RALEIGH, N.C. -- CRISPR-Cas systems are widely heralded as a new generation of genetic tools. But development of these tools requires researchers to identify the protospacer-adjacent motifs (PAMs) that unlock each systems functionality. A new set of techniques expedites PAM identification - and early testing finds that many CRISPR-Cas systems actually have multiple PAMs of varying strength. CRISPR-Cas systems protect bacteria from invaders such as viruses. They do this by creating small strands of RNA that match DNA sequences specific to a given invader. When those CRISPR RNAs find a match, they unleash proteins that chop up the invaders DNA, preventing it from replicating. However, the first step in the process isnt comparing the RNA to target DNA. The first step involves PAM recognition and binding ...

CRISPR-Cas systems are adaptive immune systems in bacteria and archaea, consisting of a clustered regularly interspaced short palindromic repeats (CRISPR) array and CRISPR associated (Cas) proteins. In this work, the type I-E CRISPR-Cas system of Escherichia coli was studied.. CRISPR-Cas immunity is divided into three stages. In the first stage, adaptation, Cas1 and Cas2 store memory of invaders in the CRISPR array as short intervening sequences, called spacers. During the expression stage, the array is transcribed, and subsequently processed into small CRISPR RNAs (crRNA), each consisting of one spacer and one repeat. The crRNAs are bound by the Cascade multi-protein complex. During the interference step, Cascade searches for DNA molecules complementary to the crRNA spacer. When a match is found, the target DNA is degraded by the recruited Cas3 nuclease.. Host factors required for integration of new spacers into the CRISPR array were first investigated. Deleting recD, involved in DNA repair, ...

CRISPR-Cas systems are common in prokaryotes and can provide small RNA-based adaptive immunity against mobile genetic elements. A CRISPR-Cas system consists of DNA loci with Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and CRISPR ass

Salmonella is recognized as one of the most common microbial pathogens worldwide. The bacterium contains the clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas) systems, providing adaptive immunity against invading foreign nucleic acids. Previous studies suggested that certain bacteria employ the Cas proteins of CRISPR-Cas systems to target their own genes, which also alters the virulence during invasion of mammals. However, whether CRISPR-Cas systems in Salmonella have similar functions during bacterial invasion of host cells remains unknown. Here, we systematically analyzed the genes that are regulated by Cas3 in a type I-E CRISPR-Cas system and the virulence changes due to the deletion of cas3 in Salmonella enterica serovar Enteritidis. Compared to the cas3 gene wild-type (cas3 WT) Salmonella strain, cas3 deletion upregulated the lsrFGBE genes in lsr (luxS regulated) operon related to quorum sensing (QS) and downregulated biofilm-forming-related genes and

CRISPR-Cas systems endow bacterial and archaeal species with adaptive immunity mechanisms to fend off invading phages and foreign genetic elements. CRISPR-Cas9 has been harnessed to confer virus interference against DNA viruses in eukaryotes, including plants. In addition, CRISPR-Cas13 systems have been used to target RNA viruses and the transcriptome in mammalian and plant cells. Recently, CRISPR-Cas13a has been shown to confer modest interference against RNA viruses. Here, we characterized a set of different Cas13 variants to identify those with the most efficient, robust, and specific interference activities against RNA viruses in planta using Nicotiana benthamiana. Our data show that LwaCas13a, PspCas13b, and CasRx variants mediate high interference activities against RNA viruses in transient assays. Moreover, CasRx mediated robust interference in both transient and stable overexpression assays when compared to the other variants tested. CasRx targets either one virus alone or two RNA viruses

Heritable and Precise Zebrafish Genome Editing Using a CRISPR-Cas System. . Biblioteca virtual para leer y descargar libros, documentos, trabajos y tesis universitarias en PDF. Material universiario, documentación y tareas realizadas por universitarios en nuestra biblioteca. Para descargar gratis y para leer online.

