人工核酸酶介导的基因组编辑技术

传统的基因组编辑技术是基于胚胎干细胞和同源重组实现生物基因组定向改造,但是该技术打靶效率低,严重制约了生命科学以及医学的研究.因此,研究新的基因组编辑技术十分重要.人工核酸酶介导的基因组编辑技术是通过特异性识别靶位点造成DNA双链断裂,引起细胞内源性的修复机制实现靶基因的修饰.与传统的基因组编辑技术相比,人工核酸酶技术打靶效率高,这对于基因功能的研究、构建人类疾病动物模型以及探索新型疾病治疗方案有着重要的意义.人工核酸酶技术有3种类型：锌指核酸酶(ZFN)、类转录激活因子核酸酶(TALEN)及规律成簇的间隔短回文重复序列(CRISPR).本文将对以上3种人工核酸酶技术的原理以及在生命科学和医学研究的应用进行综述.     

基因组编辑技术中供体DNA类型及选择

基因组编辑技术能够实现基因组的精确修饰和改造,是后基因组时代研究基因功能和遗传信息的主要手段。传统的基因打靶技术通过低效率的细胞自发同源重组实现目的基因的定点修饰。真核细胞中DNA双链断裂介导的同源重组效率远高于自发同源重组,利用人工核酸内切酶特异性地在基因组靶序列处引入双链断裂,通过提供适当形式的、含有一定长度同源臂的供体DNA,能够实现相对高效的基因组靶向编辑。本文系统总结了环状质粒、线性化质粒、聚合酶链式反应产物及单链寡聚脱氧核苷酸4种类型的供体DNA在基因组精确编辑研究中的应用及候选原则,以期为以后相关研究中供体DNA的选择、设计提供参考和借鉴。     

基因组编辑技术中供体DNA类型及选择北大核心CSCD

基因组编辑技术能够实现基因组的精确修饰和改造,是后基因组时代研究基因功能和遗传信息的主要手段。传统的基因打靶技术通过低效率的细胞自发同源重组实现目的基因的定点修饰。真核细胞中DNA双链断裂介导的同源重组效率远高于自发同源重组,利用人工核酸内切酶特异性地在基因组靶序列处引入双链断裂,通过提供适当形式的、含有一定长度同源臂的供体DNA,能够实现相对高效的基因组靶向编辑。本文系统总结了环状质粒、线性化质粒、聚合酶链式反应产物及单链寡聚脱氧核苷酸4种类型的供体DNA在基因组精确编辑研究中的应用及候选原则,以期为以后相关研究中供体DNA的选择、设计提供参考和借鉴。     

基因编辑技术在大肠杆菌中的应用

利用基因编辑技术对大肠杆菌基因组进行改造可以研究基因功能,或改变其代谢途径大量生产原本成本较高的产物,从而获得可以生产特定产物的遗传稳定性工程菌株.目前可以对细菌基因组编辑的方法有Red同源重组、CRISPR/Cas9技术等.Red同源重组是比较传统的基因编辑技术,应用广泛,但编辑效率受整合片段大小的限制,基因编辑过程...     

CRISPR/Cas9技术在植物基因功能研究和新种质创制中的应用与展望

基因组定点编辑是利用人工核酸酶,对复杂生物基因组特定位点快速而精确地进行遗传改造的一项新技术.尤其是最近从细菌和古细菌的获得性免疫防御反应中改造而来的CRISPR/Cas9系统,因其简单、廉价、高效以及通用的特性,目前已经广泛地应用于植物、动物、微生物等各种生物体和细胞的基因功能和应用研究中.CRISPR/Cas9系统的原理在于其携带的Cas9核酸酶—RNA导向的ds DNA结合蛋白,能够在靶位点对双链DNA进行定点切割,随后引发的非同源末端连接或者同源重组修复,导致了靶位点DNA的缺失、插入、替换甚至染色体大片段重排.本文对CRISPR/Cas9技术的作用机理和应用进行概括,侧重于模式植物和农作物的最新进展,探讨这一新型基因组定点编辑技术在植物基因功能研究和新种质创制中的进一步发展.     

