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GREPore-seq: A Robust Workflow to Detect Changes After Gene Editing Through Long-range PCR and Nanopore Sequencing

Genomics, Proteomics & Bioinformatics 2023年第6期21卷 1221-1236页

作者： Zi-Jun Quan Si-Ang Li zhi-xue yang Juan-Juan Zhao Guo-Hua Li Feng Zhang Wei Wen Tao Cheng Xiao-Bing Zhang State Key Laboratory of Experimental Hematology National Clinical Research Center for Blood DiseasesHaihe Laboratory of Cell EcosystemInstitute of Hematology&Blood Diseases HospitalChinese Academy of Medical Sciences&Peking Union Medical CollegeTianjin 300020China Center for Stem Cell Medicine Chinese Academy of Medical SciencesTianjin 300020China Department of Stem Cell&Regenerative Medicine Peking Union Medical CollegeTianjin 300020China

To achieve the enormous potential of gene-editing technology in clinical therapies,one needs to evaluate both the on-target efficiency and unintended editing consequences ***,there is a lack of a pipelined,large-scale,and economical workflow for detecting genome editing outcomes,in particular insertion or deletion of a large ***,we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR/Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR *** the high error rates of Oxford nanopore sequencing,we developed a novel pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing(GREPore-seq).GREPore-seq can assess nonhomologous end-joining(NHEJ)-mediated double-stranded oligodeoxynucleotide(dsODN)insertions with comparable accuracy to Illumina next-generation sequencing(NGS).GREPore-seq also reveals a full spectrum of homology-directed repair(HDR)-mediated large gene knock-in,correlating well with the fluorescence-activated cell sorting(FACS)analysis *** note,we discovered low-level fragmented and full-length plasmid backbone insertion at the CRISPR cutting ***,we have established a practical workflow to evaluate various genetic changes,including quantifying insertions of short dsODNs,knock-ins of long pieces,plasmid insertions,and large fragment deletions after CRISPR/Cas9-mediated ***-seq is freely available at GitHub(https://***/lisiang/GREPore-seq)and the National Genomics Data Center(NGDC)BioCode(https://***/biocode/tools/BT007293).

关键词： CRISPR/Cas9 Genetic change Long-range PCR Nanopore sequencing GREPore-seq

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Intracytoplasmic Sperm Injection Fertilization Rate Does Not-Depend on the Proportion of Round-headed Sperm, Small acrosomal Sperm, or Morphologically Normal Sperm in Patients with Partial Globozoospermia

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Chinese Medical Journal 2015年第12期128卷 1590-1595页

作者： Ling-Ying Jiang Ling-Yun yang Xiao-Mei Tong Hai-Yan Zhu Ya-Mei xue Wen-zhi Xu yang yang Song-Ying Zhang Department of Obstetrics and Gynecology Assisted Reproduction Unit Sir Run Run Shaw Hospital Zhejiang University School of Medicine Hangzhou Zhejiang 310016 China

Background：Generally,intracytoplasmic sperm injection （ICSI） may be the preferable method to treat partial globozoospermia,but whether there exist some correlations between ICSI fertilization rate and the proportion of round-headed sperm or morphologically normal sperm remains *** study was to explore the correlation between ICSI fertilization rate and the sperm morphology in patients with partial ***：Thirty-four patients diagnosed with partial globozoospermia accepted the following assisted fertilization treatments-2 cases accepted in-vitro fertilization （IvF） alone,26 cases accepted ICSI alone,and 6 accepted split IVF/*** morphological characteristics were described using Diff-Quik rapid *** cases accepting IVF or ICSI treatment in our reproductive center were considered as the control group after being matched by relevant *** rate,embryo quality,embryo implantation rate and clinical pregnancy rate were ***：Besides very high proportion of round-headed sperm,partial globozoospermia also showed very high proportion of small-acrosomal sperm and very low proportion of morphologically normal *** rate of IVF （IVF alone plus split IVF） was very low in partial globozoospermia （25.4% ± 17.4%）,but ICSI （ICSI alone plus split ICSI） achieved satisfying fertilization rate compared with the control group （66.2% ± 22.5% vs.68.8% ± 29.4%,P 〉 0.05）.In patients with partial globozoospermia,there were no correlations between ICSI fertilization rate and the proportion of round-headed sperm,small-acrosomal sperm,or morphologically normal ***：There was high proportion of small-acrosomal sperm in partial *** patients with partial globozoospermia,ICSI is more preferable than *** fertilization rate does not depend on the proportion of round-headed sperm,small-acrosomal sperm,or morphologically normal sperm.

