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Plant base editing and prime editing:The current status and future perspectives

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Journal of Integrative Plant Biology 2023年第2期65卷 444-467页

作者： Jingying Li Chen Zhang Yubing He Shaoya Li Lei Yan Yucai Li Ziwei Zhu Lanqin Xia Institute of Crop Sciences(ICS) Chinese Academy of Agricultural Sciences(CAAS)Beijing 100081China National Nanfan Research Institute(Sanya) Chinese Academy of Agricultural Sciences/Hainan Yazhou Bay Seed LaboratorySanya 572024China

Precise replacement of an allele with an elite allele controlling an important agronomic trait in a predefined manner by gene editing technologies is highly desirable in crop improvement.base editing and prime editing are two newly developed precision gene editing systems which can introduce the substitution of a single base and install the desired short indels to the target loci in the absence of double-strand breaks and donor repair templates,respectively.Since their discoveries,various strategies have been attempted to optimize both base editor(BE)and prime editor(PE)in order to improve the precise editing efficacy,specificity,and expand the targeting scopes.Here,we summarize the latest development of various BEs and PEs,as well as their applications in plants.based on these progresses,we recommend the appropriate BEs and PEs for both basic plant research and crop improvement.Moreover,we propose the perspectives for further optimization of these two editors.We envision that both BEs and PEs will become the routine and customized precise gene editing tools for both plant biological research and crop improvement in the near future.

关键词： base editing CRISPR/Cas plants precision genome editing prime editing

Highly efficient multiplex base editing:One-shot deactivation of eight genes in Shewanella oneidensis MR-1

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Synthetic and Systems Biotechnology 2023年第1期8卷 1-10页

作者： Yaru Chen Meijie Cheng Yan Li Lin Wang Lixia Fang Yingxiu Cao Hao Song Frontier Science Center for Synthetic Biology and Key Laboratory of Systems Bioengineering(Ministry of Education) School of Chemical Engineering and TechnologyTianjin UniversityTianjin300072China Key Laboratory of Systems Bioengineering(Ministry of Education) Tianjin UniversityTianjin300072China

Obtaining electroactive microbes capable of efficient extracellular electron transfer is a large undertaking for the scalability of bio-electrochemical systems.Inevitably,researchers need to pursue the co-modification of multiple genes rather than expecting that modification of a single gene would make a significant contribution to improving extracellular electron transfer rates.base editing has enabled highly-efficient gene deactivation in model electroactive microbe Shewanella oneidensis MR-1.Since multiplexed application of base editing is still limited by its low throughput procedure,we thus here develop a rapid and efficient multiplex base editing system in S.oneidensis.Four approaches to express multiple gRNAs were assessed firstly,and transcription of each gRNA cassette into a monocistronic unit was validated as a more favorable option than transcription of multiple gRNAs into a polycistronic cluster.Then,a smart scheme was designed to deliver one-pot assembly of multiple gRNAs.3,5,and 8 genes were deactivated using this system with editing efficiency of 83.3%,100%and 12.5%,respectively.To offer some nonrepetitive components as alternatives genetic parts of sgRNA cassette,different promoters,handles,and terminators were screened.This multiplex base editing tool was finally adopted to simultaneously deactivate eight genes that were identified as significantly downregulated targets in transcriptome analysis of riboflavin-overproducing strain and control strain.The maximum power density of the multiplex engineered strain HRF(8BE)in microbial fuel cells was 1108.1 mW/m2,which was 21.67 times higher than that of the wild-type strain.This highly efficient multiplexed base editing tool elevates our ability of genome manipulation and combinatorial engineering in Shewanella,and may provide valuable insights in fundamental and applied research of extracellular electron transfer.

关键词： Multiplex gene editing Electroactive microorganisms base editing Multiplexed engineering CRISPR Extracellular electron transfer

Systematic Exploration of Optimized base editing gRNA Design and Pleiotropic Effects with BExplorer

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Genomics, Proteomics & Bioinformatics 2023年第6期21卷 1237-1245页

