利用CRISPR/Cas9技术对人多能干细胞进行高效基因组编辑

CRISPR/Cas9技术提供了一个全新的基因组编辑体系。本文利用CRISPR/Cas9平台,在人胚胎干细胞株中对选取的一段特定基因组区域进行了多种基因组编辑：通过在基因编码框中引入移码突变进行基因敲除;通过单链DNA提供外源模板经由同源重组定点敲入FLAG序列;通过同时靶向多个位点诱导基因组大片段删除。研究结果表明CRISPR/Cas9可以对多能干细胞进行高效基因编辑,获得的突变干细胞株有助于对基因和基因组区域的功能进行分析和干细胞疾病模型的建立。     

CRISPR/Cas9系统在基因组DNA片段编辑中的应用

源于细菌和古菌的Ⅱ型成簇规律间隔短回文重复系统[Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated nuclease 9 (Cas9),CRISPR/Cas9]近年被改造成为基因组定点编辑的新技术。由于它具有设计简单、操作方便、费用低廉等巨大优势,给遗传操作领域带来了一场革命性的改变。本文重点介绍了CRISPR/Cas9系统在基因组DNA片段靶向编辑方面的研究和应用,主要包括DNA片段的删除、反转、重复、插入和易位,这一有效的DNA片段编辑方法为研究基因功能、调控元件、组织发育和疾病发生发展提供了有力手段。本文最后展望了Ⅱ型CRISPR/Cas9系统的应用前景和其他类型CRISPR系统的应用潜力,为开展利用基因组DNA片段靶向编辑进行基因调控和功能研究提供参考。     

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。     

Anti‐HIV‐1 potency of the CRISPR/Cas9 system insufficient to fully inhibit viral replication

The range of genome‐editing tools has recently been expanded. In particular, an RNA‐guided genome‐editing tool, the clustered regularly interspaced short palindromic repeat (CRISPR)‐associated 9 (Cas9) system, has many applications for human diseases. In this study, guide RNA (gRNA) to target gag, pol and a long terminal repeat of HIV‐1 was designed and used to generate gRNA‐expressing lentiviral vectors. An HIV‐1‐specific gRNA and Cas9 were stably dually transduced into a highly HIV‐1‐susceptible human T‐cell line and the inhibitory ability of the anti‐HIV‐1 CRISPR/Cas9 lentiviral vector assessed. Although clear inhibition of the early phase of HIV‐1 infection was observed, as evaluated by a VSV‐G‐pseudotyped HIV‐1 reporter system, the anti‐HIV‐1 potency in multiple rounds of wild type (WT) viral replication was insufficient, either because of generation of resistant viruses or overcoming of the activity of the WT virus. Thus, there are potential difficulties that must be addressed when considering anti‐HIV‐1 treatment with the CRISPR/Cas9 system alone.     

Increasing the efficiency of CRISPR‐Cas9‐VQR precise genome editing in rice

Clustered regularly interspaced short palindromic repeats‐associated protein 9 (CRISPR‐Cas9) is a revolutionary technology that enables efficient genomic modification in many organisms. Currently, the wide use of Streptococcus pyogenes Cas9 (SpCas9) primarily recognizes sites harbouring a canonical NGG protospacer adjacent motif (PAM). The newly developed VQR (D1135V/R1335Q/T1337R) variant of Cas9 has been shown to cleave sites containing NGA PAM in rice, which greatly expanded the range of genome editing. However, the low editing efficiency of the VQR variant remains, which limits its wide application in genome editing. In this study, by modifying the single guide RNA (sgRNA) structure and strong endogenous promoters, we significantly increased the editing efficiency of the VQR variant. The modified CRISPR‐Cas9‐VQR system provides a robust toolbox for multiplex genome editing at sites containing noncanonical NGA PAM.     

