CRISPR-Cas9驱动的基因编辑新纪元

在自然界生物长期的进化过程中,细菌和古细菌演化出了一种适应性免疫系统用以抵御外源病毒与质粒的入侵,该系统由成簇规律间隔的短回文重复序列与相关基因组成,称之为CRISPR-Cas。近年来,这一领域突飞猛进,如今已经发展成为一种功能强大的基因编辑工具并在生物学及其相关领域得到广泛应用。本文重点综述了近年来CRISPR-Cas9系统在基因编辑、基因调节以及作为体外工具酶和特异性等方面的若干前沿进展。     

CRISPR-Cas9研究进展及在基因治疗上的应用

CRISPR-Cas9[Clustered regularly interspaced short palindromic repeats（CRISPR）/CRISPR-associated （Cas）9]是近年兴起的一种高特异性和高效的基因编辑新技术,由向导RNA（single guide RNA,sgRNA）和cas9（CRISPR-associated 9）蛋白组成,引起DNA位点特异性双链断裂（double-strand breaks,DSBs）,引发同源重组修复（homology-directed repair,HDR）或非同源末端连接修复（non-homologous end joining,NHEJ）,达到靶基因修饰的作用。CRISPR-Cas9技术自发现以来,因其便于操作、花费较低、高特异性、可同时打靶任意数量基因等优点而被应用。近年研究显示,对于一些遗传性疾病,可通过CRISPR-Cas9精确的基因编辑破坏致病的内源基因、改正引起疾病的突变体或插入新的保护性基因进行治疗,该技术为基因治疗开启了一个新方向。主要从CRISPR-Cas9结构、作用机制及在疾病基因治疗上的应用等方面进行了综述。     

CRISPR-Cas9系统在疾病模型中的应用

规律成簇间隔短回文重复（CRISPR）及相关核酸内切酶（Cas）系统是最近发现的一种关于RNA指导核酸内切酶的基因编辑技术,这一技术的发现促进了生物学和医学研究的发展。CRISPR-Cas9系统的简便性使其广泛应用于细胞基因组编辑、动物模型的构建及疾病模型的基因治疗。现就CRISPR-Cas9系统的结构特点、作用机制及应用进行了综述。     

Site-directed RNA editing (SDRE): Off-target effects and their countermeasures

Site-directed RNA editing (SDRE) is invaluable to basic research and clinical applications and has emerged as a new frontier in genome editing. The past few years have witnessed a surge of interest in SDRE, with SDRE tools emerging at a breathtaking pace. However, off-target effects of SDRE remain a tough problem, which constitutes a major hurdle to their clinical applications. Here we discuss the diverse strategies for combating off-target editing, drawing lessons from the published studies as well as our ongoing research. Overall, SDRE is still at its infancy, with significant challenges and exciting opportunities ahead.     

CRISPR-Cas9技术在细菌中的研究进展及应用

CRISPR (Clustered regularly interspaced short palindromic repeats)最早发现于绝大多数细菌和古细菌中,帮助其防御和抵抗噬菌体和外来质粒的入侵。近年来,随着对该系统结构及功能的逐步深入认识和研究,CRISPR-Cas9技术已经发展成为一种简易高效的基因组定点编辑手段,但是在使用过程中仍存在一些问题。本文重点介绍了CRISPR-Cas9系统功能和分类,在细菌基因编辑中的应用和问题以及应对措施。     

CRISPR⁃Cas9技术在植物次生代谢物生产中的研究进展

基因编辑（gene editing）技术可以对目的基因进行定点插入、敲除和置换。基于CRISPR-Cas9的基因编辑技术是继锌指核酸酶和转录激活样效应物核酸酶之后的第3代基因编辑技术。近年来,CRISPR-Cas9系统作为研究的热点被广泛应用于医学、药学、植物学、动物学和微生物学等领域,但其在植物次生代谢物领域的应用还处于探索时期。阐述了基于CRISPR-Cas9基因编辑技术的发展历程、工作原理和几种常用的基因编辑方法及其应用实例,总结了CRISPR-Cas9技术在对植物次生代谢产物研究方面的应用。利用CRISPR-Cas9系统可对植物基因组进行定点敲除、突变和插入,以达到提高植物次生代谢物含量、改良作物品质和提高植物抗性等目的。该技术已在植物次生代谢物生物合成关键酶基因的编辑等方面显示出越来越重要的作用。     

A Toolkit of CRISPR-Based Genome Editing Systems in Drosophila

The last couple of years have witnessed an explosion in development of CRISPR-based genome editing technologies in cell lines as well as in model organisms. In this review, we focus on the applications of this popular system in Drosophila. We discuss the effectiveness of the CRISPR/Cas9 systems in terms of delivery, mutagenesis detection, parameters affecting efficiency, and off-target issues, with an emphasis on how to apply this powerful tool to characterize gene functions.     