作者： Gongchen Zhang Chenyu Zhu Xiaohan Chen Jifang Yan Dongyu Xue Zixuan Wei Guohui Chuai Qi Liu Translational Medical Center for Stem Cell Therapy and Institute for Regenerative Medicine Shanghai East HospitalBioinformatics DepartmentSchool of Life Sciences and TechnologyTongji UniversityShanghai 200092China

base editing technology is being increasingly applied in genome engineering,but the current strategy for designing guide RNAs(gRNAs)relies substantially on empirical experience rather than a dependable and efficient in silico design.Furthermore,the pleiotropic effect of base editing on disease treatment remains unexplored,which prevents its further clinical usage.Here,we presented BExplorer,an integrated and comprehensive computational pipeline to optimize the design of gRNAs for 26 existing types of base editors in silico.Using BExplorer,we described its results for two types of mainstream base editors,BE3 and ABE7.10,and evaluated the pleiotropic effects of the corresponding base editing loci.BExplorer revealed 524 and 900 editable pathogenic single nucleotide polymorphism(SNP)loci in the human genome together with the selected optimized gRNAs for BE3 and ABE7.10,respectively.In addition,the impact of 707 edited pathogenic SNP loci following base editing on 131 diseases was systematically explored by revealing their pleiotropic effects,indicating that base editing should be carefully utilized given the potential pleiotropic effects.Collectively,the systematic exploration of optimized base editing gRNA design and the corresponding pleiotropic effects with BExplorer provides a computational basis for applying base editing in disease treatment.

关键词： Gene editing base editing Pleiotropy CRISPR/Cas9 gRNAdesign

base editing in pigs for precision breeding

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Frontiers of Agricultural Science and Engineering 2020年第2期7卷 161-170页

作者： Ruigao SONG Yu WANG Yanfang WANG Jianguo ZHAO State Key Laboratory of Stem Cell and Reproductive Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China Medical School University of Chinese Academy of SciencesBeijing 100049China State Key Laboratory'of Animal Nutrition Institute of Animal ScienceChinese Academy of Agricultural SciencesBeijing 100081China

Pigs are one of the most important domesticated animals and have great value in agriculture and biomedicine.Single nucleotide polymorphisms(SNPs)are a dominant type of genetic variation among individual pigs and contribute to the formation of traits.Precision single base substitution provides a strategy for accurate genetic improvement in pig production with the characterization of functional SNPs and genetic variants in pigs.base editing has recently been developed as the latest gene-editing tool that can directly make changes in single nucleotides without introducing double-stranded DNA breaks(DSBs),providing a promising solution for precise genetic modification in large animals.This review summarizes gene-editing developments and highlights recent genetic dissection related to SNPs in major economic traits which may have the potential to be modified using SNP-editing applications.In addition,limitations and future directions of base editing in pig breeding are discussed.

关键词： base editing genetic improvement pigs single nucleotide polymorphisms

Generating broad-spectrum tolerance to ALS-inhibiting herbicides in rice by base editing

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Science China(Life Sciences) 2021年第10期64卷 1624-1633页

作者： Rui Zhang Sha Chen Xiangbing Meng Zhuangzhuang Chai Delin Wang Yuge Yuan Kunling Chen Linjian Jiang Jiayang Li Caixia Gao State Key Laboratory of Plant Cell and Chromosome Engineering Center for Genome EditingInstitute of Genetics and Developmental BiologyInnovation Academy for Seed DesignChinese Academy of SciencesBeijing 100101China College of Advanced Agricultural Sciences University of Chinese Academy of SciencesBeijing 100049China State Key Laboratory of Plant Genomics Institute of Genetics and Developmental BiologyInnovation Academy for Seed DesignChinese Academy of SciencesBeijing 100101China Key Lab of Pest Monitoring and Green Management MOA/Department of Plant PathologyCollege of Plant ProtectionChina Agricultural UniversityBeijing 100193China

Herbicide-tolerant rice varieties generated by genome editing are highly desirable for weed control.We have used a cytosine base editor to create a series of missense mutations in the P171 and/or G628 codons of the acetolactate synthase(ALS)gene to confer herbicide tolerance in rice.The four different missense mutations in the P171 codon,P171S,P171A,P171Yand P171F,exhibited different patterns of tolerance towards five representative herbicides from five chemical families of ALS inhibitors.For example,P171S and P171A had lower levels of tolerance than P171Y and P171F to bispyribac but not to the other herbicides.Interestingly,a novel triple mutant(P171F/G628E/G629S)had the highest tolerance to all five tested herbicides.Field trials showed that both P171F and P171F/G628E/G629S could potentially be used with nicosulfuron.Our work illustrates an effective way of using base editing to generate herbicide tolerance in elite rice varieties.

