CRISPR genotyping and regional distribution characteristics of Yersinia pestis in natural plague focus of Tibet Autonomous Region / 中华地方病学杂志

Objective:To study the clustered regularly interspaced short palindromic repeats (CRISPR) genotype of Yersinia pestis and its regional distribution characteristics in natural plague focus of Tibet Autonomous Region. Methods:A total of 125 representative Yersinia pestis strains isolated from natural plague focus in Tibet Autonomous Region at different times, regions, hosts and vectors were selected as experimental strains, and the phenol chloroform mixed extraction method was used to extract Yersinia pestis DNA. Three pairs of CRISPR primers (for YPa, YPb, YPc locus) were used to amplify the DNA of the experimental strains, and the CRISPR genotype of Yersinia pestis was determined by sequencing. Results:All 125 strains of Yersinia pestis had three CRISPR locus: YPa, YPb, and YPc. A total of 18 spacer were found, including 8 in YPa loci, 6 in YPb loci, and 4 in YPc loci. Two new types of spacers had been discovered, namely b52 and c14. CRISPR typing revealed 10 genotypes, including G1, G7, G7-b4''', G7-b52, G7-c2 -, G8, G22, G22-a4 -, G22-b4''', and G22-c14, of which 6 were newly discovered genotypes. Among the 125 experimental strains, G7 was the main genotype, accounting for 65.6% (82/125), which was distributed in 6 prefecture level citys and 1 region of Tibet Autonomous Region. Next were G22 and G7-c2 - genetypes, accounting for 14.4% (18/125) and 11.2% (14/125), respectively. G22 gene type was distributed in Nagqu, Changdu, Lhasa citys, and Ngari Prefecture, while G7-c2 - genetype was distributed in Shigatse and Shannan cities. Conclusion:The CRISPR locus of Yersinia pestis in natural plague focus of Tibet Autonomous Region is highly polymorphic, and the Yersinia pestis strains with different genotypes have obvious regional distribution characteristics.

Establishment and evaluation of a LAMP-CRISPR-based nucleic acid detection method for Schistosoma japonicum / 中国热带医学

@#Abstract: Objective To establish a sensitive and specific nucleic acid detection method for Schistosoma japonicum based on loop-mediated isothermal amplification (LAMP) and clustered regularly interspaced short palindromic repeats (CRISPR) technology. Methods The LAMP primers, gRNA and ssDNA probe that target Schistosoma japonicum SjR2 genes were designed according to the principles of LAMP and CRISPR. The LAMP-CRISPR reaction system was established and optimized. The sensitivity and specificity of the method were evaluated against the ten-fold serial dilutions of plasmid containing SjR2 target sequences, as well as genomic DNA at different stages of Schistosoma japonicum and other parasites, including Fasciola hepatica, Schistosoma mansoni, Taenia saginata, Clonorchis sinensis, Ascaris lumbricoides, Necator americanus, Paragonimus westermani, and Echinococcus granulosus. Additionally, 15 schistosome-infected snail and 30 uninfected samples were tested by LAMP-CRISPR and LAMP methods, respectively, to evaluate the potential of this method for screening for infected snails. Results　The developed LAMP-CRISPR method was able to specifically amplify and detect the SjR2 gene of S. japonicum. The optimal reaction temperature was 37 ℃, and the optimal reaction concentrations were both 40 nmol/L for gRNA and Cas12a protein. No cross-reaction was observed with genomic DNA from other parasites such as F. hepatica. The detection limit of the method was 10 copies/μL when testing 10-fold dilutions of recombinant plasmids as a template. Furthermore, the LAMP-CRISPR method was able to accurately detect genomic DNA from S. japonicum at various stages of development, including eggs, cercariae, schistosomula, juvenile worms, and adult worms. The results of testing 45 snail samples showed no significant difference between the LAMP-CRISPR and LAMP methods for detecting infected snails (χ2=0.05, P>0.05). The sensitivity and specificity of the LAMP-CRISPR method were 100.00% (15/15) and 96.67% (29/30), respectively, compared to the gold standard, while the sensitivity and specificity of the LAMP method were 100.00% (15/15) and 93.33% (28/30), respectively. Conclusions　This established LAMP-CRISPR detection method presented good sensitivity, specificity and reliability, making it a promising tool for rapid detection and risk monitoring of S. japonicum.

