遗传修饰猪模型在生物医学及农业领域研究进展及应用

猪在解剖结构、代谢、生理生化等特征方面比啮齿类动物更接近人类,因此在模拟某些人类疾病以及提供异种移植器官等方面具有其他动物不可替代的优势,是理想的人类疾病动物模型和异种器官的供体。另外,猪作为我国畜牧业最重要的物种之一,猪的品种改良、疫病防控以及动物福利等问题都与人民生活息息相关。本文主要介绍了遗传修饰猪模型在分子育种、人类疾病模型以及异种器官移植领域的研究进展及未来应用前景,希望增进相关领域研究人员对基因编辑等前沿技术的了解,理解遗传修饰猪模型在生命科学研究中的重要意义。     

利用人工核酸酶构建基因编辑猪的研究进展

猪不仅具有重要的农业生产价值,由于猪繁殖周期比灵长类动物短,产仔量多,以及猪器官大小、生理代谢过程和解剖结构,尤其是心血管系统及神经系统较啮齿类动物而言与人类更为接近,猪在生物医药领域也具有重要的用途。对猪基因组加以编辑,在农业上可以加速高生产性能猪新品种的培育;在生命科学领域可用于基因功能、胚胎发育及代谢过程等基础研究;在医学上,可提供与人体更为匹配的异种移植器官,可作为更能准确模拟人类疾病的大动物模型,用于新的药物和治疗手段的开发。人工核酸酶的出现,使基因编辑效率得到大大提高,克服了原有的基因编辑猪制备困难、成功率极低的问题。人工核酸酶技术不仅在猪上实现了高效而又精确的基因编辑,并且使各种基因突变模式,如基因敲除、基因敲入、基因组无痕点突变、多基因编辑、体内直接基因编辑以及条件性基因编辑等都成为可能。现将对利用不同人工核酸酶技术结合不同的基因编辑策略建立基因编辑猪的研究新进展进行综述。     

非人灵长类动物基因编辑技术的应用及挑战

建立有效的动物模型是研究人类疾病演进、开发新型治疗手段的重要方法。非人灵长类动物在进化发育、生理生化及病理方面和人类最接近,是研究人类疾病的理想动物模型。随着基因编辑技术的发展,研究者已经成功建立了多种模仿人类疾病的非人灵长类动物模型。但是CRISPR/Cas9的脱靶效应、嵌合突变以及基因敲入效率较低等突出问题也逐渐引起重视。本文综述了基因编辑技术在建立非人灵长类动物模型中的应用现状,提出了目前亟需解决的难点和应对策略,以期为高效、准确构建非人灵长类动物模型提供借鉴与参考。     

基因编辑技术在猪现代育种和动物模型构建中应用的研究进展

基因编辑技术是近几年兴起的一项能够对目标基因序列进行编辑的技术,该技术主要利用人工核酸酶实现对基因组上特定DNA片段的删除、插入或修饰。猪是我国优质的肉用型家畜,但随着人民生活水平的提高,对猪的需求也由过去的膘肥体重转变为能提供更优质的肉品,这就要求对猪的瘦肉率、肉质等主要经济性状方面进行育种改良,从而优化猪肉的蛋白质和脂肪含量。此外,由于猪在解剖学和生理学等方面与人类高度相似,可以用于疾病模型、药物筛选及致病机理的研究,基因编辑将在以上方面发挥显著的作用。利用基因编辑技术可以大大缩短猪在现代育种和疾病动物模型构建的时间,使猪在农业发展和生物医学研究中拥有更大的潜力。该文主要综述传统转基因技术和基因编辑技术在猪育种和动物模型构建中所采用的方法,并对比各种方法的优缺点,旨在为猪的育种和动物模型的构建提供理论依据。     

