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Generation of isogenic single and multiplex gene knockout mice by base editing-induced STOP

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Science Bulletin 2018年第17期63卷 1101-1107页

作者： Guang Yang Tianyu Zhu Zongyang Lu Guanglei Li Hao Zhang Songjie Feng Yajing Liu Jianan Li Yu Zhang Jia Chen Xuejiang Guo Xingxu Huang School of Life Science and Technology Shanghai Tech University Shanghai 201210 China State Key Laboratory of Reproductive Medicine Department of Histology and Embryology Nanjing Medical University Nanjing 211166 China

Although CRISPR/Cas9 has been widely used to generate knockout mice, two major limitations remain:the founders usually carry a mixture of genotypes, and mosaicism harboring multiple ***, it takes a long time to get homozygous mutants. Recently developed base editing(BE) system,which introduces C-to-T conversion without double strand DNA cleavage, has been used to introduce artificial stop codons(i-STOP) to prematurely terminate translation, providing a cleaner strategy for genome engineering. Using this strategy, we generated CD160 KO and VISTA/CD160 double KO mice by microinjection of a single sg RNA targeting CD160 and a mixture of sg RNAs targeting VISTA and CD160,respectively. The BE system induced STOP efficiently in mouse embryos and consequently in founder mice without detectable off-target. Most interestingly, the majority of the mutants harbor same genetic modifications, indicating we generated isogenic single and multiplex gene mutant mice by BE-induced STOP. We also obtained homozygous mutant mouse in F1 mice, demonstrating the accelerated strategy in generating animal models.

关键词： Base editing i-STOP isogenic knockout VISTA CDI60

Generation of double knockout cattle via CRISPR-Cas9 ribonucleoprotein(RNP)electroporation

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Journal of Animal Science and Biotechnology 2024年第1期15卷 456-462页

作者： Gyeong-Min Gim Kyeong-Hyeon Eom Dong-Hyeok Kwon Dae-Jin Jung Dae-Hyun Kim Jun-Koo Yi Jae-Jung Ha Ji-Hyun Lee Seong-Beom Lee Woo-Jae Son Soo-Young Yum Won-Wu Lee Goo Jang Laboratory of Theriogenology and Biotechnology Department of Veterinary Clinical ScienceCollege of Veterinary Medicine and the Research Institute of Veterinary ScienceSeoul National UniversitySeoulRepublic of Korea LARTBio Co. Ltd.SeoulRepublic of Korea Gyeongsangbukdo Livestock Research Institute YeongjuRepublic of Korea Comparative Medicine Disease Research Center Seoul National UniversitySeoulRepublic of Korea

Background Genome editing has been considered as powerful tool in agricultural ***,genome editing progress in cattle has not been fast as in other mammal species,for some disadvantages including long gestational periods,single pregnancy,and high raising ***,technically demanding methods such as microinjection and somatic cell nuclear transfer(SCNT)are needed for gene editing in *** this point of view,electroporation in embryos has been risen as an *** First,editing efficiency of our electroporation methods were tested for *** of mutation on embryo was confirmed by T7E1 *** first combination,mutation rates for MSTN and PRNP were 57.6%±13.7%and 54.6%±13.5%,*** case of MSTN/BLG,mutation rates were 83.9%±23.6%for MSTN,84.5%±18.0%for ***,the double-KO embryos were transferred to surrogates and mutation rate was identified in resultant calves by targeted deep *** recipients were transferred for MSTN/PRNP,4 calves were delivered,and one calf underwent an induction for double *** surrogates were given double-KO embryos for MSTN/BLG,and four of the six calves that were born had mutations in both *** These data demonstrated that production of genome edited cattle via electroporation of RNP could be effectively ***,MSTN and PRNP from beef cattle and MSTN and BLG from dairy cattle have been born and they will be valuable resources for future precision breeding.

