一种新型的CRISPR/Cas9-hLacI双链DNA供体适配基因编辑系统

在CRISPR/Cas9系统介导的基因编辑中,借助于双链DNA （double-stranded DNA,dsDNA）供体模板的重组效应能够实现对目标基因组靶位点的精确编辑和基因敲入,然而高等真核生物细胞中同源重组的低效性限制了该基因编辑策略的发展和应用。为提高CRISPR/Cas9系统介导dsDNA供体模板的同源重组效率,本研究利用大肠杆菌（Escherichia coli）乳糖操纵子阻遏蛋白LacI与操纵序列LacO特异性结合的特点,通过重组DNA技术将密码子人源化优化的阻遏蛋白基因LacI分别与脓链球菌（Streptococcus pyogenes）源的SpCas9和路邓葡萄球菌（Staphylococcus lugdunensis）源的SlugCas9-HF融合表达,通过PCR将操纵序列LacO与dsDNA供体嵌合,构建了新型的CRISPR/Cas9-hLacI供体适配系统（donor adapting system,DAS）。首先在报告载体水平上对Cas9核酸酶活性、DAS介导的同源引导修复（homology-directed repair,HDR）效率进行了验证和优化,其次在基因组水平对其介导的基因精确编辑进行了检测,并最终利用CRISPR/SlugCas9-hLacI DAS在HEK293T细胞中实现了VEGFA位点的精确编辑,效率高达30.5%,显著高于野生型。综上所述,本研究开发了新型的CRISPR/Cas9-hLacI供体适配基因编辑系统,丰富了CRISPR/Cas9基因编辑技术种类,为以后的基因编辑及分子设计育种研究提供了新的工具。     

基于CRISPR/Cas9的精准基因编辑技术研究进展

基因编辑技术是一种可以在基因组水平上对DNA序列进行改造的遗传操作技术。基于CRISPR/Cas9系统的精准编辑技术是一个操作方便、应用广泛的基因编辑技术,与传统的CRISPR/Cas9不同,精准基因编辑技术可以在不需要DNA模板的情况下对基因进行定点突变。本文重点介绍了近年来基于CRISPR/Cas9介导的精准基因编辑技术的发展,并深入分析了基因精准编辑技术面临的挑战和机遇。     

利用CRISPR/Cas9技术构建基因编辑大鼠模型

RNA介导的CRISPR/Cas9基因编辑系统由单链引导RNA(sgRNA)与核酸酶Cas9构成。在细胞内,sgRNA能够按照碱基互补配对的原则引导Cas9与靶点结合,由Cas9切割目标DNA,造成双链DNA断裂(double stranded break, DSB)。在随后的DNA修复过程中,细胞主要进行非同源末端连接(non-homologous end joining,NHEJ)或在有修复模板存在的情况下进行重组修复(homology directed repair, HDR)。如果将CRISPR/Cas9系统以及修复模板通过显微注射的方式导入大鼠的胚胎内,就能借助细胞的修复机制实现大鼠胚胎的基因编辑,由此构建各种基因修饰大鼠模型。本文详细介绍了利用CRISPR/Cas9基因编辑技术构建大鼠模型的具体操作步骤,以期为相关领域的科研人员提供一种大鼠基因修饰模型的构建方法。     

CRISPR/Cas9单碱基编辑技术创制抗除草剂拟南芥种质

正CRISPR/Cas9基因编辑系统因其高效、廉价、简单、通用等特点,引发了基因编辑领域的革命.但是,在植物基因组上进行定点突变,依然困难重重.目前的技术路线是,将含有突变的DNA模板通过修复Cas9产生的DNA双链断裂的方式整合到植物基因组中,从而实现目标区域中的点突变.整合机制包括同源末端修复~([1])或非同源末端修复~([2])的两种方式.但是,DNA模板整合到目标区域的效率极低,这是在植物基因组中     

