenotypes as evidenced by white stripe leaf and light green/dark green leaf pattern,respectively.Through selfing and genetic segregation,transgene-free,base edited wsl5 and z3 mutants were obtained in a short period of time.We noticed a novel mutation(V540A)in Z3 locus could also mimic the phenotype of Z3 mutation(S542P).Furthermore,we observed unexpected non-A/G or T/C mutations in the ABE editing window in a few of the edited plants.The ABE vectors and the method from this study could be used to simultaneously generate point mutations in multiple target genes in a single transformation and serve as a useful base editing tool for crop improvement as well as basic studies in plant biology.
基金supported by CAMS Innovation Fund for Medical Sciences(CIFMS,2022-I2M-1-024,2022-RC180-02 to Z.G.).
文摘Single-nucleotide variants account for about half of known pathogenic genetic variants in human.Genome editing strategies by reversing pathogenic point mutations with minimum side effects have great therapeutic potential and are now being actively pursued.The emerge of precise and effcient genome editing strategies such as base editing and prime editing provide powerful tools for nucleotide conversion without inducing double-stranded DNA breaks(DSBs),which have shown great potential for curing genetic disorders.A diverse toolkit of base editors has been devel-oped to improve the editing effciency and accuracy in different context of application.Here,we summarized the evolving of base editors(BEs),their limitations and future perspective of base editing-based therapeutic strategies.
基金funded by the National Key Research and Development Program of China(2023YFC2506100)the National Natural Science Foundation of China(31971365,32371509,32001063,and 82271688)+3 种基金the Local Innovative and Research Teams Project of Guangdong Pearl River Talents Program(2019BT02Y276)the Guangdong Basic and Applied Basic Research Foundation(2023A1515010176)the grant from MOE Key Laboratory of Gene Function and Regulation,the Guangzhou Science and Technology Planning Project(2023A04J1952)the Fundamental Research Funds for the Central Universities,Sun Yat-sen University(23ptpy59).
文摘Mutations in the Rhodopsin(RHO)gene are the main cause of autosomal dominant retinitis pigmentosa(adRP),84%of which are pathogenic gain-of-function point mutations.Treatment strategies for adRP typically involve silencing or ablating the pathogenic allele,while normal RHO protein replacement has no meaningful therapeutic benefit.Here,we present an adenine base editor(ABE)-mediated therapeutic approach for adRP caused by RHO point mutations in vivo.The correctable pathogenic mutations are screened and verified,including T17M,Q344ter,and P347L.Two adRP animal models are created carrying the class 1(Q344ter)and class 2(T17M)mutations,and dual AAV-delivered ABE can effectively repair both mutations in vivo.The early intervention of ABE8e efficiently corrects the Q344ter mutation that causes a severe form of adRP,delays photoreceptor death,and restores retinal function and visual behavior.These results suggest that ABE is a promising alternative to treat RHO mutation-associated adRP.Our work provides an effective spacer-mediated point mutation correction therapy for dominantly inherited ocular disorders.
基金financially supported by the Strategic Priority Research Program of the Chinese Academy of Sciences(XDC0110200)National Natural Science Foundation of China(31522002,32171449,3240120454)China Postdoctoral Science Foundation(2024M763495).
