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Simultaneous multiplex genome loci editing of Halomonas bluephagenesis using an engineered CRISPR-guided base editor

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Synthetic and Systems Biotechnology 2024年第3期9卷 586-593页

作者： Yulin Zhang Yang Zheng Qiwen Hu Zhen Hu Jiyuan Sun Ping Cheng Xiancai Rao Xiao-Ran Jiang Medical Research Institute Southwest UniversityChongqing400716China Department of Microbiology College of Basic Medical SciencesArmy Medical University(Third Military Medical University)Chongqing400038China

Halomonas bluephagenesis TD serves as an exceptional chassis for next generation industrial biotechnology to produce various ***,the simultaneous editing of multiple loci in *** TD remains a significant ***,we report the development of a multiple loci genome editing system,named CRISPR-deaminase-assisted base editor(CRISPR-BE)in *** *** system comprises two components:a cytidine(CRISPR-cBE)and an adenosine(CRISPR-aBE)deaminase-based base ***-cBE can introduce a cytidine to thymidine mutation with an efficiency of up to 100%within a 7-nt editing window in *** ***,CRISPR-aBE demonstrates an efficiency of up to 100%in converting adenosine to guanosine mutation within a 7-nt editing ***-cBE has been further validated and successfully employed for simultaneous multiplexed editing in *** *** findings reveal that CRISPR-cBE efficiently inactivated all six copies of the IS1086 gene simultaneously by introducing stop *** system achieved an editing efficiency of 100%and 41.67%in inactivating two genes and three genes,*** substituting the Pcas promoter with the inducible promoter PMmp1,we optimized CRISPR-cBE system and ultimately achieved 100%editing efficiency in inactivating three *** conclusion,our research offers a robust and efficient method for concurrently modifying multiple loci in *** TD,opening up vast possibilities for industrial applications in the future.

关键词： base editor Gene editing Multiplex Halomonas bluephagenesis

Human 8-cell embryos enable efficient induction of disease-preventive mutations without off-target effect by cytosine base editor

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Protein & Cell 2023年第6期14卷 416-432页

作者： Yinghui Wei Meiling Zhang Jing Hu Yingsi Zhou Mingxing Xue Jianhang Yin Yuanhua Liu Hu Feng Ling Zhou Zhifang Li Dongshuang Wang Zhiguo Zhang Yin Zhou Hongbin Liu Ning Yao Erwei Zuo Jiazhi Hu Yanzhi Du Wen Li Chunlong Xu Hui Yang Center for Reproductive Medicine International Peace Maternity and Child Health HospitalInnovative Research Team of High-level Local Universities in ShanghaiSchool of MedicineShanghai Jiao Tong UniversityShanghai 2000301.Center for Reproductive MedicineInternational Peace Maternity and Child Health HospitalInnovative Research Team of High-level Local Universities in ShanghaiSchool of MedicineShanghai Jiao Tong UniversityShanghai 200030China Institute of Neuroscience State Key Laboratory of NeuroscienceKey Laboratory of Primate NeurobiologyCAS Center for Excellence in Brain Science and Intelligence TechnologyShanghai Institutes for Biological SciencesChinese Academy of SciencesShanghai 200031China Lingang Laboratory Shanghai Research Center for Brain Science and Brain-Inspired Intelligence TechnologyShanghai 200031China Center for Reproductive Medicine Ren Ji HospitalSchool of MedicineShanghai Key Laboratory for Assisted Reproduction and Reproductive GeneticsShanghai Jiao Tong UniversityShanghai 200127China The MOE Key Laboratory of Cell Proliferation and Differentiation School of Life SciencesGenome Editing Research CenterPeking UniversityBeijing 100871China Center for Reproductive Medicine Shandong UniversityJinan 250012China Center for Reproductive Medicine Anhui Provincial Maternal and Child Health HospitalHefei 230001China Shenzhen Branch Guangdong Laboratory for Lingnan Modern AgricultureGenome Analysis Laboratory of the Ministry of AgricultureAgricultural Genomics Institute at ShenzhenChinese Academy of Agricultural SciencesShenzhen 518000China Reproductive Medicine Center Department of Obstetrics and Gynecologythe First Affiliated Hospital of Anhui Medical UniversityHefei 230022China

Approximately 140 million people worldwide are homozygous carriers of APOE4(ε4),a strong genetic risk factor for late onset familial and sporadic Alzheimer’s disease(AD),91%of whom will develop AD at earlier age than heterozygous carriers and *** to AD could be reduced by targeted editing of APOE4,but a technical basis for controlling the off-target effects of base editors is necessary to develop low-risk personalized gene ***,we first screened eight cytosine base editor variants at four injection stages(from 1-to 8-cell stage),and found that FNLS-YE1 variant in 8-cell embryos achieved the comparable base conversion rate(up to 100%)with the lowest bystander *** particular,80%of AD-susceptibleε4 allele copies were converted to the AD-neutralε3 allele in humanε4-carrying *** control measures combined with targeted deep sequencing,whole genome sequencing,and RNA sequencing showed no DNA or RNA off-target events in FNLS-YE1-treated human embryos or their derived stem ***,base editing with FNLS-YE1 showed no effects on embryo development to the blastocyst ***,we also demonstrated FNLS-YE1 could introduce known protective variants in human embryos to potentially reduce human susceptivity to systemic lupus erythematosus and familial *** study therefore suggests that base editing with FNLS-YE1 can efficiently and safely introduce known preventive variants in 8-cell human embryos,a potential approach for reducing human susceptibility to AD or other genetic diseases.