CRISPR/Cas9技术在生物医学领域的非病毒及病毒载体

规律成簇的间隔短回文重复序列(CRISPR)及CRISPR相关蛋白9(CRISPR/Cas9)系统是一种新型基因组编辑技术,能够靶向干扰或修复基因组的特定基因.来自细菌或人工改造的CRISPR/Cas9系统已经由生物学家发现或构建,Cas9核酸酶及单链导向RNA(sgRNA)是CRISPR/Cas9系统的主要组成成分.该系统被广泛应用于疾病治疗新靶点的发掘,基因功能的鉴定,动物模型的建立以及基因治疗药物的开发.CRISPR/Cas9系统已经通过突变或修正疾病相关基因来部分缓解或彻底治愈某些病症.然而,如何有效递送CRISPR/Cas9至目标细胞及靶器官仍然是运用该技术所面临的挑战之一,这影响着该系统稳定和精准的基因编辑能力.本文主要综述Cas9mRNA,Cas9蛋白或编码Cas9基因及相应sgRNA载体的递送系统.递送Cas9蛋白的非病毒载体能够维持Cas9的靶向作用,减少脱靶效应;递送sgRNA和供体模板的病毒载体能够改进基因编辑及同源修复效率.安全,有效及可规模化生产的递送载体将会推进CRISPR/Cas9技术在人类基因治疗领域中的应用.     

CRISPR/Cas9系统在植物基因组编辑中技术改进与创新的研究进展

CRISPR/Cas9基因组编辑技术是一项对基因组进行精准修饰的技术, 可实现对靶标基因的碱基插入、缺失或DNA片段替换。随着人们对CRISPR/Cas9系统的了解逐渐加深, 其在科研、农业和医疗等领域的应用也越来越广泛。该文简要介绍了CRISPR/Cas9基因组编辑技术的发展以及工作原理, 总结了近几年对该技术进行优化与改进的研究进展, 包括基因组编辑效率的提升、基因组编辑范围的扩展、单碱基精准编辑以及多基因同时编辑、基因组编辑安全性的提升以及基因片段替换与基因靶向转录调控, 以期为深入开展这一领域的研究提供参考。     

同源重组及非同源末端连接修复途径介导的基因编辑:CRISPR技术的认知、应用及展望

CRISPR系统具有精确识别及剪切特异性DNA序列功能而被开发成一种高效的基因编辑工具。它以成本低廉、操作简便、效率高及通用性广等优势,成为新一代最具代表性的基因编辑技术。在应用中,CRISPR系统可在特定靶点形成DNA双链断裂,继而诱导同源重组(HDR)或非同源末端连接修复(NHEJ),为基因组定向改造与调控带来了革命性的突破。该文将对近年来生物科学领域中发展迅猛的研究工具CRISPR/Cas系统进行介绍,包括其结构、作用原理、类型及应用等,并重点阐述同源重组或非同源末端连接修复途径介导的基因定向编辑技术及应用。     

CRISPR/Cas9系统在培育抗病毒植物新种质中的应用

随着CRISPR/Cas9系统在基因组编辑技术上的开发和完善,CRISPR/Cas9系统在应用于动物病毒感染性疾病防治并取得相当成效的同时,也逐步被应用到对植物病毒基因组进行高效靶向修饰的研究中。CRISPR/Cas9系统对基因组靶向修饰作用不仅实现了对植物DNA病毒基因组序列的编辑,还展示了其有效作用于植物RNA病毒基因组的潜力,同时CRISPR/Cas9系统还能在基因转录和转录后调控水平发挥作用,说明该系统具有通过多种途径调控植物病毒复制的潜能。相对其他植物病毒病防治策略,该系统对病毒基因组的编辑更精准、对基因表达的调控更稳定,对病毒病的抗性也更为广谱。本文将CRISPR/Cas9系统与其他植物病毒病防治策略进行了比较,概述了该系统在培育植物抗病毒病新种质中的优势,分析了其具体应用在该领域中面临的主要问题,讨论了该系统在培育抗病毒植物新种质应用中的发展趋势。     

