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。     

Global detection of DNA repair outcomes induced by CRISPR–Cas9

CRISPR–Cas9 generates double-stranded DNA breaks (DSBs) to activate cellular DNA repair pathways for genome editing. The repair of DSBs leads to small insertions or deletions (indels) and other complex byproducts, including large deletions and chromosomal translocations. Indels are well understood to disrupt target genes, while the other deleterious byproducts remain elusive. We developed a new in silico analysis pipeline for the previously described primer-extension-mediated sequencing assay to comprehensively characterize CRISPR–Cas9-induced DSB repair outcomes in human or mouse cells. We identified tremendous deleterious DSB repair byproducts of CRISPR–Cas9 editing, including large deletions, vector integrations, and chromosomal translocations. We further elucidated the important roles of microhomology, chromosomal interaction, recurrent DSBs, and DSB repair pathways in the generation of these byproducts. Our findings provide an extra dimension for genome editing safety besides off-targets. And caution should be exercised to avoid not only off-target damages but also deleterious DSB repair byproducts during genome editing.     

Construction of a One-Vector Multiplex CRISPR/Cas9 Editing System to Inhibit Nucleopolyhedrovirus Replication in Silkworms

Recently the developed single guide(sg)RNA-guided clustered regularly interspaced short palindromic repeats/associated protein 9 nuclease(CRISPR/Cas9) technology has opened a new avenue for antiviral therapy. The CRISPR/Cas9 system uniquely allows targeting of multiple genome sites simultaneously. However, there are relatively few applications of CRISPR/Cas9 multigene editing to target insect viruses. To address the need for sustained delivery of a multiplex CRISPR/Cas9-based genome-editing vehicle against insect viruses, we developed a one-vector(pSL1180-Cas9-U6-sgRNA) system that expresses multiple sgRNA and Cas9 protein to excise Bombyx mori nucleopolyhedrovirus(BmNPV) in insect cells.We screened the immediate-early-1 gene(ie-1), the major envelope glycoprotein gene(gp64), and the late expression factor gene(lef-11), and identified multiple sgRNA editing sites through flow cytometry and viral DNA replication analysis. In addition, we constructed a multiplex editing vector(PSL1180-Cas9-sgIE1-sgLEF11-sgGP64, sgMultiple) to efficiently regulate multiplex gene-editing and inhibit BmNPV replication after viral infection. This is the first report of the application of a multiplex CRISPR/Cas9 system to inhibit insect virus replication. This multiplex system can significantly enhance the potential of CRISPR/Cas9-based multiplex genome engineering in insect virus.     

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。     

Delivery of CRISPR/Cas9 for therapeutic genome editing

The clustered, regularly‐interspaced, short palindromic repeat (CRISPR)‐associated nuclease 9 (CRISPR/Cas9) is emerging as a promising genome‐editing tool for treating diseases in a precise way, and has been applied to a wide range of research in the areas of biology, genetics, and medicine. Delivery of therapeutic genome‐editing agents provides a promising platform for the treatment of genetic disorders. Although viral vectors are widely used to deliver CRISPR/Cas9 elements with high efficiency, they suffer from several drawbacks, such as mutagenesis, immunogenicity, and off‐target effects. Recently, non‐viral vectors have emerged as another class of delivery carriers in terms of their safety, simplicity, and flexibility. In this review, we discuss the modes of CRISPR/Cas9 delivery, the barriers to the delivery process and the application of CRISPR/Cas9 system for the treatment of genetic disorders. We also highlight several representative types of non‐viral vectors, including polymers, liposomes, cell‐penetrating peptides, and other synthetic vectors, for the therapeutic delivery of CRISPR/Cas9 system. The applications of CRISPR/Cas9 in treating genetic disorders mediated by the non‐viral vectors are also discussed.     

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 gene editing. A promising strategy in hematological disorders