关键词： Fertilization Rate Globozoospermia Intracytoplasmic Sperm Injection In-vitro Fertilization Sperm Acrosome

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Synthesis and Crystal Structure of a Novel Monomeric Di butyltin Dicarboxylate: nBu 2Sn O 2CCH 2CH(4 ClC 6H 4)Ge(OCH 2CH 2) 3N 2·H 2O

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Chinese Journal of Structural Chemistry 1999年第2期18卷 9-13页

作者： SONG xue Qing yang zhi Qiang XIE Qing Lan (National Laboratory of Elemento Organic Chemistry, Nankai University, Tianjin, 300071)

nBu 2Sn O 2CCH 2CH(4 ClC 6H 4)Ge(OCH 2CH 2) 3N 2·H 2O( M r =1053.66) is an air stable compound which crystallizes in the monoclinic space group C2/c with a=21.182(5), b=12.174(3), c=17.108(4) , β=99.59(2)°, V=4350(3)  3, Z=4, F(000)=2104, μ =2.104 mm -1 . The refinement of structure with I≥3σ(I) for 1819 reflections converged at R =0.045. The coordination geometry around the Sn atom is best described as an askew pentagonal bipyramid, in which four carboxylate oxygen atoms ( Sn(1)-O(5), Sn(1)-O(5a)=2.099  and Sn(1)-O(4), Sn(1)-O(4a)=2.158 ) and an oxygen atom from an aqua ligand comprise the pentagonal plane, with two butyl groups occupying axial positions.

关键词： diorganotin dicarboxylate crystal structure synthesis.

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Superior Fidelity and Distinct Editing Outcomes of SaCas9 Compared with SpCas9 in Genome Editing

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Genomics, Proteomics & Bioinformatics 2023年第6期21卷 1206-1220页

作者： zhi-xue yang Ya-Wen Fu Juan-Juan Zhao Feng Zhang Si-Ang Li Mei Zhao Wei Wen Lei Zhang Tao Cheng Jian-Ping Zhang Xiao-Bing Zhang State Key Laboratory of Experimental Hematology National Clinical Research Center for Blood DiseasesHaihe Laboratory of Cell EcosystemInstitute of Hematology&Blood Diseases HospitalChinese Academy of Medical Sciences&Peking Union Medical CollegeTianjin 300020China Center for Stem Cell Medicine Chinese Academy of Medical SciencesTianjin 300020China Department of Stem Cell&Regenerative Medicine Peking Union Medical CollegeTianjin 300020China

A series of clustered regularly interspaced short palindromic repeats(CRISPR)-CRISPR associated protein 9(Cas9)systems have been engineered for genome *** most widely used Cas9 is SpCas9 from Streptococcus pyogenes and SaCas9 from Staphylococcus ***,a comparison of their detailed gene editing outcomes is still *** characterizing the editing outcomes of 11 sites in human induced pluripotent stem cells(iPSCs)and K562 cells,we found that SaCas9 could edit the genome with greater efficiencies than *** also compared the effects of spacer lengths of single-guide RNAs(sgRNAs;18–21 nt for SpCas9 and 19–23 nt for SaCas9)and found that the optimal spacer lengths were 20 nt and 21 nt for SpCas9 and SaCas9,***,the optimal spacer length for a particular sgRNA was 18–21 nt for SpCas9 and 21–22 nt for ***,SpCas9 exhibited a more substantial bias than SaCas9 for nonhomologous end-joining(NHEJ)+1 insertion at the fourth nucleotide upstream of the protospacer adjacent motif(PAM),indicating a characteristic of a staggered ***,editing with SaCas9 led to higher efficiencies of NHEJ-mediated double-stranded oligodeoxynucleotide(dsODN)insertion or homology-directed repair(HDR)-mediated adeno-associated virus serotype 6(AAV6)donor ***,GUIDE-seq analysis revealed that SaCas9 exhibited significantly reduced off-target effects compared with *** work indicates the superior performance of SaCas9 to SpCas9 in transgene integration-based therapeutic gene editing and the necessity to identify the optimal spacer length to achieve desired editing results.

