scholarly journals Harnessing CRISPR-Cas9 for genome editing in Streptococcus pneumoniae

2020 ◽  
Author(s):  
Dimitra Synefiaridou ◽  
Jan-Willem Veening
Keyword(s):  
Streptococcus Pneumoniae ◽  
Genome Editing ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Human Pathogen ◽  
Gram Positive Bacteria ◽  
Homology Directed Repair ◽  
Dna Fragment ◽  
Opportunistic Human Pathogen ◽  
Template Dna

AbstractCRISPR systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by detection and cleavage of invading foreign DNA. Modified versions of this system can be exploited as a biotechnological tool for precise genome editing at a targeted locus. Here, we developed a novel, replicative plasmid that carries the CRISPR-Cas9 system for RNA-programmable, genome editing by counterselection in the opportunistic human pathogen Streptococcus pneumoniae. Specifically, we demonstrate an approach for making targeted, marker-less gene knockouts and large genome deletions. After a precise double-stranded break (DSB) is introduced, the cells’ DNA repair mechanism of homology-directed repair (HDR) pathway is being exploited to select successful transformants. This is achieved through the transformation of a template DNA fragment that will recombine in the genome and eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered strain, can be easily cured from the plasmid that is temperature-sensitive for replication, by growing it at the non-permissive temperature. This allows for consecutive rounds of genome editing. Using this system, we engineered a strain with three major virulence factors deleted. The here developed approaches should be readily transportable to other Gram-positive bacteria.ImportanceStreptococcus pneumoniae (the pneumococcus) is an important opportunistic human pathogen killing over a million people each year. Having the availability of a system capable of easy genome editing would significantly facilitate drug discovery and vaccine candidate efforts. Here, we introduced an easy to use system to perform multiple rounds of genome editing in the pneumococcus by putting the CRISPR-Cas9 system on a temperature-sensitive replicative plasmid. The here used approaches will advance genome editing projects in this important human pathogen.

scholarly journals Harnessing CRISPR-Cas9 for genome editing in Streptococcus pneumoniae D39V

2021 ◽  
Author(s):  
Dimitra Synefiaridou ◽  
Jan-Willem Veening
Keyword(s):  
Streptococcus Pneumoniae ◽  
Genome Editing ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Human Pathogen ◽  
Gram Positive Bacteria ◽  
Homology Directed Repair ◽  
Dna Fragment ◽  
Opportunistic Human Pathogen ◽  
Template Dna

CRISPR-Cas systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by detection and cleavage of invading foreign DNA. Modified versions of this system can be exploited as a biotechnological tool for precise genome editing at a targeted locus. Here, we developed a replicative plasmid that carries the CRISPR-Cas9 system for RNA-programmable, genome editing by counterselection in the opportunistic human pathogen Streptococcus pneumoniae. Specifically, we demonstrate an approach for making targeted, marker-less gene knockouts and large genome deletions. After a precise double-stranded break (DSB) is introduced, the cells’ DNA repair mechanism of homology-directed repair (HDR) pathway is being exploited to select successful transformants. This is achieved through the transformation of a template DNA fragment that will recombine in the genome and eliminate recognition of the target of the Cas9 endonuclease. Next, the newly engineered strain can be easily cured from the plasmid that is temperature-sensitive for replication, by growing it at the non-permissive temperature. This allows for consecutive rounds of genome editing. Using this system, we engineered a strain with three major virulence factors deleted. The here developed approaches could be potentially transported to other Gram-positive bacteria. Importance Streptococcus pneumoniae (the pneumococcus) is an important opportunistic human pathogen killing over a million people each year. Having the availability of a system capable of easy genome editing would significantly facilitate drug discovery and efforts in identifying new vaccine candidates. Here, we introduced an easy to use system to perform multiple rounds of genome editing in the pneumococcus by putting the CRISPR-Cas9 system on a temperature-sensitive replicative plasmid. The here used approaches will advance genome editing projects in this important human pathogen.

scholarly journals Refining the Pneumococcal Competence Regulon by RNA Sequencing

2019 ◽  
Vol 201 (13) ◽  
Author(s):  
Jelle Slager ◽  
Rieza Aprianto ◽  
Jan-Willem Veening
Keyword(s):  
Antibiotic Resistance ◽  
Streptococcus Pneumoniae ◽  
Rna Sequencing ◽  
Genome Annotation ◽  
Stress Factors ◽  
Microarray Technology ◽  
Human Pathogen ◽  
Exogenous Dna ◽  
Content Type ◽  
Opportunistic Human Pathogen

