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Journal of Genetics and Genomics 2024年第2期51卷 159-183页

作者： Yuxi Chen Xiao Luo Rui Kang Kaixin Cui Jianping Ou Xiya Zhang puping liang MOE Key Laboratory of Gene Function and Regulation State Key Laboratory of BiocontrolSchool of Life SciencesSun Yat-sen UniversityGuangzhouGuangdong 510275China Center for Reproductive Medicine The Third Affiliated Hospital of Sun Yat-sen UniversitySun Yat-sen UniversityGuangzhouGuangdong 510630China

Osteoarthritis(OA)is one of the most common degenerative joint diseases worldwide,causing pain,disability,and decreased quality of *** balance between regeneration and inflammation-induced degradation results in multiple etiologies and complex pathogenesis of ***,there is a lack of effective therapeutic strategies for OA *** the development of CRISPR-based genome,epigenome,and RNA editing tools,OA treatment has been improved by targeting genetic risk factors,activating chondrogenic elements,and modulating inflammatory *** by cell therapy and in vivo delivery vectors,genome,epigenome,and RNA editing tools may provide a promising approach for personalized OA *** review summarizes CRISPR-based genome,epigenome,and RNA editing tools that can be applied to the treatment of OA and provides insights into the development of CRISPR-based therapeutics for OA ***,in-depth evaluations of the efficacy and safety of these tools in human OA treatment are needed.

关键词： Osteoarthritis Genome editing Epigenone editing RNA editing

Correction of a CADASIL point mutation using adenine base editors in hiPsCsandbloodvessel organoids

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Journal of Genetics and Genomics 2024年第2期51卷 197-207页

作者： Jingwen Wang Lei Zhang Guanglan Wu Jinni Wu Xinyao Zhou Xiaolin Chen Yongxia Niu Yiren Jiao Qianyi Liu puping liang Guang Shi Xueqing Wu Junjiu Huang MOE Key Laboratory of Gene Function and Regulation State Key Laboratory of BiocontrolSchool of Life SciencesSun Yat-sen UniversityGuangzhouGuangdong 510275China Key Laboratory of Reproductive Medicine of Guangdong Province The First Affiliated Hospital and School of Life SciencesSun Yat-sen UniversityGuangzhouGuangdong 510275China Center of Reproductive Medicine Children's Hospital of Shanxi and Women Health Center of ShanxiTaiyuanShanxi 030013China

Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is a monogenic small vessel disease caused by mutations in the NOTCH3 gene. However, the pathogenesis of CADASIL remains unclear, and patients have limited treatment options. Here, we use human induced pluripotent stem cells (hiPSCs) generated from the peripheral blood mononuclear cells of a patient with CADASIL carrying a heterozygous NOTCH3 mutation (c.1261C>T, p.R421C) to develop a disease model. The correction efficiency of different adenine base editors (ABEs) is tested using the HEK293T-NOTCH3 reporter cell line. ABEmax is selected based on its higher efficiency and minimization of predicted off-target effects. Vascular smooth muscle cells (VSMCs) differentiated from CADASIL hiPSCs show NOTCH3 deposition and abnormal actin cytoskeleton structure, and the abnormalities are recovered in corrected hiPSC-derived VSMCs. Furthermore, CADASIL blood vessel organoids generated for in vivo modeling show altered expression of genes related to disease phenotypes, including the downregulation of cell adhesion, extracellular matrix organization, and vessel development. The dual adeno-associated virus (AAV) split-ABEmax system is applied to the genome editing of vascular organoids with an average editing efficiency of 8.82%. Collectively, we present potential genetic therapeutic strategies for patients with CADASIL using blood vessel organoids and the dual AAV split-ABEmax system.

关键词： CADASIL Induced pluripotent stem cells Adenine base editors AAV Blood vessel Organoid

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基因治疗在遗传代谢性肝病中的应用进展

临床内科杂志 2024年第4期41卷 249-253页

作者：张银纯梁普平郝虎中山大学附属第六医院广州510655 中山大学生命科学学院

遗传代谢性肝病(IMLD)是由于基因缺陷而导致肝脏代谢异常的一大类疾病,其病因复杂多样,临床表现差异大,以黄疸、肝脏肿大、肝酶增高和低血糖为主要表现,多数在婴幼儿和儿童期发病。目前对于IMLD较为有效的治疗方法是进行活体肝移植和肝... 详细信息

