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 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 Programmed genome editing of the omega-1 ribonuclease 1 of the blood fluke,Schistosoma mansoni

2018 ◽  
Author(s):  
Wannaporn Ittiprasert ◽  
Victoria H. Mann ◽  
Shannon E. Karinshak ◽  
Avril Coghlan ◽  
Gabriel Rinaldi ◽  
...  
Keyword(s):  
Schistosoma Mansoni ◽  
Genome Editing ◽  
Human Macrophage ◽  
Wild Type ◽  
End Joining ◽  
Chromosomal Breakage ◽  
Knock Out ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Non Homologous End Joining

AbstractCRISPR/Cas9 based genome editing has yet been reported in parasitic or indeed any species of the phylum Platyhelminthes. We tested this approach by targeting omega-1 (ω1) ofSchistosoma mansonias a proof of principle. This secreted ribonuclease is crucial for Th2 priming and granuloma formation, providing informative immuno-pathological readouts for programmed genome editing. Schistosome eggs were either exposed to Cas9 complexed with a synthetic guide RNA (sgRNA) complementary to exon 6 of ω1 by electroporation or transduced with pseudotyped lentivirus encoding Cas9 and the sgRNA. Some eggs were also transduced with a single stranded oligodeoxynucleotide donor transgene that encoded six stop codons, flanked by 50 nt-long 5’-and 3’-microhomology arms matching the predicted Cas9-catalyzed double stranded break (DSB) within ω1. CRISPResso analysis of amplicons spanning the DSB revealed ∼4.5% of the reads were mutated by insertions, deletions and/or substitutions, with an efficiency for homology directed repair of 0.19% insertion of the donor transgene. Transcripts encoding ω1 were reduced >80% and lysates of ω1-edited eggs displayed diminished ribonuclease activity indicative that programmed editing mutated the ω1 gene. Whereas lysates of wild type eggs polarized Th2 cytokine responses including IL-4 and IL-5 in human macrophage/T cell co-cultures, diminished levels of the cytokines followed the exposure to lysates of ω1-mutated schistosome eggs. Following injection of schistosome eggs into the tail vein of mice, the volume of pulmonary granulomas surrounding ω1-mutated eggs was 18-fold smaller than wild type eggs. Programmed genome editing was active in schistosomes, Cas9-catalyzed chromosomal breakage was repaired by homology directed repair and/or non-homologous end joining, and mutation of ω1 impeded the capacity of schistosome eggs both to drive Th2 polarization and to provoke formation of pulmonary circumoval granulomas. Knock-out of ω1 and the impaired immunological phenotype showcase the novel application of programmed gene editing in and functional genomics for schistosomes.

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 Scalable tagging of endogenous genes by homology-independent intron targeting

2018 ◽  
Author(s):  
Yevgeniy V Serebrenik ◽  
Stephanie E Sansbury ◽  
Saranya Santhosh Kumar ◽  
Jorge Henao-Mejia ◽  
Ophir Shalem
Keyword(s):  
Genome Editing ◽  
Gene Tagging ◽  
Gene Fusions ◽  
Homology Directed Repair ◽  
Efficient Gene ◽  
Endogenous Genes ◽  
Sgrna Design

AbstractGenome editing tools have simplified the generation of knock-in gene fusions, yet the requirement for gene-specific homology directed repair (HDR) templates still hinders the scalability of most approaches. Here, we combine intron-based protein trapping with homology independent repair-based editing and demonstrate precise and efficient gene tagging that can be easily scaled due to use of a generic donor. As editing is done in introns, this approach tolerates mutations in the unedited allele, disruptive indels, and allows for flexible donor and sgRNA design.

scholarly journals IMPROVING THE FUNCTION OF CRISPR-CAS9 FOR GENOME EDITING THERAPY: EDITING THE EDITOR

2017 ◽  
Vol 4 (1) ◽  
pp. 44
Author(s):  
Alva Sahiri Alexander Supit
Keyword(s):  
Genome Editing ◽  
Viral Vectors ◽  
Gene Editing ◽  
Genetic Disorders ◽  
Genetic Diseases ◽  
End Joining ◽  
Guide Rna ◽  
Homology Directed Repair ◽  
Short Palindromic Repeat ◽  
Active Research