关键词： Beta-lactoglobulin Cattle CRISPR-Cas9 Electroporation knockout MSTN PRNP

CRISPR Primer Designer:Design primers for knockout and chromosome imaging CRISPR-Cas system

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Journal of Integrative Plant Biology 2015年第7期57卷 613-617页

作者： Meng Yan Shi-Rong Zhou Hong-Wei Xue National Key Laboratory of Plant Molecular Genetics Shanghai Institute of Plant Physiology and Ecologythe Chinese Academy of Sciences

The clustered regularly interspaced short palin- dromic repeats （CRiSPR）-associated system enables biologists to edit genomes precisely and provides a powerful tool for perturbing endogenous gene regulation, modulation of epigenetic markers, and genome architecture. However, there are concerns about the specificity of the system, especially the usages of knocking out a gene. Previous designing tools either were mostly built-in websites or ran as command-line programs, and none of them ran locally and acquired a user- friendly interface. In addition, with the development of CRISPR-derived systems, such as chromosome imaging, there were still no tools helping users to generate specific end-user spacers. We herein present researchers to design primers CRISPR Primer Designer for for CRISPR applications. Theprogram has a user-frfendly interface, can analyze the BLAST results by using multiple parameters, score for each candidate spacer, and generate the primers when using a certain plasmid. In addition, CRISPR Primer Designer runs locally and can be used to search spacer clusters, and exports primers for the CRiSPR-Cas system-based chromosome imaging system.

关键词： Chromosome imaging CRISPR knockout primer software

Reduced prostate branching morphogenes,s in stromal fibroblast, but not in epithelial, estrogen receptor a knockout mice

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Asian Journal of Andrology 2012年第4期14卷 546-555页

作者： Ming Chen Chiuan-Ren Yeh Chih-Rong Shyr Hsiu-Hsia Lin Iun Da Shuyuan Yeh Departments of Urology and Pathology University of Rochester Medical Center Rochester New York 14642 USA Sex Hormone Research Center China Medical University Hospital Taichung 40402 Taiwan China

Early studies suggested that estrogen receptor alpha （ERa） is involved in estrogen-mediated imprinting effects in prostate development. We recently reported a more complete ERa knockout （KO） mouse model via mating β-actin Cre transgenic mice with floxed ERa mice. These ACTB-ERaKO male mice showed defects in prostatic branching morphogenesis, which demonstrates that ERa is necessary to maintain proliferative events in the prostate. However, within which prostate cell type ERa exerts those important functions remains to be elucidated. To address this, we have bred floxed ERa mice with either fibroblast-specific protein （FSP）-Cre or probasin-Cre transgenic mice to generate a mouse model that has deleted ERa gene in either stromal fibroblast （FSP-ERaKO） or epithelial （pes-ERaKO） prostate cells. We found that circulating testosterone and fertility were not altered in *** and pes-ERaKO male mice. Prostates of FSP-ERaKO mice have less branching morphogenesis compared to that of wild-type littermates. Further analyses indicated that loss of stromal ERa leads to increased stromal apoptosis, reduced expression of insulin-likegrowth factor-1 （IGF-1） and FGFIO, and increased expression of BMP4. Collectively, we have established the first in vivo prostate stromal and epithelial selective ERaKO mouse models and the results from these mice indicated that stromal fibroblast ERa plays important roles in prostatic branching morphogenesis via a paracrine fashion. Selective deletion of the ERa gene in mouse prostate epithelial cells by probasin-Cre does not affect the regular prostate development and homeostasis.

关键词： Cre-loxP estrogen receptor knockout prostate stromal-epithelial interaction

Neuropeptide Y receptor Y8b(npy8br)regulates feeding and digestion in Japanese medaka(Oryzias latipes)larvae:evidence from gene knockout

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Journal of Zhejiang University-Science B(Biomedicine & Biotechnology) 2024年第7期25卷 605-616页

作者： Xiaodan JIA Ke LU Xufang LIANG College of Fisheries Chinese Perch Research CenterHuazhong Agricultural UniversityWuhan 430070China Engineering Research Center of Green Development for Conventional Aquatic Biological Industry in the Yangtze River Economic Belt Ministry of EducationWuhan 430070China

