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用于分枝杆菌碱基编辑的PAM扩展型嗜热链球菌Cas9 C到T和C到G碱基编辑器 Article

张洪源, 张翼飞, 王卫晓, 陈未中, 张侠, 黄行许, 陈伟, 季泉江

《工程（英文）》 2022年 第15卷 第8期   页码 67-77 doi: 10.1016/j.eng.2022.02.013

摘要： 依赖于规律成簇的间隔短回文重复序列（CRISPR）的碱基编辑器能够快速有效地进行碱基编辑和基因失活，然而，目前还没有开发出可用于MTB的碱基编辑器。通过筛选不同的碱基编辑器，发现广泛使用的酿脓链球菌CRISPR相关蛋白9（SpCas9）或毛螺科菌Cpf1（LbCpf1）胞嘧啶碱基编辑器在分枝杆菌中不具有活性，而嗜热链球菌Cas9（St1Cas9）胞嘧啶碱基编辑器活性较好虽然使用St1Cas9 胞嘧啶碱基编辑器能够实现C到T的碱基编辑，但是，在碱基编辑过程中却产生了大量的副产物。将尿嘧啶-N-糖基化酶抑制剂或尿嘧啶-N-糖基化酶分别融合到St1Cas9 胞嘧啶碱基编辑器中，得到了两种新的碱基编辑器——CTBE和CGBE此外，由于野生型St1Cas9 在靶向DNA序列时需要识别严格的前间隔序列邻近基序（PAM），因此，通过结构介导的蛋白质工程设计了PAM扩展的St1Cas9，扩大了碱基编辑器的靶向范围。

关键词： CRISPR     Cas9     结核分枝杆菌     基因编辑     碱基编辑    

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Generation of CRISPR/Cas9-mediated lactoferrin-targeted mice by pronuclear injection of plasmid pX330

Mengxu GE,Fei LIU,Fei CHANG,Zhaolin SUN,Jing FEI,Ying GUO,Yunping DAI,Zhengquan YU,Yaofeng ZHAO,Ning LI,Qingyong MENG

《农业科学与工程前沿（英文）》 2015年 第2卷 第3期   页码 242-248 doi: 10.15302/J-FASE-2015059

摘要： Lactoferrin is a member of the transferrin family of multifunctional iron binding glycoproteins. While numerous physiological functions have been described for lactoferrin, the mechanisms underlying these functions are not clear. To further study the functions and mechanisms of lactoferrin, we modified the lactoferrin promoter of mice using the CRISPR/Cas9 system to reduce or eliminate lactoferrin expression. Seven mice with lactoferrin promoter mutations were obtained with an efficiency of 24% (7/29) by injecting the plasmid pX330, expressing a small guide RNA and human codon-optimized SpCas9, into fertilized eggs of mice. Plasmid integration and off-targeting of pX330 were not detected. These results confirmed that pronuclear injection of a circular plasmid is a feasible and efficient method for targeted mutagenesis in mice.

关键词： lactoferrin     promoter     CRISPR/Cas9     plasmid pX330    

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One-step generation of myostatin gene knockout sheep via the CRISPR/Cas9 system

Hongbing HAN,Yonghe MA,Tao WANG,Ling LIAN,Xiuzhi TIAN,Rui HU,Shoulong DENG,Kongpan LI,Feng WANG,Ning LI,Guoshi LIU,Yaofeng ZHAO,Zhengxing LIAN

《农业科学与工程前沿（英文）》 2014年 第1卷 第1期   页码 2-5 doi: 10.15302/J-FASE-2014007

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Mobile CRISPR-Cas9 based anti-phage system in E. coli

《化学科学与工程前沿（英文）》 2022年 第16卷 第8期   页码 1281-1289 doi: 10.1007/s11705-022-2141-7

摘要： Escherichia coli is one of the most important microbial cell factories, but infection by bacteriophages in the environment may have a huge impact on its application in industrial production. Here, we developed a mobile CRISPR-Cas9 based anti-phage system for bacteriophages defense in E. coli. Two conjugative plasmids pGM1 (phosphoglucomutase 1) and pGM2 carrying one and two guide RNAs, respectively, were designed to defend against a filamentous phage. The results showed that the pGM1 and pGM2 could decrease the phage infection rate to 1.6% and 0.2% respectively in infected cells. For preventing phage infection in E. coli, the pGM2 decreased the phage infection rate to 0.1%, while pGM1 failed to block phage infection. Sequence verification revealed that point mutations in protospacer or protospacer adjacent motif sequences of the phage genome caused loss of the defense function. These results support the potential application of MCBAS in E. coli cell factories to defend against phage infections.