Meningkatkan Fungsi CRISPR-Cas9 untuk Terapi Pengeditan GenPengeditan gen menjadi mudah dilakukan sejak ditemukannya clustered regularly interspaced short palindromic repeat (CRISPR) dan CRISPR-associated protein 9 (Cas9) sebagai alat untuk menyunting gen suatu organisme. Sebagian besar penyakit genetik tidak dapat disembuhkan secara kausal dengan terapi yang ada, maka pengeditan gen merupakan suatu cara yang prospektif dalam terapi medis di masa depan. Sayangnya, pengeditan gen dengan Cas9 yang ada saat ini masih memiliki banyak kelemahan, yaitu: 1) kurang spesifik, di mana RNA pemandu dapat berikatan dengan beberapa segmen pada genom manusia, sehingga memungkinkan terjadinya salah target; 2) kurang efisien, karena sekalipun telah berhasil memotong utas ganda DNA, kebanyakan penyambungan kembali akan dilakukan secara non-homology end joining (NHEJ), yang justru meningkatkan peluang terjadinya mutasi; 3) sulit disalurkan ke dalam inti sel karena berbagai sawar fisiologis maupun biokimiawi. Tulisan ini akan membahas perkembangan terkini dalam mengatasi ketiga masalah di atas. Untuk meningkatkan spesifisitas, dapat dilakukan modifikasi RNA pemandu dan struktur Cas9. Efisiensi dapat ditingkatkan dengan meningkatkan peluang terjadinya homology-directed repair dibandingkan NHEJ, sedangkan untuk meningkatkan distribusi ke dalam sel, dapat digunakan berbagai macam vektor, seperti virus dan nanopartikel. CRISPR-Cas9 merupakan area yang aktif diteliti dalam bidang biosains, dan dalam waktu dekat, diharapkan dapat dimanfaatkan dalam bidang klinik.Kata kunci: CRISPR, Cas9, efektivitas, spesifisitas, terapi genABSTRACTGene editing has become reasonably easy since the discovery of clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated protein 9 (Cas9). Most genetic diseases cannot be treated causally, and currently available therapies are mainly symptom-based. To treat the etiology of genetic diseases, a firm gene editing therapy is necessary to be established. This posits Cas9-facilitated gene editing as a prospective modality to become a clinically approved therapy in the future to treat genetic disorders. However, until recently, Cas9-based genome editing is still facing several hurdles, including low specificity, low effectiveness, and difficult delivery. Currently available Cas9 nucleases are able to bind to non-specific DNA sequence and produce non-specific cleavage. The efficiency has been relatively low due to the preference of non-homologous end-joining (NHEJ) over homology-directed repair (HDR) by the host cell. Furthermore, in order to deliver Cas9 into the nucleus, multiple physiological barriers have to be overcome. This review discussed recent developments in tackling these three hurdles, ranging from designing the guide RNA using multiple bioinformatics tools, modifying Cas9 structure, as well as packaging the nuclease-guide RNA complex into viral vectors and nanoparticles. Considering the active research on this area, it is expected that CRISPR/Cas9 can be utilized as a clinical therapy in the near future.Received: 02 June 2017        Accepted: 07 July 2017        Published: 19 July 2017

scholarly journals Tailor-made exopolysaccharides—CRISPR-Cas9 mediated genome editing in Paenibacillus polymyxa

2017 ◽  
Vol 2 (1) ◽  
Author(s):  
Marius Rütering ◽  
Brady F Cress ◽  
Martin Schilling ◽  
Broder Rühmann ◽  
Mattheos A G Koffas ◽  
...  
Keyword(s):  
Genome Editing ◽  
Gene Deletion ◽  
State Of The Art ◽  
Paenibacillus Polymyxa ◽  
Vector System ◽  
Wild Type ◽  
Monomer Composition ◽  
Homology Directed Repair ◽  
Highly Efficient ◽  
Micro Organisms

Abstract Application of state-of-the-art genome editing tools like CRISPR-Cas9 drastically increase the number of undomesticated micro-organisms amenable to highly efficient and rapid genetic engineering. Adaptation of these tools to new bacterial families can open up entirely new possibilities for these organisms to accelerate as biotechnologically relevant microbial factories, also making new products economically competitive. Here, we report the implementation of a CRISPR-Cas9 based vector system in Paenibacillus polymyxa, enabling fast and reliable genome editing in this host. Homology directed repair allows for highly efficient deletions of single genes and large regions as well as insertions. We used the system to investigate the yet undescribed biosynthesis machinery for exopolysaccharide (EPS) production in P. polymyxa DSM 365, enabling assignment of putative roles to several genes involved in EPS biosynthesis. Using this simple gene deletion strategy, we generated EPS variants that differ from the wild-type polymer not only in terms of monomer composition, but also in terms of their rheological behavior. The developed CRISPR-Cas9 mediated engineering approach will significantly contribute to the understanding and utilization of socially and economically relevant Paenibacillus species and extend the polymer portfolio.