ABSTRACTCompetence for genetic transformation allows the opportunistic human pathogenStreptococcus pneumoniaeto take up exogenous DNA for incorporation into its own genome. This ability may account for the extraordinary genomic plasticity of this bacterium, leading to antigenic variation, vaccine escape, and the spread of antibiotic resistance. The competence system has been thoroughly studied, and its regulation is well understood. Additionally, over the last decade, several stress factors have been shown to trigger the competent state, leading to the activation of several stress response regulons. The arrival of next-generation sequencing techniques allowed us to update the competence regulon, the latest report on which still depended on DNA microarray technology. Enabled by the availability of an up-to-date genome annotation, including transcript boundaries, we assayed time-dependent expression of all annotated features in response to competence induction, were able to identify the affected promoters, and produced a more complete overview of the various regulons activated during the competence state. We show that 4% of all annotated genes are under direct control of competence regulators ComE and ComX, while the expression of a total of up to 17% of all genes is affected, either directly or indirectly. Among the affected genes are various small RNAs with an as-yet-unknown function. Besides the ComE and ComX regulons, we were also able to refine the CiaR, VraR (LiaR), and BlpR regulons, underlining the strength of combining transcriptome sequencing (RNA-seq) with a well-annotated genome.IMPORTANCEStreptococcus pneumoniaeis an opportunistic human pathogen responsible for over a million deaths every year. Although both vaccination programs and antibiotic therapies have been effective in prevention and treatment of pneumococcal infections, respectively, the sustainability of these solutions is uncertain. The pneumococcal genome is highly flexible, leading to vaccine escape and antibiotic resistance. This flexibility is predominantly facilitated by competence, a state allowing the cell to take up and integrate exogenous DNA. Thus, it is essential to obtain a detailed overview of gene expression during competence. This is stressed by the fact that administration of several classes of antibiotics can lead to competence. Previous studies on the competence regulon were performed with microarray technology and were limited to an incomplete set of known genes. Using RNA sequencing combined with an up-to-date genome annotation, we provide an updated overview of competence-regulated genes.

scholarly journals Three New Integration Vectors and Fluorescent Proteins for Use in the Opportunistic Human Pathogen Streptococcus pneumoniae

Genes ◽  
2019 ◽  
Vol 10 (5) ◽  
pp. 394 ◽  
Author(s):  
Lance E. Keller ◽  
Anne-Stéphanie Rueff ◽  
Jun Kurushima ◽  
Jan-Willem Veening
Keyword(s):  
Streptococcus Pneumoniae ◽  
Transformation Efficiency ◽  
Fluorescent Proteins ◽  
Inducible Promoter ◽  
Multidrug Resistant ◽  
Clinical Isolates ◽  
Large Set ◽  
Human Pathogen ◽  
Wide Range ◽  
Opportunistic Human Pathogen

Here, we describe the creation of three integration vectors, pPEPX, pPEPY and pPEPZ, for use with the opportunistic human pathogen Streptococcus pneumoniae. The constructed vectors, named PEP for Pneumococcal Engineering Platform (PEP), employ an IPTG-inducible promoter and BglBrick and BglFusion compatible multiple cloning sites allowing for fast and interchangeable cloning. PEP plasmids replicate in Escherichia coli and harbor integration sites that have homology in a large set of pneumococcal strains, including recent clinical isolates. In addition, several options of antibiotic resistance markers are available, even allowing for selection in multidrug resistant clinical isolates. The transformation efficiency of these PEP vectors as well as their ability to be expressed simultaneously was tested. Two of the three PEP vectors share homology of the integration regions with over half of the S. pneumoniae genomes examined. Transformation efficiency varied among PEP vectors based on the length of the homology regions, but all were highly transformable and can be integrated simultaneously in strain D39V. Vectors used for pneumococcal cloning are an important tool for researchers for a wide range of uses. The PEP vectors described are of particular use because they have been designed to allow for easy transfer of genes between vectors as well as integrating into transcriptionally silent areas of the chromosome. In addition, we demonstrate the successful production of several new spectrally distinct fluorescent proteins (mTurquoise2, mNeonGreen and mScarlet-I) from the PEP vectors. The PEP vectors and newly described fluorescent proteins will expand the genetic toolbox for pneumococcal researchers and aid future discoveries.

scholarly journals 5' modifications improve potency and efficacy of DNA donors for precision genome editing

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Krishna S Ghanta ◽  
Zexiang Chen ◽  
Aamir Mir ◽  
Gregoriy A Dokshin ◽  
Pranathi M Krishnamurthy ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Triethylene Glycol ◽  
Great Promise ◽  
Precise Form ◽  
Homology Directed Repair ◽  
Genomic Location ◽  
Cultured Human Cells ◽  
Repair Template ◽  
Template Dna