小型猪模型在人类内分泌代谢性疾病研究中的应用进展

内分泌代谢性疾病(metabolic and endocrine diseases)是危害人类健康、降低生活质量的重要病症之一。肥胖、病毒感染、遗传易感性及免疫功能异常等多种因素均可导致内分泌代谢性疾病的发生,但其致病机制仍不明确。制备适宜动物模型是开展相关研究的关键基础。小型猪在生理解剖结构、机体代谢过程、病理诊断指标等方面与人类极为相似,是制备内分泌代谢性疾病的理想动物模型。本文对小型猪品系、构建疾病模型的方法及现状展开综述,旨在为人类内分泌代谢性疾病动物模型相关研究提供参考。     

CRISPR/Cas9基因编辑技术在阿尔茨海默病研究中的应用

阿尔茨海默病(Alzheimer's disease, AD)是一种与基因和环境等多因素相关的神经退行性疾病,其发病机理复杂,目前也鲜有针对AD的治疗药物。CRISPR/Cas9是基于DNA重组修复原理的第三代基因编辑技术,该技术已经成功地应用于斑马鱼、啮齿类动物基因组的编辑,将其应用于AD有助于推动AD发病机理和治疗方法的研究。该文在介绍CRISPR/Cas9基因编辑技术的基础上,综述了该技术在AD病理模型构建、致病风险因素筛查、治疗靶标寻找和靶向治疗等方面的研究和应用进展。     

CRISPR基因组编辑技术在传染病致病机理研究和防诊治中的应用

CRISPR基因组编辑技术在基因操作和传染病研究等方面展现出巨大的应用前景,对于有效控制和治愈传染病具有重要价值。通过其构建的细胞、类器官和动物疾病模型,为探索传染病相关分子机制提供了极大便利。CRISPR筛选技术使得高通量鉴定传染病相关风险因子成为可能。基于CRISPR的新型分子诊断工具为病原体的检测提供了更灵敏和快速的方法。利用CRISPR工具敲入抗性基因或破坏风险基因和病毒基因组,有望实现预防或治疗传染病。本综述讨论了CRISPR基因组编辑技术在疾病模型制备、传染病风险因子筛选、病原体诊断和传染病防治中的应用,以期为后续传染病的研究和防诊治提供参考。     

CRISPR/Cas9在人病毒感染相关疾病治疗研究中的应用*

病毒感染相关疾病严重威胁人类的健康,目前的抗病毒疗法难以治愈慢性病毒性感染引起的一些疾病,如获得性免疫缺陷综合征和乙型肝炎等,因此迫切需要新的治疗方法。可直接靶向遗传物质的基因编辑技术或将成为对抗病毒的有力工具。作为一种可编程的核酸酶介导的新型基因编辑技术,CRISPR/Cas9系统因其具有编辑效率高、操作简单、成本低、适用范围广等优点,而成功应用于多种人类相关疾病的研究中,也为病毒感染相关疾病的研究以及开发新的治疗方法提供了新的技术手段。主要对CRISPR/Cas9系统的作用机制以及在人类常见的病毒感染相关疾病治疗研究中的最新进展进行综述。     

CRISPR/Cas9在人病毒感染相关疾病治疗研究中的应用*

病毒感染相关疾病严重威胁人类的健康,目前的抗病毒疗法难以治愈慢性病毒性感染引起的一些疾病,如获得性免疫缺陷综合征和乙型肝炎等,因此迫切需要新的治疗方法。可直接靶向遗传物质的基因编辑技术或将成为对抗病毒的有力工具。作为一种可编程的核酸酶介导的新型基因编辑技术,CRISPR/Cas9系统因其具有编辑效率高、操作简单、成本低、适用范围广等优点,而成功应用于多种人类相关疾病的研究中,也为病毒感染相关疾病的研究以及开发新的治疗方法提供了新的技术手段。主要对CRISPR/Cas9系统的作用机制以及在人类常见的病毒感染相关疾病治疗研究中的最新进展进行综述。     

CRISPR/Cas9-mediated genome editing: From basic research to translational medicine

The recent development of the CRISPR/Cas9 system as an efficient and accessible programmable genome-editing tool has revolutionized basic science research. CRISPR/Cas9 system-based technologies have armed researchers with new powerful tools to unveil the impact of genetics on disease development by enabling the creation of precise cellular and animal models of human diseases. The therapeutic potential of these technologies is tremendous, particularly in gene therapy, in which a patient-specific mutation is genetically corrected in order to treat human diseases that are untreatable with conventional therapies. However, the translation of CRISPR/Cas9 into the clinics will be challenging, since we still need to improve the efficiency, specificity and delivery of this technology. In this review, we focus on several in vitro, in vivo and ex vivo applications of the CRISPR/Cas9 system in human disease-focused research, explore the potential of this technology in translational medicine and discuss some of the major challenges for its future use in patients.     