As CRISPR-Cas9 system is emerging as a versatile tool in genome editing it is necessary to know the complete landscape of this system. The authors in this review tried to give a perspective of CRISPR-Cas9 along with RNAi. To explain the whole phenomenon they chose several comparable aspects between CRISPR-Cas9 and RNAi with respect to efficiency, off target effects, genome screening tools, in vivo studies and molecular consequences. As off targets are a major problem in genome editing technologies the authors covered certain developments such as introducing mutations in sgRNAs, delivery of multiple sgRNAs for a single target etc. In addition to it various screening strategies, which are used in RNAi have also been explained.. CRISPR-Cas9 system does not interfere with the endogenous machinery of cell as it is edited at the level of DNA within the nucleus; sometimes it is a major problem with siRNAs or shRNAs, which may lead to cell death (Doudna et al, 2014).. ...

The clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9 (CRISPR-Cas9) system is considered as a technological revolution in targeted mutagenesis. However, a large amount of time and cost is needed to screen for the CRISPR-Cas9 induced mutants from a usual large number of initial samples. Thus, Chun Wang and Kejian Wang from the Chinese Academy of Agricultural Sciences presented a cost-effective and sensitive screening technique for identifying mutants based on conventional polymerase chain reaction (PCR). They called this new technique as annealing at critical temperature PCR (ACT-PCR). ACT-PCR needs only one PCR step and then execution of agarose gel electrophoresis. Because of its simplicity, ACT-PCR is suitable for rapid, large-scale screening of CRISPR-Cas9-induced mutants. Read more description about ACT-PCR in Plant Genome Editing with CRISPR Systems.. ...

In 2013, George Church and his colleagues at Harvard University in Cambridge, Massachusetts published RNA-Guided Human Genome Engineering via Cas 9, in which they detailed their use of RNA-guided Cas 9 to genetically modify genes in human cells. Researchers use RNA-guided Cas 9 technology to modify the genetic information of organisms, DNA, by targeting specific sequences of DNA and subsequently replacing those targeted sequences with different DNA sequences. Church and his team used RNA-guided Cas 9 technology to edit the genetic information in human cells.. Format: Articles Subject: Publications ...

Using structural knowledge of Cas9, scientists have overcome a key CRISPR-Cas9 genome editing hurdle and developed a highly specific genome-editing tool.. Researchers at the Broad Institute of MIT and Harvard and the McGovern Institute for Brain Research at MIT have engineered changes to the revolutionary CRISPR-Cas9 genome editing system that significantly cut down on "off-target" editing errors. The refined technique addresses one of the major technical issues in the use of genome editing.. The CRISPR-Cas9 system works by making a precisely targeted modification in a cells DNA. The protein Cas9 alters the DNA at a location that is specified by a short RNA whose sequence matches that of the target site. While Cas9 is known to be highly efficient at cutting its target site, a major drawback of the system has been that, once inside a cell, it can bind to and cut additional sites that are not targeted. This has the potential to produce undesired edits that can alter gene expression or knock a ...

Abstract By Brigette Corder, Sterling Ericsson, and Taylor Uhlir The CRISPR-Cas systems are a new and exciting tool for research and scientific discovery. Here we discuss and compare the various CRISPR-Cas systems and report current uses for these systems by concentrating a principal spotlight on CRISPR-Cas 10. From CRISPRs rudimentary beginnings in the form of […]. ...

Abstract By Brigette Corder, Sterling Ericsson, and Taylor Uhlir The CRISPR-Cas systems are a new and exciting tool for research and scientific discovery. Here we discuss and compare the various CRISPR-Cas systems and report current uses for these systems by concentrating a principal spotlight on CRISPR-Cas 10. From CRISPRs rudimentary beginnings in the form of […]. ...

Bacteria face a constant threat of being infected and killed by viruses, called bacteriophages, that are specially equipped to destroy them. In the Bondy-Denomy lab we are interested in the ways in which bacteria defend themselves from attack. We use a combination of genetic, molecular and biochemical approaches to characterize the arms race between bacteria and phages, with a goal to better understand microbial ecosystems. Furthermore, we hope to make discoveries that will be influential in combatting infectious disease and providing novel biotechnologies.. The CRISPR-Cas system was functionally characterized just ten years ago as a bacterial immune system that targets phages. Since then, there has been an explosion of interest in this system for its widespread presence in the microbial world as well as its facile programmability. This has formed the basis of a revolutionary gene editing technique, CRISPR-Cas9. In the lab, we are focused on studying CRISPR-Cas systems in their natural settings, ...

Are you genome editing with CRISPR-Cas9? Consider the Alt-R CRISPR-Cas9 kit-a customizable, end-to-end Cas9-CRISPR system offering best in class performance.