Come together,right now….

Editing the genome using approaches like TALEN and siRNA are already well tested. The new kid on the block is CRISPR-Cas9. CRISPR-Cas9 is rapidly evolving with impressive refinements for specificity, eliminating off-target effects, and versatility. One can adjust constructs and conditions to produce opposite effects on the genome and for a specific purpose. The nuances of the system, the means to significantly reduce off-targeting, and numerous applications are now emerging rapidly. This B&B commentary looks forward into how the CRISPR-Cas9 tool might serve the CCN field.     

曲霉高效基因编辑技术研究进展

曲霉Aspergillus spp.是自然界中分布极为广泛的一大类真菌,在工业上如黑曲霉等已经被人们广泛利用于食品添加剂等生产。因此,曲霉在工业、农业、医药领域均发挥着重要的作用。然而,有些曲霉如黄曲霉能够分泌致癌物黄曲霉毒素从而污染农产品;在临床上还有一类以烟曲霉为主的引起侵袭性真菌感染的条件致病曲霉菌。因此,曲霉就像一把双刃剑影响着人们的生活。曲霉菌丝具有发达的隔膜形成多细胞菌丝体并能在分生孢子梗上产生大量的分生孢子进行无性繁殖。研究曲霉的基因编辑技术对于控制医学和农业上有害曲霉的增殖和促进工业上有益曲霉的生长和代谢都具有非常重要的意义。长期以来,同源重组和随机整合一直是被用于研究曲霉基因功能的传统基因编辑方法,然而其操作费时费力且效率低、难以达到预期的要求。随着第三代基因编辑技术CRISPR/Cas9即成簇的规律间隔的短回文重复序列及其相关系统CRISPR/Cas9（Clustered regulatory interspaced short palindromic repeats-associated protein 9）建立以来,CRISPR/Cas9以编辑高效、操作简便等优势被广泛应用到不同物种中。目前,世界上多个研究团队已经建立了有效地用于不同曲霉物种的CRISPR-Cas9 基因编辑体系。本文概述了有关曲霉基因编辑的历史和进展,以期为尚未建立完整遗传编辑体系的其他曲霉物种或者丝状真菌引入高效CRISPR-Cas9体系提供帮助。     

靶向番茄SlACS2基因CRISPR-Cas9 sgRNA的设计和分析

番茄为呼吸跃变型果实,伴随呼吸跃变产生大量乙烯,即系统II乙烯,易使番茄果实过熟,导致腐烂变质。SlACS2是番茄系统II乙烯合成的限速酶,通过CRISPR-Cas9基因组编辑系统修饰该基因,调控系统II乙烯过量表达,将迟滞番茄过熟。本研究基于RNA-seq建立了SlACS2基因的数字表达谱,表明该基因呈果实特异性表达,在植株的根、茎、叶等部位不表达。SlACS2位于番茄1号染色体,含4个外显子和3个内含子。利用在线工具CRISPRdirect 和CRISPR-P发现第1、2、3外显子分别具有18、9和11条sgRNA。其中,sgRNA1-14和sgRNA3-8及二者的近PAM的12 nt 种子序列在番茄基因组是唯一序列,GC含量高于40%,不存在TTTT终止序列。BLAST结果表明,sgRNA1-14和sgRNA3-8与GenBank公布的8条SlACS2同源序列高度一致,位于该基因的保守区,而与SlACS4和SlACS6的同源序列存在多个SNP,预示这2条sgRNA可用于番茄不同品种SlACS2基因的靶向编辑,并可规避对SlACS家族其他同源基因的脱靶效应。     

Genome editing methods in animal models

ABSTRACT

Genetically engineered animal models that reproduce human diseases are very important for the pathological study of various conditions. The development of the clustered regularly interspaced short palindromic repeats (CRISPR) system has enabled a faster and cheaper production of animal models compared with traditional gene-targeting methods using embryonic stem cells. Genome editing tools based on the CRISPR-Cas9 system are a breakthrough technology that allows the precise introduction of mutations at the target DNA sequences. In particular, this accelerated the creation of animal models, and greatly contributed to the research that utilized them. In this review, we introduce various strategies based on the CRISPR-Cas9 system for building animal models of human diseases and describe various in vivo delivery methods of CRISPR-Cas9 that are applied to disease models for therapeutic purposes. In addition, we summarize the currently available animal models of human diseases that were generated using the CRISPR-Cas9 system and discuss future directions.