基因组编辑技术及其在昆虫研究中的应用

基因组编辑技术是进行功能基因组研究的重要工具．锌指核酸酶技术（ZFNs）、类转录激活因子核酸酶技术（TALENs）以及CRISPR／Cas技术是近年来发展起来的3种主流基因组编辑技术．这3种基因组编辑技术的原理都是通过在生物基因组特定位点制造DNA断裂损伤,从而激活机体自身的DNA损伤修复机制,在此过程中引发各种变异．ZFNs是最早发展的通用基因组编辑技术,可用以实施定点敲除和定点敲入变异,但ZFNs技术的发展受限于构建难度大、成本高等缺点．TALENs技术在ZFNs基础上发展而来,较ZFNs技术而言,TALENs技术具备构建灵活度高、成本低等优势．不同于ZFNs与TALENs技术,CRISPR／Cas技术具有独特的DNA靶向机制,这种机制使其非常适合进行多位点编辑．目前,3种技术都在多种物种中成功测试,例如小鼠、斑马鱼、果蝇、线虫和家蚕．在后基因组时代,这些新技术工具必将在未来功能基因组研究中发挥重大作用．     

CRISPR/Cas系统：RNA靶向的基因组定向编辑新技术

CRISPR/Cas系统广泛存在于细菌及古生菌中, 是机体长期进化形成的RNA指导的降解入侵病毒或噬菌体DNA的适应性免疫系统。对Ⅱ型CRISPR/Cas系统的改造使其成为继锌指核酸酶(ZFNs)和TALE核酸酶(TALENs)以来的另一种对基因组进行高效定点修饰的新技术, 与ZFNs和TALENs相比, CRISPR/Cas系统更简单, 并且更容易操作。文章重点介绍了Ⅱ型CRISPR/Cas系统的基本结构、作用原理及这一技术在基因组定点修饰中的应用, 剖析了该技术可能存在的问题, 展望了CRISPR/Cas系统的应用前景, 为开展这一领域的研究工作提供参考。     

CRISPR/Cas9的应用及脱靶效应研究进展

CRISPR/Cas9基因编辑技术在生命科学领域掀起了一场全新的技术革命,该技术可以对基因组特定位点进行靶向编辑,包括缺失、插入、修复等。CRISPR/Cas9比锌指核酸酶 (ZFNs)和转录激活因子样效应物核酸酶(TALENs)技术更易于操作,而且更高效。CRISPR/Cas9系统中的向导RNA(Single guide RNA, sgRNA)是一段与目标DNA片段匹配的RNA序列,指导Cas9蛋白对基因组进行识别。研究发现,设计的sgRNA会与非靶点DNA序列错配,引入非预期的基因突变,即脱靶效应(Off-target effects)。脱靶效应严重制约了CRISPR/Cas9基因编辑技术的广泛应用。为了避免脱靶效应,研究者对影响脱靶效应的因素进行了系统研究并提出了许多降低脱靶效应的方法。文章总结了CRISPR/Cas9系统的应用及脱靶效应研究进展,以期为相关领域的工作提供参考。     

Detection of on-target and off-target mutations generated by CRISPR/Cas9 and other sequence-specific nucleases

The development of customizable sequence-specific nucleases such as TALENs, ZFNs and the powerful CRISPR/Cas9 system has revolutionized the field of genome editing. The CRISPR/Cas9 system is particularly versatile and has been applied in numerous species representing all branches of life. Regardless of the target organism, all researchers using sequence-specific nucleases face similar challenges: confirmation of the desired on-target mutation and the detection of off-target events. Here, we evaluate the most widely-used methods for the detection of on-target and off-target mutations in terms of workflow, sensitivity, strengths and weaknesses.     

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。     

哺乳动物基因组靶向修饰阳性细胞富集的报告载体系统研究进展

基因组靶向修饰技术对基因功能研究、基因治疗以及转基因育种研究都具有重要的意义和价值。近年来发展起来的人工核酸酶如ZFNs、TALENs和CRISPR/Cas9等的应用大大提高了基因组靶向修饰的效率。但是由于核酸酶表达载体转染效率、核酸酶表达效率及活性以及基因组被打靶后的修复效率等因素在一定程度上制约着基因组靶向修饰阳性细胞的获得。因此富集和筛选基因组靶向修饰阳性细胞是一个亟待解决的问题。报告载体系统可以间接地反映核酸酶的工作效率并有效富集核酸酶修饰的阳性细胞,进而提高基因组靶向修饰阳性细胞的富集和筛选效率。本文主要针对由非同源末端连接(Non-homologous end joining,NHEJ)和单链退火(Single-strand annealing,SSA)两种修复机制分别介导的报告载体系统的原理和应用进行了详细的介绍,以期为以后的相关研究提供借鉴和参考。     

Genome editing with the CRISPR‐Cas system: an art,ethics and global regulatory perspective

Over the last three decades, the development of new genome editing techniques, such as ODM, TALENs, ZFNs and the CRISPR‐Cas system, has led to significant progress in the field of plant and animal breeding. The CRISPR‐Cas system is the most versatile genome editing tool discovered in the history of molecular biology because it can be used to alter diverse genomes (e.g. genomes from both plants and animals) including human genomes with unprecedented ease, accuracy and high efficiency. The recent development and scope of CRISPR‐Cas system have raised new regulatory challenges around the world due to moral, ethical, safety and technical concerns associated with its applications in pre‐clinical and clinical research, biomedicine and agriculture. Here, we review the art, applications and potential risks of CRISPR‐Cas system in genome editing. We also highlight the patent and ethical issues of this technology along with regulatory frameworks established by various nations to regulate CRISPR‐Cas‐modified organisms/products.     

Genome editing technologies and their applications in crop improvement

Crop improvement is very essential to meet the increasing global food demands and enhance food nutrition. Conventional crop-breeding methods have certain limitations such as taking lot of time and resources, and causing biosafety concerns. These limitations could be overcome by the recently emerged-genome editing technologies that can precisely modify DNA sequences at the genomic level using sequence-specific nucleases (SSNs). Among the artificially engineered SSNs, the CRISPR/Cas9 is the most recently developed targeted genome modification system and seems to be more efficient, inexpensive, easy, user-friendly and rapidly adopted genome-editing tool. Large-scale genome editing has not only improved the yield and quality but also has enhanced the disease resistance ability in several model and other major crops. Increasing case studies suggest that genome editing is an efficient, precise and powerful technology that can accelerate basic and applied research towards crop improvement. In this review, we briefly overviewed the structure and mechanism of genome editing tools and then emphatically reviewed the advances in the application of genome editing tools for crop improvement, including the most recent case studies with CRISPR/Cpf1 and base-editing technologies. We have also discussed the future prospects towards the improvement of agronomic traits in crops.     

CRISPR/Cas9: a promising way to exploit genetic variation in plants

Creation of variation in existing gene pool of crop plants is the foremost requirement in crop improvement programmes. Genome editing is a tool to produce knock out of target genes either by introduction of insertion or by deletion that disrupts the function of a specific gene. The CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9) system is the most recent addition to the toolbox of sequence-specific nucleases that includes ZFNs and TALENs. The CRISPR/Cas9 system allows targeted cleavage of genomic DNA guided by a small noncoding RNA, resulting in gene modifications by both non-homologous end joining and homology-directed repair mechanisms. Here, we present an overview of mechanisms of CRISPR, its potential roles in creating variation in germplasm and applications of this novel interference pathway in crop improvement. The availability of the CRISPR/Cas9 system holds promise in facilitating both forward and reverse genetics and will enhance research in crops that lack genetic resources.