Functional genomic screening is largely used for identifying the essential genes for a specific cellular process. RNA interference (RNAi) [51] has been dominantly applied for genome-wide screening; however, the off-target effects of RNAi has limited its applications [52-54]. In addition, RNAi could not be used for silencing RNAs located in nucleus. The CRISPR-Cas9 system has been successfully used in various genome-scale loss of function screening [55-58]. Using a genome-scale lentiviral sgRNA library, all expected genes of the DNA mismatch repair pathway have been identified in screening for resistance to the nucleotide analog 6-thioguanine, and numerous genes corresponding to fundamental processes have been obtained with a negative selection screening for essential genes [55]. A genome-scale CRISPR-Cas9 knockout (GeCKO) library has been developed and successfully used for screening genes essential for cell viability in cancer and pluripotent stem cells and for genes associated with the ...

GENETIC and molecular techniques to manipulate the genomes of model organisms are invaluable tools for understanding gene function. In Drosophila, chemical and insertional mutagenesis are powerful and widely utilized methods for disrupting gene function (St Johnston 2002; Venken and Bellen 2005). Imprecise excision of a transposable element inserted near a gene of interest can result in deletion of all or part of the locus. More recently, techniques that stimulate homologous recombination (HR) using an exogenous template have made precisely targeted genome modifications possible (Gloor et al. 1991; Banga and Boyd 1992; Nassif et al. 1994; Rong and Golic 2000; Gong and Golic 2003; Huang et al. 2009). The most widely used methods rely on double-strand breaks (DSBs) in the donor template to trigger HR, but these can be time consuming and labor intensive (Gao et al. 2008; Huang et al. 2009). Zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) have been ...

Just got an announcement about this from a colleague and thought it might be of interest: Science-Corps Providing an opportun... ...

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In addition to defense against foreign DNA, the CRISPR-Cas9 system of Francisella novicida represses expression of an endogenous immunostimulatory lipoprotein. We investigated the specificity and molecular mechanism of this regulation, demonstrating that Cas9 controls a highly specific regulon of four genes that must be repressed for bacterial virulence. Regulation occurs through a protospacer adjacent motif (PAM)-dependent interaction of Cas9 with its endogenous DNA targets, dependent on a non-canonical small RNA (scaRNA) and tracrRNA. The limited complementarity between scaRNA and the endogenous DNA targets precludes cleavage, highlighting the evolution of scaRNA to repress transcription without lethally targeting the chromosome. We show that scaRNA can be reprogrammed to repress other genes, and with engineered, extended complementarity to an exogenous target, the repurposed scaRNA:tracrRNA-FnoCas9 machinery can also direct DNA cleavage. Natural Cas9 transcriptional interference likely ...

https://www.fightaging.org/archives/2016/04/the-actuarial-press-interviews-aubrey-de-grey/#comment-23917 The part relevant to using CRISPR-Cas9 for in vivo gene editing: But your (Barbaras) implicit premise seems to be that the CRISPR/Cas9 system could easily be used for somatic gene therapy - ie, introducing therapeutic genes into existing tissue in situ. And if you didnt mean that, such is certainly the explicit premise of…

CRISPR-CAS9 gene editing complex from Streptococcus pyogenes. The Cas9 nuclease protein uses a guide RNA (ribonucleic acid) sequence to cut DNA (deoxyribonucleic acid) at a complementary site. Cas9 protein: red surface model. DNA fragments: orange ladder. RNA: lime green ladder. - Stock Image F010/6730

Some of our experiments from last year suggested that one could mismatch a few nucleotides at one end of the gRNA complementarity region without affecting the targeting activity," Joung explains. "That led us to wonder whether removing these nucleotides could make the system more sensitive to mismatches in the remaining sequence." Based on a natural system a species of bacteria uses against other pathogens, the CRISPR-Cas RGNs most widely used by researchers includes a 20-nucleotide targeting region within the gRNA. To test their theory, the MGH team constructed RGNs with progressively shorter gRNAs and found that, while gRNAs with targeting segments of 17 or 18 nucleotides were as or more efficient than full-length gRNAs in reaching their targets, those with 15- or 16-nucleotide targeting segments had reduced or no targeting activity. Subsequent experiments found that 17-nucleotide truncated RGNs efficiently induced the desired mutations in human cells with greatly reduced or undetectable ...

