沙门氏菌CRISPR位点的结构特征比较

成簇规律间隔的短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR),是存在于多数细菌和古菌中的遗传结构,能够有效防御外源DNA的入侵(质粒、噬菌体等),进而防御外源基因的水平转移。【目的】本研究以沙门氏菌属中常见的鸡伤寒沙门氏菌(Salmonella gallinarum)、鼠伤寒沙门氏菌(Salmonella typhimurium)、猪霍乱沙门氏菌(Salmonella choleraesuis)以及肠炎沙门氏菌(salmonella enteritidis)等30个菌株为研究对象。探索CRISPR位点在不同沙门氏菌种中的结构差异。【方法】通过生物信息学的方法比较间隔序列与插入序列的同源性以及CRISPR位点与质粒数量关系。【结果】30株沙门氏菌中均存在CRISPR结构,包括CRISPR位点61个以及可疑位点12个。重复序列和cas1基因均不能作为这4类细菌的分类依据。【结论】虽然我们发现CRISPR位点数量与间隔区数量和质粒数量之间均不存在统计学关系,但间隔序列整合子、耐药基因等移动遗传原件具有一定的同源性,说明沙门氏菌在进化过程中不断受外源基因的侵袭。     

醋酸菌中CRISPR位点的比较基因组学与进化分析

CRISPR (Clustered regularly interspaced short palindromic repeats)是近几年发现的一种广泛存在于细菌和古菌中,能够应对外源DNA干扰(噬菌体、病毒、质粒等),并提供免疫机制的重复序列结构。CRISPR系统通常由同向重复序列、前导序列、间隔序列和CRISPR相关蛋白组成。本研究以醋酸发酵中常见3个属醋杆菌属(Acetobacter)、葡糖醋杆菌属(Gluconacetobacter)和葡糖杆菌属(Gluconobacter)的48个菌株为研究对象,通过其基因组上CRISPR相关基因序列的生物信息学分析,探索CRISPR位点在醋酸菌中的多态性及其进化模式。结果表明48株醋酸菌中有32株存在CRISPR结构,大部分CRISPR-Cas结构属于type I-E和type I-C类型。除了葡糖杆菌属外,葡糖醋杆菌属和醋杆菌属中的部分菌株含有II类的CRISPR-Cas系统结构(CRISPR-Cas9)。来自不同属菌株的CRISPR结构中重复序列具有较强的保守性,而且部分菌株CRISPR结构中的前导序列具有保守的motif (与基因的转录调控有关)及启动子序列。进化树分析表明cas1适合用于醋酸菌株的分类,而不同菌株间cas1基因的进化与重复序列的保守性相关,预示它们可能受相似的功能选择压力。此外,间隔序列的数量与噬菌体数量及插入序列(Insertion sequence, IS)数量有正相关的趋势,说明醋酸菌在进化过程中可能正不断受新的外源DNA入侵。醋酸菌中CRISPR结构位点的分析,为进一步研究不同醋酸菌株对醋酸胁迫耐受性差异及其基因组稳定性的分子机制奠定了基础。     

CRISPR在食源性致病菌进化分析、检测分型及毒力耐药调控中的应用进展

规律成簇间隔的短回文重复序列 (Clustered regularly interspaced short palindromic repeats,CRISPR) 与其相关蛋白基因系统可通过限制基因的水平转移而有效防御噬菌体等外源基因元件的入侵,不同细菌之间的CRISPR结构有所差异。基于CRISPR系统的差异性,文中对近几年CRISPR在食源性致病菌进化分析、检测与分型、毒力与耐药中的应用研究进行概述,并对基于CRISPR序列特点开发的细菌检测分型方法以及CRISPR与食源性致病菌的毒力、耐药性之间的相关性进行重点总结分析。此外,文中探讨了CRISPR在进化分析、检测与分型、毒力与耐药应用中的不足,提出将CRISPR分型方法标准化、完善与扩充致病菌CRISPR数据库、进一步探究噬菌体与细菌之间的共进化关系等建议,为进一步探索CRISPR功能提供参考。     