Neuropeptide Y receptor Y8(NPY8R)is a fish-specific receptor with two subtypes,NPY8AR and *** in expression levels during physiological processes or in vivo regulation after ventricular injection suggest that NPY8BR plays an important role in feeding regulation;this has been found in only a few fish,at *** order to better understand the physiological function of npy8br,especially in digestion,we used clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9)technology to generate npy8br-/-japanese medaka(Oryzias latipes).We found that the deletion of npy8br in medaka larvae affected their feeding and digestion ability,ultimately affecting their ***,npy8br deficiency in medaka larvae resulted in decreased feed intake and decreased expression levels of orexigenic genes(npy and agrp).npy8br-/-medaka larvae fed for 10 d(10th day of feeding)still had incompletely digested brine shrimp(Artemia nauplii)in the digestive tract 8 h after feeding,the messenger RNA(mRNA)expression levels of digestion-related genes(amy,lpl,ctra,and ctrb)were significantly decreased,and the activity of amylase,trypsin,and lipase also significantly *** deletion of npy8br in medaka larvae inhibited the growth and significantly decreased the expression of growth-related genes(gh and igf1).Hematoxylin and eosin(H&E)sections of intestinal tissue showed that npy8br-/-medaka larvae had damaged intestine,thinned intestinal wall,and shortened intestinal *** far,this is the first npy8br gene knockout model established in fish and the first demonstration that npy8br plays an important role in digestion.

关键词： Neuropeptide Y receptor Y8b(npy8br) Japanese medaka(Oryzias latipes) knockout Feeding Digestion

Claudin-7 gene knockout causes destruction of intestinal structure and animal death in mice

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World Journal of Gastroenterology 2019年第5期25卷 584-599页

作者： Chang Xu Kun Wang Yu-Han Ding Wen-Jing Li Lei Ding Department of Oncology Beijing Shijitan Hospital Capital Medical University

BACKGROUND Claudin-7, one of the important components of cellular tight junctions, is currently considered to be expressed abnormally in colorectal inflammation and colorectal cancer. However, there is currently no effective animal model to study its specific mechanism. Therefore, we constructed three lines of Claudin-7 knockout mice using the Cre/LoxP *** To determine the function of the tumor suppressor gene Claudin-7 by generating three lines of Claudin-7 gene knockout *** We crossed Claudin-7-floxed mice with CMV-Cre, vil1-Cre, and villin-CreERT2 transgenic mice, and the offspring were self-crossed to obtain conventional Claudin-7 knockout mice, conditional(intestinal specific) Claudin-7 knockout mice, and inducible conditional Claudin-7 knockout mice. Intraperitoneal injection of tamoxifen into the inducible conditional Claudin-7 knockout mice can induce the knockout of Claudin-7. PCR and agarose gel electrophoresis were used to identify mouse genotypes, and Western blot was used to confirm the knockout of Claudin-7. The mental state, body length, and survival time of these mice were observed. The dying mice were sacrificed, and hematoxylin-eosin(HE) staining and immunohistochemical staining were performed to observe changes in intestinal structure and proliferation *** We generated Claudin-7-floxed mice and three lines of Claudin-7 gene knockout mice using the Cre/LoxP system successfully. Conventional and intestinal specific Claudin-7 knockout mice were stunted and died during the perinatal period, and intestinal HE staining in these mice revealed mucosal gland structure disappearance and connective tissue hyperplasia with extensive inflammatory cell infiltration. The inducible conditional Claudin-7 knockout mice had a normal phenotype at birth, but after the induction with tamoxifen, they exhibited a dying state. Intestinal HE staining showed significant inflammatory cell infiltration, and atypical hyperplasia and adenoma were als

关键词： Claudin-7 Gene knockout Inflammation Adenomas Colorectal carcinoma

knockout of TMEM206 in mice associated with a loss of corneal transparency

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International Journal of Ophthalmology(English edition) 2024年第11期17卷 1967-1972页

作者： Zi-Jian Yang Shou-Yue Huang Yu-Feng Zhou Shun-Chang Sun Department of Ophthalmology Ruijin HospitalShanghai Jiao Tong University School of MedicineShanghai 201801China Department of Laboratory Medicine Ruijin HospitalShanghai Jiao Tong University School of MedicineShanghai 201801China

AIM:To investigate the role of transmembrane protein 206(TMEM206)in corneal edema in mice by knockout the TMEM206 gene using CRISPR/Cas9 editing ***:TMEM206-knockout mice were generated using the CRISPR-Cas9 *** in ophthalmic pathology were observed using slit lamp microscope and optical coherence tomography(OCT),intraocular pressure(IOP)was measured using a TonoLab Rebound Tonometer,and the ultrastructure of the corneal was observed using a transmission electron ***:Corneal opacity was observed in 4/18 homozygous TMEM206^(-/-)mice whereas a similar change was not observed in heterozygous TMEM206^(+/-)mice and wild-type *** examination showed that the mean central cornea thickness was 125±5.4μm in 4 homozygous TMEM206^(-/-)mice developed corneal edema and 115±1.2μm in wild-type mice(t=3.468,P0.05).Transmission electron microscopy revealed a disruption in the organization of the collagen fibrils in the central part of the cornea in homozygous TMEM206^(-/-)***:TMEM206 is associated with corneal edema which caused organizational disruption of collagen fibrils in corneas of mice.