Genetic resources and precise gene editing for targeted improvement of barley abiotic stress tolerance / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Abiotic stresses, predominately drought, heat, salinity, cold, and waterlogging, adversely affect cereal crops. They limit barley production worldwide and cause huge economic losses. In barley, functional genes under various stresses have been identified over the years and genetic improvement to stress tolerance has taken a new turn with the introduction of modern gene-editing platforms. In particular, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) is a robust and versatile tool for precise mutation creation and trait improvement. In this review, we highlight the stress-affected regions and the corresponding economic losses among the main barley producers. We collate about 150 key genes associated with stress tolerance and combine them into a single physical map for potential breeding practices. We also overview the applications of precise base editing, prime editing, and multiplexing technologies for targeted trait modification, and discuss current challenges including high-throughput mutant genotyping and genotype dependency in genetic transformation to promote commercial breeding. The listed genes counteract key stresses such as drought, salinity, and nutrient deficiency, and the potential application of the respective gene-editing technologies will provide insight into barley improvement for climate resilience.

Emerging molecular diagnostic technologies for point-of-care testing: applications and prospects / 中华检验医学杂志

Molecular diagnostic technologies are critical diagnostic tools in clinical research. Point-of-care testing (POCT) presents numerous advantages such as portability, rapidity, accuracy, and cost-effectiveness, facilitating timely identification of infection sources and control of infectious disease transmission. This article explores the emerging molecular diagnostic technologies that can be used for POCT, including digital loop-mediated isothermal amplification technology and the clustered regularly interspaced short palindromic repeats and CRISPR-associated proteins system, while also discussing their future prospects.

Clustered regularly interspaced short palindromic repeats genotyping of Yersinia pestis in Yushu Tibetan Autonomous Prefecture, Qinghai Province / 中华地方病学杂志

Objective:To learn about the clustered regularly interspaced short palindromic repeats (CRISPR) genotyping of Yersinia pestis in Yushu Tibetan Autonomous Prefecture (Yushu for short), Qinghai Province, and to explore its genetic characteristics. Methods:In this study, 44 representative strains isolated from local natural plague focus in Yushu from 1963 to 2007 were selected as experimental objects to extract DNA. Primers targeting the three CRISPR loci (YPa, YPb, and YPc) were designed for PCR amplification. The amplified products were sequenced and analyzed to identify the CRISPR spacer, and to determine the CRISPR genotypes and clusters.Results:Twenty-three spacers including 14 of YPa, 6 of YPb and 3 of YPc were observed among 44 strains, of which 2 spacers (a106 and a107) were firstly identified. According to the spacer arrays, the strains were divided into 15 CRISPR genotypes and classified into 6 CRISPR clusters which were Cb4, Cc3', Ca7, Ca7', CaΔ5' and Ca35', respectively. Among them, Ca7 was the most epidemic dominant cluster (34 strains) in Yushu.Conclusion:The CRISPR loci of Yersinia pestis in Yushu have multiple genotypes, high genetic polymorphism, and complex population structure.

Construction of EF-G knockdown strain of Mycobacterium smegmatis and drug resistance analysis / 生物工程学报

As the only translational factor that plays a critical role in two translational processes (elongation and ribosome regeneration), GTPase elongation factor G (EF-G) is a potential target for antimicrobial agents. Both Mycobacterium smegmatis and Mycobacterium tuberculosis have two EF-G homologous coding genes, MsmEFG1 (MSMEG_1400) and MsmEFG2 (MSMEG_6535), fusA1 (Rv0684) and fusA2 (Rv0120c), respectively. MsmEFG1 (MSMEG_1400) and fusA1 (Rv0684) were identified as essential genes for bacterial growth by gene mutation library and bioinformatic analysis. To investigate the biological function and characteristics of EF-G in mycobacterium, two induced EF-G knockdown strains (Msm-ΔEFG1(KD) and Msm-ΔEFG2(KD)) from Mycobacterium smegmatis were constructed by clustered regularly interspaced short palindromic repeats interference (CRISPRi) technique. EF-G2 knockdown had no effect on bacterial growth, while EF-G1 knockdown significantly retarded the growth of mycobacterium, weakened the film-forming ability, changed the colony morphology, and increased the length of mycobacterium. It was speculated that EF-G might be involved in the division of bacteria. Minimal inhibitory concentration assay showed that inhibition of EF-G1 expression enhanced the sensitivity of mycobacterium to rifampicin, isoniazid, erythromycin, fucidic acid, capreomycin and other antibacterial agents, suggesting that EF-G1 might be a potential target for screening anti-tuberculosis drugs in the future.