关键词： base editing herbicide tolerance rice acetolactate synthase(ALS)

PAM-Expanded Streptococcus thermophilus Cas9 C-to-T and C-to-G base Editors for Programmable base editing in Mycobacteria

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Engineering 2022年第8期8卷 67-77页

作者： Hongyuan Zhang Yifei Zhang Wei-Xiao Wang Weizhong Chen Xia Zhang Xingxu Huang Wei Chen Quanjiang Ji School of Physical Science and Technology ShanghaiTech UniversityShanghai 201210China University of Chinese Academy of Sciences Beijing 100049China Clinical Research Center the Second Hospital of NanjingNanjing University of Chinese MedicineNanjing 210003China Department of Tuberculosis the Second Hospital of NanjingNanjing University of Chinese MedicineNanjing 210003China Gene editing Center School of Life Science and TechnologyShanghaiTech UniversityShanghai 201210China Guangzhou Laboratory Guangzhou 510120China

New therapeutic strategies for the rapid and effective treatment of drug-resistant tuberculosis are highly desirable,and their development can be drastically accelerated by facile genetic manipulation methods in Mycobacterium tuberculosis(M.tuberculosis).Clustered regularly interspaced short palindromic repeat(CRISPR)base editors allow for rapid,robust,and programmed single-base substitutions and gene inactivation,yet no such systems are currently available in M.tuberculosis.By screening distinct CRISPR base editors,we discovered that only the unusual Streptococcus thermophilus CRISPR associated protein 9(St1Cas9)cytosine base editor(CBE)-but not the widely used Streptococcus pyogenes Cas9(SpCas9)or Lachnospiraceae bacterium Cpf1(LbCpf1)CBEs-is active in mycobacteria.Despite the notable C-to-T conversions,a high proportion of undesired byproducts exists with St1Cas9 CBE.We therefore engineered St1Cas9 CBE by means of uracil DNA glycosylase inhibitor(UGI)or uracil DNA glycosylase(UNG)fusion,yielding two new base editors(CTBE and CGBE)capable of C-to-T or C-to-G conversions with dramatically enhanced editing product purity and multiplexed editing capacity in Mycobacterium smegmatis(M.smegmatis).Because wild-type St1Cas9 recognizes a relatively strict protospacer adjacent motif(PAM)sequence for DNA targeting,we engineered a PAM-expanded St1Cas9 variant by means of structureguided protein engineering for the base editors,substantially broadening the targeting scope.We first developed and characterized CTBE and CGBE in M.smegmatis,and then applied CTBE for genome editing in M.tuberculosis.Our approaches significantly reduce the efforts and time needed for precise genetic manipulation and will facilitate functional genomics,antibiotic-resistant mechanism study,and drugtarget exploration in M.tuberculosis and related organisms.

关键词： CRISPR Cas9 Mycobacterium tuberculosis Genome editing base editing

Generation of a Hutchinson-Gilford progeria syndrome monkey model by base editing

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Protein & Cell 2020年第11期11卷 809-824页

作者： Fang Wang Weiqi Zhang Qiaoyan Yang Yu Kang Yanling Fan Jingkuan Wei Zunpeng Liu Shaoxing Dai Hao Li Zifan Li Lizhu Xu Chu Chu Jing Qu Chenyang Si Weizhi Ji Guang-Hui Liu Chengzu Long Yuyu Niu Yunnan Key Laboratory of Primate Biomedical Research Institute of Primate Translational MedicineKunming University of Science and TechnologyKunming 650500China Faculty of Life Science and Technology Kunming University of Science and TechnologyKunming 650500China Institute for Stem Cell and Regeneration Chinese Academy of ScienceBeijing 100101China CAS Key Laboratory of Genomic and Precision Medicine Beijing Institute of GenomicsChinese Academy of SciencesBeijing 100101China China National Center for Bioinformation Beijing 100101China University of Chinese Academy of Sciences Beijing 100049China The Leon H Charney Division of Cardiology New York University School of MedicineNew YorkNY 10016USA State Key Laboratory of Stem Cell and Reproductive Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China State Key Laboratory of Membrane Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China Advanced Innovation Center for Human Brain Protection and National Clinical Research Center for Geriatric DisordersXuanwu Hospital Capital Medical UniversityBeijing 100053China Department of Neuroscience and Physiology New York University School of MedicineNew YorkNY 10016USA Department of Neurology New York University School of MedicineNew YorkNY 10016USA

Many human genetic diseases,including Hutchinson-Gilford progeria syndrome(HGPS),are caused by single point mutations.HGPS is a rare disorder that causes premature aging and is usually caused by a de novo point mutation in the LMNA gene.base editors(BEs)composed of a cytidine deaminase fused to CRISPR/Cas9 nickase are highly efficient at inducing C to T base conversions in a programmable manner and can be used to generate animal disease models with single amino-acid substitutions.Here,we generated the first HGPS monkey model by delivering a BE mRNA and guide RNA(gRNA)targeting the LMNA gene via microinjection into monkey zygotes.Five out of six newborn monkeys carried the mutation specifically at the target site.HGPS monkeys expressed the toxic form of lamin A,progerin,and recapitulated the typical HGPS phenotypes including growth retardation,bone alterations,and vascular abnormalities.Thus,this monkey model genetically and clinically mimics HGPS in humans,demonstrating that the BE system can efficiently and accurately generate patient-specific disease models in non-human primates.