The Alt-R|sup>®|/sup> CRISPR-Cas9 System is a complete, RiboNucleoProtein-based solution for efficient knock-in and knock-out genome editing experiments.

A new study from researchers at the University of Edinburgh succeeds in using CRISPR-Cas9 to produce pigs resistant to PRRS infection.

CRISPR-Cas9 gene editing has been around not even 4 years, and people are avidly discussing its promises and perils (see "The Public and the Gene Editing Revolution" in todays New England Journal of Medicine). Thats great. But. ...

It is the latter, the CRISPR-cas9 gene editing technique, which holds perhaps the greatest promise for changing and improving the human condition and the greatest risk for destroying both the fabric of society and the biological basis for life.

The Emmanuelle Charpentier Lab at the Max Planck Institute for Infection Biology focuses on fundamental mechanisms of regulation in pathogens (CRISPR-Cas9)

This issue profiles an innovative method to prove accuracy of CRISPR-Cas9 in human cells, herpes simplex virus molecular test that obtains FDA approval, and DNA therapeutic vaccine for human papillomavirus.

Researchers report discovering for the first time, a CRISPR-Cas9 system in archaea, as well as of simple CRISPR-Cas systems in uncultivable bacteria.

Whole genome sequencing analyses are providing compelling evidence that pro- and eukaryote microbes, like multicellular organisms, have their life threatened by parasitic attacks. Bacteria and Archea face invading viral nucleic acids with an inheritable DNA-encoded immunity (known as the CRISPR-Cas system) that recognizes foreign DNA from self DNA. In eukaryotic microbes that are exposed to invasions from both bacteria and viruses, bacteria are promptly made harmless either by digestion into food vacuoles, or by domestication as symbionts. But the defence from viral attacks is much less effective. Foreign viral sequences can randomly insert into the cell genome, and may disrupt or deactivate vital genes. To fight this threat, ciliates rely on a unique model of inheritable genomic immune mechanism based on the evolution of two genomes, a germ-line one lying in the cell micronucleus and a somatic one lying in the macronucleus. The germ-line genome characterized by an orthodox chromosomic ...

A stitched image of fluorescently-labeled DNAs on the surface of a Next-Generation Sequencing flowcell. Images were collected on a prism-TIRF microscope and used in the Chip Hybridized Affinity Mapping Platform (CHAMP), a newly described framework for high-throughput, quantitative analysis of protein-DNA interactions on synthetic and genomic DNA. With CHAMP, Jung et al. revealed the highly-specific sequence dependence for target binding and nuclease recruitment by Cascade, a Type 1 CRISPR-Cas system.

Dr. Joseph Bondy-Denomy, Faculty Fellow at UCSF, shares his experiences starting as an accounting major to finding out that working in the lab is his true calling to discovering a whole new component of the CRISPR-Cas system, which he named

No, CRISPR-Cas9 complexes do not recognize, or recognize extremely poorly, targets lacking PAM sequences [1].. The PAM sequence recognized by the S. pyogenes CRISPR-Cas9 system is NGG. If this sequence is not present in your target, you may be able to use other CRISPR systems (from other bacterial species) that recognize different PAM sequences. The following table lists examples of PAM sequences:. ...

Prokaryotic and eukaryotic cells Genetic engineering terms the targeted modifications of genetic information of living organisms/cells. Genome editing stands for targeted engineering by using specifically designed nucleases. The recent CRISPR/ Cas-9 method uses RNA-guided nuclease. That makes the process faster, easier and more specific compared to other gene editing methods. Since the DNA-binding element is RNA it is simple and cheap to manipulate and it allows sequence specific programming. The high targeting efficiency, results in significant time saving, e.g., for the generation of knockout mice. In basic research CRISPR is mainly used in cell line engineering and animal genetic engineering (e.g. of disease models) to investigate gene function. ...

Genetic engineering terms the targeted modifications of genetic information of living organisms/cells. Genome editing stands for targeted engineering by using specifically designed nucleases. The recent CRISPR/ Cas-9 method uses RNA-guided nuclease. That makes the process faster, easier and more specific compared to other gene editing methods. Since the DNA-binding element is RNA it is simple and cheap to manipulate and it allows sequence specific programming. The high targeting efficiency, results in significant time saving, e.g., for the generation of knockout mice. In basic research CRISPR is mainly used in cell line engineering and animal genetic engineering (e.g. of disease models) to investigate gene function. ...