Association of clustered regularly interspaced short palindromic repeats with genotyping and drug-resistance genes of group B Streptococcus / 中华围产医学杂志

Objective:To investigate the distribution of clustered regularly interspaced short palindromic repeats (CRISPR) in group B Streptococcus (GBS) in the genital tract of women during the third trimester and in infants with invasive infection and its relationship with multilocus sequence typing (MLST) and drug-resistance genes. Methods:This study retrospectively collected 84 GBS strains isolated from pregnant women with GBS colonization and infants with invasive GBS infection who were admitted to Children's Hospital Affiliated to Shanxi Medical University from January 2017 to January 2022. CRISPR, MLST, and drug-resistance phenotype and genes were detected and analyzed using χ 2 test or Fisher exact probability method. MEGA11 was used to construct a dendrogram. Results:There were ten sequence typing in the 84 GBS strains and ST10 was the dominant one (46.4%). GBS was sensitive to penicillin, and its resistance rates to erythromycin (75.0%) and clindamycin (73.8%) were high. Among the 17 invasive GBS strains, ST10 had 100% resistance to erythromycin, clindamycin, and levofloxacin. CRISPR1 gene was amplified in 62 strains (73.8%). CRISPR1-positive strains had a significantly higher proportion of ST10 [56.5%(35/62) vs 18.2%(4/22), χ 2=9.56, P=0.002] and ermB, gyrA, parC [54.8%(34/62) vs 22.7%(5/22), 67.7%(42/62) vs 36.4%(8/22), 71.0%(44/62) vs 36.4%(8/22); χ 2=6.73, 6.64, and 8.25, all P<0.05], and a lower proportion of ermA [6.5%(4/62) vs 31.8%(7/22), χ 2=7.09, P=0.008] than CRISPR1-negative strains. Conclusions:ST10 is the main GBS genotype among the colonized microbiota the genital tract of pregnant women and in infants with invasive GBS infection, which is also a dominant type in CRISPR1-positive strains. GBS is sensitive to penicillin and CRISPR1 gene is linked to the spread of some drug-resistance genes.

Construction of aquaporin 9 gene knockout mice using CRISPR/Cas9 gene editing system / 解剖学报

Objective To construct homozygous aquaporin 9(AQP-9)

Crispr-cas9: el debate bioético más allá de la línea germinal / Crispr-cas9: the bioethical debate beyond the germline / Crispr-cas9: o debate bioético mais além da linhagem germinativa

Resumen El sistema CRISPR-Cas9 es una tecnología de edición genética que, además de ampliar las posibilidades en investigación científica, despierta reflexiones asociadas a la dignidad humana, el control biológico, la terapia y la mejora genética. Se revisaron las discusiones bioéticas asociadas a los desafíos y las repercusiones que suscita su aplicación. Como resultado, los cuestionamientos bioéticos tienden a problematizar la aplicación en organismos no humanos, en la investigación básica y en la línea somática y germinal humana. Para concluir, falta incrementar los niveles de seguridad y efectividad para que los beneficios superen los riesgos y, de esta forma, sea posible disminuir las preocupaciones bioéticas y aumentar la credibilidad en el uso de la técnica.

