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Computer-aided engineering of CRISPR-Cas proteins for enhanced human genome editing

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Science China(life Sciences) 2023年第4期66卷 883-886页

作者： Yangcan Chen Yanping Hu Shengqiu Luo Xinge Wang Bangwei Mao Yi Chen Jing Xu zhikun li Qi Zhou Wei li State Key Laboratory of Stem Cell and Reproductive Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China Institute for Stem Cell and Regenerative Medicine Chinese Academy of SciencesBeijing 100101China Bejing Institute for Stem Cell and Regenerative Medicine Beijing 100101China

Dear editor.The remarkably diversified CRISPR-Cas systems in nature have provided unlimited valuable resources to develop genome editing tools that are revolutionizing the fields of biotechnology and medicine.However,due to their microbial origin,the activity of most naturally occurring CRISPR-Cas nucleases is relatively poor in mammalian cells(Ran et al.,2015).Thus,improving the mammalian genome editing efficiency becomes the priority for harnessing more CRISPRCas systems for widespread applications.Rational protein engineering serves as a powerful approach to enhance the catalytic activity of enzymes.Whereas,this approach largely relies on proteinic three-dimension(3D)structure information as guide for design.The fact is that of the large numbers of the CRISPR-Cas systems discovered in recent years,only a small number of them with the 3D structures were reported.To bypass this limitation,here,we report an efficient and simple strategy for rational engineering of Cas nucleases without the need of 3D structure information and successfully optimized nucleases from Cas9 and Cas12 families.

关键词： rational editor editing

Deciphering the placental abnormalities associated with somatic cell nuclear transfer at single-nucleus resolution

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Protein & Cell 2023年第12期14卷 924-928页

作者： liyuan Jiang Xin Wang Leyun Wang Sinan Ma Yali Ding Chao liu Siqi Wang Xuan Shao Ying Zhang zhikun li Wei li Guihai Feng Qi Zhou College of life Sciences Northeast Agricultural UniversityHarbin 150030China State Key Laboratory of Stem Cell and Reproductive Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China Institute for Stem Cell and Regenerative Medicine Chinese Academy of SciencesBeijing 100101China Savaid Medical School University of Chinese Academy of SciencesBeijing 100049China University of Chinese Academy of Sciences Beijing 100049China Beijing Institute for Stem Cell and Regenerative Medicine Beijing 100101China

Dear Editor,The placenta connecting the fetus to the matermal uterus provides material exchange and an immune-tolerant environment for the embryo in all eutherian mammals(Shao et al.,2022).The representative mouse placenta displays a multilayered structure with distinct characteristics and functions,including the matermal decidua,junctional zone,and labyrinth layer(Marsh and Blelloch,2020).The decidua,which is thought to be derived from the matermal endometrium(and further undergoes decidualization),provides an anchor for fetal trophoblast invasion.The junctional zone predominantly contains spongiotrophoblasts(SpT),glycogen trophoblasts(GlyT),and trophoblast giant cells(TGCs).The labyrinth is the innermost two-layer structure,which mainly consists of syncytiotrophoblast cells(SynTI and SynTII),sinusoidal TGCs(S-TGCs),and fetal endothelial cells(Simmons and Cross,2005).

关键词： fetal invasion fetus

Current advances in haploid stem cells

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Protein & Cell 2020年第1期11卷 23-33页

作者： Tongtong Cui zhikun li Qi Zhou Wei li State Key Laboratory of Stem Cell and Reproductive Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China Institute for Stem Cell and Regeneration Chinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China

Diploidy is the typical genomic mode in all mammals.Haploid stem cells are artificial cell lines experimentally derived in vitro in the form of different types of stem cells,which combine the characteristics of haploidy with a broad developmental potential and open the possibility to uncover biological mysteries at a genomic scale.To date,a multitude of haploid stem cell types from mouse,rat,monkey and humans have been derived,as more are in development.They have been applied in high-throughput genetic screens and mammalian assisted reproduction.Here,we review the generation,unique properties and broad applications of these remarkable cells.

关键词： haploidy parthenogenetic androgenetic stem cells diploidization functional genomics imprinting

Development of modified PMMA cement in spine surgery

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Engineered Regeneration

Engineered Regeneration 2023年第4期4卷 375-386页

作者： zhikun li Junwei Shi Yi Wang Yifan li Wenjun liu Ruijun Xu Silian Wang liwei Chen Xiaojian Ye Chi Zhang Wei Xu Department of Orthopedic TongRen HospitalSchool of MedicineShanghai Jiao Tong University1111 Xianxia RoadShanghai 200336China Wuxi School of Medicine Jiangnan UniversityWuxi 214122China Department of Imaging TongRen HospitalSchool of MedicineShanghai Jiao Tong University1111 Xianxia RoadShanghai 200336China Department of Orthopedics Shanghai Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine600 Yishan RoadShanghai 200233China

1.Background.Polymethyl methacrylate(PMMA)bone cement has been widely used in orthopedic clinics for over 70 years.In 1952 and 1953,Kiaer and Haboush reported their work on the use of PMMA cement to achieve bone adherence to prostheses in femoral head replacement[1,2],.In 1987,Galibert et al.first reported the use of PMMA cement in spinal surgery for vertebroplasty(PVP)to treat vertebral hemangiomas[3].By 2002,approximately 38,000 vertebroplasties and 16,000 kyphoplasties were performed in the United States[4].

