Applications of CRISPR systems in respiratory health: Entering a new ‘red pen’ era in genome editing

Respiratory diseases, such as influenza infection, acute tracheal bronchitis, pneumonia, tuberculosis, chronic obstructive pulmonary disease, asthma, lung cancer and nasopharyngeal carcinoma, continue to significantly impact human health. Diseases of the lung and respiratory tract are influenced by environmental conditions and socio‐economic factors; however, many of these serious respiratory disorders are also rooted in genetic or epigenetic causes. Clustered regularly interspaced palindromic repeats (CRISPR) and CRISPR‐associated (Cas) proteins, isolated from the immune system of prokaryotes, provide a tool to manipulate gene sequences and gene expression with significant implications for respiratory research. CRISPR/Cas systems allow preclinical modelling of causal factors involved in many respiratory diseases, providing new insights into their underlying mechanisms. CRISPR can also be used to screen for genes involved in respiratory processes, development and pathology, identifying novel disease drivers or drug targets. Finally, CRISPR/Cas systems can potentially correct genetic mutations and edit epigenetic marks that contribute to respiratory disorders, providing a form of personalized medicine that could be used in conjunction with other technologies such as stem cell reprogramming and transplantation. CRISPR gene editing is a young field of research, and concerns regarding its specificity, as well as the need for efficient and safe delivery methods, need to be addressed further. However, CRISPR/Cas systems represent a significant step forward for research and therapy in respiratory health, and it is likely we will see the breakthroughs generated from this technology continue.     

利用CRISPR/Cas9慢病毒载体构建敲除大鼠肝星状细胞COX-2基因的细胞模型

目的通过构建CRISPR/Cas9慢病毒载体,转染HSC-T6细胞,获得能稳定表达Cas9蛋白的HSC-T6细胞和COX-2基因缺陷的HSC-T6-COX-2-/-细胞,为后期的功能研究提供良好的工具和手段,为临床上治疗肝纤维化提供新策略。方法设计合成COX-2基因特异性的sgRNA(COX-2-sgRNA-1、COX-2-sgRNA-2、COX-2-sgRNA-3),并将其连接至GV371载体上,提取重组后的质粒,将其与包装质粒共同转染到293T细胞,形成慢病毒颗粒,荧光法检测病毒滴度。按照MOI值计算病毒最适用量,先将Lenti-Cas9-puro转染至HSC-T6细胞,嘌呤霉素筛选得到HSC-T6-Cas9细胞,再用Lenti-COX-2-sgRNA-EGFP转染至HSC-T6-Cas9细胞获得HSC-T6-COX-2-/-细胞,通过Cruiser酶切检测及Western Blot等方法在基因和蛋白水平上进行敲除验证。计量资料多组间比较采用方差分析,进一步两两比较采用LSD-t检验。结果通过测序验证COX-2-sgRNA表达载体构建成功。重组表达质粒和包装质粒共同转染到293T细胞,形成慢病毒颗粒,荧光法检测病毒滴度均在1×108以上。成功构建能稳定传代的Cas9蛋白的HSC-T6细胞和COX-2基因缺陷的HSC-T6-COX-2-/-细胞模型。HSC-T6-Cas9细胞中LV-Cas9-Puro mRNA相对表达量(541.93±105.76)高于CON组细胞(1.00±0.02),差异具有统计学意义(t=12.995,P<0.01)。通过Cruiser酶切检测及Western Blot试验,结果提示CRISPR/Cas9慢病毒表达载体能在靶点起作用,其中COX-2-sgRNA-2敲除作用最明显,并且COX-2蛋白表达水平较CON组和NC组相比显著下降(P值均<0.05),提示COX-2-sgRNA有活性。结论成功构建了针对COX-2靶基因的CRISPR/Cas9慢病毒载体,获得稳定的COX-2基因敲除的HSC-T6-COX-2-/-细胞。     

The promise of CRISPR/Cas9 technology in diabetes mellitus therapy: How gene editing is revolutionizing diabetes research and treatment