Recent Progress in CRISPR/Cas9 Technology

The clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 system, a simple and efficient tool for genome editing, has experienced rapid progress in its technology and applicability in the past two years. Here, we review the recent advances in CRISPR/Cas9 technology and the ways that have been adopted to expand our capacity for precise genome manipulation. First, we introduce the mechanism of CRISPR/Cas9, including its biochemical and structural implications. Second, we highlight the latest improvements in the CRISPR/Cas9 system, especially Cas9 protein modifications for customization. Third, we review its current applications, in which the versatile CRISPR/Cas9 system was employed to edit the genome, epigenome, or RNA of various organisms. Although CRISPR/Cas9 allows convenient genome editing accompanied by many benefits, we should not ignore the significant ethical and biosafety concerns that it raises. Finally, we discuss the prospective applications and challenges of several promising techniques adapted from CRISPR/Cas9.     

The new CRISPR–Cas system: RNA-guided genome engineering to efficiently produce any desired genetic alteration in animals

The CRISPR–Cas system is the newest targeted nuclease for genome engineering. In less than 1 year, the ease, robustness and efficiency of this method have facilitated an immense range of genetic modifications in most model organisms. Full and conditional gene knock-outs, knock-ins, large chromosomal deletions and subtle mutations can be obtained using combinations of clustered regularly interspaced short palindromic repeats (CRISPRs) and DNA donors. In addition, with CRISPR–Cas compounds, multiple genetic modifications can be introduced seamlessly in a single step. CRISPR–Cas not only brings genome engineering capacities to species such as rodents and livestock in which the existing toolbox was already large, but has also enabled precise genetic engineering of organisms with difficult-to-edit genomes such as zebrafish, and of technically challenging species such as non-human primates. The CRISPR–Cas system allows generation of targeted mutations in mice, even in laboratories with limited or no access to the complex, time-consuming standard technology using mouse embryonic stem cells. Here we summarize the distinct applications of CRISPR–Cas technology for obtaining a variety of genetic modifications in different model organisms, underlining their advantages and limitations relative to other genome editing nucleases. We will guide the reader through the many publications that have seen the light in the first year of CRISPR–Cas technology.     

CRISPR/Cas9-Mediated Genome Editing in Soybean Hairy Roots

As a new technology for gene editing, the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been rapidly and widely used for genome engineering in various organisms. In the present study, we successfully applied type II CRISPR/Cas9 system to generate and estimate genome editing in the desired target genes in soybean (Glycine max (L.) Merrill.). The single-guide RNA (sgRNA) and Cas9 cassettes were assembled on one vector to improve transformation efficiency, and we designed a sgRNA that targeted a transgene (bar) and six sgRNAs that targeted different sites of two endogenous soybean genes (GmFEI2 and GmSHR). The targeted DNA mutations were detected in soybean hairy roots. The results demonstrated that this customized CRISPR/Cas9 system shared the same efficiency for both endogenous and exogenous genes in soybean hairy roots. We also performed experiments to detect the potential of CRISPR/Cas9 system to simultaneously edit two endogenous soybean genes using only one customized sgRNA. Overall, generating and detecting the CRISPR/Cas9-mediated genome modifications in target genes of soybean hairy roots could rapidly assess the efficiency of each target loci. The target sites with higher efficiencies can be used for regular soybean transformation. Furthermore, this method provides a powerful tool for root-specific functional genomics studies in soybean.     

Recent advances in CRISPR/Cas9 mediated genome editing in <Emphasis Type="Italic">Bacillus subtilis</Emphasis>

Genome editing using engineered nucleases has rapidly transformed from a niche technology to a mainstream method used in various host cells. Its widespread adoption has been largely developed by the emergence of the clustered regularly interspaced short palindromic repeats (CRISPR) system, which uses an easily customizable specificity RNA-guided DNA endonuclease, such as Cas9. Recently, CRISPR/Cas9 mediated genome engineering has been widely applied to model organisms, including Bacillus subtilis, enabling facile, rapid high-fidelity modification of endogenous native genes. Here, we reviewed the recent progress in B. subtilis gene editing using CRISPR/Cas9 based tools, and highlighted state-of-the-art strategies for design of CRISPR/Cas9 system. Finally, future perspectives on the use of CRISPR/Cas9 genome engineering for sequence-specific genome editing in B. subtilis are provided.     

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.     

