The Experts below are selected from a list of 360 Experts worldwide ranked by ideXlab platform
Alan M. Lambowitz - One of the best experts on this subject based on the ideXlab platform.
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Gene targeting using randomly inserted group ii introns targetrons recovered from an escherichia coli Gene Disruption library
Nucleic Acids Research, 2005Co-Authors: Jun Yao, Jin Zhong, Alan M. LambowitzAbstract:The Lactococcus lactis Ll.LtrB group II intron retrohomes by reverse-splicing into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it to prime reverse transcription of the inserted intron RNA. The protein and intron RNA function in a ribonucleoprotein particle, with much of the DNA target sequence recognized by base-pairing of the intron RNA. Consequently, group II introns can be reprogrammed to insert into specific or random DNA sites by substituting specific or random nucleotide residues in the intron RNA. Here, we show that an Escherichia coli Gene Disruption library obtained using such randomly inserting Ll.LtrB introns contains most viable E.coli Gene Disruptions. Further, each inserted intron is targeted to a specific site by its unique base-pairing regions, and in most cases, could be recovered by PCR and used unmodified to obtain the desired single disruptant. Additionally, we identified a subset of introns that insert at sites lacking T+5, a nucleotide residue critical for second-strand cleavage. All such introns tested individually gave the desired specific Disruption, some by switching to an alternate retrohoming mechanism targeting single-stranded DNA and using a nascent lagging DNA strand to prime reverse transcription.
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targeted and random bacterial Gene Disruption using a group ii intron targetron vector containing a retrotransposition activated selectable marker
Nucleic Acids Research, 2003Co-Authors: Jin Zhong, Michael Karberg, Alan M. LambowitzAbstract:Mobile group II introns have been used to develop a novel class of Gene targeting vectors, targetrons, which employ base pairing for DNA target recognition and can thus be programmed to insert into any desired target DNA. Here, we have developed a targetron containing a retrotransposition-activated selectable marker (RAM), which enables one-step bacterial Gene Disruption at near 100% efficiency after selection. The targetron can be Generated via PCR without cloning, and after intron integration, the marker Gene can be excised by recombination between flanking Flp recombinase sites, enabling multiple sequential Disruptions. We also show that a RAM-targetron with randomized target site recognition sequences yields single insertions throughout the Escherichia coli genome, creating a Gene knockout library. Analysis of the randomly selected insertion sites provides further insight into group II intron target site recognition rules. It also suggests that a subset of retrohoming events may occur by using a primer Generated during DNA replication, and reveals a previously unsuspected bias for group II intron insertion near the chromosome replication origin. This insertional bias likely reflects at least in part the higher copy number of origin proximal Genes, but interaction with the replication machinery or other features of DNA structure or packaging may also contribute.
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targeted and random bacterial Gene Disruption using a group ii intron targetron vector containing a retrotransposition activated selectable marker
Nucleic Acids Research, 2003Co-Authors: Jin Zhong, Michael Karberg, Alan M. LambowitzAbstract:Mobile group II introns have been used to develop a novel class of Gene targeting vectors, targetrons, which employ base pairing for DNA target recognition and can thus be programmed to insert into any desired target DNA. Here, we have developed a targetron containing a retrotransposition-activated selectable marker (RAM), which enables one-step bacterial Gene Disruption at near 100% efficiency after selection. The targetron can be Generated via PCR without cloning, and after intron integration, the marker Gene can be excised by recombination between flanking Flp recombinase sites, enabling multiple sequential Disruptions. We also show that a RAM-targetron with randomized target site recognition sequences yields single insertions throughout the Escherichia coli genome, creating a Gene knockout library. Analysis of the randomly selected insertion sites provides further insight into group II intron target site recognition rules. It also suggests that a subset of retrohoming events may occur by using a primer Generated during DNA replication, and reveals a previously unsuspected bias for group II intron insertion near the chromosome replication origin. This insertional bias likely reflects at least in part the higher copy number of origin proximal Genes, but interaction with the replication machinery or other features of DNA structure or packaging may also contribute.
Edward J Rebar - One of the best experts on this subject based on the ideXlab platform.
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Site-specific genome editing in Plasmodium falciparum using engineered zinc-finger nucleases.
Nature methods, 2012Co-Authors: Judith Straimer, Philip D Gregory, Edward J Rebar, Lei Zhang, Marcus C. S. Lee, Andrew H. Lee, Bryan Zeitler, April E. Williams, Jocelynn R. Pearl, Manuel LlinásAbstract:This paper reports Genetic manipulation of the malaria parasite Plasmodium falciparum with zinc-finger nucleases. It demonstrates Gene Disruption as well as replacement and site-specific editing of both an integrated reporter and an endogenous Gene.