The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized the gene editing field, making it possible to interrupt, insert or replace a sequence of interest with high precision in the human genome. Its easy design and wide applicability open up a variety of therapeutic alternatives for the treatment of genetic diseases. Indeed, very promising approaches for the correction of hematological disorders have been developed in the recent years, based on the self-renewal and multipotent differentiation properties of hematopoietic stem and progenitor cells, which make this cell subset the ideal target for gene therapy purposes. This technology has been applied in different congenital blood disorders, such as primary immunodeficiencies, X-linked severe combined immunodeficiency, X-linked chronic granulomatous disease or Wiskott–Aldrich syndrome, and inherited bone marrow failure syndromes, such as Fanconi anemia, congenital amegakaryocytic thrombocytopenia or severe congenital neutropenia. Furthermore, CRISPR/Cas9-based gene editing has been implemented successfully as a novel therapy for cancer immunotherapy, by the development of promising strategies such as the use of oncolytic viruses or adoptive cellular therapy to the chimeric antigen receptor–T-cell therapy. Therefore, considering the variety of genes and mutations affected, we can take advantage of the different DNA repair mechanisms by CRISPR/Cas9 in different manners, from homology-directed repair to non-homologous-end-joining to the latest emerging technologies such as base and prime editing. Although the delivery systems into hematopoietic stem and progenitor cells are still the bottleneck of this technology, some of the advances in genome editing shown in this review have already reached a clinical stage and show very promising preliminary results.     

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。     

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。     

快速构建CRISPR/Cas9基因组靶向编辑系统

CRISPR/Cas9核酸酶作为一种新的基因组靶向编辑技术,已成功应用于多种动植物基因组修饰研究. CRISPR/Cas9作用后的阳性细胞筛选和富集是该技术的关键之一. 本研究以鸡EAV-HP（endogenous avian retrovirus-HP）基因和MSTN（myostatin）基因为例,从靶位点的选择、表达载体构建、双基因报告载体构建和核酸酶活性验证4个方面,系统研究了CRISPR/Cas9核酸酶技术平台. 结果表明,利用寡聚核苷酸直接退火方法,构建表达载体和报告载体的阳性率分别高达100%和89.5%. 报告载体的PuroR（puromycin resistant gene）和eGFP（enhanced green fluorescent protein）基因的成功表达表明,构建的CRISPR/Cas9系统能有效切割靶序列,并用于后续阳性克隆的筛选和富集. 本方法摒弃了传统分子克隆的PCR扩增和酶切处理目标基因的方法,而是利用寡聚核苷酸直接退火获得含有黏性末端的目标DNA,简化了载体构建过程,低成本且快速获得CRISPR/Cas9基因组靶向编辑系统.     

Improvement of baculovirus as protein expression vector and as biopesticide by CRISPR/Cas9 editing

The clustered regularly interspaced short palindromic repeats (CRISPR) system–associated Cas9 endonuclease is a molecular tool that enables specific sequence editing with high efficiency. In this study, we have explored the use of CRISPR/Cas9 system for the engineering of baculovirus. We have shown that the delivering of Cas9-single guide RNA ribonucleoprotein (RNP) complex with or without DNA repair template into Sf21 insect cells through lipofection might be efficient to produce knockouts as well as knock-ins into the baculovirus. To evaluate potential application of our CRISPR/Cas9 method to improve baculovirus as protein expression vector and as biopesticide, we attempted to knockout several genes from a recombinant AcMNPV form used in the baculovirus expression system as well as in a natural occurring viral isolate from the same virus. We have additionally confirmed the adaptation of this methodology for the generation of viral knock-ins in specific regions of the viral genome. Analysis of the generated mutants revealed that the editing efficiency and the type of changes was variable but relatively high. Depending on the targeted gene, the editing rate ranged from 10% to 40%. This study established the first report revealing the potential of CRISPR/Cas9 for genome editing in baculovirus, contributing to the engineering of baculovirus as a protein expression vector as well as a biological control agent.     

Off‐target assessment of CRISPR‐Cas9 guiding RNAs in human iPS and mouse ES cells

The CRISPR‐Cas9 system consists of a site‐specific, targetable DNA nuclease that holds great potential in gene editing and genome‐wide screening applications. To apply the CRISPR‐Cas9 system to these assays successfully, the rate at which Cas9 induces DNA breaks at undesired loci must be understood. We characterized the rate of Cas9 off‐target activity in typical Cas9 experiments in two human and one mouse cell lines. We analyzed the Cas9 cutting activity of 12 gRNAs in both their targeted sites and ～90 predicted off‐target sites per gRNA. In a Cas9‐based knockout experiment, gRNAs induced detectable Cas9 cutting activity in all on‐target sites and in only a few off‐target sites genome‐wide in human 293FT, human‐induced pluripotent stem (hiPS) cells, and mouse embryonic stem (ES) cells. Both the cutting rates and DNA repair patterns were highly correlated between the two human cell lines in both on‐target and off‐target sites. In clonal Cas9 cutting analysis in mouse ES cells, biallelic Cas9 cutting was observed with low off‐target activity. Our results show that off‐target activity of Cas9 is low and predictable by the degree of sequence identity between the gRNA and a potential off‐target site. Off‐target Cas9 activity can be minimized by selecting gRNAs with few off‐target sites of near complementarity. genesis 53:225–236, 2015. © 2014 The Authors. Genesis Published by Wiley Periodicals, Inc.     