Diabetes mellitus is a metabolic disease, characterized by chronic hyperglycemia caused by an abnormality in insulin secretion or action. Millions of people across the world are affected by diabetes mellitus which has serious implications for their health. Over the past few decades, diabetes has become a major cause of mortality and morbidity across the world due to its rapid prevalence. Treatment for diabetes that focuses on insulin secretion and sensitization can lead to unwanted side effects and/or poor compliance, as well as treatment failure. A promising way to treat diabetes is through gene-editing technologies such as clustered regularly interspaced short palindromic repeats (CRISPR/Cas9). However, issues such as efficiency and off-target effects have hindered the use of these technologies. In this review, we summarize what we know today about CRISPR/Cas9 technology's therapeutic potential for treating diabetes. We discuss how different strategies are employed, including cell-based therapies (such as stem cells and brown adipocytes), targeting critical genes involved in diabetes pathogenesis, and discussing the challenges and limitations associated with this technology. A novel and powerful treatment approach to diabetes and other diseases can be found with CRISPR/Cas9 technology, and further research should be carried out in this field.     

CRISPR/Cas9 gene drive technology to control transmission of vector-borne parasitic infections

Gene drive is the process of copying of an endonuclease-containing cassette that leads to increased frequency of inheritance of the desired traits in a targeted population. CRISPR/Cas9 technology is advancing genetic manipulation of insects in the field of gene drive experiments. The CRISPR/Cas9 drive could be engineered for genetic manipulation of parasites and/or vectors for disease control. A number of promising CRISPR/Cas9-based gene drive strategies that interfere with parasite development or impairs the reproductive capability of the insect vector have been proposed in the laboratory for blocking transmission of malaria and leishmaniasis. Still several technical and ethical challenges remain to be addressed, and none appear insuperable in this field.     

陕西省鼠疫耶尔森菌间区规律短回文重复序列基因分型研究及流行病学分析北大核心CSCD

目的通过CRISPR(间区规律短回文重复序列)对陕西省鼠疫菌进行基因分型,分析流行病学特征,为陕西省鼠疫防治工作提供科学依据。方法提取分离自陕西省鼠疫疫源地的66株鼠疫菌核酸,利用针对鼠疫菌的3对CRISPR引物进行PCR扩增,对扩增产物进行测序及分析,确定CRISPR基因分型。结果 66株鼠疫菌在3个位点上共有12种spacer,其中YPa 3种、YPb 5种、YPc 4种,具体为al′、a2′、a3′、b1′、b2′、b29′、b1″、b2″、c1、c2、c3、c3′,基因簇为Cb2′,基因型分为1′和4′两种。结论疫情发生年份不同,鼠疫菌CRISPR基因型不同,鼠疫疫情处置进行溯源应将生态分型与基因分型结合。     

CRISPR/Cas9基因编辑技术在病原微生物中的应用

规律成簇间隔短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR)广泛存在于古细菌及细菌中是细菌在长期进化过程中形成的一种获得性免疫系统。近年来以该系统为基础,经过人工改造形成的一种新型基因编辑技术—CRISPR/Cas9在基因工程领域的应用越发广泛;该技术与前两代编辑技术相比,具有结构简单、成本低廉、实用价值较高等优点。自2012年在基因研究领域成功应用后,已成为当前关注度较高的基因编辑工具;该技术在多种真核生物的基因修饰中已得到成功应用,但在病原微生物上却报道较少。本文将从CRISPR/Cas9系统的结构、作用机制及其在病原微生物基因功能研究中的应用等几个方面进行综述为其深入研究奠定基础。     

Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development

In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.     

基因多态性与炎症性肠病发病的关系

炎症性肠病(IBD)发病机制复杂,近年来多项研究显示,相关基因的多态性在IBD发病中起重要作用,本文对其中主要基因与IBD的关系作一综述.     

