Repurposing the Streptococcus mutans CRISPR-Cas9 System to Understand Essential Gene Function

AbstractA recent genome-wide screen identified ∼300 essential or growth-supporting genes in the dental caries pathogen Streptococcus mutans. To be able to study these genes, we built a CRISPR interference tool around the Cas9 nuclease (Cas9Smu) encoded in the S. mutans UA159 genome. Using a xylose-inducible dead Cas9Smu with a constitutively active single-guide RNA (sgRNA), we observed titratable repression of GFP fluorescence that compared favorably to that of Streptococcus pyogenes dCas9 (Cas9Spy). We then investigated sgRNA specificity and proto-spacer adjacent motif (PAM) requirements. Interference by sgRNAs did not occur with double or triple base-pair mutations, or if single base-pair mutations were in the 3’ end of the sgRNA. Bioinformatic analysis of >450 S. mutans genomes allied with in vivo assays revealed a similar PAM recognition sequence as the Cas9Spy. Next, we created a comprehensive library of sgRNA plasmids that were directed at essential and growth-supporting genes. We discovered growth defects for 77% of the CRISPRi strains expressing sgRNAs. Phenotypes of CRISPRi strains, across several biological pathways, were assessed using fluorescence microscopy. A variety of cell structure anomalies were observed, including segregational instability of the chromosome, enlarged cells, and ovococci-to-rod shape transitions. CRISPRi was also employed to observe how silencing of cell wall glycopolysaccharide biosynthesis (rhamnose-glucose polysaccharide, RGP) affected both cell division and pathogenesis in a wax worm model. The CRISPRi tool and sgRNA library are valuable resources for characterizing essential genes in S. mutans, some of which could prove to be promising therapeutic targets.

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Prime-editors (nickases), hRad51–Cas9 nickase fusions and dCas9 have the same problem as conventional CRISPR-Cas9 of plasmid/Cas9 integration after making a double stranded break

10.31219/osf.io/jf6pe ◽

2019 ◽

Author(s):

Sandeep Chakraborty

Keyword(s):

Cellular Response ◽

Sequencing Data ◽

Dna Breaks ◽

Precise Integration ◽

Guide Rna ◽

Double Strand Dna Breaks ◽

Human Therapy ◽

P53 Activation ◽

Programmable Nucleases

‘Prime-editing’ proposes to replace traditional programmable nucleases (CRISPR-Cas9) using a catalytically impaired Cas9 (dCas9) connected to a engineered reverse transcriptase, and a guide RNA encoding both the target site and the desired change. With just a ‘nick’ on one strand, it is hypothe- sized, the negative, uncontrollable effects arising from double-strand DNA breaks (DSBs) - translocations, complex proteins, integrations and p53 activation - will be eliminated. However, sequencing data pro- vided (Accid:PRJNA565979) reveal plasmid integration, indicating that DSBs occur. Also, looking at only 16 off-targets is inadequate to assert that Prime-editing is more precise. Integration of plasmid occurs in all three versions (PE1/2/3). Interestingly, dCas9 which is known to be toxic in E. coli and yeast, is shown to have residual endonuclease activity. This also affects studies that use dCas9, like base- editors and de/methylations systems. Previous work using hRad51–Cas9 nickases also show significant integration in on-targets, as well as off-target integration [1]. Thus, we show that cellular response to nicking involves DSBs, and subsequent plasmid/Cas9 integration. This is an unacceptable outcome for any in vivo application in human therapy.

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Genome wide effects of oleic acid on cultured bovine granulosa cells: evidence for the activation of pathways favoring folliculo-luteal transition

BMC Genomics ◽

10.1186/s12864-021-07817-6 ◽

2021 ◽

Vol 22 (1) ◽

Author(s):

Vengala Rao Yenuganti ◽

Dirk Koczan ◽

Jens Vanselow

Keyword(s):

