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Improve meat production and virus resistance by simultaneously editing multiple genes in livestock using Cas12i^(Max)

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Science China(Life Sciences) 2024年第3期67卷 555-564页

作者： Jilong Ren Tang Hai Yangcan Chen Ke Sun Zhiqiang Han Jing Wang Chongyang Li Qingwei Wang Leyun Wang Huabing Zhu Dawei Yu Wei Li Shanjiang Zhao State Key laboratory of animal Nutrition Key Laboratory of Animal GeneticsBreeding and Reproduction of Ministry of Agriculture and Rural AffairsInstitute of Animal ScienceChinese Academy of Agricultural SciencesBeijing 100193China State Key laboratoryf stem cell and Reproductive biology Instituteof ZooogyChinese Academyof SciencesBeijing 100101China Beijing Institute for stem cell and Regenerative Medicine Beijing 100101China Beijing Farm animal Research Center Institute of ZoologyChinese Academy of SciencesBeijing 100101China

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated gene(Cas) system is continually optimized to achieve the most efficient gene editing effect. The Cas12i^(Max), a Cas12i variant, exhibits powerful DNA editing activity and enriches the gene editing toolbox. However, the application of Cas12i^(Max)in large domestic animals has not yet been reported. To verify the efficiency and feasibility of multiple gene editing in large animals, we generated porcine fibroblasts with simultaneous knockouts of IGF2, ANPEP, CD163,and MSTN via Cas12i^(Max)in one step. Phenotypically stable pigs were created through somatic cell nuclear transfer technology. They exhibited improved growth performance and muscle quality. Furthermore, we simultaneously edited three genes in bovine fibroblasts. A knockout of MSTN and PRNP was created and the amino acid Q-G in CD18 was precisely substituted. Meanwhile, no off-target phenomenon was observed by sum-type analysis or off-target detection. These results verified the effectiveness of Cas12i^(Max)for gene editing in livestock animals and demonstrated the potential application of Cas12i^(Max)in the field of animal trait improvement for agricultural production.

关键词： Cas12i^(Max) gene editing livestock multiple trait improvement

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Genome-edited rabbits:Unleashing the potential of a promising experimental animal model across diverse diseases

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Zoological Research 2024年第2期45卷 253-262页

作者： Yang Han Jiale Zhou Renquan Zhang Yuru Liang Liangxue Lai Zhanjun Li Key laboratory of Zoonosis Research Ministry of EducationCollege of Animal ScienceJilin UniversityChangchunJilin 130062China CAS Key laboratory of Regenerative biology Guangdong Provincial Key Laboratory of Stem Cell and Regenerative MedicineSouth China Institute for Stem Cell Biology and Regenerative MedicineGuangzhou Institutes of Biomedicine and HealthChinese Academy of SciencesGuangzhouGuangdong 510530China Guangzhou Regenerative Medicine and Health Guang Dong laboratory(GRMH-GDL) GuangzhouGuangdong 510005China Institute for stem cell and Regeneration Chinese Academy of SciencesBeijing 100101China

animal models are extensively used in all aspects of biomedical research,with substantial contributions to our understanding of diseases,the development of pharmaceuticals,and the exploration of gene *** field of genome modification in rabbits has progressed ***,recent advancements,particularly in CRISPR/Cas9-related technologies,have catalyzed the successful development of various genome-edited rabbit models to mimic diverse diseases,including cardiovascular disorders,immunodeficiencies,agingrelated ailments,neurological diseases,and ophthalmic *** models hold great promise in advancing biomedical research due to their closer physiological and biochemical resemblance to humans compared to *** review aims to summarize the novel gene-editing approaches currently available for rabbits and present the applications and prospects of such models in biomedicine,underscoring their impact and future potential in translational medicine.