Upgraded CRISPR/Cas9 tools for tissue-specific mutagenesis in Drosophila

CRISPR/Cas9 has emerged as a powerful technology for tissue-specific mutagenesis. However, tissue-specific CRISPR/Cas9 tools currently available in Drosophila remain deficient in three significant ways. First, many existing gRNAs are inefficient, such that further improvements of gRNA expression constructs are needed for more efficient and predictable mutagenesis in both somatic and germline tissues. Second, it has been difficult to label mutant cells in target tissues with current methods. Lastly, application of tissue-specific mutagenesis at present often relies on Gal4-driven Cas9, which hampers the flexibility and effectiveness of the system. Here, we tackle these deficiencies by building upon our previous CRISPR-mediated tissue-restricted mutagenesis (CRISPR-TRiM) tools. First, we significantly improved gRNA efficiency in somatic tissues by optimizing multiplexed gRNA design. Similarly, we also designed efficient dual-gRNA vectors for the germline. Second, we developed methods to positively and negatively label mutant cells in tissue-specific mutagenesis by incorporating co-CRISPR reporters into gRNA expression vectors. Lastly, we generated genetic reagents for convenient conversion of existing Gal4 drivers into tissue-specific Cas9 lines based on homology-assisted CRISPR knock-in. In this way, we expand the choices of Cas9 for CRISPR-TRiM analysis to broader tissues and developmental stages. Overall, our upgraded CRISPR/Cas9 tools make tissue-specific mutagenesis more versatile, reliable, and effective in Drosophila. These improvements may be also applied to other model systems.

The ability to characterize gene function in a tissue-specific manner has been critical for studying developmental and disease mechanisms of essential genes. The CRISPR/Cas9 system has recently provided powerful tools for inducing tissue-specific gene loss of function (LOF). In this system, the endonuclease Cas9 is directed by a small guide RNA (gRNA) to a specific DNA sequence to create double-strand breaks (DSBs) (1). In the absence of homologous repair templates, DSBs are primarily repaired by nonhomologous end joining, an error-prone process that often introduces mutations in the form of insertions or deletions (indels) (2, 3). Because the protospacer adjacent motif required for Cas9 action is ubiquitous in genomes (1, 4), by targeting the expression of Cas9 and gRNAs to specific tissues, mutations can be induced at virtually any gene in a tissue-specific manner. However, current tissue-specific CRISPR/Cas9 tools in Drosophila are still deficient in three areas, limiting the power of CRISPR/Cas9 in analyzing gene functions in broad tissues and biological processes.     

Inflammatory bowel disease and the genes for the natural resistance-associated macrophage protein-1 and the interferon-Á receptor 1

The genes for the interferon-γ receptor1 and the natural resistance-associated macrophage protein1 (NRAMP1) control the immune response to intracellular microbial pathogens. Such pathogens, in particular Mycobacterium paratuberculosis, have been implicated in the pathogenesis of Crohn's disease. We studied markers in the genes for NRAMP1 and two mutations in the interferon-γ receptor in relation to inflammatory bowel disease (IBD) in the following groups: 270 healthy individuals, 74 patients with Crohn's disease, 72 patients with ulcerative colitis, and 40 patients with primary sclerosing cholangitis. We studied the allele frequencies of two restriction fragment length polymorphisms in the gene for NRAMP1 and the prevalence of two mutations in the interferon-γ receptor1 gene. The markers in the NRAMP1 gene were not associated with inflammatory bowel disease. Also, the mutations in the interferon-γ receptor1 were not found in the 186 IBD patients. Genetic markers in NRAMP1 are thus not associated with IBD. Therefore this gene is not likely to play a role in the pathogenesis of IBD. The mutation in the interferon-γ receptor was not found in our IBD patients group. Accepted: 13 October 1998     