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.     

Biallelic CCM3 mutations cause a clonogenic survival advantage and endothelial cell stiffening

CCM3, originally described as PDCD10, regulates blood‐brain barrier integrity and vascular maturation in vivo. CCM3 loss‐of‐function variants predispose to cerebral cavernous malformations (CCM). Using CRISPR/Cas9 genome editing, we here present a model which mimics complete CCM3 inactivation in cavernous endothelial cells (ECs) of heterozygous mutation carriers. Notably, we established a viral‐ and plasmid‐free crRNA:tracrRNA:Cas9 ribonucleoprotein approach to introduce homozygous or compound heterozygous loss‐of‐function CCM3 variants into human ECs and studied the molecular and functional effects of long‐term CCM3 inactivation. Induction of apoptosis, sprouting, migration, network and spheroid formation were significantly impaired upon prolonged CCM3 deficiency. Real‐time deformability cytometry demonstrated that loss of CCM3 induces profound changes in cell morphology and mechanics: CCM3‐deficient ECs have an increased cell area and elastic modulus. Small RNA profiling disclosed that CCM3 modulates the expression of miRNAs that are associated with endothelial ageing. In conclusion, the use of CRISPR/Cas9 genome editing provides new insight into the consequences of long‐term CCM3 inactivation in human ECs and supports the hypothesis that clonal expansion of CCM3‐deficient dysfunctional ECs contributes to CCM formation.     

Establishing RNA virus resistance in plants by harnessing CRISPR immune system

Recently, CRISPR‐Cas (clustered, regularly interspaced short palindromic repeats–CRISPR‐associated proteins) system has been used to produce plants resistant to DNA virus infections. However, there is no RNA virus control method in plants that uses CRISPR‐Cas system to target the viral genome directly. Here, we reprogrammed the CRISPR‐Cas9 system from Francisella novicida to confer molecular immunity against RNA viruses in Nicotiana benthamiana and Arabidopsis plants. Plants expressing FnCas9 and sgRNA specific for the cucumber mosaic virus (CMV) or tobacco mosaic virus (TMV) exhibited significantly attenuated virus infection symptoms and reduced viral RNA accumulation. Furthermore, in the transgenic virus‐targeting plants, the resistance was inheritable and the progenies showed significantly less virus accumulation. These data reveal that the CRISPR/Cas9 system can be used to produce plant that stable resistant to RNA viruses, thereby broadening the use of such technology for virus control in agricultural field.     

CRISPR/Cas9 System as an Agent for Eliminating Polyomavirus JC Infection

Progressive multifocal leukoencephalopathy (PML) is a fatal demyelinating disease of the central nervous system (CNS) caused by reactivation of the human polyomavirus JCV gene expression and its replication in oligodendrocytes, the myelin producing cells in the brain. Once a rare disease seen in patients with lymphotproliferative and myeloproliferative disorders, PML has been seen more frequently in HIV-1 positive/AIDS patients as well as patients undergoing immunomodulatory therapy due for autoimmune disorders including multiple sclerosis, rheumatoid arthritis, and others. As of now there is no cure for PML and in most cases disease progression leads to death within two years. Similar to other polyomaviruses, the JCV genome is small circular double stranded DNA that includes coding sequences for the viral early protein, T-antigen, which is critical for directing viral reactivation and lytic infection. Here, we employ a newly developed gene editing strategy, CRISPR/Cas9, to introduce mutations in the viral genome and, by inactivating the gene encoding T-antigen, inhibit viral replication. We first used bioinformatics screening and identified several potential targets within the JCV T-antigen gene that can serve as sites for the creation of guide RNAs (gRNAs) for positioning the Cas9 nuclease on the designated area of the viral genome for editing. Results from a series of integrated genetic and functional studies showed that transient or conditional expression of Cas9 and gRNAs specifically targets the DNA sequences corresponding to the N-terminal region of T-antigen, and by introducing mutation, interferes with expression and function of of the viral protein, hence suppressing viral replication in permissive cells. Results from SURVEYOR assay revealed no off-target effects of the JCV-specific CRISPR/Cas9 editing apparatus. These observations provide the first evidence for the employment of a gene editing strategy as a promising tool for the elimination of the JCV genome and a potential cure for PML.     

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。     

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。