关键词： Genome editing animal model Rabbit CRISPR/Cas9 genetic diseases

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Programmable synthetic receptors:the next-generation of cell and gene therapies

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Signal Transduction and Targeted Therapy 2024年第2期9卷 376-400页

作者： Fei Teng Tongtong Cui Li Zhou Qingqin Gao Qi Zhou Wei Li University of Chinese Academy of Sciences Beijing 101408China State Key laboratory of stem cell and Regenerative biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China Institute for stem cell and Regeneration Chinese Academy of SciencesBeijing 100101China Beijing Institute for stem cell and Regenerative Medicine Beijing 100101China

cell and gene therapies hold tremendous promise for treating a range of difficult-to-treat ***,concerns over the safety and efficacy require to be further addressed in order to realize their full *** receptors,a synthetic biology tool that can precisely control the function of therapeutic cells and genetic modules,have been rapidly developed and applied as a powerful *** designed and engineered,they can be applied to finetune the therapeutic activities,i.e.,to regulate production of dosed,bioactive payloads by sensing and processing user-defined signals or *** review provides an overview of diverse synthetic receptor systems being used to reprogram therapeutic cells and their wide applications in biomedical *** a special focus on four synthetic receptor systems at the forefront,including chimeric antigen receptors(CARs)and synthetic Notch(synNotch)receptors,we address the generalized strategies to design,construct and improve synthetic ***,we also highlight the expanding landscape of therapeutic applications of the synthetic receptor systems as well as current challenges in their clinical translation.

关键词： synthetic landscape payload

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genetically modified pigs:Emerging animal models for hereditary hearing loss

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Zoological Research 2024年第2期45卷 284-291页

作者： Xiao Wang Tian-Xia Liu Ying Zhang Liang-Wei Xu Shuo-Long Yuan A-Long Cui Wei-Wei Guo Yan-Fang Wang Shi-Ming Yang Jian-Guo Zhao State Key laboratory of stem cell and Reproductive biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China Savaid Medical School University of Chinese Academy of SciencesBeijing 100049China Institute for stem cell and Regeneration Chinese Academy of SciencesBeijing 100101China Beijing Institute for stem cell and Regenerative Medicine Beijing 100101China Department of Otolaryngology-Head and Neck Surgery Institute of OtolaryngologyChinese PLA General HospitalBeijing 100853China Division of Life Sciences and Medicine University of Science and Technology of ChinaHefeiAnhui 230022China State Key laboratory of animal Biotech Breeding Institute of Animal ScienceChinese Academy of Agricultural SciencesBeijing100193China

Hereditary hearing loss(HHL),a genetic disorder that impairs auditory function,significantly affects quality of life and incurs substantial economic losses for *** investigate the underlying causes of HHL and evaluate therapeutic outcomes,appropriate animal models are *** have been extensively used as valuable large animal models in biomedical *** this review,we highlight the advantages of pig models in terms of ear anatomy,inner ear morphology,and electrophysiological characteristics,as well as recent advancements in the development of distinct genetically modified porcine models of hearing ***,we discuss the prospects,challenges,and recommendations regarding the use pig models in HHL ***,this review provides insights and perspectives for future studies on HHL using porcine models.

关键词： Pigs animal models Hereditary hearing loss genetic modification Inner ear

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Exploring the impact of pericentric inversion of chromosome 9 on fertility in sperm donors

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Asian Journal of Andrology 2024年第3期26卷 302-307页

作者： Qian Liu Wen-Jun Zhou Zeng-Hui Huang Xiu-Hai Huang Jian Wu Xi-Ren Ji Xue-Feng Luo Yu-Ling Tang Rui-Jun Wang Li-Qing Fan Chuan Huang Wen-Bing Zhu Institute of Reproductive and stem cell engineering Basic Medicine CollegeCentral South UniversityChangsha 410013China Clinical Research Center for Reproduction and genetics in Hunan Province Reproductive and Genetic Hospital of CITIC-XiangyaChangsha 410005China

Pericentric inversion of chromosome 9(inv[9])is a common chromosomal structural variant,but its impact on clinical outcomesremains *** screening criteria of sperm banks are rarely mentioned to individuals with inv(9).In this study,we evaluatedthe fertility of sperm donors with inv(9)who met eligibility criteria for sperm banks(inv[9]-eligible donors).From March 2004 toMay 2022,chromosomal analysis of 16124 sperm donors at CITIC-Xiangya Human Sperm Bank in Hunan Province(Changsha,China)found that 251(1.6%)had chromosome variations,with inv(9)being the most prevalent at 1.1%.All 169 inv(9)-eligibledonors were contacted to collect fertility outcome data,along with 206 eligible donors without inv(9)as *** addition,semen samples from inv(9)-eligible donors and eligible donors underwent assessments of sperm fluorescence in situ hybridization(FISH),mitochondrial membrane potential,DNA fragmentation index,acrosome integrity,reactive oxygen species(ROS),andsperm *** showed that inv(9)did not significantly increase reproductive risks *** detecting ROSlevel differences,the clinical impact may be *** study provides new data on the inv(9)population that can serveas a valuable reference for decision-making by sperm banks as well as for genetic counseling and clinical guidance for individualscarrying inv(9)variant.