遗传代谢性肝病(IMLD)是由于基因缺陷而导致肝脏代谢异常的一大类疾病,其病因复杂多样,临床表现差异大,以黄疸、肝脏肿大、肝酶增高和低血糖为主要表现,多数在婴幼儿和儿童期发病。目前对于IMLD较为有效的治疗方法是进行活体肝移植和肝再生疗法,但供体肝脏稀缺、手术风险高、免疫排斥反应等风险导致其临床应用存在较大限制。基因治疗是指通过导入外源正常基因或修饰细胞内源突变基因来治疗疾病的一种生物医学手段。随着科技与医疗上的发展与进步,日益成熟的基因治疗手段被认为是除肝移植外的一种更高效、更安全地彻底治愈IMLD的方法。本文将重点对IMLD基因治疗的进展和现状进行论述。

关键词：遗传代谢性肝病基因治疗现状和进展

Homology-based repair induced by CRISPR-Cas nucleases in mammalian embryo genome editing

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Protein & Cell 2022年第5期13卷 316-335页

作者： Xiya Zhang Tao Li Jianping Ou Junjiu Huang puping liang Center for Reproductive Medicine the Third Affiliated Hospital of Sun Yat-sen UniversitySun Yat-sen UniversityGuangzhou 510630China MOE Key Laboratory of Gene Function and Regulation State Key Laboratory of BiocontrolSchool of Life SciencesSun Yat-sen UniversityGuangzhou 510275China Key Laboratory of Reproductive Medicine of Guangdong Province the First Affiliated Hospital and School of Life SciencesSun Yat-sen UniversityGuangzhou 510275China

Recent advances in genome editing,especially CRISPR-Cas nucleases,have revolutionized both laboratory research and clinical ***-Cas nucleases,together with the DNA damage repair pathway in cells,enable both genetic diversification by classical non-homologous end joining(c-NHEJ)and precise genome modification by homology-based repair(HBR).Genome editing in zygotes is a convenient way to edit the germline,paving the way for animal disease model generation,as well as human embryo genome editing therapy for some life-threatening and incurable *** efficiency is highly dependent on the DNA donor that is utilized as a repair ***,we review recent progress in improving CRISPR-Cas nuclease-induced HBR in mammalian embryos by designing a suitable DNA ***,we want to provide a guide for producing animal disease models and correcting genetic mutations through CRISPR-Cas nuclease-induced HBR in mammalian ***,we discuss recent developments in precise genome-modification technology based on the CRISPR-Cas system.

关键词： homology-based repair(HBR) genome editing disease modeling embryo precision medicine

Systematic identification of CRISPR off-target effects by CROss-seq

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Protein & Cell 2023年第4期14卷 299-303页

作者： Yan Li Shengyao Zhi Tong Wu Hong-Xuan Chen Rui Kang Dong-Zhao Mai Zhou Songyang Chuan He puping liang Guan-Zheng Luo IMOE Key Laboratory of Gene Function and Regulation Guangdong Province Key Laboratory of Pharmaceutical Functional GenesState Key Laboratory of BiocontrolSchool of Life SciencesSun Yat-sen UniversityGuangzhou 510275China Department of Chemistry University of ChicagoChicagoIL 60637USA Institute for Biophysical Dynamics Department of Biochemistry and Molecular BiologyHoward Hughes Medical InstituteUniversity of ChicagoChicagoIL 60637 USA

DearEditor,The CRISPR-mediated genome editing tools,including nucleases,base editors(ABE/CBE),transposases/recombinases,and prime editor(PE),have been extensively applied in basic and clinical researches,although the off-target effect remains a major concern(Anzalone et al.,2020).Recently,various methods have been developed to assess the specificity and accuracy of different tools(Zhang et al.,2021),yet each method is designed for limited editing systems,and none of them can simultaneously detect off-target sites in vivo and in vitro.A versatile method for profiling genome-wide off-target effects of various tools remains lacking.

