CRISPR/Cas系统在病原体核酸检测中的应用进展

快速、灵敏、特异的检测方法对于传染病防控至关重要。聚合酶链式反应、等温扩增等体外核酸扩增技术已广泛应用于病原体检测。近年来,基于规则成簇的间隔短回文重复序列及其相关蛋白(CRISPR/Cas)系统的核酸检测方法显示出快速、高灵敏度、高特异性与便携性等优点。本文对CRISPR/Cas系统类型、原理及其在病原体检测中的应用进展进行综述,并对其应用前景进行展望。     

基于CRISPR/Cas12a的室温稳定鼠疫耶尔森氏菌检测体系的建立

目的 筛选并优化冻干保护剂组合，建立可室温稳定储存的、不影响试剂检测灵敏度的Cas12a鼠疫菌核酸检测冻干体系，为其应用于动物鼠疫监测的现场快速检测奠定基础。方法 以鼠疫耶尔森氏菌pla基因为靶基因，选择9种常用冻干保护剂加入核酸检测试剂中，筛选有明显保护效果的4种保护剂进行优化。按照正交实验设计原则设计保护剂组合，通过37℃加速破坏实验及26℃室温稳定性实验对复合保护剂进行保护效果和灵敏度评价。结果 加入蔗糖、山梨醇和海藻糖3种保护剂的冻干检测体系可在37℃稳定存放2周。室温下存放3个月后体系检测信号强度与37℃加速破坏1周时基本相当，同时检测方法的灵敏度依然能达到10^-5 ng/μL,说明Cas12a检测试剂加入筛选得到的保护剂冻干后，可在室温下稳定保存至少6个月。结论 本研究构建的室温稳定CRISPR/Cas12a鼠疫菌检测体系可在室温下稳定存放至少半年，能够满足鼠疫菌现场快速检测中对试剂稳定性的需求。同时，本研究获得的复合保护剂组分对构建其它室温稳定CRISPR/Cas12a核酸检测体系具有重要参考价值。     

基于CRISPR的埃博拉病毒核酸检测方法的建立

目的建立一种基于CRISPR-Cas13a的埃博拉病毒快速、现场核酸检测方法。方法根据埃博拉病毒基因组保守区序列设计合成特异性RT-RAA引物及crRNA,采用荧光法CRISPR检测技术筛选灵敏度高的crRNA;利用"消线法"CRISPR试纸读取CRISPR检测结果;通过检测梯度稀释的埃博拉病毒核酸及其他病原体核酸评价该方法的灵敏度及特异性。结果从5条靶向埃博拉病毒NP基因保守序列的crRNA中筛选出1条检测灵敏度高的crRNA。利用该crRNA建立了基于"消线法"试纸的CRISPR-Cas13a埃博拉病毒核酸检测方法,其灵敏度为1拷贝/μL,与荧光法CRISPR和RT-qPCR法一致,优于普通RT-PCR法(10~1拷贝/μl)和RT-RAA扩增法(10~2拷贝/μl)。采用该方法检测7种其他病原体核酸,结果均为阴性。结论建立的试纸法CRISPR埃博拉病毒核酸检测方法快速、灵敏、特异,且无需专业核酸检测设备,为实现痕量埃博拉病毒核酸的现场检测提供了新方法。     

CRISPR/Cas9基因编辑技术在炎症性肠病中的研究进展

炎症性肠病(IBD)是一类严重影响全球人口健康的疾病,而CRISPR/Cas9基因编辑技术作为一种革命性的基因工具,正在为IBD的治疗带来新的希望。本文综述了CRISPR/Cas9基因编辑技术在炎症性肠病(IBD)研究中的最新进展。介绍了CRISPR/Cas9基因编辑技术的原理和方法,并对其在IBD基因研究中的应用进行了探讨。尽管CRISPR/Cas9技术在IBD领域显示出潜在的巨大优势,但在递送效率、特异性和安全性等方面仍然面临一些挑战。未来的研究需要进一步解决这些问题,以深入探索CRISPR/Cas9技术在IBD治疗中的潜力和应用前景。     