关键词： transmembrane protein 206 knockout cornea edema mouse

knockout of lipoprotein lipase with CRISPR/Cas9 causes severe developmental defects and affects lipid deposition in Japanese medaka(Oryzias latipes)

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Water Biology and Security 2022年第2期1卷 105-111页

作者： Hexiong Feng Xu-Fang Liang College of Fisheries and Chinese Perch Research Center Huazhong Agricultural UniversityWuhan430070China Engineering Research Center of Green Development for Conventional Aquatic Biological Industry in the Yangtze River Economic Belt Ministry of EducationWuhan430070China

In mammals,lipoprotein lipase(LPL)has been found to play an important role in lipid mentalismand *** deficiency in humans(Homo sapiens)and mice(Mus musculus)tends to cause *** lpl gene is not expressed in adult mammalian liver,but is in adult fish *** functions provided by the lpl gene are diverse in ***,we knocked out the lpl gene in Japanese medaka(Oryzias latipes)with the CRISPR/Cas9 *** lpl-knockout(KO)homozygous individuals showed severe developmental defects with an extremely emaciated and deformedbody and onlyaccounted for about5%of the *** is consistentwiththefindings inmicebut disaccords with the results for zebrafish(Danio rerio).Compared with wild-type(WT)madaka,the mRNA level of lpl in lpl-KO heterozygous mutant was significantly higher in the muscle,showed no significant difference in the liver,and was significantly lower in the *** lpl heterozygous deficiency,the relative area of Oil RedO and triglycerides(TG)level in the liver,heart and muscle tissue covaried with levels of lpl mRNA in *** lpl heterozygous deficiency did not affect the levelsofTG,low-density lipoprotein cholesterol(LDL-C),high-density lipoprotein cholesterol(HDL-C)and total cholesterol(TC)in the plasma of medaka,which is inconsistent with the findings in *** general,the lpl gene plays an important role in the growth and development and is closely related to lipid deposition of medaka.

关键词： Lipoprotein lipase knockout Lipid Japanese medaka(Oryzias latipes)

Molecular mechanisms of liver ischemia reperfusion injury:Insights from transgenic knockout models

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World Journal of Gastroenterology 2013年第11期19卷 1683-1698页

作者： Gourab Datta Barry J Fuller Brian R Davidson Liver Transplantation and Hepatobiliary Unit Division of Surgery and Interventional ScienceRoyal Free CampusUniversity College LondonRoyal Free Hampstead NHS Trust HospitalLondon NW 2QGUnited Kingdom

Ischemia reperfusion injury is a major obstacle in liver resection and liver transplantation *** the mechanisms of liver ischemia reperfusion injury(IRI) and developing strategies to counteract this injury will therefore reduce acute complications in hepatic resection and transplantation,as well as expanding the potential pool of usable donor *** initial liver injury is initiated by reactive oxygen species which cause direct cellular injury and also activate a cascade of molecular mediators leading to microvascular changes,increased apoptosis and acute inflammatory changes with increased hepatocyte *** adaptive pathways are activated during reperfusion that reduce the reperfusion *** involves a complex interplay between neutrophils,natural killer T-cells cells,CD4+ T cell subtypes,cytokines,nitric oxide synthases,haem oxygenase-1,survival kinases such as the signal transducer and activator of transcription,Phosphatidylinositol 3-kinases/Akt and nuclear factor κβ *** animals,particularly genetic knockout models,have become a powerful tool at elucidating mechanisms of liver ischaemia reperfusion injury and are complementary to pharmacological *** disruption of the protein at the genetic level is more specific and maintained than pharmacological inhibitors or stimulants of the same *** article reviews the evidence from knockout models of liver IRI about the cellular and molecular mechanisms underlying liver IRI.

关键词： Liver Ischemia/reperfusion Transgenic knockout Nitric oxide synthase Haem oxygenase Mitogen-activated protein kinase T cell receptor