Distribution Of CRISPR-Cas Systems Research Articles

CRISPR-Cas-Based Engineering of Probiotics.

Probiotics are the treasure of the microbiology fields. They have been widely used in the food industry, clinical treatment, and other fields. The equivocal health-promoting effects and the unknown action mechanism were the largest obstacles for further probiotic's developed applications. In recent years, various genome editing techniques have been developed and applied to explore the mechanisms and functional modifications of probiotics. As important genome editing tools, CRISPR-Cas systems that have opened new improvements in genome editing dedicated to probiotics. The high efficiency, flexibility, and specificity are the advantages of using CRISPR-Cas systems. Here, we summarize the classification and distribution of CRISPR-Cas systems in probiotics, as well as the editing tools developed on the basis of them. Then, we discuss the genome editing of probiotics based on CRISPR-Cas systems and the applications of the engineered probiotics through CRISPR-Cas systems. Finally, we proposed a design route for CRISPR systems that related to the genetically engineered probiotics.

Distribution of CRISPR-Cas systems in the Burkholderiaceae family and its biological implications.

CRISPR-Cas systems are composed of repeated sequences separated by non-repeated sequences that are near genes coding for Cas proteins, which are involved in the function of these systems. Their function has been mostly related to "genetic immunity" against foreign genetic material, among other roles. Interest in them increased after their use in genetic manipulation was uncovered and surveys to find and classify them have been done in several bacterial groups. To determine the presence of these genetic elements in the Burkholderiaceae family members, a bioinformatic approach was followed. Attention in this family comes as it is formed by a great diversity of microorganisms that include opportunistic and true pathogens, and symbiotic and saprophytic organisms, among others. Results show that, in contrast to other bacterial groups, only 8.4% of family members harbor complete CRISPR-Cas systems and the rest either do not have one or have remains or sections of one. Analyses of the spacer sequences indicated that most of them have identity to sections of the same genomes they were found, while a few had identities with either plasmids or phages. The genus with the higher proportion of self-directed spacers is Ralstonia, and their possible roles are discussed. Most of the systems (60%) belong to the class I subtype I-E and a few to subtypes I-C (13.3%), I-F (18.3%), II-C (5%), IV-A (1.7%) and V-C (1.7%). To the best of our knowledge, this is the first study to uncover the CRISPR-Cas system for the whole Burkholderiaceae family.

Distribution of CRISPR-Cas Systems in Clinical Carbapenem-Resistant Klebsiella pneumoniae Strains in a Chinese Tertiary Hospital and Its Potential Relationship with Virulence.

Aim: In this study, we aimed to characterize the CRISPR-Cas systems in clinical carbapenem-resistant Klebsiella pneumoniae (CRKP) isolates and to investigate the potential association of CRISPR-Cas systems with bacterial virulence. Methods: A total of 168 CRKP strains were collected from inpatients in a teaching hospital in Jiangxi Province. Five common carbapenemase genes, subtype genes of the CRISPR-Cas system, and 13 virulence genes were amplified by PCR using specific primers. The potential virulence of all the clinical CRKP strains was tested in a Galleria mellonella infection model. Results: PCR analysis of five common carbapenemase genes revealed the frequency of carbapenemase gene KPC-2 was the highest in the CRISPR-negative strains, compared to CRISPR type I-E* strains or CRISPR type I-E strains (p < 0.01). Isolates having the subtype I-E* CRISPR-Cas system tended to have more virulence genes such as magA, kfu, wcaG, and allS, compared to CRISPR-negative isolates and type I-E CRISPR-Cas isolates (p < 0.01). The average survival time of the larvae infected with the isolates having the subtype I-E* CRISPR-Cas system was significantly shorter than the other two group isolates (p < 0.05). Conclusion: The CRKP strains, which had the subtype I-E CRISPR-Cas system or the subtype I-E* CRISPR-Cas system, showed reduced acquisition of carbapenemase genes compared to CRISPR-negative isolates. Importantly, we first found that a small portion of "CR-hvKP" strains were selected from the CRKP clones, which had the type I-E* CRISPR-Cas systems.

Composition and Diversity of CRISPR-Cas13a Systems in the Genus Leptotrichia.

Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13a, previously known as CRISPR-C2c2, is the most recently identified RNA-guided RNA-targeting CRISPR-Cas system that has the unique characteristics of both targeted and collateral single-stranded RNA (ssRNA) cleavage activities. This system was first identified in Leptotrichia shahii. Here, the complete whole genome sequences of 11 Leptotrichia strains were determined and compared with 18 publicly available Leptotrichia genomes in regard to the composition, occurrence and diversity of the CRISPR-Cas13a, and other CRISPR-Cas systems. Various types of CRISPR-Cas systems were found to be unevenly distributed among the Leptotrichia genomes, including types I-B (10/29, 34.4%), II-C (1/29, 2.6%), III-A (6/29, 15.4%), III-D (6/29, 15.4%), III-like (3/29, 7.7%), and VI-A (11/29, 37.9%), while 8 strains (20.5%) had no CRISPR-Cas system at all. The Cas13a effectors were found to be highly divergent with amino acid sequence similarities ranging from 61% to 90% to that of L. shahii, but their collateral ssRNA cleavage activities leading to impediment of bacterial growth were conserved. CRISPR-Cas spacers represent a sequential achievement of former intruder encounters, and the retained spacers reflect the evolutionary phylogeny or relatedness of strains. Analysis of spacer contents and numbers among Leptotrichia species showed considerable diversity with only 4.4% of spacers (40/889) were shared by two strains. The organization and distribution of CRISPR-Cas systems (type I-VI) encoded by all registered Leptotrichia species revealed that effector or spacer sequences of the CRISPR-Cas systems were very divergent, and the prevalence of types I, III, and VI was almost equal. There was only one strain carrying type II, while none carried type IV or V. These results provide new insights into the characteristics and divergences of CRISPR-Cas systems among Leptotrichia species.

Characterization and distribution of CRISPR-Cas systems in Lactobacillus sakei.

Clustered regularly interspaced palindromic repeats (CRISPR)-Cas (CRISPR-associated) structures, known as prokaryotes 'immune system', have been successfully applied for genetic engineering and genotyping purposes for a variety of microorganisms. Here we investigated 50 Lactobacillus (L.) sakei genomes and found 13 of them as CRISPR-Cas positive. The majority of positive genomes contain type II-A system, which appears to be widespread across food born lactic acid bacteria. However, a type II-C system with low similarity in Cas protein sequence to related II-C structures is rarely present in the genomes. We depicted a correlation between prophages integrated in the genomes and the presence/absence of CRISPR-Cas systems and identified the novel protospacer adjunction motifs (PAMs) (a/g)AAA for the II-A and (g/a)(c/t)AC for the II-C system including the corresponding tracrRNAs, creating the basis for the development of new Cas-mediated genome editing tools. Moreover, we performed a PCR screening for 81 selected L. sakei isolates and identified 25 (31%) isolates as CRISPR-Cas positive with hypervariable spacer content. Comparative sequence analysis of 33 repeat-spacer arrays resulted in 18 CRISPR genotypes, revealing insights into evolutionary relationships between different strains and illustrating possible applications for the research and development of starter cultures, e.g., the usage for strain differentiation in assertiveness experiments or the development of bacteriophage-resistant strains.

CRISPR-Cas systems in the marine actinomycete Salinispora: linkages with phage defense, microdiversity and biogeography.

BackgroundProkaryotic CRISPR-Cas systems confer resistance to viral infection and thus mediate bacteria-phage interactions. However, the distribution and functional diversity of CRISPRs among environmental bacteria remains largely unknown. Here, comparative genomics of 75 Salinispora strains provided insight into the diversity and distribution of CRISPR-Cas systems in a cosmopolitan marine actinomycete genus.ResultsCRISPRs were found in all Salinispora strains, with the majority containing multiple loci and different Cas array subtypes. Of the six subtypes identified, three have not been previously described. A lower prophage frequency in S. arenicola was associated with a higher fraction of spacers matching Salinispora prophages compared to S. tropica, suggesting differing defensive capacities between Salinispora species. The occurrence of related prophages in strains from distant locations, as well as spacers matching those prophages inserted throughout spacer arrays, indicate recurring encounters with widely distributed phages over time. Linkages of CRISPR features with Salinispora microdiversity pointed to subclade-specific contacts with mobile genetic elements (MGEs). This included lineage-specific spacer deletions or insertions, which may reflect weak selective pressures to maintain immunity or distinct temporal interactions with MGEs, respectively. Biogeographic patterns in spacer and prophage distributions support the concept that Salinispora spp. encounter localized MGEs. Moreover, the presence of spacers matching housekeeping genes suggests that CRISPRs may have functions outside of viral defense.ConclusionsThis study provides a comprehensive examination of CRISPR-Cas systems in a broadly distributed group of environmental bacteria. The ubiquity and diversity of CRISPRs in Salinispora suggests that CRISPR-mediated interactions with MGEs represent a major force in the ecology and evolution of this cosmopolitan marine actinomycete genus.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-936) contains supplementary material, which is available to authorized users.