Abstract The CRISPR-Cas9 system is a genetic editing technology that, in addition to expanding the possibilities for scientific research, promotes reflections associated with human dignity, biological control, therapy, and genetic improvement. Bioethical discussions on the challenges and repercussions of the CRISPR-Cas9 system are reviewed. As a result, bioethical questions tend to problematize the application to non-human organisms, primary research, and the human somatic and germline. In brief, it is necessary to increase the levels of safety and effectiveness so that the benefits outweigh the risks, while reducing bioethical concerns and increasing the credibility of the technique.

Resumo O sistema CRISPR-Cas9 é uma tecnologia de edição de genes que, além de ampliar as possibilidades em pesquisa científica, desperta reflexões associadas com a dignidade humana, o controle biológico, a terapia e o aperfeiçoamento genético. Foram revisadas as discussões bioéticas relacionadas aos desafios e às repercussões que sua aplicação suscita. Como resultado, os questionamentos bioéticos tendem a problematizar a aplicação em organismos não humanos, na pesquisa básica e na linhagem somática e germinativa humana. Para concluir, falta aumentar os níveis de segurança e efetividade para que os benefícios sejam maiores do que os riscos, e assim, seja possível diminuir as preocupações bioéticas e aumentar a credibilidade no uso da técnica.

Cell membrane-derived vesicles for delivery of therapeutic agents

Cell membranes have recently emerged as a new source of materials for molecular delivery systems. Cell membranes have been extruded or sonicated to make nanoscale vesicles. Unlike synthetic lipid or polymeric nanoparticles, cell membrane-derived vesicles have a unique multicomponent feature, comprising lipids, proteins, and carbohydrates. Because cell membrane-derived vesicles contain the intrinsic functionalities and signaling networks of their parent cells, they can overcome various obstacles encountered

Application of CRISPR/Cas9 lentiviral vector in construction of rat hepatic stellate cells with COX-2 gene knockout / 临床肝胆病杂志

ObjectiveTo obtain HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2-/- cells with COX-2 gene defect by transfecting HSC-T6 cells with CRISPR/Cas9 lentiviral vector, and to provide a good method for further functional research and new strategies for the clinical treatment of liver fibrosis. MethodsThe COX-2 gene-specific sgRNAs (COX-2-sgRNA-1, COX-2-sgRNA-2, COX-2-sgRNA-3) were designed, synthesized, and connected to the GV371 vector, and the recombinant plasmid and the packaging plasmid were transfected into 293T cells to form lentivirus particles; the fluorescence method was used to measure virus titer. The most appropriate amount of the virus was calculated based on MOI. Lenti-Cas9-puro was transfected into HSC-T6 cells, and HSC-T6-Cas9 cells were screened out by puromycin; Lenti-COX-2-sgRNA-EGFP was transfected into HSC-T6-Cas9 cells to obtain HSC-T6-COX-2-/- cells. Cruiser enzyme digestion and Western blot were used to verify gene knockout at the gene and protein levels. An analysis of variance was used for comparison of continuous data between multiple groups, and the least significant difference t-test was used for further comparison between two groups. ResultsSequencing verified that the COX-2-sgRNA expression vector was constructed successfully. Recombinant expression plasmids and packaging plasmids were transfected into 293T cells to form lentivirus particles, and the fluorescence method showed a virus titer of ＞1×108. HSC-T6 cells with stable expression of Cas9 protein and HSC-T6-COX-2-/- cells with COX-2 gene defect were successfully constructed. The HSC-T6-Cas9 group had significantly higher relative mRNA expression of LV-Cas9-Puro than the CON group (541.93±105.76 vs 1.00±0.02, t=12.995, P＜0.01). Cruiser enzyme digestion and Western blot showed that the CRISPR/Cas9 lentivirus expression vectors played a role in the target, among which COX-2-sgRNA-2 knockout had the most significant effect, and this group had a significant reduction in the protein expression level of COX-2 compared with the CON group and the NC group (both P＜0.05), suggesting that COX-2-sgRNA was active. ConclusionA CRISPR/Cas9 lentivirus vector is successfully constructed for COX-2 target gene, and HSC-T6-COX-2-/- cells with stable COX-2 gene knockout are obtained.