CRISPR Editing in Biological and Biomedical Investigation

The CRISPR (clustered regularly interspaced short palindromic repeat)‐Cas (CRISPR‐associated protein) system, a prokaryotic RNA‐based adaptive immune system against viral infection, is emerging as a powerful genome editing tool in broad research areas. To further improve and expand its functionality, various CRISPR delivery strategies have been tested and optimized, and key CRISPR system components such as Cas protein have been engineered with different purposes. Benefiting from more in‐depth understanding and further development of CRISPR, versatile CRISPR‐based platforms for genome editing have been rapidly developed to advance investigations in biology and biomedicine. In biological research area, CRISPR has been widely adopted in both fundamental and applied research fields, such as genomic and epigenomic modification, genome‐wide screening, cell and animal research, agriculture transforming, livestock breeding, food manufacture, industrial biotechnology, and gene drives in disease agents control. In biomedical research area, CRISPR has also shown its extensive applicability in the establishment of animal models for genetic disorders, generation of tissue donors, implementation of antimicrobial and antiviral studies, identification and assessment of new drugs, and even treatment for clinical diseases. However, there are still several problems to consider, and the biggest concerns are the off‐target effects and ethical issues of this technology. In this prospect article, after highlighting recent development of CRISPR systems, we outline different applications and current limitations of CRISPR in biological and biomedical investigation. Finally, we provide a perspective on future development and potential risks of this multifunctional technology. J. Cell. Biochem. 119: 52–61, 2018. © 2017 Wiley Periodicals, Inc.     

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.     

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.     

CRISPR/Cas及其衍生编辑技术在基因治疗中的应用进展

基因治疗是指利用基因编辑技术对细胞基因进行“修饰”而达到治疗的目的。CRISPR/Cas的出现为基因编辑提供了简单、高效和多功能的平台,同时,为克服DNA双链断裂产生的不良影响,基于CRISPR/Cas的新型技术,如碱基编辑器（base editors,BE）、Prime Editors（PE）和Cas13效应器,被相继开发出来。目前,CRISPR/Cas及其衍生编辑技术已被广泛应用于动物细胞模型构建、药物靶点筛查和基因功能研究等领域,在基因治疗领域也展现出广阔的应用前景。基于此,简要介绍了CRISPR/Cas及其衍生编辑技术,综述了其在单基因遗传病、肿瘤和其他疾病的基因治疗中的应用进展,并分析了其当下面临的挑战,以期为基因编辑在单基因遗传病、肿瘤和其他疾病治疗领域提供理论参考。     

Creating cell and animal models of human disease by genome editing using CRISPR/Cas9

A set of unique sequences in bacterial genomes, responsible for protecting bacteria against bacteriophages, has recently been used for the genetic manipulation of specific points in the genome. These systems consist of one RNA component and one enzyme component, known as CRISPR (“clustered regularly interspaced short palindromic repeats”) and Cas9, respectively. The present review focuses on the applications of CRISPR/Cas9 technology in the development of cellular and animal models of human disease. Making a desired genetic alteration depends on the design of RNA molecules that guide endonucleases to a favorable genomic location. With the discovery of CRISPR/Cas9 technology, researchers are able to achieve higher levels of accuracy because of its advantages over alternative methods for editing genome, including a simple design, a high targeting efficiency and the ability to create simultaneous alterations in multiple sequences. These factors allow the researchers to apply this technology to creating cellular and animal models of human diseases by knock‐in, knock‐out and Indel mutation strategies, such as for Huntington's disease, cardiovascular disorders and cancers. Optimized CRISPR/Cas9 technology will facilitate access to valuable novel cellular and animal genetic models with respect to the development of innovative drug discovery and gene therapy.     