副溶血性弧菌群中规律成簇间隔短回文重复序列的比较分析

规律成簇间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)是大多数细菌和古细菌在生存压力下进化出的一套抵抗噬菌体干扰的防御系统。本研究主要采用生物信息学的方法,对24株分离自人体且已完成全基因组测序的副溶血性弧菌内CRISPR结构进行了分析,结果发现:只有16株细菌包含1个及以上的CRISPR结构,共计29个CRISPR;仅11个具有真座位特征的CRISPR结构含有前导序列;CRISPR结构中的重复序列所形成的RNA二级结构具有一大一小共两环或一大二小共三环的特征;目前未找到与区间序列高度同源的外源遗传物质;仅含前导序列的CRISPR结构侧翼区才存在cas基因。副溶血性弧菌的CRISPR结构可能以水平基因转移的方式整合到细菌的染色体中,CRISPR结构不适合作为细菌分类的一项指标。     

蜡状芽孢杆菌群中规律成簇间隔短回文重复序列的生物信息学分析

规律成簇间隔短回文重复序列(clustered regularly interspaced short palindromic repeats,CRISPR)是最近发现针对噬菌体等外源遗传物质的获得性和可遗传性的新型原核生物防御系统。通过BLAST、多序列比对、RNA二级结构预测等生物信息学方法对已经完成全基因组测序的蜡状芽孢杆菌群24个菌株进行CRISPR的系统分析,结果表明:42%的菌株含有该结构;8个CRISPR座位的正向重复序列可以形成RNA二级结构,提示正向重复序列可能介导外源DNA或RNA与CAS编码蛋白的相互作用;31%的间区序列与噬菌体、质粒、蜡状芽孢杆菌群基因组序列具有同源性,进一步验证间区序列很可能来源于外源可移动遗传因子。由于大部分蜡状芽孢杆菌群菌株含有多个前噬菌体和质粒,通过对蜡状芽孢杆菌群CRISPR的分析,为揭示其对宿主菌与噬菌体,以及宿主菌与质粒间的关系奠定基础。     

CRISPR在细菌分型和进化中的研究进展

规律成簇的间隔的短回文重复序列（CRISPR）是近年发现的一类存在于古细菌和细菌基因组内的结构,该结构可以使细菌获得对外源DNA如质粒和噬菌体的免疫,同时由于其结构的多态性,也可作为细菌分型和进化研究的位点。简要综述了CRSIPR系统的基本结构,及其在分型和进化应用方面的研究进展。     

规律成簇的间隔短回文重复：结构，功能与应用

规律成簇的间隔短回文重复(Clustered regularly interspaced short palindromic repeats,CRISPRs)是一类广泛分布于细菌和古菌基因组中的重复结构.最近研究表明,CRISPR与一系列相关蛋白、前导序列一起,为原核生物提供对噬菌体等外源基因的获得性免疫能力,其作用机制可能与真核生物的RNA干扰过程类似.作为基因组中高度可变的区域,CRISPR非常适合成为研究细菌种内分型和微进化的分子靶标.本文综述了CRISPR系统的结构、功能及其应用概况,并对CRISPR研究的前景进行了展望.     