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heritable targeted Gene Disruption in zebrafish using designed zinc finger nucleases
Nature Biotechnology, 2008Co-Authors: Yannick Doyon, Jeffrey C Miller, Jasmine M Mccammon, Farhoud Faraji, Catherine Ngo, George E Katibah, Rainier Amora, Toby Dylan Hocking, Lei Zhang, Edward J RebarAbstract:We describe the use of zinc-finger nucleases (ZFNs) for somatic and germline Disruption of Genes in zebrafish (Danio rerio), in which targeted mutaGenesis was previously intractable. ZFNs induce a targeted double-strand break in the genome that is repaired to Generate small insertions and deletions. We designed ZFNs targeting the zebrafish golden and no tail/Brachyury (ntl) Genes and developed a budding yeast-based assay to identify the most active ZFNs for use in vivo. Injection of ZFN-encoding mRNA into one-cell embryos yielded a high percentage of animals carrying distinct mutations at the ZFN-specified position and exhibiting expected loss-of-function phenotypes. Over half the ZFN mRNA-injected founder animals transmitted disrupted ntl alleles at frequencies averaging 20%. The frequency and precision of Gene-Disruption events observed suggest that this approach should be applicable to any loci in zebrafish or in other organisms that allow mRNA delivery into the fertilized egg.
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targeted Gene knockout in mammalian cells by using engineered zinc finger nucleases
Proceedings of the National Academy of Sciences of the United States of America, 2008Co-Authors: Yolanda Santiago, D. Y. Guschin, Michael C Holmes, Edmond Chan, Salvatore Orlando, Lin Zhang, Fyodor D Urnov, Adam James Waite, Jeffrey C Miller, Edward J RebarAbstract:Gene knockout is the most powerful tool for determining Gene function or permanently modifying the phenotypic characteristics of a cell. Existing methods for Gene Disruption are limited by their efficiency, time to completion, and/or the potential for confounding off-target effects. Here, we demonstrate a rapid single-step approach to targeted Gene knockout in mammalian cells, using engineered zinc-finger nucleases (ZFNs). ZFNs can be designed to target a chosen locus with high specificity. Upon transient expression of these nucleases the target Gene is first cleaved by the ZFNs and then repaired by a natural—but imperfect—DNA repair process, nonhomologous end joining. This often results in the Generation of mutant (null) alleles. As proof of concept for this approach we designed ZFNs to target the dihydrofolate reductase (DHFR) Gene in a Chinese hamster ovary (CHO) cell line. We observed biallelic Gene Disruption at frequencies >1%, thus obviating the need for selection markers. Three new Genetically distinct DHFR−/− cell lines were Generated. Each new line exhibited growth and functional properties consistent with the specific knockout of the DHFR Gene. Importantly, target Gene Disruption is complete within 2–3 days of transient ZFN delivery, thus enabling the isolation of the resultant DHFR−/− cell lines within 1 month. These data demonstrate further the utility of ZFNs for rapid mammalian cell line engineering and establish a new method for Gene knockout with application to reverse Genetics, functional genomics, drug discovery, and therapeutic recombinant protein production.
Jin Zhong - One of the best experts on this subject based on the ideXlab platform.
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Gene targeting using randomly inserted group ii introns targetrons recovered from an escherichia coli Gene Disruption library
Nucleic Acids Research, 2005Co-Authors: Jun Yao, Jin Zhong, Alan M. LambowitzAbstract:The Lactococcus lactis Ll.LtrB group II intron retrohomes by reverse-splicing into one strand of a double-stranded DNA target site, while the intron-encoded protein cleaves the opposite strand and uses it to prime reverse transcription of the inserted intron RNA. The protein and intron RNA function in a ribonucleoprotein particle, with much of the DNA target sequence recognized by base-pairing of the intron RNA. Consequently, group II introns can be reprogrammed to insert into specific or random DNA sites by substituting specific or random nucleotide residues in the intron RNA. Here, we show that an Escherichia coli Gene Disruption library obtained using such randomly inserting Ll.LtrB introns contains most viable E.coli Gene Disruptions. Further, each inserted intron is targeted to a specific site by its unique base-pairing regions, and in most cases, could be recovered by PCR and used unmodified to obtain the desired single disruptant. Additionally, we identified a subset of introns that insert at sites lacking T+5, a nucleotide residue critical for second-strand cleavage. All such introns tested individually gave the desired specific Disruption, some by switching to an alternate retrohoming mechanism targeting single-stranded DNA and using a nascent lagging DNA strand to prime reverse transcription.