关键词： CRISPR versatile editor

CRISPR-assisted transcription activation by phaseseparation proteins

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Protein & Cell 2023年第12期14卷 874-887页

作者： Jiaqi Liu Yuxi Chen Baoting Nong Xiao Luo Kaixin Cui Zhan Li Pengfei Zhang Wenqiong Tan Yue Yang Wenbin Ma puping liang Zhou Songyang State Key Laboratory of Biocontrol MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging ResearchSchool of Life SciencesSun Yat-senUniversityGuangzhou510275China Sun Yat-sen Memorial Hospital Sun Yat-sen UniversityGuangzhou 510275China Lumiere Therapeutics Suzhou215000China

The clustered regularly interspaced short palindromic repeats(CRISPR)-Cas9 system has been widely used for genome engineering and transcriptional regulation in many different *** CRISPR-activation(CRISPRa)platforms often require multiple components because of inefficient transcriptional ***,we fused different phase-separation proteins to dCas9-VPR(dCas9-VP64-P65-RTA)and observed robust increases in transcriptional activation ***,human NUP98(nucleoporin 98)and FUS(fused in sarcoma)IDR domains were best at enhancing dCas9-VPR activity,with dCas9-VPR-FUS IDR(VPRF)outperforming the other CRISPRa systems tested in this study in both activation efficiency and system ***9-VPRF overcomes the target strand bias and widens gRNA designing windows without affecting the off-target effect of *** findings demonstrate the feasibility of using phase-separation proteins to assist in the regulation of gene expression and support the broad appeal of the dCas9-VPRF system in basic and clinical applications.

关键词： CRISPR transcriptional activation phase-separation proteins

Off-target effects of cytidine base editor and adenine base editor:What can we do?

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Journal of Genetics and Genomics 2019年第11期46卷 509-512页

作者： puping liang Jinkun Wen Junjiu Huang Key Laboratory of Reproductive Medicine of Guangdong Province School of Life Sciencesand the First Affiliated HospitalSun Yat-sen UniversityGuangzhou510275China MOE Key Laboratory of Gene Function and Regulation and Guangzhou Key Laboratory of Healthy Aging Research SYSU-BCM Joint Research CenterSchool of Life SciencesSun Yat-sen UniversityGuangzhou510275China Jiangmen Central Hospital Affiliated Jiangmen Hospital of Sun Yat-sen UniversityGuangzhou510275China Key Laboratory of Reproductive Medicine of Guangdong Province The Third Affiliated Hospital of Guangzhou Medical UniversityGuangzhou510275China

DNA is the blueprint of life,instructing the growth,development,and reproduction of an *** sequencing uncovers the codes of life,whereas genome editing could rewrite the codes,thus then leads to revolutionary advances in all aspects of life sciences,such as uncovering regulatory network of life,increasing the production of crops,producing new breeds of livestock,and curing genetic disorders(Liu,2017).

关键词： Off-target effects editor

Telomere regulation in pluripotent stem cells

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Protein & Cell 2014年第3期5卷 194-202页

作者： Yan Huang puping liang Dan Liu Junjiu Huang Zhou Songyang Key Laboratory of Reproductive Medicine of Guangdong Province the First Affiliated Hospital and Key Laboratory of Gene_ Engineering of the Ministry of Education School of Life Sciences Sun Yat-sen University Guangzhou 510275 China Cell-Based Assay Screening Core Baylor College of Medicine One Baylor Plaza Houston TX 77030 USA Dan L. Duncan Cancer Center Baylor'College of Medicine one Baylor'Plaza Houston TX 77030 uSA Verna and Marrs Mclean Department of Biochemistry and Molecular Biology Baylor College of Medicine One Baylor Plaza Houston TX 77030 USA

Pluripotent stem cells （PSCs） have the potential to pro- duce any types of cells from all three basic germ layers and the capacity to self-renew and proliferate indefinitely in vitro. The two main types of PSCs, embryonic stem cells （ESCs） and induced pluripotent stem cells （iPSCs）, share common features such as colony morphology, high expression of Oct4 and Nanog, and strong alkaline phosphatase activity. In recent years, increasing evi- dences suggest that telomere length represents another important internal factor in maintaining stem cell pluri- potency. Telomere length homeostasis and its structural integrity help to protect chromosome ends from recom- bination, end fusion, and DNA damage responses, ensuring the divisional ability of mammalian cells. PSCs generally exhibit high telomerase activity to maintain their extremely long and stable telomeres, and emerging data indicate the alternative lengthening of telomeres （ALT） pathway may play an important role in telomere functions too. Such characteristics are likely key to their abilities to differentiate into diverse cell types in v/vo. In this review, we will focus on the function and regulation of telomeres in ESCs and iPSCs, thereby shedding light on the importance of telomere length to pluripotency and the mechanisms that regulate telomeres in PSCs.