文摘Base editors(BEs)are a promising tool for precise base conversion in human cells and animals,while the adeno-associated virus(AAV)is the major vector for human gene therapy.However,the size of the DNA cassette required for BE expression exceeds the 4.7 kb packing capacity of the AAV vector,making dual-AAV approaches based on trans-splicing intein necessary.Even with this approach,current split DNA cassettes are still larger than the AAV packing limit,posing a challenge for cellular production of AAV.Moreover,some split strategies yield variable editing results and target coverage.To address these limitations,25 different split sets for BE4max and A3A-BE4max were tested at two target sites respectively,with splitting sites ranging from 493rd to 517th amino acids on the Cas9 peptide.Fortunately,the best Cas9 split site was identified between His511 and Ser512 and the arrangement of the AAV expression cassette was further manipulated to create evenly distributed CBE and ABE intein systems within 4.7 kb.These novel dual-AAV systems,designated 4.6AAV-CBE and 4.7AAV-ABE,were found to have base editing efficiencies similar to wild-type BEs,with a narrower editing window than the current 573 split system.Notably,4.6AAV-CBE yield a higher AAV production titer,up to 2.1-fold in AAV-N and 1.5-fold in AAV-C,compared to the split-573BE system,likely due to the reduction of DNA cassette size within the AAV packaging capacity.Moreover,after packaging and infecting cells with AAV-N and AAV-C at the same volume and number of cells,the multiplicities of infection(MOI)and editing efficiency of 4.6 AAV-CBE were both higher than those of the split-573BE system.This study present advanced dual-AAV systems for ABE and CBE delivery,establishing a basis for safe and efficient BE therapies.
基金supported by grants from the National Natural Science Foundation of China (Nos. 31600619 and 31600654)Shanghai Municipal Science and Technology Commission (Nos. 16PJ1407000 and 16PJ1407500)
文摘APOBECs（apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like） are a family of cytidine deaminases that prefer single-stranded nucleic acids as substrates. Besides their physiological functions,APOBEC family members have been found to cause hypermutations of cancer genomes, which could be correlated with cancer development and poor prognosis. Recently, APOBEC family members have been combined with the versatile CRISPR/Cas9 system to perform targeted base editing or induce hypermutagenesis. This combination improved the CRISPR/Cas9-mediated gene editing at single-base precision, greatly enhancing its usefulness. Here, we review the physiological functions and structural characteristics of APOBEC family members and their roles as endogenous mutators that contribute to hypermutations during carcinogenesis. We also review the various iterations of the APOBEC-CRISPR/Cas9 gene-editing tools, pointing out their features and limitations as well as the possibilities for future developments.
基金the China Priority Program-Breeding of Seven Major Crops(Grant No.2017YFD01100100)the Innovation Program of Chinese Academy of Agricultural Sciences(Grant No.01-ICS)the Talented Young Scientist Program of China(Grant No.India-17-01).
文摘The emerging pests and phytopathogens have reduced the crop yield and quality, which hasthreatened the global food security. Traditional breeding methods, molecular marker-based breedingapproaches and use of genetically modified crops have played a crucial role in strengthening the foodsecurity worldwide. However, their usages in crop improvement have been highly limited due to multiplecaveats. Genome editing tools like transcriptional activator-like effector nucleases and clustered regularlyinterspaced short palindromic repeats (CRISPR)-associated endonuclease Cas9 (CRISPR/Cas9) haveeffectively overcome limitations of the conventional breeding methods and are being widely accepted forimprovement of crops. Among the genome editing tools, the CRISPR/Cas9 system has emerged as themost powerful tool of genome editing because of its efficiency, amicability, flexibility, low cost andadaptability. Accumulated evidences indicate that genome editing has great potential in improving thedisease resistance in crop plants. In this review, we offered a brief introduction to the mechanisms of differentgenome editing systems and then discussed recent developments in CRISPR/Cas9 system-based genomeediting towards enhancement of rice disease resistance by different strategies. This review also discussed thepossible applications of recently developed genome editing approaches like CRISPR/Cas12a (formerlyknown as Cpf1) and base editors for enhancement of rice disease resistance.
基金This work was supported by grants from the National Natural Science Foundation of China(31871948)the Fundamental Research Funds,and the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences to H.Z.a grant from the Fundamental Research Funds for the Central Universities to S.L.