产甲烷古菌中CRISPR簇的研究

【目的】通过对51个产甲烷古菌基因组中成簇的规律间隔短回文重复序列(Clustered regularly interspaced short palindromic repeats,CRISPR)的组成和来源进行研究,推测产甲烷古菌与环境中其他微生物的物质交换和相互作用,在基因组水平上阐述产甲烷古菌之间的遗传差异。【方法】利用CRISPRdb和CRISPRFinder,找出产甲烷古菌基因组中所有潜在的CRISPR簇。对CRISPR簇的基本组成部分进行分析:利用BLASTCLUST对重复序列(Repeat)进行分类;分别将间隔序列(Spacer)与Refseq病毒基因组、Refseq质粒基因组和Refseq产甲烷古菌基因组进行比对,从而获得间隔序列的物种来源和功能信息的注释。【结果】在51个产甲烷古菌中共找到了196个CRISPR簇,这些CRISPR簇中包含了总共4 355条间隔序列。在这些产甲烷古菌中,CRISPR簇的分布是不均匀的,且每个物种的间隔序列数量与其CRISPR簇数量是不成正比的。在对重复序列进行分类之后,发现Mclu1是分布最广且最具代表性的一类重复序列。在4 355条间隔序列中有388条具有物种注释信息,266条具有功能注释信息。从CRISPR簇间隔序列的来源来看,产甲烷古菌曾受到来自Poxiviridae、Siphoviridae以及Myoviridae属病毒的攻击,并且产甲烷古菌之间存在比较广泛的遗传物质交换。【结论】产甲烷古菌基因组中的CRISPR簇在组成和来源上存在较大的差异,这些差异与它们的生存环境有较大的关系。从CRISPR簇的角度阐述了产甲烷古菌之间基因组序列的差异。     

肠球菌CRISPR-Cas系统基因结构的分析及其与耐药基因的关系

CRISPR-Cas系统是一种细菌的适应性免疫系统,参与特异性防御不同类型的可移动遗传元件,如质粒、噬菌体、转座子等的入侵。旨在分析肠球菌（Enterococcus）基因组中该系统的基因结构,并探讨其与细菌耐药基因之间的关系。NCBI数据库中下载10种肠球菌的全基因组信息,利用软件对CRISPR-Cas系统的分布、cas1基因、重复序列、间隔序列等进行比对分析;查找耐药相关基因,分析其与CRISPR-Cas系统之间的关系。235株肠球菌中含完整CRISPR-cas系统的有35株（14.9%）,含确定CRISPR阵列196个和cas基因簇46个。肠球菌基因组中CRISPR系统主要为II-A型（80.4%）,其次是II-C型（15.2%）,cas1基因序列的系统发育分析结果与CRISPR-cas系统的分型基本一致。肠球菌CRISPR-Cas系统的分布在不同菌种之间差异较大;CRISPR-Cas系统可能阻碍肠球菌某些耐药基因的水平转移。     

保加利亚乳杆菌CRISPR位点的序列分析

乳酸菌基因组的CRISPR序列是乳酸菌识别并抵御噬菌体侵染的重要元件,其与cas蛋白共同构成了乳酸菌的获得性免疫系统。而目前对乳酸菌CRISPR序列信息研究较少。因此本文对8株不同来源的保加利亚乳杆菌的CRISPR序列进行了克隆测序,对其重复序列进行了遗传进化分析并预测了二级结构,同时进行了间隔序列的同源性分析。结果显示:8株保加利亚乳杆菌均含有长度不一的CRISPR区,最长的CRISPR序列片段长度为1 820 bp,含有30条间隔序列,最小的CRISPR片段仅有408 bp,含5个间隔序列。经分析发现CRISPR重复序列的二级结构预测有两类不同的结构,一类以"环"为主的典型RNA二级结构,一类以"茎"为主,前者剪切后具有典型crRNA结构,而后者的功能还有待进一步研究。通过分析保加利亚乳杆菌的CRISPR序列结构,可为保加利亚乳杆菌抗噬菌体的分子机制奠定基础,也对乳品工业筛选抗噬菌体的发酵剂菌株具有指导意义。     

Detection of a Phage Genome Carrying a Zonula Occludens like Toxin Gene (zot) in clinical isolates of Stenotrophomonas maltophilia