scholarly journals First genome edited poinsettias: targeted mutagenesis of flavonoid 3′-hydroxylase using CRISPR/Cas9 results in a colour shift

2021 ◽  
Author(s):  
Daria Nitarska ◽  
Robert Boehm ◽  
Thomas Debener ◽  
Rares Calin Lucaciu ◽  
Heidi Halbwirth
Keyword(s):  
Transgenic Plants ◽  
Genome Editing ◽  
Ornamental Plants ◽  
Targeted Mutagenesis ◽  
Cloning And Expression ◽  
Euphorbia Pulcherrima ◽  
Loss Of Function ◽  
Wild Type ◽  
Promising Tool ◽  
Reddish Orange

AbstractThe CRISPR/Cas9 system is a remarkably promising tool for targeted gene mutagenesis, and becoming ever more popular for modification of ornamental plants. In this study we performed the knockout of flavonoid 3′-hydroxylase (F3′H) with application of CRISPR/Cas9 in the red flowering poinsettia (Euphorbia pulcherrima) cultivar ‘Christmas Eve’, in order to obtain plants with orange bract colour, which accumulate prevalently pelargonidin. F3′H is an enzyme that is necessary for formation of cyanidin type anthocyanins, which are responsible for the red colour of poinsettia bracts. Even though F3′H was not completely inactivated, the bract colour of transgenic plants changed from vivid red (RHS 45B) to vivid reddish orange (RHS 33A), and cyanidin levels decreased significantly compared with the wild type. In the genetically modified plants, an increased ratio of pelargonidin to cyanidin was observed. By cloning and expression of mutated proteins, the lack of F3′H activity was confirmed. This confirms that a loss of function mutation in the poinsettia F3′H gene is sufficient for obtaining poinsettia with orange bract colour. This is the first report of successful use of CRISPR/Cas9 for genome editing in poinsettia.

scholarly journals Role of SpoVA Proteins in Release of Dipicolinic Acid during Germination of Bacillus subtilis Spores Triggered by Dodecylamine or Lysozyme

2006 ◽  
Vol 189 (5) ◽  
pp. 1565-1572 ◽  
Author(s):  
Venkata Ramana Vepachedu ◽  
Peter Setlow
Keyword(s):  
Bacillus Subtilis ◽  
Small Molecules ◽  
Spore Germination ◽  
Dipicolinic Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Ph Optimum ◽  
Inner Membrane ◽  
Temperature Sensitive Mutants

ABSTRACT The release of dipicolinic acid (DPA) during the germination of Bacillus subtilis spores by the cationic surfactant dodecylamine exhibited a pH optimum of ∼9 and a temperature optimum of 60°C. DPA release during dodecylamine germination of B. subtilis spores with fourfold-elevated levels of the SpoVA proteins that have been suggested to be involved in the release of DPA during nutrient germination was about fourfold faster than DPA release during dodecylamine germination of wild-type spores and was inhibited by HgCl2. Spores carrying temperature-sensitive mutants in the spoVA operon were also temperature sensitive in DPA release during dodecylamine germination as well as in lysozyme germination of decoated spores. In addition to DPA, dodecylamine triggered the release of amounts of Ca2+ almost equivalent to those of DPA, and at least one other abundant spore small molecule, glutamic acid, was released in parallel with Ca2+ and DPA. These data indicate that (i) dodecylamine triggers spore germination by opening a channel in the inner membrane for Ca2+-DPA and other small molecules, (ii) this channel is composed at least in part of proteins, and (iii) SpoVA proteins are involved in the release of Ca2+-DPA and other small molecules during spore germination, perhaps by being a part of a channel in the spore's inner membrane.

scholarly journals Lethal Action of Quinolones against a Temperature-Sensitive dnaB Replication Mutant of Escherichia coli

2006 ◽  
Vol 50 (1) ◽  
pp. 362-364 ◽  
Author(s):  
Xilin Zhao ◽  
Muhammad Malik ◽  
Nymph Chan ◽  
Alex Drlica-Wagner ◽  
Jian-Ying Wang ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Protein Synthesis ◽  
Dna Replication ◽  
Nalidixic Acid ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Lethal Action ◽  
Inhibition Of Protein Synthesis

ABSTRACT Inhibition of DNA replication in an Escherichia coli dnaB-22 mutant failed to block quinolone-mediated lethality. Inhibition of protein synthesis by chloramphenicol inhibited nalidixic acid lethality and, to a lesser extent, ciprofloxacin lethality in both dnaB-22 and wild-type cells. Thus, major features of quinolone-mediated lethality do not depend on ongoing replication.

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