关键词： phage infections     anti-phage     CRISPR-Cas9     conjugative transfer     synthetic biology    

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CRISPR-Cas9 mediated LAG-3 disruption in CAR-T cells

null

《医学前沿（英文）》 2017年 第11卷 第4期   页码 554-562 doi: 10.1007/s11684-017-0543-6

摘要：

T cells engineered with chimeric antigen receptor (CAR) have been successfully applied to treat advanced refractory B cell malignancy. However, many challenges remain in extending its application toward the treatment of solid tumors. The immunosuppressive nature of tumor microenvironment is considered one of the key factors limiting CAR-T efficacy. One negative regulator of T cell activity is lymphocyte activation gene-3 (LAG-3). We successfully generated LAG-3 knockout T and CAR-T cells with high efficiency using CRISPR-Cas9 mediated gene editing and found that the viability and immune phenotype were not dramatically changed during in vitro culture. LAG-3 knockout CAR-T cells displayed robust antigen-specific antitumor activity in cell culture and in murine xenograft model, which is comparable to standard CAR-T cells. Our study demonstrates an efficient approach to silence immune checkpoint in CAR-T cells via gene editing.

关键词： CAR-T     CRISPR-Cas9     LAG-3    

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Embryo-mediated genome editing for accelerated genetic improvement of livestock

Zachariah MCLEAN, Björn OBACK, Götz LAIBLE

《农业科学与工程前沿（英文）》 2020年 第7卷 第2期   页码 148-160 doi: 10.15302/J-FASE-2019305

摘要：

Selecting beneficial DNA variants is the main goal of animal breeding. However, this process is inherently inefficient because each animal only carries a fraction of all desirable variants. Genome editing technology with its ability to directly introduce beneficial sequence variants offers new opportunities to modernize animal breeding by overcoming this biological limitation and accelerating genetic gains. To realize rapid genetic gain, precise edits need to be introduced into genomically-selected embryos, which minimizes the genetic lag. However, embryo-mediated precision editing by homology-directed repair (HDR) mechanisms is currently an inefficient process that often produces mosaic embryos and greatly limits the numbers of available edited embryos. This review provides a summary of genome editing in bovine embryos and proposes an embryo-mediated accelerated breeding scheme that overcomes the present efficiency limitations of HDR editing in bovine embryos. It integrates embryo-based genomic selection with precise multi-editing and uses embryonic cloning with elite edited blastomeres or embryonic pluripotent stem cells to resolve mosaicism, enable multiplex editing and multiply rare elite genotypes. Such a breeding strategy would enable a more targeted, accelerated approach for livestock improvement that allows stacking of beneficial variants, even including novel traits from outside the breeding population, in the most recent elite genetic background, essentially within a single generation.

关键词： animal breeding     cattle     cloning     CRISPR/Cas9     cytoplasmic injection     embryo     genome editing     germline chimaeras     HDR     livestock improvement     TALENs    

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is essential for the integrity of stereociliary rootlet in cochlear hair cells in mice

Yuqin Men, Xiujuan Li, Hailong Tu, Aizhen Zhang, Xiaolong Fu, Zhishuo Wang, Yecheng Jin, Congzhe Hou, Tingting Zhang, Sen Zhang, Yichen Zhou, Boqin Li, Jianfeng Li, Xiaoyang Sun, Haibo Wang, Jiangang Gao

《医学前沿（英文）》 2019年 第13卷 第6期   页码 690-704 doi: 10.1007/s11684-018-0638-8

摘要： encodes the taperin protein, which is concentrated in the tapered region of hair cell stereocilia in the inner ear. In humans, mutations cause autosomal recessive nonsyndromic deafness (DFNB79) by an unknown mechanism. To determine the role of in hearing, we generated -null mice by clustered regularly interspaced short palindromic repeat/Cas9 genome-editing technology from a CBA/CaJ background. We observed significant hearing loss and progressive degeneration of stereocilia in the outer hair cells of -null mice starting from postnatal day 30. Transmission electron microscopy images of stereociliary bundles in the mutant mice showed some stereociliary rootlets with curved shafts. The central cores of the stereociliary rootlets possessed hollow structures with surrounding loose peripheral dense rings. Radixin, a protein expressed at stereocilia tapering, was abnormally dispersed along the stereocilia shafts in null mice. The expression levels of radixin and -actin significantly decreased. We propose that is critical to the retention of the integrity of the stereociliary rootlet. Loss of in -null mice caused the disruption of the stereociliary rootlet, which resulted in damage to stereociliary bundles and hearing impairments. The generated -null mice are ideal models of human hereditary deafness DFNB79.

