CRISPR Elements Research Articles

Candida albicans Gene Deletion with a Transient CRISPR-Cas9 System.

Clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated gene 9 (CRISPR-Cas9) systems are used for a wide array of genome-editing applications in organisms ranging from fungi to plants and animals. Recently, a CRISPR-Cas9 system has been developed for the diploid fungal pathogen Candida albicans; the system accelerates genetic manipulation dramatically [V. K. Vyas, M. I. Barrasa, and G. R. Fink, Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. We show here that the CRISPR-Cas9 genetic elements can function transiently, without stable integration into the genome, to enable the introduction of a gene deletion construct. We describe a transient CRISPR-Cas9 system for efficient gene deletion in C.albicans. Our observations suggest that there are two mechanisms that lead to homozygous deletions: (i) independent recombination of transforming DNA into each allele and (ii) recombination of transforming DNA into one allele, followed by gene conversion of the second allele. Our approach will streamline gene function analysis in C.albicans, and our results indicate that DNA can function transiently after transformation of this organism. IMPORTANCE The fungus Candida albicans is a major pathogen. Genetic analysis of this organism has revealed determinants of pathogenicity, drug resistance, and other unique biological features, as well as the identities of prospective drug targets. The creation of targeted mutations has been greatly accelerated recently through the implementation of CRISPR genome-editing technology by Vyas et al. [Sci Adv 1(3):e1500248, 2015, http://dx.doi.org/10.1126/sciadv.1500248]. In this study, we find that CRISPR elements can be expressed from genes that are present only transiently, and we develop a transient CRISPR system that further accelerates C.albicans genetic manipulation.

CRISPR/Cas9 genome editing of rubber producing dandelion Taraxacum kok-saghyz using Agrobacterium rhizogenes without selection

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) is a highly accessible genome editing tool. Here we demonstrate its potential for use in Taraxacum species. Taraxacum kok-saghyz (TK, Rubber Dandelion) is notable for its ability to produce high molecular weight rubber in its roots and promise as an alternative source of natural rubber. In order to accelerate the domestication of TK, we have established a simple strategy to deploy CRISPR/Cas9 in this species. A critical gene encoding fructan:fructan 1-fructosyltransferase (1-FFT), implicated in inulin biosynthesis was selected as the target, as inulin is an expected antagonist of rubber production. TK plantlets were inoculated with Agrobacterium rhizogenes harboring a plasmid encoding a Cas9 and sgRNA (single guide RNA) targeting TK 1-FFT. We were able to rapidly induce hairy roots harboring knockout alleles without the selection of stable, herbicide or antibiotic resistant transformants. Mutagenesis was affirmed by observing a loss of restriction sites within 1-FFT, followed by sequencing. Of 11 hairy root samples tested, 10 showed the presence of genome editing, with mutation rates as high as 88.9%, suggesting a high efficiency mutagenesis induced by CRISPR/Cas9 via A. rhizogenes-mediated transformation. Whole TK plants were regenerated from hairy roots harboring knockout alleles. The regenerated plants contained knockout alleles, with mutation rates as high as 80.0%. TK plants with edited genomes were obtained within 10 weeks. By omitting a selection step, it was possible to generate edited TK plants lacking stably transformed CRISPR elements, which may potentially reduce off-target mutagenesis. Application of high efficiency CRISPR/Cas9 genome editing will facilitate the domestication and commercialization of TK as a rubber producing crop, and may accelerate basic research on the regulation of rubber biosynthesis.

The standard operating procedure of the DOE-JGI Metagenome Annotation Pipeline (MAP v.4)

The DOE-JGI Metagenome Annotation Pipeline (MAP v.4) performs structural and functional annotation for metagenomic sequences that are submitted to the Integrated Microbial Genomes with Microbiomes (IMG/M) system for comparative analysis. The pipeline runs on nucleotide sequences provided via the IMG submission site. Users must first define their analysis projects in GOLD and then submit the associated sequence datasets consisting of scaffolds/contigs with optional coverage information and/or unassembled reads in fasta and fastq file formats. The MAP processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNAs, as well as CRISPR elements. Structural annotation is followed by functional annotation including assignment of protein product names and connection to various protein family databases.

A Comprehensive Curation Shows the Dynamic Evolutionary Patterns of Prokaryotic CRISPRs.