文摘Recently developed CRISPR-mediated base editors,which enable the generation of num erous nucleotide changes in target genomic regions,have been widely adopted for gene correction and generation of crop germ plasms containing im portant gain-of-function genetic variations.How ever,to engineer target genes with unknown functional SNPs remains challenging.To address this issue,we present here abase-e diting-mediated gene evolution(BEMGE)m ethod,employing both Cas9n-based cytosine and adenine base editors as well as a single-guide RNA(sgRNA)library tiling the full-length coding region,for developing novel rice germ plasm swith mutations in any endogenous gene.To this end,OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bom bardment-mediated transform ation.Four different types of amino acid substitutions in the evolved OsALS1,derived from two sites that have never been targeted by natural or human selection during rice dom estication,were identified,conferring varying levels of tolerance to the herbicide bispyribac-sodium.Furtherm ore,the P171F substitution identified in a strong OsALS1 allele was quickly introduced into the commercial rice cultivar Nangeng 46 through precise base editing w ith the corresponding base editor and sgRNA.Collectively,these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.
基金This work was supported by the National Natural Science Foundation of China （Grant Nos. 91640119, 31601196, 81330055, 31371508, and 31671540）, the Natural Science Foundation of Guangdong Province （2016A030310206 and 2014A030312011）, the Science and Technology Planning Project of Guangdong Province （2015B020228002 and 2015A020212005）, the Guangzhou Science and Technology Project （201605030012 and 201707010085）, and the Fundamental Research Funds for the Central Universities （161gzd13 and 161gpy31）. We would also like to acknowledge the support of CA211653, CPRIT RP160462, the Welch Foundation Q-1673, and the C-BASS Shared Resource at the Dan L. Duncan Cancer Center （DLDCC） of Baylor College of Medicine （P30CA125123）.
文摘Targeted point mutagenesis through homologous recombination has been widely used in genetic studies and holds considerable promise for repairing disease- causing mutations in patients. However, problems such as mosaicism and low mutagenesis efficiency continue to pose challenges to clinical applicaUon of such approaches. Recently, a base editor （BE） system built on cytidine （C） deaminase and CRISPR/Cas9 technology was developed as an alternative method for targeted point mutagenesis in plant, yeast, and human cells. Base editors convert C in the deamination window to thymidine （T） efficiently, however, it remains unclear whether targeted base editing in mouse embryos is feasible. In this report, we generated a modified high- fidelity version of base editor 2 （HF2-BE2）, and investigated its base editing efficacy in mouse embryos. We found that HF2-BE2 could convert C to T efficiently, with up to 100% biallelic mutation efficiency in mouse embryos. Unlike BE3, HF2-BE2 could convert C to T on both the target and non-target strand, expanding the editing scope of base editors. Surprisingly, we found HF2-BE2 could also deaminate C that was proximal to the gRNA-binding region. Taken together, our work demonstrates the feasibility of generating point mutations in mouse by base editing, and underscores the need to carefully optimize base editing systems in order to eliminate proximal-site deamination.
基金partly funded by the National Natural Science Foundation of China (32188102 to LX)Hainan Yazhou Bay Seed Lab (B21HJ0215 to LX)+2 种基金the Central Public-Interest Scientific Institution-Based Research Fund (S2023ZD03 to LX)Key Laboratory of Gene Editing Technologies (Hainan), ChinaNational Engineering Research Center of Crop Molecular Breeding。
文摘Exploiting novel endogenous glyphosate-tolerant alleles is highly desirable and has promising potential for weed control in rice breeding. Here,through fusions of different effective cytosine and adenine deaminases with nCas9-NG, we engineered an effective surrogate two-component composite base editing system, STCBE-2, with improved C-to-T and A-to-G base editing efficiency and expanded the editing window. Furthermore,we targeted a rice endogenous OsEPSPS gene for artificial evolution through STCBE-2-mediated near-saturated mutagenesis. After hygromycin and glyphosate selection, we identified a novel OsEPSPS allele with an Asp-213-Asn(D213N)mutation(OsEPSPS-D213N) in the predicted glyphosate-binding domain, which conferred rice plants reliable glyphosate tolerance and had not been reported or applied in rice breeding. Collectively, we developed a novel dual base editor which will be valuable for artificial evolution of important genes in crops. And the novel glyphosate-tolerant rice germplasm generated in this study will benefit weeds management in rice paddy fields.