应用CRISPR/Cas13a快速鉴定布鲁氏菌

目的 建立一种基于CRISPR/Cas13a的布鲁氏菌鉴定方法.方法 以布鲁氏菌BCSP31基因的保守区为靶标区域设计特异的引物和crRNA,建立鉴定布鲁氏菌的CRISPR/Cas13a方法.通过对纯菌株以及临床需氧血培养阳性菌液检测评价该方法的特异性和敏感性;通过梯度稀释法评估其检测下限.结果 建立了基于CRISPR...     

规律簇集间断的短回文重复序列及其相关核酸酶9系统应用于糖尿病领域的研究进展

规律簇集间断的短回文重复序列及其相关核酸酶9(CRISPR/Cas9)系统是一种新型的基因编辑工具,可同时研究多个基因的功能及相互关系,已在多种疾病中显示出巨大的潜在价值。与其他基因编辑技术,如锌指核酸酶(ZFNs)和转录激活样核酸酶(TALENs)相比,CRISPR/Cas9更简单、高效。随着CRISPR/Cas9系统技术的广泛应用,在糖尿病领域有不少相关研究出现。本文从基础到临床对CRISPR/Cas9在糖尿病领域的研究进展作文献回顾,期待为相关人员提供参考。     

基于CRISPR/Cas9技术对刚地弓形虫假定蛋白TGGT1_310420的研究

目的鉴定刚地弓形虫假定蛋白TGGT1_310420在速殖子阶段的功能及其蛋白N端序列的亚细胞定位作用。方法在线设计针对TGGT1_310420的sgRNA,通过定点突变弓形虫CRISPR/Cas9载体pSAG1::CAS9-GFP-U6::sgUPRT上的sgRNA序列获得针对TGGT1_310420的CRISPR/Cas9载体。利用CRISPR/Cas9编辑技术将含有荧光蛋白mCherry和弓形虫次黄嘌呤-黄嘌呤-鸟嘌呤磷酸核糖转移酶(HXGPRT)的PCR片段m CherryTy_HXGPRT插入至TGGT1_310420内源基因编码N端前20个氨基酸残基之后。通过PCR鉴定片段是否正确插入;十二烷基硫酸钠-聚丙烯酰胺凝胶电泳(SDS-PAGE)和蛋白质印迹分析检测融合蛋白的表达情况。间接免疫荧光分析和共聚焦显微镜观察融合蛋白亚细胞定位;空斑实验检测基因缺失株虫体表型的变化。结果 PCR和测序结果显示,成功构建了针对TGGT1_310420的CRISPR/Cas9载体, mCherry-Ty_HXGPRT序列定点插入至靶点位置。蛋白质印迹分析结果显示, mCherry融合蛋白的相对分子质量(Mr)约为36 000;间接免疫荧光实验表明mCherry融合蛋白与弓形虫滑行相关蛋白45 (Tg GAP45)共定位于虫体的表膜。空斑实验检测结果显示, TGGT1_310420的缺失并未引起虫体出现可测的表型变化。结论 TGGT1_310420编码蛋白前20个氨基酸残基序列具有定位到弓形虫表膜的功能, TGGT1_310420在弓形虫速殖子阶段为非必需基因。     