Motivation. Clustered regularly interspaced short palindromic repeat (CRISPR) is a genetic element with active regulation roles for foreign invasive genes in the prokaryotic genomes and has been engineered to work with the CRISPR-associated sequence (Cas) gene Cas9 as one of the modern genome editing technologies. Due to inconsistent definitions, the existing CRISPR detection programs seem to have missed some weak CRISPR signals. Results. This study manually curates all the currently annotated CRISPR elements in the prokaryotic genomes and proposes 95 updates to the annotations. A new definition is proposed to cover all the CRISPRs. The comprehensive comparison of CRISPR numbers on the taxonomic levels of both domains and genus shows high variations for closely related species even in the same genus. The detailed investigation of how CRISPRs are evolutionarily manipulated in the 8 completely sequenced species in the genus Thermoanaerobacter demonstrates that transposons act as a frequent tool for splitting long CRISPRs into shorter ones along a long evolutionary history.

The standard operating procedure of the DOE-JGI Microbial Genome Annotation Pipeline (MGAP v.4)

The DOE-JGI Microbial Genome Annotation Pipeline performs structural and functional annotation of microbial genomes that are further included into the Integrated Microbial Genome comparative analysis system. MGAP is applied to assembled nucleotide sequence datasets that are provided via the IMG submission site. Dataset submission for annotation first requires project and associated metadata description in GOLD. The MGAP sequence data processing consists of feature prediction including identification of protein-coding genes, non-coding RNAs and regulatory RNA features, as well as CRISPR elements. Structural annotation is followed by assignment of protein product names and functions.

Transcriptomic and proteomic dynamics in the metabolism of a diazotrophic cyanobacterium, Cyanothece sp. PCC 7822 during a diurnal light-dark cycle.

BackgroundCyanothece sp. PCC 7822 is an excellent cyanobacterial model organism with great potential to be applied as a biocatalyst for the production of high value compounds. Like other unicellular diazotrophic cyanobacterial species, it has a tightly regulated metabolism synchronized to the light–dark cycle. Utilizing transcriptomic and proteomic methods, we quantified the relationships between transcription and translation underlying central and secondary metabolism in response to nitrogen free, 12 hour light and 12 hour dark conditions.ResultsBy combining mass-spectrometry based proteomics and RNA-sequencing transcriptomics, we quantitatively measured a total of 6766 mRNAs and 1322 proteins at four time points across a 24 hour light–dark cycle. Photosynthesis, nitrogen fixation, and carbon storage relevant genes were expressed during the preceding light or dark period, concurrent with measured nitrogenase activity in the late light period. We describe many instances of disparity in peak mRNA and protein abundances, and strong correlation of light dependent expression of both antisense and CRISPR-related gene expression. The proteins for nitrogenase and the pentose phosphate pathway were highest in the dark, whereas those for glycolysis and the TCA cycle were more prominent in the light. Interestingly, one copy of the psbA gene encoding the photosystem II (PSII) reaction center protein D1 (psbA4) was highly upregulated only in the dark. This protein likely cannot catalyze O2 evolution and so may be used by the cell to keep PSII intact during N2 fixation. The CRISPR elements were found exclusively at the ends of the large plasmid and we speculate that their presence is crucial to the maintenance of this plasmid.ConclusionsThis investigation of parallel transcriptional and translational activity within Cyanothece sp. PCC 7822 provided quantitative information on expression levels of metabolic pathways relevant to engineering efforts. The identification of expression patterns for both mRNA and protein affords a basis for improving biofuel production in this strain and for further genetic manipulations. Expression analysis of the genes encoded on the 6 plasmids provided insight into the possible acquisition and maintenance of some of these extra-chromosomal elements.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-1185) contains supplementary material, which is available to authorized users.

Genome analysis of environmental and clinical P. aeruginosa isolates from sequence type-1146.

The genomes of Pseudomonas aeruginosa isolates of the new sequence type ST-1146, three environmental (P37, P47 and P49) and one clinical (SD9) isolates, with differences in their antibiotic susceptibility profiles have been sequenced and analysed. The genomes were mapped against P. aeruginosa PAO1-UW and UCBPP-PA14. The allelic profiles showed that the highest number of differences were in “Related to phage, transposon or plasmid” and “Secreted factors” categories. The clinical isolate showed a number of exclusive alleles greater than that for the environmental isolates. The phage Pf1 region in isolate SD9 accumulated the highest number of nucleotide substitutions. The ORF analysis of the four genomes assembled de novo indicated that the number of isolate-specific genes was higher in isolate SD9 (132 genes) than in isolates P37 (24 genes), P47 (16 genes) and P49 (21 genes). CRISPR elements were found in all isolates and SD9 showed differences in the spacer region. Genes related to bacteriophages F116 and H66 were found only in isolate SD9. Genome comparisons indicated that the isolates of ST-1146 are close related, and most genes implicated in pathogenicity are highly conserved, suggesting a genetic potential for infectivity in the environmental isolates similar to the clinical one. Phage-related genes are responsible of the main differences among the genomes of ST-1146 isolates. The role of bacteriophages has to be considered in the adaptation processes of isolates to the host and in microevolution studies.

