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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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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基因编辑作物有望走上消费者的餐桌

Sarah C.P. Williams

《工程（英文）》 2023年第20卷第1期页码 6-8 doi: 10.1016/j.eng.2022.11.002

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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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Genome reprogramming for synthetic biology

Kylie Standage-Beier,Xiao Wang

《化学科学与工程前沿（英文）》 2017年第11卷第1期页码 37-45 doi: 10.1007/s11705-017-1618-2

摘要： The ability to go from a digitized DNA sequence to a predictable biological function is central to synthetic biology. Genome engineering tools facilitate rewriting and implementation of engineered DNA sequences. Recent development of new programmable tools to reengineer genomes has spurred myriad advances in synthetic biology. Tools such as clustered regularly interspace short palindromic repeats enable RNA-guided rational redesign of organisms and implementation of synthetic gene systems. New directed evolution methods generate organisms with radically restructured genomes. These restructured organisms have useful new phenotypes for biotechnology, such as bacteriophage resistance and increased genetic stability. Advanced DNA synthesis and assembly methods have also enabled the construction of fully synthetic organisms, such as J. Craig Venter Institute (JCVI)-syn 3.0. Here we summarize the recent advances in programmable genome engineering tools.

关键词： CRISPR genome engineering synthetic biology rational design

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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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人类基因编辑技术及其伦理问题

Peter Weiss

《工程（英文）》 2020年第6卷第7期页码 719-722 doi: 10.1016/j.eng.2020.05.010

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Advances in genetic engineering of domestic animals

Shaohua WANG,Kun ZHANG,Yunping DAI

《农业科学与工程前沿（英文）》 2016年第3卷第1期页码 1-10 doi: 10.15302/J-FASE-2016085

摘要： Global population will increase to over nine billion by 2050 with the doubling in demand for meat and milk. To overcome this challenge, it is necessary to breed highly efficient and productive livestock. Furthermore, livestock are also excellent models for human diseases and ideal bioreactors to produce pharmaceutical proteins. Thus, genetic engineering of domestic animals presents a critical and valuable tool to address these agricultural and biomedical applications. Overall, genetic engineering has evolved through three stages in history: transgenesis, gene targeting, and gene editing. Since the birth of the first transgenic pig, genetic engineering in livestock has been advancing slowly due to inherent technical limitations. A major breakthrough has been the advent of somatic cell nuclear transfer, which, for the first time, provided the technical ability to produce site-specific genome-modified domestic animals. However, the low efficiency of gene targeting events in somatic cells prohibits its wide use in agricultural and biomedical applications. Recently, rapid progress in tools and methods of genome engineering has been made, allowing genetic editing from mutation of a single base pair to the deletion of entire chromosomes. Here, we review the major advances of genetic engineering in domestic animals with emphasis placed on the introduction of latest designer nucleases.

关键词： CRISPR TALEN ZFN gene editing livestock pig cattle

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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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Livestock breeding for the 21st century: the promise of the editing revolution

Chris PROUDFOOT, Gus MCFARLANE, Bruce WHITELAW, Simon LILLICO

《农业科学与工程前沿（英文）》 2020年第7卷第2期页码 129-135 doi: 10.15302/J-FASE-2019304

摘要：

In recent years there has been a veritable explosion in the use of genome editors to create site-specific changes, both and , to the genomes of a multitude of species for both basic research and biotechnology. Livestock, which form a vital component of most societies, are no exception. While selective breeding has been hugely successful at enhancing some production traits, the rate of progress is often slow and is limited to variants that exist within the breeding population. Genome editing provides the potential to move traits between breeds, in a single generation, with no impact on existing productivity or to develop phenotypes that tackle intractable issues such as disease. As such, genome editors provide huge potential for ongoing livestock development programs in light of increased demand and disease challenge. This review will highlight some of the more notable agricultural applications of this technology in livestock.

关键词： cattle pig sheep chicken aquaculture CRISPR

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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

关键词： CRISPR Cas9 结核分枝杆菌基因编辑碱基编辑

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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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A brief review of genome editing technology for generating animal models

Haoyi WANG, Sen WU, Mario R. CAPECCHI, Rudolf JAENISCH

《农业科学与工程前沿（英文）》 2020年第7卷第2期页码 123-128 doi: 10.15302/J-FASE-2019309

摘要：

The recent development of genome editing technologies has given researchers unprecedented power to alter DNA sequences at chosen genomic loci, thereby generating various genetically edited animal models. This mini-review briefly summarizes the development of major genome editing tools, focusing on the application of these tools to generate animal models in multiple species.

关键词： animal model CRISPR genome editing TALEN ZFN

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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