CRISPR-Cas9: A method for establishing rat models of drug metabolism and pharmacokinetics

The 2020 Nobel Prize in Chemistry recognized CRISPR-Cas9, a super-selective and precise gene editing tool. CRISPR-Cas9 has an obvious advantage in editing multiple genes in the same cell, and presents great potential in disease treatment and animal model construction. In recent years, CRISPR-Cas9 has been used to establish a series of rat models of drug metabolism and pharmacokinetics (DMPK), such as

Advances in the regulation of type Ⅰ CRISPR system on biofilm formation and antibiotic resistance / 中华检验医学杂志

In recent years, bacterial antibiotic resistance is becoming a huge threaten for human health. It′s getting even more serious after biofilm formation. In addition to physical and chemical factors such as barrier function and microenvironmental changes, the regulation of some genes in biofilm also specifically improves the level of antibiotic resistance. There might be a common regulatory mechanism between antibiotic resistance and the ability of biofilm formation. Type Ⅰ clustered regularly interspaced short palindromic repeats (CRISPR) system can affect the acquisition and spread of antibiotic resistance. The effect varies with species, evolution process and environmental pressure. Recently, it has been reported that type Ⅰ CRISPR system could not only regulate biofilm formation, but could also participate in the interaction between bacteriophages and biofilm. It might become a breakthrough point in the study of phage therapy. This article reviews the situation of antibiotic resistance in recent years, the new progress of biofilm-specific antibiotic resistance, and the effects of type Ⅰ CRISPR system on biofilm formation and antibiotic resistance, so as to provide new ideas for the prevention and treatment of bacterial infection.

CRISPR/Cas: a Nobel Prize award-winning precise genome editing technology for gene therapy and crop improvement / 浙江大学学报（英文版）（B辑：生物医学和生物技术）

Since it was first recognized in bacteria and archaea as a mechanism for innate viral immunity in the early 2010s, clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein (Cas) has rapidly been developed into a robust, multifunctional genome editing tool with many uses. Following the discovery of the initial CRISPR/Cas-based system, the technology has been advanced to facilitate a multitude of different functions. These include development as a base editor, prime editor, epigenetic editor, and CRISPR interference (CRISPRi) and CRISPR activator (CRISPRa) gene regulators. It can also be used for chromatin and RNA targeting and imaging. Its applications have proved revolutionary across numerous biological fields, especially in biomedical and agricultural improvement. As a diagnostic tool, CRISPR has been developed to aid the detection and screening of both human and plant diseases, and has even been applied during the current coronavirus disease 2019 (COVID-19) pandemic. CRISPR/Cas is also being trialed as a new form of gene therapy for treating various human diseases, including cancers, and has aided drug development. In terms of agricultural breeding, precise targeting of biological pathways via CRISPR/Cas has been key to regulating molecular biosynthesis and allowing modification of proteins, starch, oil, and other functional components for crop improvement. Adding to this, CRISPR/Cas has been shown capable of significantly enhancing both plant tolerance to environmental stresses and overall crop yield via the targeting of various agronomically important gene regulators. Looking to the future, increasing the efficiency and precision of CRISPR/Cas delivery systems and limiting off-target activity are two major challenges for wider application of the technology. This review provides an in-depth overview of current CRISPR development, including the advantages and disadvantages of the technology, recent applications, and future considerations.

Effect of inhibition of heat shock protein Gp96 expression on alcoholic liver fibrosis in mice by CRISPR/Cas-9 technique / 解剖学报