Investigating the Interplay between Nucleoid-Associated Proteins, DNA Curvature, and CRISPR Elements Using Comparative Genomics

Many prokaryotic and eukaryotic genomes feature a characteristic periodic signal in distribution of short runs of A or T (A-tracts) phased with the DNA helical period of ∼10–11 bp. Such periodic spacing of A-tracts has been associated with intrinsic DNA curvature. In eukaryotes, this periodicity is a major component of the nucleosome positioning signal but its physiological role in prokaryotes is not clear. One hypothesis centers on possible role of intrinsic DNA bends in nucleoid compaction. We use comparative genomics to investigate possible relationship between the A-tract periodicity and nucleoid-associated proteins in prokaryotes. We found that genomes with DNA-bridging proteins tend to exhibit stronger A-tract periodicity, presumably indicative of more prevalent intrinsic DNA curvature. A weaker relationship was detected for nucleoid-associated proteins that do not form DNA bridges. We consider these results an indication that intrinsic DNA curvature acts collaboratively with DNA-bridging proteins in maintaining the compact structure of the nucleoid, and that previously observed differences among prokaryotic genomes in terms DNA curvature-related sequence periodicity may reflect differences in nucleoid organization. We subsequently investigated the relationship between A-tract periodicity and presence of CRISPR elements and we found that genomes with CRISPR tend to have stronger A-tract periodicity. This result is consistent with our earlier hypothesis that extensive A-tract periodicity could help protect the chromosome against integration of prophages, possibly due to its role in compaction of the nucleoid.

Comparison of genotypic and phenotypic cluster analyses of virulence determinants and possible role of CRISPR elements towards their incidence in Enterococcus faecalis and Enterococcus faecium

Enterococcus faecalis and Enterococcus faecium are human commensals frequently found in fermented foods or used as probiotics, but also recognized as opportunistic pathogens. We investigated 62 Enterococcus strains isolated from clinical, food and environmental origins towards a rationale for safety evaluation of strains in food or probiotic applications. All isolates were characterised with respect to the presence of the virulence determinants fsrB, sprE, gelE, ace, efaAfs/fm, as, esp, cob and the cytolysin operon. In addition RAPD-PCR was used to obtain genomic fingerprints that were clustered and compared to phenotypic profiles generated by MALDI-TOF-MS. The gelatinase phenotype (GelE) and the haemolytic activity (β-haemolysis) were analysed. E. faecium strains contained esp and efaAfm only, and none of them contained any CRISPR elements. The amenability of E. faecalis strains to acquisition of virulence factors was investigated along the occurrence of CRISPR associated ( cas) genes. While distribution of most virulence factors, and RAPD versus MALDI-TOF-MS typing patterns were unrelated, 2 out of 5 RAPD clusters almost exclusively contained clinical E. faecalis isolates, and an occurrence of CRISPR elements versus reduced number of virulence factors was observed. The presence of the cytolysin operon, cob and as encoding pheromone and aggregation substance, respectively, significantly corresponded to absence of cas. As their production promote genetic exchange, their absence limits further gene acquisition and distribution. Thus, absence of the cytolysin operon, cob and as in a cas positive environment suggests itself as promising candidate for E. faecalis evaluation towards their occurrence in food fermentation or use as probiotics.

PILER-CR: Fast and accurate identification of CRISPR repeats

BackgroundSequencing of prokaryotic genomes has recently revealed the presence of CRISPR elements: short, highly conserved repeats separated by unique sequences of similar length. The distinctive sequence signature of CRISPR repeats can be found using general-purpose repeat- or pattern-finding software tools. However, the output of such tools is not always ideal for studying these repeats, and significant effort is sometimes needed to build additional tools and perform manual analysis of the output.ResultsWe present PILER-CR, a program specifically designed for the identification and analysis of CRISPR repeats. The program executes rapidly, completing a 5 Mb genome in around 5 seconds on a current desktop computer. We validate the algorithm by manual curation and by comparison with published surveys of these repeats, finding that PILER-CR has both high sensitivity and high specificity. We also present a catalogue of putative CRISPR repeats identified in a comprehensive analysis of 346 prokaryotic genomes.ConclusionPILER-CR is a useful tool for rapid identification and classification of CRISPR repeats. The software is donated to the public domain. Source code and a Linux binary are freely available at .