关键词： telomere pluripotent stem cells alternativelengthening of telomeres (ALT) regulators

Production of non-mosaic genome edited porcine embryos by injection of CRISPR/Cas9 into germinal vesicle oocytes

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Journal of Genetics and Genomics 2019年第7期46卷 335-342页

作者： Xiaohu Su Wei Chen Qingqing Cai puping liang Yaosheng Chen Peiqing Cong Junjiu Huang Key Laboratory of Reproductive Medicine of Guangdong Province First Affiliated Hospital and School of Life Sciences Sun Yat-sen University Guangzhou 510275 China MOE Key Laboratory of Gene Function and Regulation State Key Laboratory of Biocontrol School of Life Sciences Sun Yat-sen University Guangzhou 510275 China Key Laboratory of Reproductive Medicine of Guangdong Province Third Affiliated Hospital of Guangzhou Medical University Guangzhou 510150 China

Genetically modified pigs represent a great promise for generating models of human diseases and producing new *** of genetically edited pigs using somatic cell nuclear transfer(SCNT)or zygote cytoplasmic microinjection is a tedious process due to the low developmental rate or mosaicism of the founder(FO).Herein,we developed a method termed germinal vesicle oocyte gene editing(GVGE)to produce non-mosaic porcine embryos by editing maternal alleles during the GV to MII *** of Cas9 mRNA and X-linked Dmd gene-specific gRNA into GV oocytes did not affect their developmental *** MII oocytes edited during in vitro maturation(IVM)could develop into blastocysts after parthenogenetic activation(PA)or in vitro fertilization(IVF).Genotyping results indicated that the maternal gene X-linked Dmd could be efficiently edited during oocyte *** to81.3% of the edited IVF embryos were non-mosaic Dmd gene mutant *** conclusion,GVGE might be a valuable method for the generation of non-mosaic maternal allele edited FO embryos in a short simple step.

关键词： Porcine Germinal vesicle(GV) In vitro maturation CRISPR/Cas9 Dmd

Effective generation of maternal genome point mutated porcine embryos by injection of cytosine base editor into germinal vesicle oocytes

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Science China(Life Sciences) 2020年第7期63卷 996-1005页

作者： Xiaohu Su Wei Chen Qingqing Cai puping liang Yaosheng Chen Peiqing Cong Junjiu Huang Key Laboratory'of Reproductive Medicine of Guangdong Province the First Affiliated Hospital and School of Life SciencesSun Yat-sen UniversityGuangzhou 510275China MOE Key Laboratory of Gene Function and Regulation State Key Laboratory of BiocontrolSchool of Life SciencesSun Yat-sen UniversityGuangzhou 510275China Key Laboratory of Reproductive Medicine of Guangdong Province the Third Affiliated Hospital of Guangzhou Medical University'Guangzhou 510150China

Cytosine and adenine base editors are promising new tools for introducing precise genetic modifications that are required to generate disease models and to improve traits in pigs. Base editors can catalyze the conversion of C→T(C>T) or A→G(A>G) in the target site through a single guide RNA. Injection of base editors into the zygote cytoplasm can result in the production of offspring with precise point mutations, but most F0 are mosaic, and breeding of F1 heterozygous pigs is time-intensive. Here, we developed a method called germinal vesicle oocyte base editing(GVBE) to produce point mutant F0 porcine embryos by editing the maternal alleles during the GV to MⅡ transition. Injection of cytosine base editor 3(BE3) mRNA and X-linked Dmdspecific guide RNAs into GVoocytes efficiently edited maternal Dmd during in vitro maturation and did not affect the maturation potential of the oocytes. The edited MⅡ oocytes developed into blastocysts after parthenogenetic activation(PA) or in vitro fertilization(IVF). However, BE3 may reduce the developmental potential of IVF blastocysts from 31.5%±0.8% to 20.4%±2.1%. There 40%–78.3% diploid PA blastocysts had no more than two different alleles, including up to 10% embryos that had only C>T mutation alleles. Genotyping of IVF blastocysts indicated that over 70% of the edited embryos had one allele or two different alleles of Dmd. Since the male embryos had only a copy of Dmd allele, all five(5/19) F0 male embryos are homozygous and three of them were Dmd precise C>T mutation. Nine(9/19) female IVF embryos had two different alleles including a WT and a C>T mutation. DNA sequencing showed that some of them might be heterozygous embryos. In conclusion, the GVBE method is a valuable method for generating F0 embryos with maternal point mutated alleles in a single step.

关键词： cytosine base editor point mutation porcine germinal vesicle oocyte maternal genome Dmd