关键词： surgery femoral

Treatment of glutaric aciduria type I(GA-I)via intracerebroventricular delivery of GCDH

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Fundamental Research 2022年第6期2卷 836-842页

作者： Lu Guo zhikun li Yuhuan li Bin Qu Guanyi Jiao Chen liang Zongbao Lu Xin-Ge Wang Cheng Huang Hongwei Du Jianmin liang Qi Zhou Wei li State Key Laboratory of Stem Cell and Reproductive Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China Beijing Institute for Stem Cell and Regenerative Medicine Beijing 100101China University of Chinese Academy of Sciences Beijing 100049China The First Hospital of Jilin University ChangchunJilin 130021China

Glutaric aciduria type I(GA-I)is an autosomal recessive genetic disorder caused by a deficiency in glutaryl-CoA dehydrogenase(GCDH).Patients who do not receive proper treatment may die from acute encephalopathic crisis.Current treatments for GA-I include a low-lysine diet combined with oral supplementation of L-carnitine.A mouse model of Gcdh^(c.422_428del/c.422_428del)(Gcdh^(−/−))was generated in our laboratory using CRISPR/Cas9.Gcdh^(−/−)mice had significantly higher levels of glutaric acid(GA)in the plasma,liver,and brain than those in wild-type C57BL/6 mice.When given a high-protein diet(HPD)for two days,approximately 60%of Gcdh^(−/−)mice did not survive the metabolic stress.To evaluate whether GCDH gene replacement therapy could be used to provide sustained treatment for patients with GA-1,we prepared a recombinant adeno-associated virus(rAAV)carrying a human GCDH expression cassette and injected it into Gcdh^(−/−)neonates for a proof-of-concept(PoC)study.Our study demonstrated that delivering rAAV to the central nervous system(CNS),but not the peripheral system,significantly increased the survival rate under HPD exposure.Our study also demonstrated that rAAVPHP.eB mediated a higher efficiency than that of rAAV9 in increasing the survival rate.Surviving mice showed dose-dependent GCDH protein expression in the CNS and downregulation of GA levels.Our study demonstrated that AAV-based gene replacement therapy was effective for GA-I treatment and provided a feasible solution for this unmet medical need.

关键词： Glutaric aciduria type I(GA-I) Glutaryl-CoA dehydrogenase(GCDH) Adeno-associated virus(AAV) Gene therapy Neurometabolic disease

Synergistic engineering of CRISPR-Cas nucleases enables robust mammalian genome editing

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The Innovation 2022年第4期3卷 74-82页

作者： Yangcan Chen Yanping Hu Xinge Wang Shengqiu Luo Ning Yang Yi Chen zhikun li Qi Zhou Wei li State Key Laboratory of Stem Cell and Reproductive Biology Institute of ZoologyChinese Academy of SciencesBeijing 100101China University of Chinese Academy of Sciences Beijing 100049China Institute for Stem Cell and Regenerative Medicine Chinese Academy of SciencesBeijing 100101China Bejing Institute for Stem Cell and Regenerative Medicine Beijing 100101China

The naturally occurring prokaryotic CRISPR-Cas systems provide valuable resources for the development of new genome-editing tools.However,the majority of prokaryotic Cas nucleases exhibit poor editing efficiency in mammalian cells,which significantly limits their utility.Here,we have developed a method termed Improving Editing Activity by Synergistic Engineering(MIDAS).This method exerts a synergistic effect to improve mammalian genome-editing efficiency of a wide range of CRISPR-Cas systems by enhancing the interactions of Cas nuclease with the protospacer adjacent motif(PAM)and the single-stranded DNA(ssDNA)substrate in the catalytic pocket simultaneously.MIDAS robustly and significantly increased the gene-editing efficiency of Cas12i,Cas12b,and CasX in human cells.Notably,a Cas12i variant,Cas12iMax,exhibited robust activity with a very broad PAM range(NTNN,NNTN,NAAN,and NCAN)and higher efficiency than the current widely used Cas nucleases.A high-fidelity version of Cas12iMax(Cas12iHiFi)has been further engineered to minimize off-target effects.Our work provides an expandable and efficacious method for engineering Cas nucleases for robust mammalian genome editing.