基金supported by the National Key R&D Program of China (2017YFC1001901 and 2017YFA0102801)the National Natural Science Foundation (31671540)+3 种基金the National Transgenic Major Program (2016ZX08006003-006)the Natural Science Foundation of Guangdong Province (2015A020212005 and 2014A030312011)the Key R&D Program of Guangdong Province (2018B020203003)the Guangzhou Science and Technology Project (201803010020)。
文摘Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T(C>T) or A→G(A>G) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F0 are mosaic, and breeding of F1 heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing(GVBE) to produce point mutant F0 porcine embryos by editing the maternal alleles during the GV to MⅡ transition. Injection of cytosine base editor 3(BE3) mRNA and X-linked Dmdspecific guide RNAs into GVoocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MⅡ oocytes developed into blastocysts after parthenogenetic activation(PA) or in vitro fertilization(IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4%±2.1%. There 40%–78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only C>T mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five(5/19) F0 male embryos are homozygous and three of them were Dmd precise C>T mutation. Nine(9/19) female IVF embryos had two different alleles including a WT and a C>T mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F0 embryos with maternal point mutated alleles in a single step.
基金funded by the National Natural Science Foundation of China(Grant No.32188102 to L.X),Hainan Yazhou Bay Seed Lab(Grant No.B23CJ0208 to L.X)the Central Public-interest Scientific Institution Basal Research Fund(Grant No.ZDXM2308 to L.X)National Engineering Research Centre of Crop Molecular Breeding.
文摘Engineering of a new type of plant base editor for simultaneous adenine transition and transversion within the editing window will greatly expand the scope and potential of base editing in directed evolution and crop improvement.Here,we isolated a rice endogenous hypoxanthine excision protein,N-methylpurine DNA glycosylase(OsMPG),and engineered two plant A-to-K(K=G or T)base editors,rAKBE01 and rAKBE02,for simultaneous adenine transition and transversion base editing in rice by fusing OsMPG or its mutant mOsMPG to a plant adenine transition base editor,ABE8e.We further coupled either OsMPG or mOsMPG with a transactivation factor VP64 to generate rAKBE03 and rAKBE04,respectively.Testing these four rAKBEs,at five endogenous loci in rice protoplasts,indicated that rAKBE03 and rAKBE04 enabled higher levels of A-to-G base transitions when compared to ABE8e and ABE8e-VP64.Furthermore,whereas rAKBE01 only enabled A-to-C/T editing at one endogenous locus,in comparison with rAKBE02 and rAKBE03,rAKBE04 could significantly improve the A-to-C/T base transversion efficiencies by up to 6.57-and 1.75-fold in the rice protoplasts,respectively.Moreover,although no stable lines with A-to-C transversion were induced by rAKBE01 and rAKBE04,rAKBE04 could enable simultaneous A-to-G and A-to-T transition and transversion base editing,at all the five target loci,with the efficiencies of A-to-G transition and A-to-T transversion editing ranging from 70.97 to 92.31%and 1.67 to 4.84%in rice stable lines,respectively.Together,these rAKBEs enable different portfolios of editing products and,thus,now expands the potential of base editing in diverse application scenario for crop improvement.
基金This work was sponsored by National Key R&D Program of China(2018YFA0901200)Science and Technology Commission of Shanghai Municipality(18JC1413600)National Natural Science Foundation of China(31870071).