白念珠菌SUMO结合酶UBC9基因缺失株的构建及其在形态调控中的作用研究

目的构建白念珠菌UBC9基因缺失株,探索UBC9基因缺失对细胞形态转换和生物被膜形成的影响。方法采用瞬时CRISPR/Cas9基因敲除系统,分别于体外扩增Cas9基因、sgRNA以及修复DNA,通过醋酸锂法转化白念珠菌野生型菌株SN148,于SD-His选择培养基筛选转化子,通过菌落PCR验证基因型,构建UBC9双等位基因缺失株。分别检测UBC9缺失株在液体培养基中的细胞形态、固体YPD平板上的菌落形态和浸入生长情况以及生物被膜形成能力。结果 PCR检测发现UBC9双等位基因缺失株中能扩出约2.8 kb的单一条带,UBC9基因缺失导致液体培养基中细胞伸长主要以菌丝态形式存在,固体培养基上菌落表面粗糙且深入到培养基内呈典型的浸入生长,并且生物被膜形成能力显著增高(A值为10.31,约为野生型的10.3倍)。结论利用CRISPR/Cas9系统快速构建了白念珠菌UBC9纯合子缺失株,且证明UBC9参与细胞的形态调控和生物被膜形成。     

CRISPR/Cas9基因编辑系统中两种gRNA活性检测方法比较

目的:比较CRISPR/Cas9系统中gRNA活性检测的两种方法,即利用Surveyor/T7E1核酸酶检测方法或利用Cas9蛋白的体外检测方法。方法:一方面构建表达Cas9和特异gRNA的质粒,转染NIH3T3细胞,然后提取DNA,利用T7E1核酸内切酶检测gRNA介导Cas9切割靶DNA并产生indel突变的效率;另一方面,设计引物并利用PCR扩增出gRNA的DNA模板片段,通过体外转录获得gRNA,利用Cas9蛋白及gRNA进行体外反应检测切割效率。结果:利用Cas9蛋白体外酶切可以检测到较高的gRNA活性,然而通过T7E1核酸酶方法检测gRNA在细胞中活性整体偏低,且在不同基因之间差异较大。结论:细胞Surveyor/T7E1法与Cas9蛋白体外酶切法检测到的gRNA活性不完全一致,体外活性检测法不能替代。     

CRISPR/Cas9系统在寄生虫领域的研究进展

CRISPR/Cas9是一种有效的基因组靶向修饰系统,能够实现对基因组特定位点的基因敲除、定点插入和基因修复等遗传操作,为寄生虫的基因功能分析、药物靶点和疫苗分子筛选等研究提供了技术创新平台。本文对CRISPR/Cas9系统的原理及其在刚地弓形虫(Toxoplasma gondii)、疟原虫(Plasmodium)、锥虫(Trypanosoma)和利什曼原虫(Leishmania)等重要寄生虫研究中的新进展进行综述,分析讨论现阶段该系统在寄生虫研究应用方面存在的问题和优化策略。     

Construction of Baculovirus-Inducible CRISPR/Cas9 Antiviral System Targeting BmNPV in Bombyx mori

The silkworm Bombyx mori is an economically important insect. The sericulture industry is seriously affected by pathogen infections. Of these pathogens, Bombyx mori nucleopolyhedrovirus (BmNPV) causes approximately 80% of the total economic losses due to pathogen infections. We previously constructed a BmNPV-specific CRISPR/Cas9 silkworm line with significantly enhanced resistance to BmNPV. In order to optimize the resistance properties and minimize its impact on economic traits, we constructed an inducible CRISPR/Cas9 system for use in transgenic silkworms. We used the 39k promoter, which is induced by viral infection, to express Cas9 and the U6 promoter to express four small guide RNA targeting the genes encoding BmNPV late expression factors 1 and 3 (lef-1 and lef-3, respectively), which are essential for viral DNA replication. The system was rapidly activated when the silkworm was infected and showed considerably higher resistance to BmNPV infection than the wild-type silkworm. The inducible system significantly reduced the development effects due to the constitutive expression of Cas9. No obvious differences in developmental processes or economically important characteristics were observed between the resulting transgenic silkworms and wild-type silkworms. Adoption of this accurate and highly efficient inducible CRISPR/Cas9 system targeting BmNPV DNA replication will result in enhanced antivirus measures during sericulture, and our work also provides insights into the broader application of the CRISPR/Cas9 system in the control of infectious diseases and insect pests.     