关键词： SpCas9 SaCas9 Spacer length Indel pattern Knock-in efficiency Off-target

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CP-25 reverses prostaglandin E4 receptor desensitization-induced fibroblast-like synoviocyte dysfunction via the G protein-coupled receptor kinase 2 in autoimmune arthritis

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Acta Pharmacologica Sinica 2019年第8期40卷 1029-1039页

作者： Xiao-yi Jia Yan Chang Fang Wei Xing Dai Yu-jing Wu Xiao-jing Sun Shu Xu Hua-xun Wu Chun Wang xue-zhi yang Wei Wei Institute of Clinical Pharmacology Anhui Medical University Key Laboratory of Anti-inflammatory and Immune Medicine Ministry of Education Anhui Collaborative Innovation Center of Anti-inflammatory and Immune Medicine School of Pharmacy Anhui University of Chinese Medicine

Paeoniflorin-6’-O-benzene sulfonate(CP-25)is a novel compound derived from paeoniflorin that has been demonstrated to have therapeutic effects in a rat model of rheumatoid arthritis(RA).However,the underlying mechanism has not been elucidated to *** explored this mechanism in the present study by treating rats with adjuvant arthritis(AA)with *** found that the membrane EP4 protein level was downregulated;whereas,GRK2 was upregulated,in fibroblast-like synoviocyte(FLS)s of AA ***(PGE)2 stimulated FLS proliferation and enhanced the membrane EP4 receptor protein level;the latter was reversed by the administration of an EP4 receptor agonist,whereas the membrane GRK2 protein level gradually *** changes in the EP4 receptor and GRK2 expression were enhanced by TNF-α,and the former was accompanied by an alteration in the cyclic(c)AMP *** EP4 receptor agonist stimulation increased the association between GRK2 and the EP4 ***2 knockdown abrogated the abnormalities in FLS *** CP-25 treatment(100 mg/kg)suppressed joint inflammation with an efficacy that was similar to that of *** finding was associated with EP4 upregulation and GRK2 downregulation in ***,GRK2 plays an important role in the abnormal FLS proliferation observed in AA possibly by promoting EP4 receptor desensitization and decreasing the cAMP *** results demonstrate that CP-25 has therapeutic potential for the treatment of human RA via GRK2 regulation.

关键词： adjuvant-induced arthritis paeoniflorin-6’-O-benzene sulfonate fibroblast-like synoviocytes G protein-coupled receptor kinase 2 prostaglandin E2

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HDAC inhibitors improve CRISPR-mediated HDR editing efficiency in iPSCs

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Science China(Life Sciences) 2021年第9期64卷 1449-1462页

作者： Jian-Ping Zhang zhi-xue yang Feng Zhang Ya-Wen Fu Xin-Yue Dai Wei Wen Beldon Zhang Hannah Choi Wanqiu Chen Meredith Brown David Baylink Lei Zhang Hongyu Qiu Charles Wang Tao Cheng Xiao-Bing Zhang State Key Laboratory of Experimental Hematology National Clinical Research Center for Blood DiseasesInstitute of Hematology&Blood Diseases HospitalChinese Academy of Medical Sciences&Peking Union Medical CollegeTianjin 300020China School of Medicine Loma Linda UniversityLoma LindaCA 92354USA Center for Genomics School of MedicineLoma Linda UniversityLoma LindaCA 92350USA CAMS Key Laboratory of Gene Therapy for Blood Diseases Tianjin 300020China Center for Stem Cell Medicine Chinese Academy of Medical SciencesTianjin 300020China Department of Stem Cell&Regenerative Medicine Peking Union Medical CollegeTianjin 300020China Tianjin Key Laboratory of Gene Therapy for Blood Diseases Tianjin 300020China Center of Molecular and Translational Medicine Institution of Biomedical ScienceGeorgia State UniversityAtlantaGA 30303USA

Genome-edited human induced pluripotent stem cells(iPSCs)hold great promise for therapeutic ***,low editing efficiency has hampered the applications of CRISPR-Cas9 technology in creating knockout and homology-directed repair(HDR)-edited iPSC lines,particularly for silent *** is partially due to chromatin compaction,inevitably limiting Cas9 access to the target *** the six HDAC inhibitors we examined,vorinostat,or suberoylanilide hydroxamic acid(SAHA),led to the highest HDR efficiency at both open and closed loci,with acceptable *** inhibitors equally increased non-homologous end joining(NHEJ)editing efficiencies(~50%)at both open and closed loci,due to the considerable HDAC inhibitor-mediated increase in Cas9 and sgRNA ***,we observed more substantial HDR efficiency improvement at closed loci relative to open chromatin(2.8 vs.1.7-fold change).These studies provide a new strategy for HDRediting of silent genes in iPSCs.