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targeted and random bacterial Gene Disruption using a group ii intron targetron vector containing a retrotransposition activated selectable marker
Nucleic Acids Research, 2003Co-Authors: Jin Zhong, Michael Karberg, Alan M. LambowitzAbstract:Mobile group II introns have been used to develop a novel class of Gene targeting vectors, targetrons, which employ base pairing for DNA target recognition and can thus be programmed to insert into any desired target DNA. Here, we have developed a targetron containing a retrotransposition-activated selectable marker (RAM), which enables one-step bacterial Gene Disruption at near 100% efficiency after selection. The targetron can be Generated via PCR without cloning, and after intron integration, the marker Gene can be excised by recombination between flanking Flp recombinase sites, enabling multiple sequential Disruptions. We also show that a RAM-targetron with randomized target site recognition sequences yields single insertions throughout the Escherichia coli genome, creating a Gene knockout library. Analysis of the randomly selected insertion sites provides further insight into group II intron target site recognition rules. It also suggests that a subset of retrohoming events may occur by using a primer Generated during DNA replication, and reveals a previously unsuspected bias for group II intron insertion near the chromosome replication origin. This insertional bias likely reflects at least in part the higher copy number of origin proximal Genes, but interaction with the replication machinery or other features of DNA structure or packaging may also contribute.
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targeted and random bacterial Gene Disruption using a group ii intron targetron vector containing a retrotransposition activated selectable marker
Nucleic Acids Research, 2003Co-Authors: Jin Zhong, Michael Karberg, Alan M. LambowitzAbstract:Mobile group II introns have been used to develop a novel class of Gene targeting vectors, targetrons, which employ base pairing for DNA target recognition and can thus be programmed to insert into any desired target DNA. Here, we have developed a targetron containing a retrotransposition-activated selectable marker (RAM), which enables one-step bacterial Gene Disruption at near 100% efficiency after selection. The targetron can be Generated via PCR without cloning, and after intron integration, the marker Gene can be excised by recombination between flanking Flp recombinase sites, enabling multiple sequential Disruptions. We also show that a RAM-targetron with randomized target site recognition sequences yields single insertions throughout the Escherichia coli genome, creating a Gene knockout library. Analysis of the randomly selected insertion sites provides further insight into group II intron target site recognition rules. It also suggests that a subset of retrohoming events may occur by using a primer Generated during DNA replication, and reveals a previously unsuspected bias for group II intron insertion near the chromosome replication origin. This insertional bias likely reflects at least in part the higher copy number of origin proximal Genes, but interaction with the replication machinery or other features of DNA structure or packaging may also contribute.
Marcel Wuthrich - One of the best experts on this subject based on the ideXlab platform.
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crispr cas9 mediated Gene Disruption reveals the importance of zinc metabolism for fitness of the dimorphic fungal pathogen blastomyces dermatitidis
Mbio, 2018Co-Authors: Gregory C Kujoth, Richard M Merkhofer, Taekjin Lee, Huafeng Wang, Tristan T Brandhorst, Marcel WuthrichAbstract:Blastomyces dermatitidis is a human fungal pathogen of the lung that can lead to disseminated disease in healthy and immunocompromised individuals. Genetic analysis of this fungus is hampered by the relative inefficiency of traditional recombination-based Gene-targeting approaches. Here, we demonstrate the feasibility of applying CRISPR/Cas9-mediated Gene editing to Blastomyces, including to simultaneously target multiple Genes. We created targeting plasmid vectors expressing Cas9 and either one or two single guide RNAs and introduced these plasmids into Blastomyces via Agrobacterium Gene transfer. We succeeded in disrupting several fungal Genes, including PRA1 and ZRT1, which are involved in scavenging and uptake of zinc from the extracellular environment. Single-Gene-targeting efficiencies varied by locus (median, 60% across four loci) but were approximately 100-fold greater than traditional methods of Blastomyces Gene Disruption. Simultaneous dual-Gene targeting proceeded with efficiencies similar to those of single-Gene-targeting frequencies for the respective targets. CRISPR/Cas9 Disruption of PRA1 or ZRT1 had a variable impact on growth under zinc-limiting conditions, showing reduced growth at early time points in low-passage-number cultures and growth similar to wild-type levels by later passage. Individual impairment of PRA1 or ZRT1 resulted in a reduction of the fungal burden in a mouse model of Blastomyces infection by a factor of ~1 log (range, up to 3 logs), and combined Disruption of both Genes had no additional impact on the fungal burden. These results underscore the utility of CRISPR/Cas9 for efficient Gene Disruption in dimorphic fungi and reveal a role for zinc metabolism in Blastomyces fitness in vivoIMPORTANCEBlastomyces is a human fungal pathogen that can cause serious, even fatal, lung infections. Genetic analysis of this fungus is possible but inefficient. We applied a recently developed Gene editing technology, CRISPR/Cas9, to dramatically improve the efficiency with which Gene Disruptions are introduced into Blastomyces We used this system to disrupt Genes involved in zinc uptake and found that this reduced the fitness of the fungus upon infection.