Plant Viruses: From Targets to Tools for CRISPR

Plant viruses cause devastating diseases in many agriculture systems, being a serious threat for the provision of adequate nourishment to a continuous growing population. At the present, there are no chemical products that directly target the viruses, and their control rely mainly on preventive sanitary measures to reduce viral infections that, although important, have proved to be far from enough. The current most effective and sustainable solution is the use of virus-resistant varieties, but which require too much work and time to obtain. In the recent years, the versatile gene editing technology known as CRISPR/Cas has simplified the engineering of crops and has successfully been used for the development of viral resistant plants. CRISPR stands for ‘clustered regularly interspaced short palindromic repeats’ and CRISPR-associated (Cas) proteins, and is based on a natural adaptive immune system that most archaeal and some bacterial species present to defend themselves against invading bacteriophages. Plant viral resistance using CRISPR/Cas technology can been achieved either through manipulation of plant genome (plant-mediated resistance), by mutating host factors required for viral infection; or through manipulation of virus genome (virus-mediated resistance), for which CRISPR/Cas systems must specifically target and cleave viral DNA or RNA. Viruses present an efficient machinery and comprehensive genome structure and, in a different, beneficial perspective, they have been used as biotechnological tools in several areas such as medicine, materials industry, and agriculture with several purposes. Due to all this potential, it is not surprising that viruses have also been used as vectors for CRISPR technology; namely, to deliver CRISPR components into plants, a crucial step for the success of CRISPR technology. Here we discuss the basic principles of CRISPR/Cas technology, with a special focus on the advances of CRISPR/Cas to engineer plant resistance against DNA and RNA viruses. We also describe several strategies for the delivery of these systems into plant cells, focusing on the advantages and disadvantages of the use of plant viruses as vectors. We conclude by discussing some of the constrains faced by the application of CRISPR/Cas technology in agriculture and future prospects.     

From the Cover: Multigeneration analysis reveals the inheritance,specificity, and patterns of CRISPR/Cas-induced gene modifications in Arabidopsis

The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has emerged as a powerful tool for targeted gene editing in many organisms, including plants. However, all of the reported studies in plants focused on either transient systems or the first generation after the CRISPR/Cas system was stably transformed into plants. In this study we examined several plant generations with seven genes at 12 different target sites to determine the patterns, efficiency, specificity, and heritability of CRISPR/Cas-induced gene mutations or corrections in Arabidopsis. The proportion of plants bearing any mutations (chimeric, heterozygous, biallelic, or homozygous) was 71.2% at T1, 58.3% at T2, and 79.4% at T3 generations. CRISPR/Cas-induced mutations were predominantly 1 bp insertion and short deletions. Gene modifications detected in T1 plants occurred mostly in somatic cells, and consequently there were no T1 plants that were homozygous for a gene modification event. In contrast, ∼22% of T2 plants were found to be homozygous for a modified gene. All homozygotes were stable to the next generation, without any new modifications at the target sites. There was no indication of any off-target mutations by examining the target sites and sequences highly homologous to the target sites and by in-depth whole-genome sequencing. Together our results show that the CRISPR/Cas system is a useful tool for generating versatile and heritable modifications specifically at target genes in plants.Genome engineering tools are important for plant functional genomics research and plant biotechnology. The CRISPR (clustered regularly interspaced short palindromic repeat)/Cas (CRISPR-associated) system has been successfully used for efficient genome editing in human cell lines, zebrafish, and mouse (1–3) and recently applied to gene modification in plants (4–10). In this system a short RNA molecule guides the associated endonuclease Cas9 to generate double strand breaks (DSBs) in the target genomic DNA, which lead to sequence mutations as a result of error-prone nonhomologous end-joining (NHEJ) DNA damage repair or to gene correction or replacement as a result of homology-dependent recombination (HR) (11). It was shown that engineered CRISPR/Cas caused mutations in target genes or corrections in transgenes in transient expression assays in plant protoplasts and tobacco leaves (10). Importantly, stable expression of the CRISPR/Cas in transgenic Arabidopsis, tobacco, and rice plants led to mutations (mostly indels) in target genes and correction of a transgene (4–9). However, it was not known whether the gene mutations and corrections occurred in somatic cells only or whether some of the mutations and corrections happened in germ-line cells and thus may be heritable. Additionally, it is unclear how specific the CRISPR/Cas is in plants. Previous studies in human cell lines indicated a high frequency of off-target effect of CRISPR/Cas-induced mutagenesis (12, 13) but a lower off-target effect in mice and zebrafish (14, 15). Here we show that the CRISPR/Cas-induced transgene correction or mutations in endogenous plant genes and transgenes detected in Arabidopsis T1 plants occurred mostly in somatic cells. However, some of the gene modifications were transmitted through the germ line and were heritable in Arabidopsis T2 and T3 plants following the classic Mendelian model. Mutations caused during DSB repair were predominantly 1 bp insertion and short deletions. Furthermore, our deep sequencing and analysis did not detect any off-targets in multiple CRISPR/Cas transgenic Arabidopsis lines, indicating that the mutagenesis effect of CRISPR/Cas is highly specific in plants.     