Objective To investigate the effects of heat shock protein Gp96 on alcoholic liver fibrosis in mice. Methods A total of 220 male healthy C57BL/6 J mice were randomly divided into four groups; normal control group (n = 10), saline+alcohol induced liver fibrosis group (n = 70), the injection of CRISPR expression Gp96-sgRNA3 by tail vein+ alcohol induced liver fibrosis group (n = 70), the intraperitoneal injection of nuclear factor kappa B(NF-κB) inhibitors PDTC+alcohol induced liver fibrosis group (n = 70). The blood was got from eyeballs and the mice were killed after 8 weeks of ethanol induction. We detected the activity of serum aspartate aminotransferase (AST) in mice of different groups. The pathological changes were detected by HE staining, sirius red staining and periodic acid-Schiff (PAS) staining in the liver of mice. The expression of Gp96 and transforming growth factor βl ( TGF-βl ) were detected by Western blotting. Results Compared with the normal control group, the AST enzyme activity and liver fibrosis increased significantly, glycogen decreased significantly in other three groups (P<0.01). Compared with the saline+alcohol group, the AST enzyme activity and liver fibrosis increased more significantly, glycogen decreased more significantly, Gp96 expression decreased significantly and TGF-βl expression increased significantly in Gp96-sgRNA3+ alcohol group and NF-κB inhibitors PDTC+ alcohol group (P<0.01 or P<0.05). Conclusion The injection of CRISPR expression plasmid Gp96-sgRNA3 by tail vein significantly inhibited the Gp96 expression, promoted the degree of alcoholic liver fibrosis in mice, and NF-κB signaling pathway played a certain role in regulating the expression of Gp96.

Construction of forkhead box J2 gene knockout plasmids by CRISPR / Cas9 and the effects on expression of transforming growth factor￣β/ Smads and proliferation in hepatocellular carcinoma / 解剖学报

Objective To construct the clustered regularly interspaced short palindromic repeats / associated protein 9 (CRISPR/ Cas9) plasmid targeting forkhead box J2 (FOXJ2) gene and investigate the effects of FOXJ2 interference on the expression of transforming growth factor-β(TGF-β) / Smads and proliferation in hepatocellular carcinoma cells of mouse. Methods Small guide RNA(sgRNA) sequence of FOXJ2 was designed, linked with PX458 vector and transfected into competent E. coli for proliferation. The recombinant plasmids were sent for sequencing to confirm the accuracy of the sgRNA sequence. The PX458-FOXJ2-sgRNAs plasmids were transfected into Hepa1-6 cells by liposome transfection, respectively. The empty vectors of PX458 were transfected as control group. After 48 hours, the expression of FOXJ2, TGF-β and Smads were obtained by RT-PCR and agarose gel electrophoresis, respectively. The cell proliferation was detected by methylthio tetrazole (MTT) method . Results The CRISPR/ Cas9 plasmids of PX458-FOXJ2-sgRNAs were successfully constructed. The recombinant plasmid of PX458-FOXJ2-sgRNA2 could effectively inhibit FOXJ2 gene expression which induced increasing expression of TGF-β, Smad2 and Smad4 in Hepa1-6 cells comparing to the control group transfected with PX458 only. And the proliferation of Hepa1-6 was promoted in PX458-FOXJ2-sgRNA2 interference group. Conclusion In hepatocellular carcinoma cells of mouse, FOXJ2 gene inhibits the expression of TGF-β, Smad2, Smad4 and cell proliferation partially, which indicates the relationship between FOXJ2 and TGF-β signal pathway. The result provides the target molecule of FOXJ2 for the prevention and treatment of hepatocellular carcinoma.

Construction of Rho GDIa-sgRNAs plasmids by clustered regularly interspaced short palindromic repeats/associated protein 9 and the effect on migration of Hepa 1-6 cell line in mouse / 解剖学报

Objective To construct the recombinant plasmids of knocking down Rho guanine dissociation inhibitor α (GDIα ) gene by using clustered regularly interspaced short palindromic repeats/associated protein 9 (CRISPR/Cas9) technique, and investigate the effect of Rho GDIα interference on the migration of Hepa 1-6 cells of mouse in order to provide the method of prevention and treatment of liver cancer. Methods To construct and identify the PX458-Rho GDIα-single guide (sg) RNAs by using CRISPR/Cas9 technique. And the Hepa 1-6 cells were transfected by liposomes with PX458-Rho GDIα-sgRNAs for 48 hours respectively, and cells treated with PX458 plasmids were used as control. The migration ability of Hepa 1-6 was checked by wound healing assay and Transwell assay, respectively. Results The expression of Rho GDIα was depressed in group of PX458-Rho GDIα-sgRNAl transfection which was detected by using RT-PCR. The migration distance of Hepa 1-6 in PX458-Rho GDIα-sgRNAl transfection group was significantly promoted comparing with the control group which was transfected with PX458 only, and the cell number of PX458-Rho GDIα-sgRNAl group was more than that in control group by using transwell assay, indicating concluded that knocking down of Rho GDIα promoted the migration ability of Hepal-6 cells. Conclusion The result is explicit that in vivo, Rho GDIα may inhibit the migration of Hepal-6 partially. Overexpression of Rho GDIα might be used as an important method to prevent the metastasize of carcinoma.