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach to correct mutations that cause disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit HDR efficacy. Here, we explore chemical modifications to both double-stranded and single-stranded DNA-repair templates. We describe 5′-terminal modifications, including in its simplest form the incorporation of triethylene glycol (TEG) moieties, that consistently increase the frequency of precision editing in the germlines of three animal models (Caenorhabditis elegans, zebrafish, mice) and in cultured human cells.

scholarly journals CRISPR Co-Editing Strategy for Scarless Homology-Directed Genome Editing

2021 ◽  
Vol 22 (7) ◽  
pp. 3741
Author(s):  
Nina Reuven ◽  
Julia Adler ◽  
Nadav Myers ◽  
Yosef Shaul
Keyword(s):  
Genome Editing ◽  
Selectable Marker ◽  
Small Subset ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Biologically Relevant ◽  
Homology Directed Repair ◽  
Endogenous Genes ◽  
Short Palindromic Repeat ◽  
Single Stranded Oligonucleotide

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 has revolutionized genome editing by providing a simple and robust means to cleave specific genomic sequences. However, introducing templated changes at the targeted site usually requires homology-directed repair (HDR), active in only a small subset of cells in culture. To enrich for HDR-dependent edited cells, we employed a co-editing strategy, editing a gene of interest (GOI) concomitantly with rescuing an endogenous pre-made temperature-sensitive (ts) mutation. By using the repair of the ts mutation as a selectable marker, the selection is “scarless” since editing restores the wild-type (wt) sequence. As proof of principle, we used HEK293 and HeLa cells with a ts mutation in the essential TAF1 gene. CRISPR co-editing of TAF1ts and a GOI resulted in up to 90% of the temperature-resistant cells bearing the desired mutation in the GOI. We used this system to insert large cassettes encoded by plasmid donors and smaller changes encoded by single-stranded oligonucleotide donors (ssODN). Of note, among the genes we edited was the introduction of a T35A mutation in the proteasome subunit PSMB6, which eliminates its caspase-like activity. The edited cells showed a specific reduction in this activity, demonstrating this system’s utility in generating cell lines with biologically relevant mutations in endogenous genes. This approach offers a rapid, efficient, and scarless method for selecting genome-edited cells requiring HDR.

scholarly journals 5′ Modifications Improve Potency and Efficacy of DNA Donors for Precision Genome Editing

2018 ◽  
Author(s):  
Krishna S. Ghanta ◽  
Gregoriy A. Dokshin ◽  
Aamir Mir ◽  
Pranathi Meda Krishnamurthy ◽  
Hassan Gneid ◽  
...  
Keyword(s):  
Dna Repair ◽  
Genome Editing ◽  
Genetic Basis ◽  
Cell Types ◽  
Great Promise ◽  
Precise Form ◽  
Homology Directed Repair ◽  
Genomic Location ◽  
Repair Template ◽  
Template Dna

Nuclease-directed genome editing is a powerful tool for investigating physiology and has great promise as a therapeutic approach that directly addresses the underlying genetic basis of disease. In its most precise form, genome editing can use cellular homology-directed repair (HDR) pathways to insert information from an exogenously supplied DNA repair template (donor) directly into a targeted genomic location. Unfortunately, particularly for long insertions, toxicity and delivery considerations associated with repair template DNA can limit the number of donor molecules available to the HDR machinery, thus limiting HDR efficacy. Here, we explore modifications to both double-stranded and single-stranded repair template DNAs and describe simple 5′ end modifications that consistently and dramatically increase donor potency and HDR efficacy across cell types and species.

scholarly journals Female-to-male sex conversion in Ceratitis capitata by CRISPR/Cas9 HDR-induced point mutations in the sex determination gene transformer-2

2020 ◽  
Vol 10 (1) ◽  
Author(s):  
Roswitha A. Aumann ◽  
Irina Häcker ◽  
Marc F. Schetelig
Keyword(s):  
Sex Determination ◽  
Ceratitis Capitata ◽  
Point Mutations ◽  
Cost Effective ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Homology Directed Repair ◽  
Homozygous Line ◽  
Male Sex ◽  
The Cost