CRISPR/Cas9:基因编辑的新时代

基因编辑技术是通过核酸内切酶对基因组DNA进行定向改造的技术,可以实现对特定DNA碱基的缺失、替换等,常用的四种基因编辑工具分别是: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CRISPR/Cas9系统。其中CRISPR/Cas9系统作为一种新型的基因组编辑技术具有组成简单、特异性好、切割效率高的优点。该文对CRISPR/Cas9系统的结构组成和功能机制,动植物基因靶向编辑和人类在遗传性疾病、病毒感染性疾病以及肿瘤方面进行综述,旨在对CRISPR/Cas9系统的现状和发展进行总结和展望。     

CRISPR/Cas system: An emerging technology in stem cell research

The identification of new and even more precise technologies for modifying and manipulating the genome has been a challenge since the discovery of the DNA double helix. The ability to modify selectively specific genes provides a powerful tool for characterizing gene functions, performing gene therapy, correcting specific genetic mutations, eradicating diseases, engineering cells and organisms to achieve new and different functions and obtaining transgenic animals as models for studying specific diseases. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology has recently revolutionized genome engineering. The application of this new technology to stem cell research allows disease models to be developed to explore new therapeutic tools. The possibility of translating new systems of molecular knowledge to clinical research is particularly appealing for addressing degenerative diseases. In this review, we describe several applications of CRISPR/Cas9 to stem cells related to degenerative diseases. In addition, we address the challenges and future perspectives regarding the use of CRISPR/Cas9 as an important technology in the medical sciences.     

CRISPR/Cas9-mediated mutagenesis at microhomologous regions of human mitochondrial genome

Genetic manipulation of mitochondrial DNA(mtDNA) could be harnessed for deciphering the gene function of mitochondria; it also acts as a promising approach for the therapeutic correction of pathogenic mutation in mtDNA. However, there is still a lack of direct evidence showing the edited mutagenesis within human mtDNA by clustered regularly interspaced short palindromic repeats-associated protein 9(CRISPR/Cas9). Here, using engineered CRISPR/Cas9, we observed numerous insertion/deletion(InDel) events at several mtDNA microhomologous regions, which were triggered specifically by double-strand break(DSB)lesions within mtDNA. InDel mutagenesis was significantly improved by sgRNA multiplexing and a DSB repair inhibitor,iniparib, demonstrating the evidence of rewiring DSB repair status to manipulate mtDNA using CRISPR/Cas9. These findings would provide novel insights into mtDNA mutagenesis and mitochondrial gene therapy for diseases involving pathogenic mtDNA.     

Leptin gene-targeted editing in ob/ob mouse adipose tissue based on the CRISPR/Cas9 system

Gene therapy has become the most effective treatment for monogenic diseases. Congenital LEPTIN deficiency is a rare autosomal recessive monogenic obesity syndrome caused by mutations in the Leptin gene. Ob/ob mouse is a monogenic obesity model, which carries a homozygous point mutation of C to T in Exon 2 of the Leptin gene. Here, we attempted to edit the mutated Leptin gene in ob/ob mice preadipocytes and inguinal adipose tissues using CRISPR/Cas9 to correct the C to T mutation and restore the production of LEPTIN protein by adipocytes. The edited preadipocytes exhibit a correction of 5.5% of Leptin alleles and produce normal LEPTIN protein when differentiated into mature adipocytes. The ob/ob mice display correction of 1.67% of Leptin alleles, which is sufficient to restore the production and physiological functions of LEPTIN protein, such as suppressing appetite and alleviating insulin resistance. Our study suggests CRISPR/Cas9-mediated in situ genome editing as a feasible therapeutic strategy for human monogenic diseases, and paves the way for further research on efficient delivery system in potential future clinical application.