Relationship of clustered regularly interspaced short palindromic repeats with virulence genes and resistance genes in Shigella / 西安交通大学学报(医学版)

Objective: To investigate the relationship of the clustered regularly interspaced short palindromic repeats (CRISPR) with resistance genes and virulence genes. Methods: The Shigella genome sequences, resistance genes and virulence genes sequences were obtained from GenBank Database. Then we obtained the distribution of CRISPR1, CRISPR2 and CRISPR-Q1 and the information of repeat and spacers in Shigella by multiple sequences alignment. Meanwhile, the distribution of resistance genes and virulence genes in Shigella were obtained by BLAST and the relationship of the CRISPR with resistance genes and virulence genes was computed by SPSS 17.0. Results: The spacers of CRISPR1 and CRISPR2 showed obvious differences in different Shigella strains, and the CRISPR-Q1 was more widely distributed (over 99%). The virulence genes were widely distributed in Shigella (more than 56%); the distribution of resistance genes (blaOXA-1, camr, Sul2, and tetB) was also wide (over 50%). There were significant differences between the CRISPR1/CRISPR2 and 9 kinds of virulence genes and 4 kinds of resistance genes (P<0.05). There were significant differences between the CRISPR-Q1 and 3 kinds of virulence genes and 4 kinds of resistance genes (P<0.05). There were significant differences between the CRISPR1/CRISPR2, CRISPR-Q1 and the number of virulence genes and resistance genes (P<0.05). There were significant differences between the CRISPR1/CRISPR2 and the number of spacers of CRISPR-Q1 (χ2=61.6, P<0.001). Conclusion: The distribution of CRISPR was quite different in Shigella, and the number of spacers was also different. There were negative correlation of CRISPR of Shigella with some resistance genes and virulence genes. The CRISPR1/CRISPR2 had a positive relationship with the number of the spacers.

Charactercs of clustered regularly interspaced short palindromic repeats in Shigella and their correlation analysis / 吉林大学学报(医学版)

Oetive: To analyze the characteristics of clustered regularly interspaced short palindromic repeats (CRISPR) in Shigella from the database and clinic, and to explore the characteristics of CRISPR-Q1 and the correlations among CRISPRs. Methods: According to the genome database of Shigella, the primer sequences and PCR amplication were used to obtain the CRISPR sequences of Shigella, and the multiple sequence alignment was used to analyze the CRISPR distribution, the number and features of spacer; BLAST was used to analyze the repeats and spacers in CRISPR-Q1; the distribution characteristics of the repeats and spacers in Shigella and the location and features of CRISPR-Q1 in the strain were detected; the relationships between the spacer number of CRISPR-Q1 and CRISPR1/CRISPR2 in the database and laboratory were analyzed. Results: Among 107 Shigella strains in the database, 106 Shigella strains contained CRISPR1 or CRISPR2, 8 Shigella strains contained severial spacers; 106 Shigella strains contained CRISPR-Q1, and 13 Shigella strains contained severial spacers. A total of 6 among 12 Shigella strains in the laboratory contained CRISPR1 or CRISPR2, all of the 12 strains contained CRISPR-Ql. A portion of the CRISP R-Ql spacer (about 35 bp) widely distributed in the genome of the same strain of chromosome, as was the repeat sequence; CRISPR-Q1 contained the repetitive extragenic palindromic elements (REP). The spacer number of CRISPR-Q1 was related to the CRISPR1 or CRISPR2 of Shigella in the database (χ2 = 61. 60, P<0. 01) and in the laboratory (P=0. 015). Conclusion: CRISPR-Q1 widely distributes in Shigella and the REP elements can be found in CRISPR-Q1. There are correlations between CRISPR1 or CRISPR2 and CRISPR-Q1 in Shigella.