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CRISPR/Cas9-Mediated Gene Disruption Reveals the Importance of Zinc Metabolism for Fitness of the Dimorphic Fungal Pathogen Blastomyces dermatitidis
American Society for Microbiology, 2018Co-Authors: Gregory C Kujoth, Richard M Merkhofer, Taekjin Lee, Huafeng Wang, Marcel Wuthrich, Tristan Brandhorst, Joseph HeitmanAbstract:Blastomyces dermatitidis is a human fungal pathogen of the lung that can lead to disseminated disease in healthy and immunocompromised individuals. Genetic analysis of this fungus is hampered by the relative inefficiency of traditional recombination-based Gene-targeting approaches. Here, we demonstrate the feasibility of applying CRISPR/Cas9-mediated Gene editing to Blastomyces, including to simultaneously target multiple Genes. We created targeting plasmid vectors expressing Cas9 and either one or two single guide RNAs and introduced these plasmids into Blastomyces via Agrobacterium Gene transfer. We succeeded in disrupting several fungal Genes, including PRA1 and ZRT1, which are involved in scavenging and uptake of zinc from the extracellular environment. Single-Gene-targeting efficiencies varied by locus (median, 60% across four loci) but were approximately 100-fold greater than traditional methods of Blastomyces Gene Disruption. Simultaneous dual-Gene targeting proceeded with efficiencies similar to those of single-Gene-targeting frequencies for the respective targets. CRISPR/Cas9 Disruption of PRA1 or ZRT1 had a variable impact on growth under zinc-limiting conditions, showing reduced growth at early time points in low-passage-number cultures and growth similar to wild-type levels by later passage. Individual impairment of PRA1 or ZRT1 resulted in a reduction of the fungal burden in a mouse model of Blastomyces infection by a factor of ~1 log (range, up to 3 logs), and combined Disruption of both Genes had no additional impact on the fungal burden. These results underscore the utility of CRISPR/Cas9 for efficient Gene Disruption in dimorphic fungi and reveal a role for zinc metabolism in Blastomyces fitness in vivo
Jacob E Corn - One of the best experts on this subject based on the ideXlab platform.
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non homologous dna increases Gene Disruption efficiency by altering dna repair outcomes
Nature Communications, 2016Co-Authors: Christopher D Richardson, Graham J Ray, Nicolas Bray, Jacob E CornAbstract:The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9–sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting Genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated Gene Disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous Gene Disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced Gene Disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of Gene Disruption. CRISPR-Cas9 mediated Gene editing has begun to revolutionize molecular biology, but editing efficiencies can vary greatly between reagents. The authors show that the addition of single-stranded non-homologous DNA stimulates Gene Disruption by favouring error-prone DNA repair.
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non homologous dna increases Gene Disruption efficiency by altering dna repair outcomes
Nature Communications, 2016Co-Authors: Christopher D Richardson, Graham J Ray, Nicolas Bray, Jacob E CornAbstract:The Cas9 endonuclease can be targeted to genomic sequences by programming the sequence of an associated single guide RNA (sgRNA). For unknown reasons, the activity of these Cas9-sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting Genes in polyploid cell lines or when using poorly performing sgRNAs can require extensive downstream screening to identify homozygous clones. Here we find that non-homologous single-stranded DNA greatly stimulates Cas9-mediated Gene Disruption in the absence of homology-directed repair. This stimulation increases the frequency of clones with homozygous Gene Disruptions and rescues otherwise ineffective sgRNAs. The molecular outcome of enhanced Gene Disruption depends upon cellular context, stimulating deletion of genomic sequence or insertion of non-homologous DNA at the edited locus in a cell line specific manner. Non-homologous DNA appears to divert cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of Gene Disruption.
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non homologous dna increases Gene Disruption efficiency by altering dna repair outcomes
bioRxiv, 2016Co-Authors: Christopher D Richardson, Graham J Ray, Jacob E CornAbstract:Cas9 endonuclease can be targeted to genomic sequences by varying the sequence of the single guide RNA (sgRNA). The activity of these Cas9-sgRNA combinations varies widely at different genomic loci and in different cell types. Thus, disrupting Genes in polyploid cell lines, or using inefficient sgRNAs, can require extensive downstream screening to identify homozygous clones. We have found that linear, non-homologous oligonucleotide DNA greatly stimulates Cas9-mediated Gene Disruption in the absence of homology-directed repair. This stimulation greatly increases the frequency of clones with homozygous Gene Disruptions, even in polyploid cell lines, and rescues otherwise ineffective sgRNAs. The mechanism of enhanced Gene Disruption differs between human cell lines, stimulating deletion of genomic sequence and/or insertion of non-homologous oligonucleotide DNA at the edited locus in a cell line specific manner. Thus, the addition of non-homologous DNA appears to drive cells towards error-prone instead of error-free repair pathways, dramatically increasing the frequency of Gene Disruption.