基因修饰小鼠构建的革命:CRISPR/Cas9技术

基因修饰小鼠在研究人类疾病致病机理和治疗手段中起到了关键作用。传统的小鼠基因组编辑使用胚胎干细胞(ES细胞),虽然可以对内源基因进行精细的修改,但是复杂、繁琐并且耗时。近几年发展的人工核酸酶可以在靶位点切割DNA双链,诱发细胞内源性修复机制,发生同源重组修复或非同源末端连接,从而提高了基因组编辑的效率。从ZFN到TALEN再到CRISPR/Cas9技术,新型基因组编辑技术正以惊人的速度渗入到生命科学的各项研究工作中。将对新型基因组编辑技术进行介绍,着重阐述CRISPR/Cas9系统介导的基因编辑技术在基因修饰小鼠中的应用,比较该系统与其他方法的优越性和不足,并对该系统进行展望。     

序列特异性核酸酶及其在植物基因组定向修饰中的应用

通过对基因组特定区域进行精确定向遗传修饰,一方面可以针对目标序列进行精确突变,获得突变材料,对目标基因功能进行明确鉴定;另一方面可以进行目标序列的精确置换或插入,将外源基因随机导入造成的表达及遗传的不确定性降至最低。传统的基因定向修饰技术仅依赖于细胞自身的同源重组,修饰效率低下,而且还存在位置效应和遗传不稳定等诸多问题。通过引入序列特异性核酸酶（sequence—specificnucleases,SSN）,可以在基因组特定位点造成DNA双链断裂（doublestrandbreak,OSB）,促进依赖于细胞内源“同源重组”及“非同源末端连接”的DNA修复事件的定向发生,实现基因组定向遗传修饰效率的大幅提升。迄今为止,在基因组定向遗传修饰研究及应用领域,已经有多种不同类型的序列特异性核酸酶被有效使用,在多种生物中实现了不同类型的基因组定向遗传修饰。该文首先综述了SSN的结构特征及技术原理,然后对SSN技术在植物基因组定向遗传修饰中的研究进展和应用前景进行了重点介绍。     

CRISPR/Cas9系统在疾病研究和治疗中的应用

基因组编辑技术(Genomeeditingtechnology)是一种通过人工手段在基因组水平对DNA序列进行改造的遗传操作技术,包括特定DNA片段的插入、敲除、替换和点突变。其中,依赖核酸酶的基因组编辑技术的基本原理是在基因组的特定位置产生双链DNA断裂(Double-strandedbreak,DSB)后通过非同源末端连接(Non-homologous end joining,NHEJ)或同源重组(Homologous recombination,HR)的方式进行修复。随着对核酸酶更深入的研究,基因组编辑技术也得到了快速发展,其中最常使用的核酸酶主要包括巨型核酸酶、锌指核酸酶、转录激活因子样效应物核酸酶以及成簇的规律间隔的短回文重复序列相关蛋白(Clusteredregularly interspaced short palindromic repeats (CRISPR)-associated proteins (Cas)。文中在介绍上述基因组编辑技术的发展及作用原理的基础上,主要综述了CRISPR/Cas9系统在基因功能鉴定、疾病模型建立、基因治疗和免疫治疗等应用领域的研究进展,并对其未来发展进行了展望。     

A Unique Protease Cleavage Site Predicted in the Spike Protein of the Novel Pneumonia Coronavirus (2019-nCoV) Potentially Related to Viral Transmissibility

正Dear Editor,In December 2019, a novel human coronavirus caused an epidemic of severe pneumonia(Coronavirus Disease 2019,COVID-19) in Wuhan, Hubei, China(Wu et al. 2020; Zhu et al. 2020). So far, this virus has spread to all areas of China and even to other countries. The epidemic has caused 67,102 confirmed infections with 1526 fatal cases     

Curcuminoids as inhibitors of thioredoxin reductase: A receptor based pharmacophore study with distance mapping of the active site

Curcumin is the yellow pigment of turmeric that interacts irreversibly forming an adduct with thioredoxin reductase (TrxR), an enzyme responsible for redox control of cell and defence against oxidative stress. Docking at both the active sites of TrxR was performed to compare the potency of three naturally occurring curcuminoids, namely curcumin, demethoxy curcumin and bis-demethoxy curcumin. Results show that active sites of TrxR occur at the junction of E and F chains. Volume and area of both cavities is predicted. It has been concluded by distance mapping of the most active conformations that Se atom of catalytic residue SeCYS498, is at a distance of 3.56 from C13 of demethoxy curcumin at the E chain active site, whereas C13 carbon atom forms adduct with Se atom of SeCys 498. We report that at least one methoxy group in curcuminoids is necessary for interation with catalytic residues of thioredoxin. Pharmacophore of both active sites of the TrxR receptor for curcumin and demethoxy curcumin molecules has been drawn and proposed for design and synthesis of most probable potent antiproliferative synthetic drugs.     