Oleic Acid ◽

Granulosa Cells ◽

System Development ◽

Circulatory System ◽

Bioinformatic Analysis ◽

Negative Energy ◽

Tissue Remodelling ◽

Gonadotropin Secretion ◽

Genome Wide

Abstract Background Metabolic stress, as negative energy balance on one hand or obesity on the other hand can lead to increased levels of free fatty acids in the plasma and follicular fluid of animals and humans. In an earlier study, we showed that increased oleic acid (OA) concentrations affected the function of cultured bovine granulosa cells (GCs). Here, we focus on genome wide effects of increased OA concentrations. Results Our data showed that 413 genes were affected, of which 197 were down- and 216 up-regulated. Specifically, the expression of FSH-regulated functional key genes, CCND2, LHCGR, INHA and CYP19A1 and 17-β-estradiol (E2) production were reduced by OA treatment, whereas the expression of the fatty acid transporter CD36 was increased and the morphology of the cells was changed due to lipid droplet accumulation. Bioinformatic analysis revealed that associated pathways of the putative upstream regulators “FSH” and “Cg (choriogonadotropin)” were inhibited and activated, respectively. Down-regulated genes are over-represented in GO terms “reproductive structure/system development”, “ovulation cycle process”, and “(positive) regulation of gonadotropin secretion”, whereas up-regulated genes are involved in “circulatory system development”, “vasculature development”, “angiogenesis” or “extracellular matrix/structure organization”. Conclusions From these data we conclude that besides inhibiting GC functionality, increased OA levels seemingly promote angiogenesis and tissue remodelling, thus suggestively initiating a premature fulliculo-luteal transition. In vivo this may lead to impeded folliculogenesis and ovulation, and cause sub-fertility.

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miR-379 deletion ameliorates features of diabetic kidney disease by enhancing adaptive mitophagy via FIS1

Communications Biology ◽

10.1038/s42003-020-01516-w ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Mitsuo Kato ◽

Maryam Abdollahi ◽

Ragadeepthi Tunduguru ◽

Walter Tsark ◽

Zhuo Chen ◽

...

Keyword(s):

Kidney Disease ◽

Diabetic Kidney Disease ◽

Mesangial Cells ◽

Mitochondrial Fission ◽

Major Complication ◽

Body Weight Loss ◽

Diabetic Kidney ◽

Guide Rna ◽

And Oxidative Stress

AbstractDiabetic kidney disease (DKD) is a major complication of diabetes. Expression of members of the microRNA (miRNA) miR-379 cluster is increased in DKD. miR-379, the most upstream 5′-miRNA in the cluster, functions in endoplasmic reticulum (ER) stress by targeting EDEM3. However, the in vivo functions of miR-379 remain unclear. We created miR-379 knockout (KO) mice using CRISPR-Cas9 nickase and dual guide RNA technique and characterized their phenotype in diabetes. We screened for miR-379 targets in renal mesangial cells from WT vs. miR-379KO mice using AGO2-immunopreciptation and CLASH (cross-linking, ligation, sequencing hybrids) and identified the redox protein thioredoxin and mitochondrial fission-1 protein. miR-379KO mice were protected from features of DKD as well as body weight loss associated with mitochondrial dysfunction, ER- and oxidative stress. These results reveal a role for miR-379 in DKD and metabolic processes via reducing adaptive mitophagy. Strategies targeting miR-379 could offer therapeutic options for DKD.

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Development of a rabies virus-based retrograde tracer with high trans-monosynaptic efficiency by reshuffling glycoprotein

Molecular Brain ◽

10.1186/s13041-021-00821-7 ◽

2021 ◽

Vol 14 (1) ◽

Author(s):

Fan Jia ◽

Li Li ◽

Haizhou Liu ◽

Pei Lv ◽

Xiangwei Shi ◽

...

Keyword(s):

Experimental Data ◽

Rabies Virus ◽

Oncolytic Virus ◽

Neural Circuits ◽

Bioinformatic Analysis ◽

Cell Engineering ◽

Bias Score ◽

Codon Pair Bias ◽

Codon Pair

AbstractRabies virus (RV) is the most widely used vector for mapping neural circuits. Previous studies have shown that the RV glycoprotein can be a target to improve the retrograde transsynaptic tracing efficiency. However, the current versions still label only a small portion of all presynaptic neurons. Here, we reshuffled the oG sequence, a chimeric glycoprotein, with positive codon pair bias score (CPBS) based on bioinformatic analysis of mouse codon pair bias, generating ooG, a further optimized glycoprotein. Our experimental data reveal that the ooG has a higher expression level than the oG in vivo, which significantly increases the tracing efficiency by up to 12.6 and 62.1-fold compared to oG and B19G, respectively. The new tool can be used for labeling neural circuits Therefore, the approach reported here provides a convenient, efficient and universal strategy to improve protein expression for various application scenarios such as trans-synaptic tracing efficiency, cell engineering, and vaccine and oncolytic virus designs.