关键词： TPRN     stereocilia     stereociliary rootlet     actin filament     CRISPR/Cas9     hearing    

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转录因子HNF1A、HNF4A和FOXA2调节肝细胞蛋白质N-糖基化 Article

Vedrana Vičić Bočkor,Nika Foglar,Goran Josipović,Marija Klasić,Ana Vujić,Branimir Plavša,Toma Keser,Samira Smajlović,Aleksandar Vojta,Vlatka Zoldoš

《工程（英文）》 2024年 第32卷 第1期   页码 58-69 doi: 10.1016/j.eng.2023.09.019

摘要：

Hepatocyte nuclear factor 1 alpha (HNF1A), hepatocyte nuclear factor 4 alpha (HNF4A), and forkhead box protein A2 (FOXA2) are key transcription factors that regulate a complex gene network in the liver, creating a regulatory transcriptional loop. The Encode and ChIP-Atlas databases identify the recognition sites of these transcription factors in many glycosyltransferase genes. Our in silico analysis of HNF1A, HNF4A, and FOXA2 binding to the 10 candidate glyco-genes studied in this work confirms a significant enrichment of these transcription factors specifically in the liver. Our previous studies identified HNF1A as a master regulator of fucosylation, glycan branching, and galactosylation of plasma glycoproteins. Here, we aimed to functionally validate the role of the three transcription factors on downstream glyco-gene transcriptional expression and the possible effect on glycan phenotype. We used the state-of-the-art clustered regularly interspaced short palindromic repeats/dead Cas9 (CRISPR/dCas9) molecular tool for the downregulation of the HNF1A, HNF4A, and FOXA2 genes in HepG2 cells—a human liver cancer cell line. The results show that the downregulation of all three genes individually and in pairs affects the transcriptional activity of many glyco-genes, although downregulation of glyco-genes was not always followed by an unambiguous change in the corresponding glycan structures. The effect is better seen as an overall change in the total HepG2 N-glycome, primarily due to the extension of biantennary glycans. We propose an alternative way to evaluate the N-glycome composition via estimating the overall complexity of the glycome by quantifying the number of monomers in each glycan structure. We also propose a model showing feedback loops with the mutual activation of HNF1A–FOXA2 and HNF4A–FOXA2 affecting glyco-genes and protein glycosylation in HepG2 cells.

关键词： Clustered regularly interspaced short palindromic repeats/dead Cas9 (CRISPR/dCas9)     Epigenetics     Hepatocyte nuclear factor 1 alpha (HNF1A)     Hepatocyte nuclear factor 4 alpha (HNF4A)     Forkhead box protein A2 (FOXA2)     N-glycosylation     HepG2 cells    

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Genome-edited crops: how to move them from laboratory to market

Kunling CHEN, Caixia GAO

《农业科学与工程前沿（英文）》 2020年 第7卷 第2期   页码 181-187 doi: 10.15302/J-FASE-2020332

摘要：

Recent breakthroughs in CRISPR technology allow specific genome manipulation of almost all crops and have initiated a revolution in precision crop breeding. Rationally-based regulation and widespread public acceptance are needed to propel genome-edited crops from laboratory to market and to translate this innovative technology into agricultural productivity.

关键词： CRISPR/Cas     genome editing     base editing     precision breeding     regulation    

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全球首个CRISPR基因编辑疗法有望造福患者

Jennifer Welsh

《工程（英文）》 2023年 第30卷 第11期   页码 3-6 doi: 10.1016/j.eng.2023.09.005

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Acetylated HOXB9 at lysine 27 is of differential diagnostic value in patients with pancreatic ductal