Comparative morphology of the carpel in the Liliaceae: Hewardieae, Petrosavieae, and Tricyrteae

The young pistils in the melanthioid tribes, Hewardieae, Petrosavieae and Tricyrteae, are uniformly tricarpellate and syncarpous. They lack raphide idioblasts. All are multiovulate, with bitegmic ovules. The Petrosavieae are marked by the presence of septal glands and incomplete syncarpy. Tepals and stamens adhere to the ovary in the Hewardieae and the Petrosavieae but not in the Tricyrteae. Two vascular bundles occur in the stamens of the Hewartlieae and Tricyrtis latifolia. Ventral bundles in the upper part of the ovary of the Hewardieae are continuous with compound septal bundles and placental bundles in the lower part. Putative ventral bundles occur in the alternate position in the Tricyrteae and putative placental bundles in the opposite. position in the Petrosavieae. The dichtomously branched stigma in each carpel of the Tricyrteae is supplied by a bifurcated dorsal bundle.     

Enterovirus 71 3C proteolytically processes the histone H3 N-terminal tail during infection

Highlights
1. The N-terminal tail of histone H3 is specifically cleaved during EV71 infection.
2. Viral protease 3C is identified as a protease responsible for proteolytically processing the N-terminal H3 tail.
3. Our finding reveals a new epigenetic regulatory mechanism for Enterovirus 71 in virus-host interactions.     

Detection of EBV and HHV6 in the Brain Tissue of Patients with Rasmussen’s Encephalitis

Rasmussen’s encephalitis (RE) is a rare pediatric neurological disorder, and the exact etiology is not clear. Viral infection may be involved in the pathogenesis of RE, but conflicting results have reported. In this study, we evaluated the expression of both Epstein-Barr virus (EBV) and human herpes virus (HHV) 6 antigens in brain sections from 30 patients with RE and 16 control individuals by immunohistochemistry. In the RE group, EBV and HHV6 antigens were detected in 56.7% (17/30) and 50% (15/30) of individuals, respectively. In contrast, no detectable EBV and HHV6 antigen expression was found in brain tissues of the control group. The co-expression of EBV and HHV6 was detected in 20.0% (6/30) of individuals. In particular, a 4-year-old boy had a typical clinical course, including a medical history of viral encephalitis, intractable epilepsy, and hemispheric atrophy. The co-expression of EBV and HHV6 was detected in neurons and astrocytes in the brain tissue, accompanied by a high frequency of CD8⁺ T cells. Our results suggest that EBV and HHV6 infection and the activation of CD8⁺ T cells are involved in the pathogenesis of RE.     

Perinatal Vertical Transmission of Chikungunya Virus in Ruili,a Town on the Border between China and Myanmar

正Dear Editor,Chikungunya virus (CHIKV), an arbovirus in the family of Togaviridae, genus Alphavirus, is transmitted by the A.aegyptii or A. albopictus mosquito, and causes disease in humans characterized by fever, rash, and arthralgia (Silva and Dermody 2017; Suhrbier 2019). It was first reported in 1953 in Tanzania, and caused only a few outbreaks and sporadic cases in Africa and Asia in last century. However, in the epidemic in 2004, CHIKV acquired mutations that conferred enhanced transmission by the A. albopictus mosquito(Schuffenecker et al. 2006). Since then, it has successively caused outbreaks in Africa, the Indian Ocean, South East Asia, the South America, and Europe (Zeller et al. 2016).     

Development of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification Method for Rapid Detection of SARS-CoV-2

In conclusion, the novel visual RT-LAMP assay is a simple, rapid, and sensitive approach for detection of SARS-CoV-2, and it is ready for application in primary care and community hospitals or health care centers, and even patients' own houses in response to the current SARS-CoV-2 epidemic because the assay does not require sophisticated equipment and skilled personnel. Furthermore, it is also ready to be used in fields for screening samples from wild animals and environments to facilitate the identification of potential intermediate hosts that mediate the cross-species transmission of SARS-CoV-2 from bats to humans.