关键词： male fertility pericentric inversion of chromosome 9 sperm bank sperm functional parameters

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Enhanced axonal regeneration and functional recovery of the injured sciatic nerve in a rat model by lithium-loaded electrospun nanofibrous scaffolds

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Bio-Design and Manufacturing 2024年第5期7卷 701-720页

作者： Banafsheh Dolatyar Bahman Zeynali Iman Shabani Azita Parvaneh Tafreshi Reza Karimi-Soflou Developmental biology laboratory School of BiologyCollege of ScienceUniversity of TehranTehran ***Iran Department of Biomedical engineering Amirkabir University of Technology(Tehran Polytechnic)Tehran ***Iran Department of Molecular Medicine Faculty of Medical BiotechnologyNational Institute of Genetic Engineering and BiotechnologyTehran ***Iran 123

Increasing evidence indicates that engineered nerve grafts have great potential for the regeneration of peripheral nerve injuries(PNIs).While most studies have focused only on the topographical features of the grafts,we have considered both the biophysical and biochemical manipulations in our applied *** achieve this,we fabricated an electrospun nanofibrous scaffold(ENS)containing polylactide nanofibers loaded with lithium(Li)ions,a Wnt/β-catenin signaling *** addition,we seeded human adipose-derived mesenchymal stem cells(hADMSCs)onto this engineered scaffold to examine if their differentiation toward Schwann-like cells was *** further examined the efficacy of the scaffolds for nerve regeneration in vivo via grafting in a PNI rat *** results showed that Li-loaded ENSs gradually released Li within 11 d,at concentrations ranging from 0.02 to(3.64±0.10)mmol/L,and upregulated the expression of Wnt/β-catenin target genes(cyclinD1 and c-Myc)as well as those of Schwann cell markers(growth-associated protein 43(GAP43),S100 calcium binding protein B(S100B),glial fibrillary acidic protein(GFAP),and SRY-box transcription factor 10(SOX10))in differentiated *** the PNI rat model,implantation of Li-loaded ENSs with/without cells improved behavioral features such as sensory and motor functions as well as the electrophysiological characteristics of the injured *** improved function was further validated by histological analysis of sciatic nerves grafted with Li-loaded ENSs,which showed no fibrous connective tissue but enhanced organized myelinated *** potential of Li-loaded ENSs in promoting Schwann cell differentiation of hADMSCs and axonal regeneration of injured sciatic nerves suggests their potential for application in peripheral nerve tissue engineering.

关键词： stem cell Schwann cell differentiation Electrospun nanofibrous scaffold Lithium ion Nerve regeneration

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Gene-knockout by iSTOP enables rapid reproductive disease modeling and phenotyping in germ cells of the founder generation

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Science China(Life Sciences) 2024年第5期67卷 1035-1050页

作者： Yaling Wang Jingwen Chen Xueying Huang Bangguo Wu Peng Dai Feng Zhang Jinsong Li Lingbo Wang State Key laboratory of genetic engineering Shanghai Key Laboratory of Metabolic Remodeling and HealthInstitute of Metabolism and Integrative BiologyFudan UniversityShanghai 200438China Institute of Reproduction and Development Obstetrics and Gynecology HospitalFudan UniversityShanghai 200011China NHC Key Lab of Reproduction Regulation(Shanghai Institute for Biomedical and Pharmaceutical Technologies) School of PharmacyFudan UniversityShanghai 200433China Shanghai Key laboratoryof Maternal and Fetal Medicine Clinical and Translational Research Center of Shanghai First Maternity and Infant HospitalFrontier Science Center for Stem Cell ResearchSchoolof Life Sciences and TechnologyTongji UniversityShanghai 200092China State Key laboratory of cell biology CAS Center for Excellence in Molecular Cell ScienceShanghai Institute of Biochemistry and Cell BiologyChinese Academy of SciencesShanghai 200031China