CRISPR-Cas9基因编辑技术在病毒感染疾病治疗中的应用

CRISPR-Cas9基因编辑技术是基于细菌或古细菌CRISPR介导的获得性免疫系统衍生而来,由一段RNA通过碱基互补配对识别DNA,指导Cas9核酸酶切割识别的双链DNA,诱发同源重组或非同源末端链接,进而实现在目的DNA上进行编辑。病毒通过特异的受体侵染细胞,其基因组在细胞内发生复制、转录、翻译等过程完成其生活周期,某些DNA病毒或逆转录病毒基因组会整合到宿主基因组中。基因治疗是病毒感染疾病治疗的新趋势。因此,基因编辑技术在持续感染的病毒或潜伏感染病毒疾病治疗中具有重大的潜在意义。文章主要从CRISPR-Cas9作用机制以及在病毒感染疾病治疗中的应用等方面进行了综述。     

Connecting genotypes,phenotypes and fitness: harnessing the power of CRISPR/Cas9 genome editing

One of the fundamental goals in evolution and ecology is to identify the genetic basis of adaptive phenotypes. Unfortunately, progress towards this goal has been hampered by a lack of genetic tools available for nonmodel organisms. The exciting new development of the CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 (CRISPR‐associated nuclease 9) genome‐editing system now promises to transform the field of molecular ecology by providing a versatile toolkit for manipulating the genome of a wide variety of organisms. Here, we review the numerous applications of this groundbreaking technology and provide a practical guide to the creation of genetic knockouts, transgenics and other related forms of gene manipulation in nonmodel organisms. We also specifically discuss the potential uses of the CRISPR/Cas9 system in ecological and evolutionary studies, which will further advance the field towards the long‐standing goal of connecting genotypes, phenotypes and fitness.     

Rapid generation of genetic diversity by multiplex CRISPR/Cas9 genome editing in rice

The clustered regularly interspaced short palindromic repeats(CRISPR)-associated endonuclease 9(CRISPR/Cas9) system has emerged as a promising technology for specific genome editing in many species. Here we constructed one vector targeting eight agronomic genes in rice using the CRISPR/Cas9 multiplex genome editing system. By subsequent genetic transformation and DNA sequencing, we found that the eight target genes have high mutation efficiencies in the T_0 generation. Both heterozygous and homozygous mutations of all editing genes were obtained in T_0 plants. In addition, homozygous sextuple, septuple, and octuple mutants were identified. As the abundant genotypes in T_0 transgenic plants, various phenotypes related to the editing genes were observed. The findings demonstrate the potential of the CRISPR/Cas9 system for rapid introduction of genetic diversity during crop breeding.     

CRISPR/Cas9技术在感染性疾病中的应用

成簇的规律间隔的短回文重复序列及其相关蛋白9〔clustered regularly interspaced short palindromic repeat(CRISPR)/CRISPR-associated protein 9(Cas9),CRISPR/Cas9〕是一种新兴的基因编辑技术,与以前的三大基因编辑技术——归巢核酸内切酶、锌指核酸酶和转录激活因子样效应物核酸酶技术相比,其在靶向特异性、操作简便性、治疗彻底性、应用广泛性等方面具有更大的优势和发展潜力。艾滋病、乙型肝炎、疟疾等感染性疾病的治疗一直是医学上的重大难题,科学家正努力尝试利用CRISPR/Cas9技术解决这些医学难题。本文主要综述了CRISPR/Cas9技术在这些感染性疾病中应用的研究进展。     

The CRISPR/Cas9 revolution continues: From base editing to prime editing in plant science

The ability to precisely inactivate or modify genes in model organisms helps us understand the mysteries of life. Clustered regularly interspaced short palindromic repeats(CRISPR)/CRISPR-associated protein 9(Cas9), a revolutionary technology that could generate targeted mutants, has facilitated notable advances in plant science. Genome editing with CRISPR/Cas9 has gained great popularity and enabled several technical breakthroughs. Herein, we briefly introduce the CRISPR/Cas9, with a focus on the latest breakthroughs in precise genome editing(e.g., base editing and prime editing), and we summarize various platforms that developed to increase the editing efficiency, expand the targeting scope, and improve the specificity of base editing in plants. In addition, we emphasize the recent applications of these technologies to plants. Finally, we predict that CRISPR/Cas9 and CRISPR/Cas9-based genome editing will continue to revolutionize plant science and provide technical support for sustainable agricultural development.