CRISPR/Cas9系统在植物基因组编辑中技术改进与创新的研究进展

CRISPR/Cas9基因组编辑技术是一项对基因组进行精准修饰的技术,可实现对靶标基因的碱基插入、缺失或DNA片段替换。随着人们对CRISPR/Cas9系统的了解逐渐加深,其在科研、农业和医疗等领域的应用也越来越广泛。该文简要介绍了CRISPR/Cas9基因组编辑技术的发展以及工作原理,总结了近几年对该技术进行优化与改进的研究进展,包括基因组编辑效率的提升、基因组编辑范围的扩展、单碱基精准编辑以及多基因同时编辑、基因组编辑安全性的提升以及基因片段替换与基因靶向转录调控,以期为深入开展这一领域的研究提供参考。     

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。     

Expanding the CRISPR/Cas9 toolkit for Pichia pastoris with efficient donor integration and alternative resistance markers

Komagataella phaffii (syn. Pichia pastoris) is one of the most commonly used host systems for recombinant protein expression. Achieving targeted genetic modifications had been hindered by low frequencies of homologous recombination (HR). Recently, a CRISPR/Cas9 genome editing system has been implemented for P. pastoris enabling gene knockouts based on indels (insertion, deletions) via non‐homologous end joining (NHEJ) at near 100% efficiency. However, specifically integrating homologous donor cassettes via HR for replacement studies had proven difficult resulting at most in ～20% correct integration using CRISPR/Cas9. Here, we demonstrate the CRISPR/Cas9 mediated integration of markerless donor cassettes at efficiencies approaching 100% using a ku70 deletion strain. The Ku70p is involved in NHEJ repair and lack of the protein appears to favor repair via HR near exclusively. While the absolute number of transformants in the Δku70 strain is reduced, virtually all surviving transformants showed correct integration. In the wildtype strain, markerless donor cassette integration was also improved up to 25‐fold by placing an autonomously replicating sequence (ARS) on the donor cassette. Alternative strategies for improving donor cassette integration using a Cas9 nickase variant or reducing off targeting associated toxicity using a high fidelity Cas9 variant were so far not successful in our hands in P. pastoris. Furthermore we provide Cas9/gRNA expression plasmids with a Geneticin resistance marker which proved to be versatile tools for marker recycling. The reported CRSIPR‐Cas9 tools can be applied for modifying existing production strains and also pave the way for markerless whole genome modification studies in P. pastoris.     

CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes

Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.     

利用CRISPR/Cas9技术构建定点突变小鼠品系

CRISPR/Cas9技术是新近发展起来的对细胞和动物模型进行基因编辑的重要方法。本文利用DNA双链断裂(Double-strand breaks, DSBs)引起的同源重组(Homologous recombination, HR)依赖与非依赖的修复机制,建立基于CRISPR/Cas9核酸酶技术构建定点突变小鼠品系的技术体系。针对赖氨酸特异脱甲基化酶2b(Lysine (K)-specific demethylase 2b, Kdm2b)酶活关键位点对应的基因组DNA序列设计单一导向RNA(Single-guide RNA, sgRNA),通过与Cas9 mRNA共显微注射,分别得到Kdm2b基因发生移码突变的基因失活品系及关键位点氨基酸缺失的酶活突变型小鼠品系。此外,利用HR介导的修复机理,将黄素单加氧酶3(Flavin containing monooxygenases3, Fmo3)基因的sgRNA序列及对应的点突变单链寡脱氧核苷(Single strand oligonucleotides, ssODN)修复模板共注射到小鼠受精卵雄原核。对F₀小鼠基因测序分析显示,成功构建了Fmo3基因移码突变的基因敲除和单碱基定点突变的基因敲入小鼠,这些突变能够稳定遗传给子代。本研究利用CRISPR/Cas9技术,通过同源重组依赖与非依赖两种DNA损伤修复方式,成功构建了特定位点突变的小鼠品系。     

Homology Directed Knockin of Point Mutations in the Zebrafish tardbp and fus Genes in ALS Using the CRISPR/Cas9 System

The methodology for site-directed editing of single nucleotides in the vertebrate genome is of considerable interest for research in biology and medicine. The clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 type II (Cas9) system has emerged as a simple and inexpensive tool for editing genomic loci of interest in a variety of animal models. In zebrafish, error-prone non-homologous end joining (NHEJ) has been used as a simple method to disrupt gene function. We sought to develop a method to easily create site-specific SNPs in the zebrafish genome. Here, we report simple methodologies for using CRISPR/Cas9-mediated homology directed repair using single-stranded oligodeoxynucleotide donor templates (ssODN) for site-directed single nucleotide editing, for the first time in two disease-related genes, tardbp and fus.     