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。     

CRISPR/Cas9-Mediated Disruption of the lef8 and lef9 to Inhibit Nucleopolyhedrovirus Replication in Silkworms

Bombyx mori nucleopolyhedrovirus (BmNPV) is a pathogen that causes severe disease in silkworms. In a previous study, we demonstrated that by using the CRISPR/Cas9 system to disrupt the BmNPV ie-1 and me53 genes, transgenic silkworms showed resistance to BmNPV infection. Here, we used the same strategy to simultaneously target lef8 and lef9, which are essential for BmNPV replication. A PCR assay confirmed that double-stranded breaks were induced in viral DNA at targeted sequences in BmNPV-infected transgenic silkworms that expressed small guide RNAs (sgRNAs) and Cas9. Bioassays and qPCR showed that replication of BmNPV and mortality were significantly reduced in the transgenic silkworms in comparison with the control groups. Microscopy showed degradation of midgut cells in the BmNPV-infected wild type silkworms, but not in the transgenic silkworms. These results demonstrated that transgenic silkworms using the CRISPR/Cas9 system to disrupt BmNPV lef8 and lef9 genes could successfully prevent BmNPV infection. Our research not only provides more alternative targets for the CRISPR antiviral system, but also aims to provide new ideas for the application of virus infection research and the control of insect pests.     

CRISPR-Associated (CAS) Effectors Delivery via Microfluidic Cell-Deformation Chip

Identifying new and even more precise technologies for modifying and manipulating selectively specific genes has provided a powerful tool for characterizing gene functions in basic research and potential therapeutics for genome regulation. The rapid development of nuclease-based techniques such as CRISPR/Cas systems has revolutionized new genome engineering and medicine possibilities. Additionally, the appropriate delivery procedures regarding CRISPR/Cas systems are critical, and a large number of previous reviews have focused on the CRISPR/Cas9–12 and 13 delivery methods. Still, despite all efforts, the in vivo delivery of the CAS gene systems remains challenging. The transfection of CRISPR components can often be inefficient when applying conventional delivery tools including viral elements and chemical vectors because of the restricted packaging size and incompetency of some cell types. Therefore, physical methods such as microfluidic systems are more applicable for in vitro delivery. This review focuses on the recent advancements of microfluidic systems to deliver CRISPR/Cas systems in clinical and therapy investigations.     

Rapid Detection of Genotype II African Swine Fever Virus Using CRISPR Cas13a-Based Lateral Flow Strip

The African swine fever virus (ASFV) is a dsDNA virus that can cause serious, highly infectious, and fatal diseases in wild boars and domestic pigs. The ASFV has brought enormous economic loss to many countries, and no effective vaccine or treatment for the ASFV is currently available. Therefore, the on-site rapid and accurate detection of the ASFV is key to the timely implementation of control. The RNA-guided, RNA-targeting CRISPR effector CRISPR-associated 13 (Cas13a; previously known as C2c2) exhibits a “collateral effect” of promiscuous RNase activity upon the target recognition. The collateral cleavage activity of LwCas13a is activated to degrade the non-targeted RNA, when the crRNA of LwCas13a binds to the target RNA. In this study, we developed a rapid and sensitive ASFV detection method based on the collateral cleavage activity of LwCas13a, which combines recombinase-aided amplification (RAA) and a lateral flow strip (named CRISPR/Cas13a-LFD). The method was an isothermal detection at 37 °C, and the detection can be used for visual readout. The detection limit of the CRISPR/Cas13a-LFD was 10¹ copies/µL of p72 gene per reaction, and the detection process can be completed within an hour. The assay showed no cross-reactivity to eight other swine viruses, including classical swine fever virus (CSFV), and has a 100% coincidence rate with real-time PCR detection of the ASFV in 83 clinical samples. Overall, this method is sensitive, specific, and practicable onsite for the ASFV detection, showing a great application potential for monitoring the ASFV in the field.