关键词： base editing non-human primate HGPS

Engineered DNase-inactive Cpf1 variants to improve targeting scope for base editing in E.coli

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Synthetic and Systems Biotechnology 2021年第4期6卷 326-334页

作者： Zehua Chen Jinyuan Sun Ying Guan Ming Li Chunbo Lou Bian Wu CAS Key Laboratory of Microbial Physiological&Metabolic Engineering and State Key Laboratory of Microbial Resources Institute of MicrobiologyChinese Academy of SciencesBeijing100101China College of Life Sciences University of Chinese Academy of SciencesBeijing100149China Tsinghua University Beijing100084China

The development of base editing(BE)technology has opened a new avenue for research studies in bacteriology,particularly for bacterial species in which the DNA double-strand breaks(DSBs)introduced by CRISPR/Cas system would lead to cell death.However,a major limitation of BE-mediated gene editing is the restricted editable sites in the target bacterial genome due to highly diverse genomic compositions,such as GC content.Herein,we developed a broad-spectrum DNase-inactive Cpf1(dCpf1)variant from Francisella novicida(bsdFnCpf1)through directed evolution.The resulting optimized mutant showed a substantially expanded targeting range,including previously non-canonical protospacer-adjacent motifs(PAMs),especially the GC-rich PAMs.Cytidine deaminase APOBEC1 and uracil DNA glycosylase inhibitor(UGI)were fused with bsdFnCpf1 to achieve specific C to T mutations at multiple target sites with canonical or non-canonical PAMs in the E.coli genome without compromising cell growth.We anticipate that bsdFnCpf1 could be applied for multiplex gene regulation and BE in species that have been reported to be suitable for Cpf1.

关键词： base editing Directed evolution Broad-spectrum dFnCpf1(bsdFnCpf1) Multiple gene editing

Three novel alleles of OsGS1 developed by base-editing-mediated artificial evolution confer glufosinate tolerance in rice

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The Crop Journal 2023年第2期11卷 661-665页

作者： Bin Ren Yongjie Kuang Ziyan Xu Xuemei Wu Dawei Zhang Fang Yan Xiangju Li Xueping Zhou Guirong Wang Huanbin Zhou Shenzhen Branch Guangdong Laboratory for Lingnan Modern AgricultureGenome Analysis Laboratory of the Ministry of Agriculture and Rural AffairsAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen 440307GuangdongChina State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193China Scientific Observing and Experimental Station of Crop Pests in Guilin Ministry of Agriculture and Rural AffairsGuilin 541399GuangxiChina State Key Laboratory of Rice Biology Institute of BiotechnologyZhejiang UniversityHangzhou 310058ZhejiangChina Ministry of Education Key Laboratory of Bio-Resource and Eco-Environment College of Life SciencesSichuan UniversityChengdu 610065SichuanChina

Only few glufosinate-tolerant genes,such as phosphinothricin acetyltransferase(PAT)and bialaphos resistance(bar)identified from Streptomyces,are currently available for developing genetically modified rice in agricultural application.Following the rapid development of genome editing technology,generation of novel glufosinate-tolerant gene resources through artificial evolution of endogenous genes is more promising and highly desirable in rice molecular breeding program.In this study,the endogenous Glutamine synthetase1(OsGS1)was artificially evolved by base-editing-mediated gene evolution(BEMGE)in rice cells to create novel alleles conferring glufosinate tolerance in rice germplasms.Two novel glufosinate-tolerant OsGS1 alleles(OsGS1-AVPS and OsGS1-+AF)and one reported tolerant allele(OsGS1-SGTA)were successfully identified from approximately 4200 independent hygromycin-tolerant calli.Germination assays and spray tests revealed that these three OsGS1 alleles confer glufosinate tolerance in rice.Furthermore,OsGS1-AVPS and OsGS1-SGTA were quickly deployed into the elite rice cultivar Nangeng 46 through precise base editing.Overall,our results demonstrate the feasibility of developing glufosinate-tolerant rice by editing an endogenous rice gene in molecular breeding programs.

关键词： base editing Gene evolution Glutamine synthetase Glufosinate tolerance Rice