Non-viral and viral delivery systems for CRISPR-Cas9 technology in the biomedical field

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated protein 9(CRISPR-Cas9) system provides a novel genome editing technology that can precisely target a genomic site to disrupt or repair a specific gene. Some CRISPR-Cas9 systems from different bacteria or artificial variants have been discovered or constructed by biologists, and Cas9 nucleases and single guide RNAs(sgRNA) are the major components of the CRISPR-Cas9 system. These Cas9 systems have been extensively applied for identifying therapeutic targets, identifying gene functions, generating animal models, and developing gene therapies.Moreover, CRISPR-Cas9 systems have been used to partially or completely alleviate disease symptoms by mutating or correcting related genes. However, the efficient transfer of CRISPR-Cas9 system into cells and target organs remains a challenge that affects the robust and precise genome editing activity. The current review focuses on delivery systems for Cas9 mRNA, Cas9 protein, or vectors encoding the Cas9 gene and corresponding sgRNA. Non-viral delivery of Cas9 appears to help Cas9 maintain its on-target effect and reduce off-target effects, and viral vectors for sgRNA and donor template can improve the efficacy of genome editing and homology-directed repair. Safe, efficient, and producible delivery systems will promote the application of CRISPR-Cas9 technology in human gene therapy.     

应用SSA报告载体提高ZFN和CRISPR/Cas9对猪IGF2基因的打靶效率

IGF2(Insulin-like growth factor 2)基因作为最复杂多样的生长因子之一,对猪胎儿发育以及出生后生长发育和肌肉生成起着非常重要的作用。通过基因组编辑技术对我国本地猪种的IGF2基因作精确的遗传修饰,对于提高本地猪种的瘦肉率具有重要的育种意义。文章在蓝塘猪胎儿成纤维细胞(Porcine fetal fibroblasts, PEF)中检测了锌指核酸酶(Zinc finger nucleases, ZFN)和CRISPR/Cas9对IGF2基因的打靶效率,结果表明CRISPR/Cas9对IGF2基因的切割效率最高可达9.2%,显著高于ZFN的切割效率(<1%),但两者均未达到作为体细胞核移植(Somatic nuclear transfer, SCNT)供体细胞所需的打靶效率。应用SSA (Single-strand annealing)报告载体筛选技术来富集IGF2基因被ZFN和CRISPR/Cas9修饰过的PEF细胞,结果表明,该技术可使CRISPR/Cas9的打靶效率提高5倍左右,对ZFN的打靶效率具有更大的增强作用。     

CRISPR/Cas9-mediated correction of human genetic disease

The clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated(Cas) protein 9 system(CRISPR/Cas9) provides a powerful tool for targeted genetic editing. Directed by programmable sequence-specific RNAs,this system introduces cleavage and double-stranded breaks at target sites precisely. Compared to previously developed targeted nucleases, the CRISPR/Cas9 system demonstrates several promising advantages, including simplicity, high specificity,and efficiency. Several broad genome-editing studies with the CRISPR/Cas9 system in different species in vivo and ex vivo have indicated its strong potential, raising hopes for therapeutic genome editing in clinical settings. Taking advantage of non-homologous end-joining(NHEJ) and homology directed repair(HDR)-mediated DNA repair, several studies have recently reported the use of CRISPR/Cas9 to successfully correct disease-causing alleles ranging from single base mutations to large insertions. In this review, we summarize and discuss recent preclinical studies involving the CRISPR/Cas9-mediated correction of human genetic diseases.