Xiaoran Sun, Jiagui Song, Jing Zhang, Jun Zhan, Weigang Fang, Hongquan Zhang

《医学前沿（英文）》 2020年 第14卷 第1期   页码 91-100 doi: 10.1007/s11684-019-0696-6

摘要： Pancreatic ductal adenocarcinoma (PDAC) is the ninth most common human malignancy and the sixth leading cause of cancer-related death in China. AcK27-HOXB9 is a newly identified HOXB9 post-transcriptional modification that can predict the outcome in lung adenocarcinoma and colon cancer well. However, the role of AcK27-HOXB9 in PDAC is unclear. The present study aims to investigate the differential diagnostic role of patients with AcK27-HOXB9 PDAC. Tissue microarrays consisting of 162 pancreatic tumor tissue samples from patients with PDAC and paired normal subjects were used to examine HOXB9 and AcK27-HOXB9 levels and localizations by immunohistochemical analysis and Western blot assay, respectively. HOXB9 was upregulated ( <0.0001), and AcK27-HOXB9 ( =0.0023) was downregulated in patients with PDAC. HOXB9 promoted ( =0.0115), while AcK27-HOXB9 ( =0.0279) inhibited PDAC progression. AcK27-HOXB9 predicted favorable outcome in patients with PDAC ( =0.0412). AcK27-HOXB9 also suppressed PDAC cell migration in a cell migration assay. The results of this study showed that HOXB9 promoted and AcK27-HOXB9 suppressed PDAC progression. The determination of ratio between HOXB9 and AcK27-HOXB9 exhibited potential diagnostic value in patients with PDAC.

关键词： HOXB9     AcK27-HOXB9     PDAC    

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662 A/G gene variation in human tumor necrosis factor receptor superfamily, member 9 (TNFRSF9)

QU Yanchun, YANG Ze, SUN Liang, JI Linong

《医学前沿（英文）》 2008年 第2卷 第3期   页码 283-285 doi: 10.1007/s11684-008-0053-7

摘要： The aim of this paper is to report a new coding variance of the gene, a candidate for autoimmune diseases. We found the variation in two families with type 2 diabetes mellitus by D-HPLC mutation screening method and confirmed our results by direct sequencing and PCR-RFLP. Although without changing the amino acid coding, the variance may have an effect on codon usage and play a role in disease development, such as type 2 diabetes mellitus. However, we cannot define the role of this variance because the frequency of the minor allele is low in the Chinese population and no homozygote of the variance was found. More research in multiple populations will be necessary to define the role of this variance.

关键词： D-HPLC mutation     development     autoimmune     PCR-RFLP     candidate    

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The 9th China Engineering Management Forum Guangzhou Consensus

《工程管理前沿（英文）》 2015年 第2卷 第2期   页码 105-107 doi: 10.15302/J-FEM-2015019

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REDUCTION OF NON-POINT SOURCE POLLUTION IN THE YONG’AN RIVER BY CONSTRUCTED WETLAND BASED ON 9 YEARS

《农业科学与工程前沿（英文）》 2023年 第10卷 第4期   页码 627-638 doi: 10.15302/J-FASE-2023516

摘要：

The agricultural and livestock activities surrounding the rivers flowing into the lakes have caused non-point source pollution, leading to excessive amounts of nutrient salts in downstream rivers. Introducing river water into constructed wetlands along river course has proven to be an effective solution for decreasing nitrogen (N) and phosphorus (P) loads. This paper reports 9 years of monitoring the Yong’an River and its surrounding constructed wetlands in the upper reaches of Erhai Lake, located in Yunnan Province, China. This study analyzed the main types of pollutants in the river, and evaluated the removal efficiency of pollutants by the constructed wetlands. The findings indicate that total nitrogen (TN) and nitrate nitrogen (NO3–-N) are the primary pollutants in the Yong’an River, which exhibit variation throughout the year corresponding to the alternating wet and dry seasons. Although constructed wetlands are effective in removing NO3–-N and P, their efficacy in removing ammonium nitrogen (NH4+-N) and organic pollutants is limited. This limitation can be attributed to the lack of timely disposal of aquatic plant residues. This research contributes to the understanding of the potential issues that may arise during the extended use of constructed wetlands and provides solutions to address them.

关键词： inflowing rivers     surface-flow constructed wetland     nutrients     long-term monitoring    

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Construction of lentiviral vector carrying Rab9 gene and its expression in mouse brain