Abstract The Sterile Insect Technique (SIT) is based on the mass release of sterilized male insects to reduce the pest population size via infertile mating. Critical for all SIT programs is a conditional sexing strain to enable the cost-effective production of male-only populations. Compared to current female-elimination strategies based on killing or sex sorting, generating male-only offspring via sex conversion would be economically beneficial by doubling the male output. Temperature-sensitive mutations known from the D. melanogaster transformer-2 gene (tra2ts) induce sex conversion at restrictive temperatures, while regular breeding of mutant strains is possible at permissive temperatures. Since tra2 is a conserved sex determination gene in many Diptera, including the major agricultural pest Ceratitis capitata, it is a promising candidate for the creation of a conditional sex conversion strategy in this Tephritid. Here, CRISPR/Cas9 homology-directed repair was used to induce the D. melanogaster-specific tra2ts SNPs in Cctra2. 100% female to male conversion was successfully achieved in flies homozygous for the tra2ts2 mutation. However, it was not possible, to identify a permissive temperature for the mutation allowing the rearing of a tra2ts2 homozygous line, as lowering the temperature below 18.5 °C interferes with regular breeding of the flies.

scholarly journals A Yeast taf17 Mutant Requires the Swi6 Transcriptional Activator for Viability and Shows Defects in Cell Cycle-Regulated Transcription

Genetics ◽  
2000 ◽  
Vol 154 (4) ◽  
pp. 1561-1576
Author(s):  
Neil Macpherson ◽  
Vivien Measday ◽  
Lynda Moore ◽  
Brenda Andrews
Keyword(s):  
Cell Cycle ◽  
Transcriptional Activation ◽  
Essential Gene ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Synthetic Lethal ◽  
Cycle Arrest ◽  
A Cell ◽  
Allelism Tests ◽  
Complementation Groups

Abstract In Saccharomyces cerevisiae, the Swi6 protein is a component of two transcription factors, SBF and MBF, that promote expression of a large group of genes in the late G1 phase of the cell cycle. Although SBF is required for cell viability, SWI6 is not an essential gene. We performed a synthetic lethal screen to identify genes required for viability in the absence of SWI6 and identified 10 complementation groups of swi6-dependent lethal mutants, designated SLM1 through SLM10. We were most interested in mutants showing a cell cycle arrest phenotype; both slm7-1 swi6Δ and slm8-1 swi6Δ double mutants accumulated as large, unbudded cells with increased 1N DNA content and showed a temperature-sensitive growth arrest in the presence of Swi6. Analysis of the transcript levels of cell cycle-regulated genes in slm7-1 SWI6 mutant strains at the permissive temperature revealed defects in regulation of a subset of cyclin-encoding genes. Complementation and allelism tests showed that SLM7 is allelic with the TAF17 gene, which encodes a histone-like component of the general transcription factor TFIID and the SAGA histone acetyltransferase complex. Sequencing showed that the slm7-1 allele of TAF17 is predicted to encode a version of Taf17 that is truncated within a highly conserved region. The cell cycle and transcriptional defects caused by taf17slm7-1 are consistent with the role of TAFIIs as modulators of transcriptional activation and may reflect a role for TAF17 in regulating activation by SBF and MBF.

scholarly journals A Screen for Genes Involved in the Anaphase Proteolytic Pathway Identifies tsm1+, a Novel Schizosaccharomyces pombe Gene Important for Microtubule Integrity

Genetics ◽  
1998 ◽  
Vol 149 (3) ◽  
pp. 1251-1264
Author(s):  
Ekaterina L Grishchuk ◽  
James L Howe ◽  
J Richard McIntosh
Keyword(s):  
Schizosaccharomyces Pombe ◽  
Nuclear Division ◽  
Mitotic Division ◽  
Temperature Sensitive ◽  
Permissive Temperature ◽  
Anaphase Promoting Complex ◽  
Chloromethyl Ketone ◽  
Proteolytic Pathway ◽  
Functional Involvement ◽  
Spindle Elongation

Abstract The growth of several mitotic mutants of Schizosaccharomyces pombe, including nuc2-663, is inhibited by the protease inhibitor N-Tosyl-L-Phenylalanine Chloromethyl Ketone (TPCK). Because nuc2+ encodes a presumptive component of the Anaphase Promoting Complex, which is required for the ubiquitin-dependent proteolysis of certain proteins during exit from mitosis, we have used sensitivity to TPCK as a criterion by which to search for novel S. pombe mutants defective in the anaphase-promoting pathway. In a genetic screen for temperature-sensitive mitotic mutants that were also sensitive to TPCK at a permissive temperature, we isolated three tsm (TPCK-sensitive mitotic) strains. Two of these are alleles of cut1+, but tsm1-512 maps to a novel genetic location. The tsm1-512 mutation leads to delayed nuclear division at restrictive temperatures, apparently as a result of an impaired ability to form a metaphase spindle. After shift of early G2 cells to 36°, tsm1-512 arrests transiently in the second mitotic division and then exits mitosis, as judged by spindle elongation and septation. The chromosomes, however, often fail to segregate properly. Genetic interactions between tsm1-512 and components of the anaphase proteolytic pathway suggest a functional involvement of the Tsm1 protein in this pathway.

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