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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

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

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

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

Precise genome editing with base editors

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Medical Review 2023年第1期3卷 75-84页

作者： Hongcai Liu Yao Zhu Minjie Li Zhimin Gu Institute of Systems Medicine Chinese Academy of Medical Sciences&Peking Union Medical CollegeBeijingChina Suzhou Institute of Systems Medicine SuzhouJiangsu ProvinceChina Institute of Systems Medicine Chinese Academy of Medical Sciences&Peking Union Medical CollegeBeijing 100005China Suzhou Institute of Systems Medicine Suzhou 215123Jiangsu ProvinceChina

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.

关键词： adenine base editor base editing clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein cytosine base editor

base editing:a brief review and a practical example

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The Journal of Biomedical Research 2021年第2期35卷 107-114页

作者： Dongwook C.Choe Kiran Musunuru Division of Cardiology and Cardiovascular Institute Department of MedicineDepartment of GeneticsPerelman School of Medicine at the University of PennsylvaniaPhiladelphiaPA 19104USA

Genome editing has undergone rapid development in recent years,yielding new approaches to make precise changes in genes.In this review,we discuss the development of various adenine and cytosine base-editing technologies,which share the ability to make specific base changes at specific sites in the genome.We also describe multiple applications of base editing in vitro and in vivo.Finally,as a practical example,we demonstrate the use of a cytosine base editor and an adenine base editor in human cells to introduce and then correct a prevalent mutation responsible for hereditary tyrosinemia type 1.

关键词： base editing CRISPR genetics genome editing mutation

Plant genome editing:CRISPR,base editing,prime editing,and beyond

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Grassland Research 2022年第4期1卷 234-243页

作者： Yujie Xie Syed Inzimam Ul Haq Xingyu Jiang Dianfeng Zheng Naijie Feng Wenying Wang Jin-Sheng He Quan-Sheng Qiu MOE Key Laboratory of Cell Activities and Stress Adaptations School of Life SciencesLanzhou UniversityLanzhouGansuChina College of Coastal Agricultural Sciences Guangdong Ocean UniversityZhanjiangGuangdongChina Academy of Plateau Science and Sustainability School of Life SciencesQinghai Normal UniversityXiningQinghaiChina Key Laboratory of Biodiversity Formation Mechanism and Comprehensive Utilization in Qinghai Tibet Plateau Qinghai Normal UniversityXiningQinghaiChina State Key Laboratory of Herbage Improvement and Grassland Agro-ecosystems Lanzhou UniversityLanzhouGansuChina

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)system is a fast-growing,genome editing technology that has wide applications in identifying gene functions as well as improving agricultural production and crop breeding.Here,we summarized recent advances in the development and applications of genome editing technologies in plants.We briefly described CRISPR/Cas9 technology and examined the base and prime editing techniques that have been developed from CRISPR technology.Some new prime editing-derived techniques were assessed.

关键词： base editing CRISPR/Cas9 plant genome editing prime editing prime editing-derived techniques

The CRISPR/Cas9 revolution continues: From base editing to prime editing in plant science

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Journal of Genetics and Genomics 2021年第8期48卷 661-670页

作者： Yan Li Wenjing Li Jun Li State Key Laboratory of North China Crop Improvement and Regulation College of Life SciencesHebei Agricultural UniversityBaodingHebei 071001China College of Life Sciences Capital Normal UniversityBeijing Key Laboratory of Plant Gene Resources and Biotechnology for Carbon Reduction and Environmental ImprovementBeijing 100048China

The ability to precisely inactivate or modify genes in model organisms helps us understand the mysteries of life. Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9), a revolutionary technology that could generate targeted mutants, has facilitated notable advances in plant science. Genome editing with CRISPR/Cas9 has gained great popularity and enabled several technical breakthroughs. Herein, we briefly introduce the CRISPR/Cas9, with a focus on the latest breakthroughs in precise genome editing(e.g., base editing and prime editing), and we summarize various platforms that developed to increase the editing efficiency, expand the targeting scope, and improve the specificity of base editing in plants. In addition, we emphasize the recent applications of these technologies to plants. Finally, we predict that CRISPR/Cas9 and CRISPR/Cas9-based genome editing will continue to revolutionize plant science and provide technical support for sustainable agricultural development.

关键词： CRISPR/Cas9 base editing Prime editing Loss-of-function Gain-of-function DNA free

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