文摘CRISPR-Cas(Clustered Regularly Interspaced Short Palindromic Repeats-CRISPR associated)has been extensively exploited as a genetic tool for genome editing.The RNA guided Cas nucleases generate DNA doublestrand break(DSB),triggering cellular repair systems mainly Non-homologous end-joining(NHEJ,imprecise repair)or Homology-directed repair(HDR,precise repair).However,DSB typically leads to unexpected DNA changes and lethality in some organisms.The establishment of bacteria and plants into major bio-production platforms require efficient and precise editing tools.Hence,in this review,we focus on the non-DSB and template-free genome editing,i.e.,base editing(BE)and prime editing(PE)in bacteria and plants.We first highlight the development of base and prime editors and summarize their studies in bacteria and plants.We then discuss current and future applications of BE/PE in synthetic biology,crop improvement,evolutionary engineering,and metabolic engineering.Lastly,we critically consider the challenges and prospects of BE/PE in PAM specificity,editing efficiency,off-targeting,sequence specification,and editing window.
基金This work was supported by grants from the Novo Nordisk Foundation[NNF10CC1016517,NNF15OC0016226,NNF16OC0021746].
文摘CRISPR/Cas9 systems are an established tool in genome engineering.As double strand breaks caused by the standard Cas9-based knock-out techniques can be problematic in some organisms,new systems were developed that can efficiently create knock-outs without causing double strand breaks to elegantly sidestep these issues.The recently published CRISPR-BEST base editor system for actinobacteria is built around a C to T or A to G base exchange.These base editing systems however require additional constraints to be considered for designing the sgRNAs.Here,we present an updated version of the interactive CRISPy-web single guide RNA design tool https://crispy.secondarymetabolites.org/that was built to support“classical”CRISPR and now also CRISPRBEST workflows.
基金supported by the National Key Research and Development Program of China (2018YFA0901500)the National Natural Science Foundation of China (U2032210)。
文摘Base editing,the targeted introduction of point mutations into cellular DNA,holds promise for improving genome-scale functional genome screening to single-nucleotide resolution.Current efforts in prokaryotes,however,remain confined to loss-of-function screens using the premature stop codons-mediated gene inactivation library,which falls far short of fully releasing the potential of base editors.Here,we developed a base editor-mediated functional single nucleotide variant screening pipeline in Escherichia coli.We constructed a library with 31,123 sgRNAs targeting 462 stress response-related genes in E.coli,and screened for adaptive mutations under isobutanol and furfural selective conditions.Guided by the screening results,we successfully identified several known and novel functional mutations.Our pipeline might be expanded to the optimization of other phenotypes or the strain engineering in other microorganisms.
基金supported by the STI 2030-Major Projects(2023ZD04074)the National Key Research and Development Program of China(2023YFD1202900)+2 种基金the Nanfan special project of the Chinese Academy of Agricultural Sciences(YBXM2313)the Hainan Seed Industry Laboratory(project of B23CJ0208)the Agricultural Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences.
文摘CRISPR-mediated base editors have been widely used to correct defective alleles and create novel alleles by artificial evolution for the rapid genetic improvement of crops.The editing capabilities of base editors strictly rely on the performance of various nucleotide modification enzymes.Compared with the welldeveloped adenine base editors(ABEs),cytosine base editors(CBEs)and dual base editors suffer from unstable editing efficiency and patterns at different genomic loci in rice,significantly limiting their application.Here,we comprehensively examined the base editing activities of multiple evolved TadA8e variants in rice.We found that both TadA-CDd and TadA-E27R/N46L achieved more robust C-to-T editing than previously reported hyperactive hAID*D,and TadA-CDd outperformed TadA-E27R/N46L.A C-to-G base editor(CGBE)engineered with TadA-CDd and OsUNG performed highly efficient C-to-G editing in rice compared with that of TadA-N46P.In addition,a dual base editor constructed with a single protein,TadDE,enabled simultaneous,highly efficient C-to-T and A-to-G editing in rice.Collectively,our results demonstrate that TadA8e derivatives improve both CBEs and dual base editors in rice,providing a powerful way to induce diverse nucleotide substitutions for plant genome editing.