关键词： editing engineering mammalian

Generation of an LncRNA Gtl2-GFP Reporter for Rapid Assessment of Pluripotency in Mouse Induced Pluripotent Stem Cells

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Journal of Genetics and Genomics 2015年第3期42卷 125-128页

作者： zhikun li libin Wang Yukai Wang Lei liu liu Wang Wei li Qi Zhou State Key Laboratory of Reproductive Biology Institute of ZoologyChinese Academy of Sciences University of Chinese Academy of Sciences

Epigenetic reprogramming of somatic cells into induced pluripotent stem cells （iPSCs） by overexpression of defined factors holds great promise for disease modeling and regen- erative medicine （Takahashi and Yamanaka, 2006; Robinton and Daley, 2012）. However, the stochastic reprogramming process often results in variable pluripotency levels of iPSC lines as measured by their in vivo developmental potential, which poses a huge challenge to the applications of high quality iPSCs （Hanna et al., 2010）. The activation status of an imprinted Dlkl-Dio3 region has been identified as a molecular marker for pluripotency （liu et al., 2010; Stadtfeld et al.,

关键词： GFP RNA Generation of an LncRNA Gtl2-GFP Reporter for Rapid Assessment of Pluripotency in Mouse Induced Pluripotent Stem Cells PSCs

聚丙烯腈/二硫化钼复合薄膜的挠曲电效应分析及其应用

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纺织学报 2024年第5期45卷 27-34页

作者：栗志坤于影左雨欣史豪秦金玉珍陈洪立浙江理工大学机械工程学院浙江杭州310018 嘉兴学院信息科学与工程学院浙江嘉兴314001 嘉兴南湖学院浙江嘉兴314001

复合薄膜中挠曲电材料的质量分数对薄膜的挠曲电效应影响显著,为探究聚丙烯腈(PAN)/MoS_(2)复合薄膜中MoS_(2)质量分数对其挠曲电压及挠曲电系数的影响规律,采用静电纺丝方法制备PAN/MoS_(2)复合薄膜,借助扫描电子显微镜和X射线衍射仪... 详细信息

复合薄膜中挠曲电材料的质量分数对薄膜的挠曲电效应影响显著,为探究聚丙烯腈(PAN)/MoS_(2)复合薄膜中MoS_(2)质量分数对其挠曲电压及挠曲电系数的影响规律,采用静电纺丝方法制备PAN/MoS_(2)复合薄膜,借助扫描电子显微镜和X射线衍射仪对复合薄膜的结构、形貌、元素组成等进行表征,基于悬臂梁方法测试不同MoS_(2)质量分数对PAN/MoS_(2)复合薄膜挠曲电效应的影响,通过间歇式挠曲电效应测试检测复合薄膜的稳定性。结果表明:挠曲电压和挠曲电系数随着MoS_(2)质量分数的增大而提高,且在MoS_(2)质量分数为50%时挠曲电压和挠曲电系数达到最优,分别为0.8 V和1.96 nC/m;该薄膜的挠曲电效应稳定性优异,串联多个PAN/MoS_(2)复合薄膜可获得较高的挠曲电压,且电压衰减极弱,可广泛适用于微小型可穿戴电子设备。

关键词：挠曲电效应 MoS_(2) 聚丙烯腈静电纺丝复合薄膜挠曲电系数可穿戴电子设备

Super enhancers targeting ZBTB16 in osteogenesis protect against osteoporosis

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骨研究:英文版 2023年第3期11卷 532-547页

作者： Wenhui Yu Zhongyu Xie Jinteng li Jiajie lin Zepeng Su Yunshu Che Feng Ye Zhaoqiang Zhang Peitao Xu Yipeng Zeng Xiaojun Xu zhikun li Pei Feng Rujia Mi Yanfeng Wu Huiyong Shen Department of Orthopedics The Eighth Affiliated HospitalSun Yat-sen UniversityShenzhen 518003PR China Shenzhen Key Laboratory of Ankylosing Spondylitis Shenzhen 518003PR China Department of Orthopedics Sun Yat-sen Memorial HospitalSun Yat-sen UniversityGuangzhou 510120PR China Center for Biotherapy The Eighth Affiliated HospitalSun Yat-sen UniversityShenzhen 518003PR China

As the major cell precursors in osteogenesis, mesenchymal stem cells(MSCs) are indispensable for bone homeostasis and development. However, the primary mechanisms regulating osteogenic differentiation are controversial. Composed of multiple constituent enhancers, super enhancers(SEs) are powerful cis-regulatory elements that identify genes that ensure sequential differentiation. The present study demonstrated that SEs were indispensable for MSC osteogenesis and involved in osteoporosis development. Through integrated analysis, we identified the most common SE-targeted and osteoporosis-related osteogenic gene,ZBTB16. ZBTB16, positively regulated by SEs, promoted MSC osteogenesis but was expressed at lower levels in osteoporosis.Mechanistically, SEs recruited bromodomain containing 4(BRD4) at the site of ZBTB16, which then bound to RNA polymerase IIassociated protein 2(RPAP2) that transported RNA polymerase Ⅱ(POL Ⅱ) into the nucleus. The subsequent synergistic regulation of POL Ⅱ carboxyterminal domain(CTD) phosphorylation by BRD4 and RPAP2 initiated ZBTB16 transcriptional elongation, which facilitated MSC osteogenesis via the key osteogenic transcription factor SP7. Bone-targeting ZBTB16 overexpression had a therapeutic effect on the decreased bone density and remodeling capacity of Brd4^(fl/fl)Prx1-cre mice and osteoporosis(OP) models.Therefore, our study shows that SEs orchestrate the osteogenesis of MSCs by targeting ZBTB16 expression, which provides an attractive focus and therapeutic target for osteoporosis.

关键词： B16 osteoporosis osteogenesis