Youguo HAO, Min ZHANG, Jinzhi XU, Bitao BU, Jiajun WEI

《医学前沿（英文）》 2009年 第3卷 第2期   页码 141-147 doi: 10.1007/s11684-009-0041-6

摘要： Rab proteins and their effectors facilitate vesicular transport by tethering donor vesicles to their respective target membranes. Rab9 mediates late endosome-to- -Golgi-network trafficking. To explore the possibility of Rab9-related gene therapy for neurodegenerative diseases, we packed Lentivirus encoding Rab9. The expressing plasmid pCDH1-MCF1-Rab9-EF1-copGFP was constructed by using molecular biological techniques. The Lentivirus encoding Rab9 cDNA was packed by Lifectamine-2000 mediated co-transfection of the plasmid pPACKH1- , pPACKH1- and pVSV- into 293T cells. DNA sequencing proved the successful construction of pCDH1-MCF1-Rab9-EF1-copGFP. After 72 hours, the expression of GFP could be detected in BV-2 cells. Western blotting revealed that the Rab9 gene expression in BALB/c mice brain was up-regulated significantly 4 weeks after injection with Lentivirus encoding Rab9, which evidenced a satisfactory increasing effect of this virus. Administration of Lenti-Rab9 to postnatal day 3 Niemann-Pick disease type C (NPC) mice reduced motor defects and prevented the weight loss associated with female NPC mice, as well as modulating the death rate of Purkinje neurons. It is concluded that the packaging of Lentivirus encoding Rab9 was successful. Lentivirus encoding Rab9 can increase the expression of Rab9 gene effectively, which might offer a novel means for the treatment of neurodegenerative diseases.

关键词： Rab9     lentivirus     gene therapy     gene transfer    

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标题 作者 时间 类型 操作

用于分枝杆菌碱基编辑的PAM扩展型嗜热链球菌Cas9 C到T和C到G碱基编辑器

张洪源, 张翼飞, 王卫晓, 陈未中, 张侠, 黄行许, 陈伟, 季泉江

期刊论文

Generation of CRISPR/Cas9-mediated lactoferrin-targeted mice by pronuclear injection of plasmid pX330

Mengxu GE,Fei LIU,Fei CHANG,Zhaolin SUN,Jing FEI,Ying GUO,Yunping DAI,Zhengquan YU,Yaofeng ZHAO,Ning LI,Qingyong MENG

期刊论文

One-step generation of myostatin gene knockout sheep via the CRISPR/Cas9 system

Hongbing HAN,Yonghe MA,Tao WANG,Ling LIAN,Xiuzhi TIAN,Rui HU,Shoulong DENG,Kongpan LI,Feng WANG,Ning LI,Guoshi LIU,Yaofeng ZHAO,Zhengxing LIAN

期刊论文

Mobile CRISPR-Cas9 based anti-phage system in E. coli

期刊论文

CRISPR-Cas9 mediated LAG-3 disruption in CAR-T cells

null

期刊论文

Embryo-mediated genome editing for accelerated genetic improvement of livestock

Zachariah MCLEAN, Björn OBACK, Götz LAIBLE

期刊论文

is essential for the integrity of stereociliary rootlet in cochlear hair cells in mice

Yuqin Men, Xiujuan Li, Hailong Tu, Aizhen Zhang, Xiaolong Fu, Zhishuo Wang, Yecheng Jin, Congzhe Hou, Tingting Zhang, Sen Zhang, Yichen Zhou, Boqin Li, Jianfeng Li, Xiaoyang Sun, Haibo Wang, Jiangang Gao

期刊论文

转录因子HNF1A、HNF4A和FOXA2调节肝细胞蛋白质N-糖基化

Vedrana Vičić Bočkor,Nika Foglar,Goran Josipović,Marija Klasić,Ana Vujić,Branimir Plavša,Toma Keser,Samira Smajlović,Aleksandar Vojta,Vlatka Zoldoš

期刊论文

Genome-edited crops: how to move them from laboratory to market

Kunling CHEN, Caixia GAO

期刊论文

全球首个CRISPR基因编辑疗法有望造福患者

Jennifer Welsh

期刊论文

Acetylated HOXB9 at lysine 27 is of differential diagnostic value in patients with pancreatic ductal

Xiaoran Sun, Jiagui Song, Jing Zhang, Jun Zhan, Weigang Fang, Hongquan Zhang

期刊论文

662 A/G gene variation in human tumor necrosis factor receptor superfamily, member 9 (TNFRSF9)

QU Yanchun, YANG Ze, SUN Liang, JI Linong

期刊论文

The 9th China Engineering Management Forum Guangzhou Consensus

期刊论文

REDUCTION OF NON-POINT SOURCE POLLUTION IN THE YONG’AN RIVER BY CONSTRUCTED WETLAND BASED ON 9 YEARS

期刊论文

Construction of lentiviral vector carrying Rab9 gene and its expression in mouse brain

Youguo HAO, Min ZHANG, Jinzhi XU, Bitao BU, Jiajun WEI

期刊论文