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SBA1 Encodes a Yeast Hsp90 Cochaperone That Is Homologous to Vertebrate p23 Proteins

Molecular and Cellular Biology ◽

10.1128/mcb.18.7.3727 ◽

1998 ◽

Vol 18 (7) ◽

pp. 3727-3734 ◽

Cited By ~ 117

Author(s):

Yifang Fang ◽

Albert E. Fliss ◽

Jie Rao ◽

Avrom J. Caplan

Keyword(s):

Hormone Receptors ◽

Pcr Amplification ◽

Steroid Hormone Receptors ◽

Loss Of Function ◽

Vitro Assay ◽

Growth Defects ◽

Double Deletion ◽

Affinity Isolation

ABSTRACT The Saccharomyces cerevisiae SBA1 gene was cloned by PCR amplification from yeast genomic DNA following its identification as encoding an ortholog of human p23, an Hsp90 cochaperone. TheSBA1 gene product is constitutively expressed and nonessential, although a disruption mutant grew more slowly than the wild type at both 18 and 37°C. A double deletion of SBA1and STI1, encoding an Hsp90 cochaperone, displayed synthetic growth defects. Affinity isolation of histidine-tagged Sba1p (Sba1His6) after expression in yeast led to coisolation of Hsp90 and the cyclophilin homolog Cpr6. Using an in vitro assembly assay, purified Sba1His6 bound to Hsp90 only in the presence of adenosine 5′-O-(3-thiotriphosphate) or adenyl-imidodiphosphate. Furthermore, interaction between purified Sba1His6 and Hsp90 in yeast extracts was inhibited by the benzoquinoid ansamycins geldanamycin and macbecin. The in vitro assay was also used to identify residues in Hsp90 that are important for complex formation with Sba1His6, and residues in both the N-terminal nucleotide binding domain and C-terminal half were characterized. In vivo analysis of known Hsp90 substrate proteins revealed that Sba1 loss of function had only a mild effect on the activity of the tyrosine kinase v-Src and steroid hormone receptors.

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Function-adaptive clustered nanoparticles reverse Streptococcus mutans dental biofilm and maintain microbiota balance

Communications Biology ◽

10.1038/s42003-021-02372-y ◽

2021 ◽

Vol 4 (1) ◽

Author(s):

Fatemeh Ostadhossein ◽

Parikshit Moitra ◽

Esra Altun ◽

Debapriya Dutta ◽

Dinabandhu Sar ◽

...

Keyword(s):

Streptococcus Mutans ◽

Ex Vivo ◽

Bacterial Species ◽

Reactive Oxygen Species Generation ◽

Biofilm Inhibition ◽

16S Rrna Analysis ◽

Dental Biofilm ◽

Dental Plaques

AbstractDental plaques are biofilms that cause dental caries by demineralization with acidogenic bacteria. These bacteria reside inside a protective sheath which makes any curative treatment challenging. We propose an antibiotic-free strategy to disrupt the biofilm by engineered clustered carbon dot nanoparticles that function in the acidic environment of the biofilms. In vitro and ex vivo studies on the mature biofilms of Streptococcus mutans revealed >90% biofilm inhibition associated with the contact-mediated interaction of nanoparticles with the bacterial membrane, excessive reactive oxygen species generation, and DNA fragmentation. An in vivo examination showed that these nanoparticles could effectively suppress the growth of S. mutans. Importantly, 16S rRNA analysis of the dental microbiota showed that the diversity and richness of bacterial species did not substantially change with nanoparticle treatment. Overall, this study presents a safe and effective approach to decrease the dental biofilm formation without disrupting the ecological balance of the oral cavity.

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ATTAAA as well as downstream sequences are required for RNA 3'-end formation in the E3 complex transcription unit of adenovirus

Molecular and Cellular Biology ◽

10.1128/mcb.5.11.3183-3193.1985 ◽

1985 ◽

Vol 5 (11) ◽

pp. 3183-3193

Author(s):

B M Bhat ◽

W S Wold

Keyword(s):

Base Pair ◽

Transcription Unit ◽

Rna Probes ◽

Labeled Rna

We mapped the location of the E3A RNA 3' end site in the E3 transcription unit of adenovirus 2. The procedure used was nuclease-gel analysis with 32P-labeled RNA probes. The poly(A) addition sites were microheterogeneous and were located approximately 17 to 29 nucleotides downstream from an ATTAAA sequence. To identify the sequences that make up the E3A RNA 3' end signal, we constructed five viable virus mutants with deletions in or near the E3A RNA 3' end site. The mutants were analyzed for E3A RNA 3' end formation in vivo. No effect was observed from a 47-base-pair (bp) deletion (dl716) or a 72-bp deletion (dl714) located 22 and 19 nucleotides, respectively, upstream of the ATTAAA. In contrast, E3A RNA 3' end formation was abolished by a 554-bp deletion (dl708) that removes both the ATTAAA and the poly(A) addition sites, a 124-bp deletion (dl713) that removes the ATTAAA but leaves the poly(A) addition sites, and a 65-bp deletion (dl719) that leaves the ATTAAA but removes the poly(A) addition sites. These results indicate that the ATTAAA, as well as downstream sequences, including the poly(A) addition sites, are required for E3A RNA 3' end formation.