关键词： HDAC inhibitors CRISPR-Cas9 genome editing iPSC

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Specific role of synovial macrophages in rheumatoid arthritis

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中国药理学与毒理学杂志 2017年第10期31卷 1026-1026页

作者： xue-zhi yang Yan CHANG Wei WEI Institute of Clinical Pharmacology Anhui Medical UniversityKey Laboratory of Anti-inflammatory and Immune MedicineMinistry of EducationAnhui Collaborative Innovation Center of Anti-inflammatory and Immune Medicine

Rheumatoid arthritis(RA)is an autoimmune disease,which is characterized by synovial *** sublining macrophages found in synovium is an early hallmark of RA and effective treatment results in their ***,the origin of these sublining macrophages in synovium(including infiltrated macrophages and tissue-resident macrophages)are still unknown both in animal models of arthritis and RA patients,let alone the differences and feature of these *** rheumatic synovium,macrophages are submitted to a large variety of micro-environmental signals which influence the phenotypic polarization and activation of *** the mechanisms and functional consequences of the heterogeneous macrophages will contribute to confirm their potential role in synovial inflammation ***,research on macrophage plasticity to soft-control their phenotypic polarization could lead to novel therapeutic approaches.

关键词： synovial macrophage polarization heterogeneity apoptosis rheumatoid arthritis

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Real-world evidence on the efficacy and safety of vonoprazanamoxicillin dual therapy for Helicobacter pylori treatment in elderly patients

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World Journal of Gastroenterology 2025年第1期31卷 68-78页

作者： Wen Gao Jing-Wen Li Hui Ye xue-zhi Zhang Jian-Xiang Liu Hong Cheng Department of Gastroenterology Peking University First HospitalBeijing 100034China School of Medicine Tsinghua MedicineTsinghua UniversityBeijing 100034China Department of Traditional Chinese Medicine and Integrative Medicine Peking University First HospitalBeijing 100034China

BACKGROUND A dual therapy regimen containing amoxicillin is a common treatment option for the eradication of Helicobacter pylori(***).While substantial research supports the efficacy and safety of vonoprazan and amoxicillin(VA)dual therapy in the general population,there is still a lack of studies specifically focusing on its safety in elderly *** To evaluate efficacy and safety of VA dual therapy as first-line or rescue treatment for *** in elderly *** As a real-world retrospective study,data were collected from elderly patients aged 60 years and above who accepted VA dual therapy(vonoprazan 20 mg twice daily+amoxicillin 1000 mg thrice daily for 14 days)for *** eradication in the Department of Gastroenterology at Peking University First Hospital between June 2020 and January *** status was evaluated by^(13)C-urease breath test 6 weeks after *** adverse events(AEs)during treatment were *** In total,401 cases were ***-one cases were excluded due to loss to follow-up,lack of re-examination,or unwillingness to take *** total of 380 included cases comprised 250 who received VA dual therapy as first-line treatment and 130 who received VA dual therapy as rescue *** was successfully eradicated in 239 cases(95.6%)in the first-line treatment group and 116 cases(89.2%)in the rescue treatment *** overall incidence of AEs was 9.5%for both ***,9.2%of patients experienced an AE in the first-line treatment group and 10.0%in the rescue treatment *** patients discontinued treatment due to AE,with a discontinuation rate of 1.3%.No serious AE *** The VA dual therapy regimen as a first-line treatment and a rescue therapy was effective and safe for elderly patients aged 60 and older.

关键词： Helicobacter pylori Vonoprazan Amoxicillin Dual therapy Elderly

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Poorly controlled type II diabetes mellitus significantly enhances postoperative chemoresistance in patients with stage III colon cancer

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World Journal of Gastroenterology 2025年第3期31卷 29-41页

作者： Ruo-Yu Guan Jia-Wei Wu Zi-Yun Yuan zhi-Yuan Liu Zi-Zhu Liu zhi-Cong Xiao Jing-Hui Li Cheng-zhi Huang Jun-Jiang Wang xue-Qing Yao Guangdong Cardiovascular Institute Guangdong Provincial People’s HospitalGuangdong Academy of Medical SciencesSouthern Medical UniversityGuangzhou 510080Guangdong ProvinceChina Department of Gastrointestinal Surgery Department of General SurgeryGuangdong Provincial People’s Hospital(Guangdong Academy of Medical Sciences)Southern Medical UniversityGuangzhou 510080Guangdong ProvinceChina Department of General Surgery Guangdong Provincial People’s Hospital Ganzhou Hospital(Ganzhou Municipal Hospital)Ganzhou 341000Jiangxi ProvinceChina Shantou University Medical College Shantou 515041Guangdong ProvinceChina School of Medicine South China University of TechnologyGuangzhou 510006Guangdong ProvinceChina Gannan Medical University Ganzhou 341000Jiangxi ProvinceChina