Cytosine base editing achieves C·G-to-T·A substitutions and can convert four codons(CAA/CAG/CGA/TGG)into STOP-codons(induction of STOP-codons,iSTOP)to knock out genes with reduced *** enables direct phenotyping in founders’somatic cells,but it remains unknown whether this works in founders’germ cells so as to rapidly reveal novel genes for ***,we initially establish that iSTOP in mouse zygotes enables functional characterization of known genes in founders’germ cells:Cfap43-iSTOP male founders manifest expected sperm features resembling human“multiple morphological abnormalities of the flagella”syndrome(i.e.,MMAF-like features),while oocytes of Zp3-iSTOP female founders have no zona *** further illustrate iSTOP’s utility for dissecting the functions of unknown genes with Ccdc183,observing MMAF-like features and male infertility in Ccdc183-iSTOP founders,phenotypes concordant with those of Ccdc183-KO *** ultimately establish that CCDC183 is essential for sperm morphogenesis through regulating the assembly of outer dynein arms and participating in the intra-flagellar *** study demonstrates iSTOP as an efficient tool for direct reproductive disease modeling and phenotyping in germ cells of the founder generation,and rapidly reveals the essentiality of Ccdc183 in fertility,thus providing a time-saving approach for validating genetic defects(like nonsense mutations)for human infertility.

关键词： disease modeling infertility multiple morphological abnormalities of the flagella(MMAF) induction of STOP-codons(iSTOP CRISPR-STOP) sperm motility

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A repertoire of intronic lariat RNAs reveals tissue-specific regulation and target mimicry potential in plants

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Science China(Life Sciences) 2024年第6期67卷 1280-1291页

作者： Yong Zhang Xiaotuo Zhang Qi Tang Lei Li Ting Jiang Yixiao Fang Hong Zhang Jixian Zhai Guodong Ren Binglian Zheng State Key laboratory of genetic engineering Institute of Plant BiologySchool of Life SciencesFudan UniversityShanghai 200438China Department of biology School of Life SciencesSouthern University of Science and TechnologyShenzhen 518055China

Lariat RNA is concomitantly produced by excised intron during RNA splicing,which is usually debranched by DBR1,an RNA debranching ***,increasing evidence showed that some lariat RNA could escape *** is known about how and why these lariat RNAs could be *** comparing the atlas of lariat RNAs between the non-dividing cell(mature pollen)and three actively dividing tissues(young shoot apex,young seeds,and young roots),we identified hundreds to thousands of lariat RNA naturally retained in each tissue,and the incidence of lariat RNA retention is much less in shoot apex while much more in *** lariat RNAs derived from the same intron or different lariat RNAs from the same pre-m RNA could be retained in one tissue while degraded in the other *** deciphering lariat RNA sequences,we identified an AG-rich(RAAAAVAAAR)motif and a UC-rich(UCUCUYUCUC)motif for pollen-specific and the other three tissues-retained lariat RNAs,*** of the pollen-specific AG-rich motif indeed enhanced lariat RNA retention in ***,hundreds of lariat RNAs harbored mi RNA binding sites,and dual-luciferase reporter assay showed that these natural lariat RNAs had the potential to protect expression of mi RNA target ***,our results uncover that selective retention of lariat RNA is an actively regulatory process,and provide new insights into understanding how lariat RNA metabolism may impact mi RNA activity.