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Mechanism of initiator-mediated transcription: evidence for a functional interaction between the TATA-binding protein and DNA in the absence of a specific recognition sequence

Molecular and Cellular Biology ◽

10.1128/mcb.13.7.3841-3849.1993 ◽

1993 ◽

Vol 13 (7) ◽

pp. 3841-3849

Author(s):

B Zenzie-Gregory ◽

A Khachi ◽

I P Garraway ◽

S T Smale

Keyword(s):

Transcription Initiation ◽

Binding Protein ◽

Tata Binding Protein ◽

Upstream Region ◽

Promoter Strength ◽

Recognition Sequence ◽

Specific Recognition ◽

Rate Limiting

Promoters containing Sp1 binding sites and an initiator element but lacking a TATA box direct high levels of accurate transcription initiation by using a mechanism that requires the TATA-binding protein (TBP). We have begun to address the role of TBP during transcription from Sp1-initiator promoters by varying the nucleotide sequence between -14 and -33 relative to the start site. With each of several promoters containing different upstream sequences, we detected accurate transcription both in vitro and in vivo, but the promoter strengths varied widely, particularly with the in vitro assay. The variable promoter activities correlated with, but were not proportional to, the abilities of the upstream sequences to function as TATA boxes, as assessed by multiple criteria. These results confirm that accurate transcription can proceed in the presence of an initiator, regardless of the sequence present in the -30 region. However, the results reveal a role for this upstream region, most consistent with a model in which initiator-mediated transcription requires binding of TBP to the upstream DNA in the absence of a specific recognition sequence. Moreover, in vivo it appears that the promoter strength is modulated less severely by altering the -30 sequence, consistent with a previous suggestion that TBP is not rate limiting in vivo for TATA-less promoters. Taken together, these results suggest that variations in the structure of a core promoter might alter the rate-limiting step for transcription initiation and thereby alter the potential modes of transcriptional regulation, without severely changing the pathway used to assemble a functional preinitiation complex.

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Individual Mycobacterium tuberculosis Resuscitation-Promoting Factor Homologues Are Dispensable for Growth In Vitro and In Vivo

Infection and Immunity ◽

10.1128/iai.72.1.515-526.2004 ◽

2004 ◽

Vol 72 (1) ◽

pp. 515-526 ◽

Cited By ~ 84

Author(s):

JoAnn M. Tufariello ◽

William R. Jacobs, ◽

John Chan

Keyword(s):

Mycobacterium Tuberculosis ◽

Stationary Phase ◽

Essential Gene ◽

Micrococcus Luteus ◽

Expression Patterns ◽

Active Growth ◽

Prolonged Incubation ◽

Aerosol Infection

ABSTRACT Mycobacterium tuberculosis possesses five genes with significant homology to the resuscitation-promoting factor (Rpf) of Micrococcus luteus. The M. luteus Rpf is a secreted ∼16-kDa protein which restores active growth to cultures of M. luteus rendered dormant by prolonged incubation in stationary phase. More recently, the Rpf-like proteins of M. tuberculosis have been shown to stimulate the growth of extended-stationary-phase cultures of Mycobacterium bovis BCG. These data suggest that the Rpf proteins can influence the growth of mycobacteria; however, the studies do not demonstrate specific functions for the various members of this protein family, nor do they assess the function of M. tuberculosis Rpf homologues in vivo. To address these questions, we have disrupted each of the five rpf-like genes in M. tuberculosis Erdman, and analyzed the mutants for their growth in vitro and in vivo. In contrast to M. luteus, for which rpf is an essential gene, we find that all of the M. tuberculosis rpf deletion mutant strains are viable; in addition, all show growth kinetics similar to Erdman wild type both in vitro and in mouse organs following aerosol infection. Analysis of rpf expression in M. tuberculosis cultures from early log phase through late stationary phase indicates that expression of the rpf-like genes is growth phase-dependent, and that the expression patterns of the five M. tuberculosis rpf genes, while overlapping to various degrees, are not uniform. We also provide evidence that mycobacterial rpf genes are expressed in vivo in the lungs of mice acutely infected with virulent M. tuberculosis.

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