During a study of the genetic diversity of Stenotrophomonas strains, we found an autonomous replicating DNA molecule in chromosomal DNA preparations of the clinical Stenotrophomonas maltophilia strain c5. The entire sequence of 6,907 bp of the isolated DNA molecule was determined, which was called φSMA9. Seven ORFs, which code for proteins with considerable similarity to proteins in databases, were identified in the DNA sequence. The largest ORF shows high sequence similarities to the pI protein of the filamentous phage φLf, which was later shown to be identical to toxin Zot of Vibrio cholerae. Beside the Zot-like protein, six other proteins with similarities to known phage proteins such as a phage replication protein RstA and phage absorption or coat protein are encoded on φSMA9, which indicate that this circular DNA molecule represents the replicative form of a linear phage genome. A PCR-based screening showed that only five from the totally investigated 47 Stenotrophomonas strains of clinical and environmental origin harbor these genes. Altogether, we describe the first genome of a phage for the nosocomial pathogen Stenotrophomonas, which contains a Zot toxin like gene and might be regarded as the first Stenotrophomonas virulence factor.     

Genetics of CRISPR arrays in <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium 14028 associated with foreign DNA decay

Clustered regularly interspaced short palindromic repeats (CRISPRs) are a genetic locus of prokaryotes and contain highly conserved direct repeats, spacers, and CRISPR-associated genes. Spacers in CRISPRs are known as adaptive immune markers and reveal what types of phage or foreign DNA have been introduced in the past. The primary objective of this study was to analyze spacer sequences in CRISPR arrays of 15 Salmonella enterica subspecies and to determine if Salmonella CRISPRs are indeed involved in resistance to foreign DNAs. Using a bioinformatics algorithm, the CRISPR arrays of 15 subspecies of S. enterica were predicted. The transformation efficiencies of the wild-type and mutant strains lacking a space were determined using the plasmid harboring the same sequences with the space. Analysis of the CRISPR arrays indicated that S. Typhimurium encoded three possible CRISPR regions in the genome. Notably, 48 or 55 spacers were predicted in the genomes of S. Typhimurium 14028 and LT2 strains, respectively, and 39 were precisely identical. To confirm this prediction, the predicted CRISPR regions of S. Typhimurium 14028 were sequenced using the specific primers. Interestingly, a homology search of individual spacers found that the 2nd spacer of CRISPR 2 was nearly identical to a partial genome region of phage FSL SP-016. The mutant strain showed two to threefold increased transformation efficiency compared to that of the wild-type strain. These results demonstrate that the spacer sequence is dependent on genetic relations, especially for adaptive immunity against phage or foreign DNAs.     

In-silico whole-genome sequence analysis of a halotolerant filamentous mangrove cyanobacterium revealed CRISPR-Cas systems with unique properties

Novel CRISPR systems capable of cleaving both DNA and RNA are progressively emerging as attractive tools for genome manipulation of prokaryotic and eukaryotic organisms. We report specific characteristics of CRISPR systems present in Oxynema aestuarii AP17, a halotolerant, filamentous cyanobacterium and the second known member of the Oxynema genus. In-silico analyses of its whole-genome sequence revealed the presence of multiple Type I and Type III CRISPR loci with one Type I-G system previously unreported in cyanobacteria. We further identified the leader sequences at the 5′ end of multiple CRISPR loci, many of which were distinct from previously reported cyanobacterial CRISPR leaders. Phylogenetic analyses of the O. aestuarii AP17 Cas1 proteins revealed two protein sequences that were unique and distantly related to other cyanobacterial Cas1 protein sequences. Our findings are significant because novel Class 1 CRISPR systems possess multi-subunit effectors and are highly flexible for repurposing by protein domain fusions made to the effector complex. Additionally, Type III CRISPRs are particularly useful for genome editing in certain extremophiles for which mesophilic Type II CRISPRs are ineffective.     