BACKGROUND Type II diabetes mellitus(T2DM)has been associated with increased risk of colon cancer(CC)and worse prognosis in patients with *** effects of T2DM on postoperative chemoresistance rate(CRR)and long-term disease-free survival(DFS)and overall survival(OS)in patients with stage III CC who receive curative resection remain *** To investigate whether T2DM or glycemic control is associated with worse postoperative survival outcomes in stage III *** This retrospective cohort study included 278 patients aged 40-75 years who underwent surgery for stage III CC from 2018 to *** on preoperative T2DM history,the patients were categorized into non-DM(n=160)and DM groups(n=118).The latter was further divided into well-controlled(n=73)and poorly controlled(n=45)groups depending on the status of glycemic ***,OS,and CRR were compared between the groups and Cox regression analysis was used to identify risk *** Patients in the DM and non-DM groups demonstrated similar DFS,OS,and CRR(DFS:72.03%vs 78.75%,P=0.178;OS:81.36%vs 83.12%,P=0.638;CRR:14.41%vs 7.5%,P=0.063).Poorly controlled DM was associated with a significantly worse prognosis and higher CRR than well-controlled DM(DFS:62.22%vs 78.07%,P=0.021;OS:71.11%vs 87.67%,P=0.011;CRR:24.40%vs 8.22%,P=0.015).High preoperative fasting plasma glucose[DFS:Hazard ratio(HR)=2.684,P<0.001;OS:HR=2.105,P=0.019;CRR:HR=2.214,P=0.005]and glycosylated hemoglobin levels(DFS:HR=2.344,P=0.006;OS:HR=2.119,P=0.021;CRR:HR=2.449,P=0.009)indicated significantly poor prognosis and high CRR,while T2DM history did not(DFS:HR=1.178,P=0.327;OS:HR=0.933,P=0.739;CRR:HR=0.997,P=0.581).CONCLUSION Increased preoperative fasting plasma glucose and glycosylated hemoglobin levels,but not T2DM history,were identified as risk factors associated with poor postoperative outcomes and high CRR in patients with stage III CC.

关键词： Colon cancer Chemoresistance Diabetes mellitus Prognosis Type II diabetes mellitus

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飞轮组件微振动对高分辨率光学卫星光轴的影响

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光学精密工程 2016年第10期24卷 2515-2522页

作者：李林王栋徐婧谭陆洋孔林程龙贾学志杨洪波中国科学院长春光学精密机械与物理研究所吉林长春130033 中国科学院大学北京100039 长光卫星技术有限公司吉林长春130033

为了研究高分辨率光学卫星星上飞轮的微振动对卫星成像质量的影响,分别建立了飞轮扰动模型和整星结构动力学模型。首先,对飞轮组件系统进行了地面扰动测试,对实测扰动数据的分析表明,飞轮组件在与转速相关的一阶频率50Hz处产生一次谐波,... 详细信息

为了研究高分辨率光学卫星星上飞轮的微振动对卫星成像质量的影响,分别建立了飞轮扰动模型和整星结构动力学模型。首先,对飞轮组件系统进行了地面扰动测试,对实测扰动数据的分析表明,飞轮组件在与转速相关的一阶频率50Hz处产生一次谐波,在190Hz与280Hz左右存在与转速无关的一系列峰值。然后,对整星进行了单位正弦激励,获得了光轴角位移响应,并对其与飞轮实测扰动数据进行了集成分析。分析结果表明：整星在50-80 Hz和230-280Hz的角位移响应有较多的谐振响应频率成分,沿光轴方向和垂直光轴方向整星光轴的角位移最大谐振响应幅值分别为2.718″、2.739″,在245Hz左右存在较多幅值为0.5″量级的谐波。分析显示飞轮组件微振动对高分辨率光学卫星成像质量影响较大,得到的结果可为整星系统的优化设计和隔振补偿措施提供参考依据。

关键词：高分辨率光学卫星飞轮微振动光轴角位移像质

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