关键词： intron turnover lariat RNA miRNA RNA splicing DBR1

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Human CD56+CD39+ dNK cells support fetal survival through controlling trophoblastic cell fate:immune mechanisms of recurrent early pregnancy loss

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National Science Review 2024年第6期11卷 129-145页

作者： Wentong Jia Liyang Ma Xin Yu Feiyang Wang Qian Yang Xiaoye Wang Mengjie Fan Yan Gu Ran Meng Jian Wang Yuxia Li Rong Li Xuan Shao Yan-Ling Wang State Key laboratory of stem cell and Reproductive biology Key Laboratory of Organ Regeneration and ReconstructionInstitute of ZoologyChinese Academy of Sciences Beijing Institute for stem cell and Regenerative Medicine University of the Chinese Academy of Sciences NHC Key Lab of Reproduction Regulation Shanghai Engineering Research Center of Reproductive Health Drug and Devices Shanghai Institute for Biomedical and Pharmaceutical Technologies National Clinical Center for Obstetrics and Gynecology Peking University Third Hospital Department of Family Planning The Second Hospital of Tianjin Medical University Department of Prenatal Screening Haidian Maternal and Child Health Hospital

Decidual natural killer（dNK） cells are the most abundant immune cells at the maternal-fetal interface during early pregnancy in both mice and humans, and emerging single-cell transcriptomic studies have uncovered various human dNK subsets that are disrupted in patients experiencing recurrent early pregnancy loss（RPL） at early gestational stage, suggesting a connection between abnormal proportions or characteristics of dNK subsets and RPL pathogenesis. However, the functional mechanisms underlying this association remain unclear. Here, we established a mouse model by adoptively transferring human dNK cells into pregnant NOG（NOD/Shi-scid/IL-2Rγnull） mice, where human dNK cells predominantly homed into the uteri of recipients. Using this model, we observed a strong correlation between the properties of human dNK cells and pregnancy outcome. The transfer of dNK cells from RPL patients（dNK-RPL）remarkably worsened early pregnancy loss and impaired placental trophoblast cell differentiation in the recipients. These adverse effects were effectively reversed by transferring CD56+CD39+dNK *** studies revealed that CD56+CD39+dNK subset facilitates early differentiation of mouse trophoblast stem cells（m TSCs） towards both invasive and syncytial pathways through secreting macrophage colony-stimulating factor（M-CSF）. Administration of recombinant M-CSF to NOG mice transferred with dNK-RPL efficiently rescued the exacerbated pregnancy outcomes and fetal/placental development. Collectively, this study established a novel humanized mouse model featuring functional human dNK cells homing into the uteri of recipients and uncovered the pivotal role of M-CSF in fetal-supporting function of CD56+CD39+dNK cells during early pregnancy, highlighting that M-CSF may be a previously unappreciated therapeutic target for intervening RPL.

关键词： recurrent pregnancy loss CD56+CD39+ dNK subset NOG mice adoptive transfer placental trophoblast cell fate M-CSF

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engineering pluripotent stem cells with synthetic biology for regenerative medicine

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Medical Review 2024年第2期4卷 90-109页

作者： Yihuan Mao Siqi Wang Jiazhen Yu Wei Li State Key laboratory of stem cell and Reproductive biology Institute of ZoologyChinese Academy of SciencesBeijingChina Key laboratory of Organ Regeneration and Reconstruction Chinese Academy of SciencesBeijingChina Beijing Institute for stem cell and Regenerative Medicine BeijingChina University of Chinese Academy of Sciences BeijingChina State Key laboratory of stem cell and Reproductive biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China Key laboratory of Organ Regeneration and Reconstruction Chinese Academy of SciencesBeijing 100101China Beijing Institute for stem cell and Regenerative Medicine Beijing 100101China University of Chinese Academy of Sciences Beijing 100049China

Pluripotent stem cells(PSCs),characterized by self-renewal and capacity of differentiating into three germ layers,are the programmable building blocks of ***-derived cells and multicellular systems,particularly organoids,exhibit great potential for regenerative ***,this field is still in its infancy,partly due to limited strategies to robustly and precisely control stem cell behaviors,which are tightly regulated by inner gene regulatory networks in response to stimuli from the extracellular *** receptors and genetic circuits are powerful tools to customize the cellular sense-and-response process,suggesting their underlying roles in precise control of cell fate decision and function ***,we review the progress and challenges needed to be overcome in the fields of PSC-based cell therapy and multicellular system generation,***,we summarize several well-established synthetic biology tools and their applications in PSC ***,we highlight the challenges and perspectives of harnessing synthetic biology to PSC engineering for regenerative medicine.

关键词： pluripotent stem cell regenerative medicine PSC-based cell therapy multicellular systems synthetic biology

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