Novel genetic markers in inflammatory bowel disease

Genetic factors play a significant role in determining inflammatory bowel disease （IBD） susceptibility. Epidemiologic data support genetic contribution to the pathogenesis of IBD, which include familial aggregation, twin studies, racial and ethnic differences in disease prevalence. Linkage studies have identified several susceptibility genes contained in different genomic regions named IBD1 to IBD9. Nucleotide oligomerization domain （NOD2） and human leukocyte antigen （HLA） genes are the most extensively studied genetic regions （IBD1 and IBD3 respectively） in IBD. Mutations of the NOD2 gene are associated with Crohn＇s disease （CD） and several HLA genes are associated with ulcerative colitis （UC） and CD. Toll like receptors （TLRs） have an important role in the innate immune response against infections by mediating recognition of pathogen-associated microbial patterns. Studying single-nucleotide polymorphisms （SNPs） in molecules involved in bacterial recognition seems to be essential to define genetic backgrounds at risk of IBD. Recently, numerous new genes have been identified to be involved in the genetic susceptibility to IBD： NOD1/Caspase-activation recruitment domains 4 （CARD4）, Chemokine ligand 20 （CCL20）, IL-11, and IL-18 among others. The characterization of these novel genes potentially will lead to the identification of therapeutic agents and clinical assessment of phenotype and prognosis in patients with IBD.     

Use of blood based biomarkers in the evaluation of Crohn&rsquo;s disease and ulcerative colitis

Despite significant improvements in our understanding of Crohn’s disease (CD) and ulcerative colitis (UC) in recent years, questions remain regarding the best approaches to assessment and management of these chronic diseases during periods of both relapse and remission. Various serologic biomarkers have been used in the evaluation of patients with both suspected and documented inflammatory bowel disease (IBD), and while each has potential utility in the assessment of patients with IBD, potential limitation remain with each method of assessment. Given these potential shortcomings, there has been increased interest in other means of evaluation of patients with IBD, including an expanding interest in the role of gene expression profiling. Among patients with IBD, gene expression profiles obtained from whole blood have been used to differentiate active from inactive CD, as well as to differentiate between CD, UC, and non-inflammatory diarrheal conditions. There are many opportunities for a non-invasive, blood based test to aid in the assessment of patients with IBD, particularly when considering more invasive means of evaluation including endoscopy with biopsy. Furthermore, as the emphasis on personalized medicine continues to increase, the potential ability of gene expression analysis to predict patient response to individual therapies offers great promise. While whole blood gene expression analysis may not completely replace more traditional means of evaluating patients with suspected or known IBD, it does offer significant potential to expand our knowledge of the underlying genes involved in the development of these diseases.     

Therapeutic gene editing in haematological disorders with CRISPR/Cas9

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR-associated (Cas)9 platform offers an efficient way of making precise genetic changes to the human genome. This can be employed for disruption, addition and correction of genes, thereby enabling a new class of genetic therapies that can be applied to haematological disorders. Here we review recent technological advances in the CRISPR/Cas9 methodology and applications in haematology for curing monogenic genetic disorders and for engineering novel chimeric antigen receptor (CAR) T cells to treat haematological malignancies. Furthermore, we discuss current challenges for full clinical implementation of CRISPR/Cas9, and reflect on future trajectories of the technology.