关键词： human embryo APOE4 disease-preventive mutations base editor

Effective gene editing by high-fidelity base editor 2 in mouse zygotes

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Protein & Cell 2017年第8期8卷 601-611页

作者： Puping Liang Hongwei Sun Ying Sun Xiya Zhang Xiaowei Xie Jinran Zhang zhen Zhang Yuxi Chen Chenhui Ding Yuanyan Xiong Wenbin Ma Dan Liu Junjiu Huang Zhou Songyang Key Laboratory of Gene Engineering of the Ministry of Education Guangzhou Key Laboratory of Healthy Aging Researchand State Key Laboratory of Biocontrol SYSU-BCM Joint Research Center School of Life Sciences Sun Yat-sen UniversityGuangzhou 510275 China State Key Laboratory of Ophthalmology Zhongshan Ophthalmic Center Sun Yat-sen University Guangzhou 510275 China Key Laboratory of Reproductive Medicine of Guangdong Province School of Life Sciences and the First Affiliated HospitalSun Yat-sen University Guangzhou 510275 China Guangzhou Magigen Biotechnology Co.Ltd Guangzhou 510320 China 5 Vernaand Marrs Mclean Department of Biochemistry and Molecular Biology Baylor College of Medicine One Baylor Plaza Houston TX 77030 USA

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.

关键词： base editor high-fidelity mouse embryos,proximal-site deamination whole-genome sequencing

Programmable deaminase-free base editors for G-to-Y conversion by engineered glycosylase

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National Science Review 2023年第8期10卷 78-87页

作者： Huawei Tong Nana Liu Yinghui Wei Yingsi Zhou Yun Li Danni Wu Ming Jin Shuna Cui Hengbin Li Guoling Li Jingxing Zhou Yuan Yuan Hainan Zhang Linyu Shi Xuan Yao Hui Yang HuidaGene Therapeutics Co. Ltd. Department of Neurology and Institute of Neurology of First Affiliated Hospital Institute of Neuroscience and Fujian Key Laboratory of Molecular Neurology Fujian Medical University Institute of Neuroscience Center for Excellence in Brain Science and Intelligence Technology Chinese Academy of Sciences Shanghai Center for Brain Science and Brain-Inspired Intelligence

Current DNA base editors contain nuclease and DNA deaminase that enables deamination of cytosine(C)or adenine(A), but no method for guanine(G) or thymine(T) editing is available at present. Here we developed a deaminase-free glycosylase-based guanine base editor(gGBE) with G editing ability, by fusing Cas9 nickase with engineered N-methylpurine DNA glycosylase protein(MPG). By several rounds of MPG mutagenesis via unbiased and rational screening using an intron-split EGFP reporter, we demonstrated that gGBE with engineered MPG could increase G editing efficiency by more than 1500 ***, this gGBE exhibited high base editing efficiency(up to 81.2%) and high G-to-T or G-to-C(i.e. G-to-Y) conversion ratio(up to 0.95) in both cultured human cells and mouse embryos. Thus, we have provided a proof-of-concept of a new base editing approach by endowing the engineered DNA glycosylase the capability to selectively excise a new type of substrate.

关键词： glycosylase base excision repair base editor depurination deaminase-free transversion

Engineered biocontainable RNA virus vectors for non-transgenic genome editing across crop species and genotypes

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Molecular Plant 2023年第3期16卷 616-631页

作者： Qian Liu Chenglu Zhao Kai Sun Yinlu Deng Zhenghe Li State Key Laboratory of Rice Biology Institute of BiotechnologyZhejang UniversityHangzhouChina Ministry of Agricutture Key Laboratory of Molecular Biology of Crop Pathogens and Insect Pests Zhejiang UniversityHangzhouChina Key Laboratory of Biology of Crop Pathogens and Insects of Zhejang Province Zhejang UniversityHangzhouChina

CRISPR/Cas genome-editing tools provide unprecedented opportunities for basic plant biology research and crop ***,the lack of robust delivery methods has limited the widespread adoption of these revolutionary technologies in plant ***,we report an efficient,non-transgenic CRISPR/Cas delivery platform based on the engineered tomato spotted wilt virus(TSWV),an RNA virus with a host range of over 1000 plant *** eliminated viral elements essential for insect transmission to liberate genome space for accommodating large genetic cargoes without sacrificing the ability to infect plant *** resulting non-insect-transmissible viral vectors enabled effective and stable in planta delivery of Cas12a and Cas9 nucleases as well as adenine and cytosine base *** systemically infected plant tissues,the deconstructed TSWV-derived vectors induced efficient somatic gene mutations and base conversions in multiple crop species with little genotype *** with heritable,bi-allelic mutations could be readily regenerated by culturing the virus-infected tissues in vitro without antibiotic ***,we showed that antiviral treatment with ribavirin during tissue culture cleared the viral vectors in 100%of regenerated plants and further augmented the recovery of heritable *** many plants are recalcitrant to stable transformation,the viral delivery system developed in this work provides a promising tool to overcome gene delivery bottlenecks for genome editing in various crop species and elite varieties.