CRISPR/Cas9技术在非模式植物中的应用进展

基因组编辑技术的出现对植物遗传育种及作物性状的改良产生了深远意义。CRISPR/Cas(clustered regularly interspaced short palindromic repeat)是由成簇规律间隔短回文重复序列及其关联蛋白组成的免疫系统,其作用是原核生物(40%细菌和90%古细菌)用来抵抗外源遗传物质(噬菌体和病毒)的入侵。该技术实现了对基因组中多个靶基因同时进行编辑,与前两代基因编辑技术:锌指核酶(ZFNs)和转录激活因子样效应物核酶(TALENs)相比更加简单、廉价、高效。目前CRISPR/Cas9基因编辑技术已在拟南芥(Arabidopsis thaliana)、烟草(Nicotiana benthamiana)、水稻(Oryza sativa)、小麦(Triticum aestivum)、玉米(Zea mays)、番茄(tomato)等模式植物和多数大作物中实现了定点基因组编辑,其应用范围不断地向各类植物扩展。但与模式植物和一些大作物相比,CRISPR/Cas9基因编辑技术在非模式植物,尤其在一些小作物的应用中存在如载体构建、靶点设计、脱靶检测、同源重组等问题有待进一步完善。该文对CRISPR/Cas9技术在非模式植物与小作物研究的最新研究进展进行了总结,讨论了该技术目前在非模式植物、小作物应用的局限性,在此基础上提出了相关改进策略,并对CRISPR/Cas9系统在非模式植物中的研究前景进行了展望。     

Analysis of CRISPR–Cas system and antimicrobial resistance in Staphylococcus coagulans isolates

CRISPR–Cas system contributes adaptive immunity to protect the bacterial and archaeal genome against invading mobile genetic elements. In this study, an attempt was made to characterize the CRISPR–Cas system in Staphylococcus coagulans, the second most prevalent coagulase positive staphylococci causing skin infections in dogs. Out of 45 S. coagulans isolates, 42/45 (93·33%) strains contained CRISPR–Cas system and 45 confirmed CRISPR system was identified in 42 S. coagulans isolates. The length of CRISPR loci ranged from 167 to 2477 bp, and the number of spacers in each CRISPR was varied from two spacers to as high as 37 numbers. Direct repeat (DR) sequences were between 30 and 37, but most (35/45) of the DRs contained 36 sequences. The predominant S. coagulans strains 29/45 did not possess any antimicrobial resistant genes (ARG); 26/29 strains contained Type IIC CRISPR–Cas system. Three isolates from Antarctica seals neither contain CRISPR–Cas system nor ARG. Only 15/45 S. coagulans strains (33·33%) harboured at least one ARG and 13/15 of them were having mecA gene. All the methicillin susceptible S. coagulans isolates contained Type IIC CRISPR–Cas system. In contrast, many (10/13) S. coagulans isolates which were methicillin resistant had Type IIIA CRISPR–Cas system, and this Type IIIA CRISPR–Cas system was present within the SCCmec mobile genetic element. Hence, this study suggests that Type II CRISPR–Cas in S. coagulans isolates might have played a possible role in preventing acquisition of plasmid/phage invasion and Type IIIA CRISPR–Cas system may have an insignificant role in the prevention of horizontal gene transfer of antimicrobial resistance genes in S. coagulans species.     

IS<Emphasis Type="Italic">Rm31</Emphasis>, a new insertion sequence of the IS<Emphasis Type="Italic">66</Emphasis> family in<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti pSymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, ISRm31. ISRm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that ISRm31 is a member of the IS66 family. ISRm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying ISRm31 suggested that ISRm31 inserts randomly into S. meliloti genomes. Moreover, analysis of ISRm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, ISRm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.     

嗜热厌氧杆菌热稳定CRISPR/Cas9基因组编辑技术及在细胞工厂构建中的应用研究进展

嗜热厌氧杆菌属(Thermoanaerobacter)来源的菌株作为一个高温发酵的细胞工厂,具有生产生物燃料和化学品的潜力,已引起了研究者的广泛兴趣.随着嗜热厌氧杆菌属来源的多个菌株全基因组测序的完成以及相关的生理生化实验的开展,建立一个简便、快捷的基因操作技术将有助于进一步深入理解和认识嗜热厌氧杆菌有关的代谢途径.最...