关键词： genome editing CRISPR-Cas base editor RNA virus vector tomato spotted wilt virus bunyavirus delivery non-transgenic

base-Editing-Mediated Artificial Evolution of OsALS1 In Planta to Develop Novel Herbicide-Tolerant Rice Germplasms

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Molecular Plant 2020年第4期13卷 565-572页

作者： Yongjie Kuang Shaofang Li Bin Ren Fang Yan Carl Spetz Xiangju Li Xueping Zhou Huanbin Zhou State Key Laboratory for Biology of Plant Diseases and Insect Pests Institute of Plant ProtectionChinese Academy of Agricultural SciencesBeijing 100193China College of Biological Sciences China Agricultural UniversityBeijing 100193China Scientific Observing and Experimental Station of Crop Pests in Guilin Ministry of Agriculture and Rural AffairsGuilin 541399China Norwegian Institute of Bioeconomy Research 1432 AasNorway State Key Laboratory of Rice Biology Institute of BiotechnologyZhejiang UniversityHangzhou 310058China

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 *** ever,to engineer target genes with unknown functional SNPs remains *** 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 *** this end,OsALS1 was artificially evolved in rice cells using BEMGE through both Agrobacterium-mediated and particle-bom bardment-mediated transform *** 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 *** 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 ***,these data indicate great potential of BEMGE in creating important genetic variants of target genes for crop improvement.

关键词： CRISPR base editor gene evolution OsALS1 herbicide resistance Otyza sativa L

Genome Editing Strategies Towards Enhancement of Rice Disease Resistance

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Rice science 2021年第2期28卷 133-145页

作者： Rukmini MISHRA WEI Zheng Raj Kumar JOSHI ZHAO Kaijun School of Applied Sciences Centurion University of Technology and ManagementBhubaneswar 752050OdishaIndia National Key Facility for Crop Gene Resources and Genetic Improvement/Institute of Crop Science Chinese Academy of Agriculture SciencesBeijing 100081China Department of Biotechnology Rama Devi Women’s UniversityBhubaneswar 751022OdishaIndia

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.

关键词： genome editing technology rice improvement CRISPR/Cas9 CRISPR/Cas12a base editor disease resistance

Designing sgRNAs for CRISPR-BEST base editing applications with CRISPyweb 2.0

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Synthetic and Systems Biotechnology 2020年第2期5卷 99-102页

作者： Kai Blin Simon Shaw Yaojun Tong Tilmann Weber The Novo Nordisk Foundation Center for Biosustainability Technical University of DenmarkKgs.LyngbyDenmark

CRISPR/Cas9 systems are an established tool in genome *** 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 *** recently published CRISPR-BEST base editor system for actinobacteria is built around a C to T or A to G base *** base editing systems however require additional constraints to be considered for designing the ***,we present an updated version of the interactive CRISPy-web single guide RNA design tool https://***/that was built to support“classical”CRISPR and now also CRISPRBEST workflows.

关键词： CRISPR base editor sgRNA Webserver CRISPR-BEST Genome editing

Genome editing in grass plants

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aBIOTECH 2020年第1期1卷 41-57页

作者： Si Nian Char Bing Yang Division of Plant Sciences University of MissouriColumbiaMO 65211USA Donald Danforth Plant Science Center St.LouisMO 63132USA

Cereal crops including maize,rice,wheat,sorghum,barley,millet,oats and rye are the major calorie sources in our daily life and also important bioenergy sources of the *** rapidly advancing and state-of-the-art genome-editing tools such as zinc finger nucleases,TAL effector nucleases,and clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated systems(CRISPR-Cas9-,CRISPR-Cas12a-and CRISPR/Cas-derived base editors)have accelerated the functional genomics and have promising potential for precision breeding of grass *** the availability of annotated genomes of the major cereal crops,application of these established genome-editing toolkits to grass plants holds promise to increase the nutritional value and ***,these easy-to-use and robust genome-editing toolkits have advanced the reverse genetics for discovery of novel gene functions in crop *** this review,we document some of important progress in development and utilization of genome-editing tool sets in grass *** also highlight present and future uses of genome-editing toolkits that can sustain and improve the quality of cereal grain for food consumption.

关键词： Genome editing Cereal crops ZFNs TALENs CRISPR/Cas9 CRISPR/Cas12a base editor