Researchers from Skoltech, the Massachusetts Institute of Technology (MIT) and the University of Massachusetts (UMass) have developed novel nanoparticles that can deliver the CRISPR-Cas9 genome editing system and specifically modify genes directly within the liver of a mouse. Researchers were able to remove target genes in approximately 80% of hepatocytes - the best rate ever achieved with CRISPR in adult animals.. CRISPR-Cas9 is a gene editing technology that makes it possible to modify genes within a living being. Researchers in laboratories around the globe are hard at work attempting to develop safe and efficient delivery methods for the CRISPR gene-editing system, which consists of DNA-cutting enzyme Cas9 and a short sgRNA that guides Cas9 to a specific region in the genome.. A joint study, published this week in Nature Biotechnology, describes a new system developed by researchers from Skoltech, MIT and UMass that delivers both Cas9 mRNA and sgRNA using lipid nanoparticles, without any ...

From BioPortfolio: Preliminary results from one of the earliest clinical trials of CRISPR—Cas9 provide evidence that the technique is safe and feasible to use for treati...

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Now two teams of scientists, one led by Timothy Lu of the Massachusetts Institute of Technology and the other by Luciano Marriffini of Rockefeller University, each used the CRISPR-Cas9 system to generate their own version of a prototype technology that turns a bacterias defence mechanism into a self-destructing weapon. The main idea behind their work was to use genetic engineering to rewire the bacterias immunity to produce "boomerang" antibiotic targets only bugs carrying specific genes.. To do this, their teams built an artificial CRISPR-Cas9 system - that could cut out specific genes - by assembling pieces in the lab before reintroducing it back into bacteria using viruses. Once injected into the bug, the guide RNA recruits the Cas9 protein to target genes that endow the bug antibiotic resistance or other harmful properties by embedding viral DNA. After those genes are removed, the superbug either dies or turns into an harmless one.. Although the method still needs improving to become ...

Considerations for design of an optimal ssDNA donor for CRISPR-Cas9-mediated precise gene editing. Insertion of an epitope tag, mutation or other genomic modification can be readily accomplished by using CRISPR-Cas9 to cause a double-strand break (DSB) followed by homology-directed repair (HDR) using a repair template (donor oligo or donor plasmid). Knock-in of shorter sequences such as a single nucleotide (nt) change, or insertion of exogenous DNA up to ~100 nts can be performed with a single-stranded, synthetic DNA oligo (Figure 1). The design of a single-stranded donor template plays a big role in your knock-in efficiency; important parameters include homology arm length, homology arm symmetry and chemical modifications. Our top recommendations for each of these, thanks to our great R&D team, are provided here to help you obtain the best results possible for your experiment.. ...

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CRISPR-Cas-9 gene editing system could be revolutionary for genetics and fighting superbugs and diseases. School yourself on the latest biotech!

A major revolution in disease treatment is breaking: Gene editing as a way to treat hereditary and other diseases by epigenetic modification rather than DNA ch…

Introduction: Cancer is known to be the second leading cause of death in the world after the cardiovascular disease. Therefore, it is important to study the various factors involved in cancer. In this regard, animal and cell models can be used to examine the different stages of cancer, as well ...

The CRISPR-Cas9 system is an RNA-guided genome-editing tool that provides researchers a simple, easy, and quick way to modify the genomes of various organisms. Using this system, Cas9 is gudied to a target sequence where it cleaves the DNA to form a double-stranded DNA break (DSB). Cells repair the break through one of two approaches, non-homologous end joining (NHEJ) or by homology-directed repair (HDR). Most commonly, cells will utilize NHEJ, which has been shown to have high efficiencies in Cas9-mediated genome modification. However, NHEJ is notably imprecise, typically resutling in insertions or deletions (INDELS) that result in unpredictable outcomes. Many investigators desire precision targeting to modify exact DNA sequences (including SNPs, knock-ins, conditional knock-outs, etc) in a controlled setting. In these cases HDR from a provided DNA template is the preferred mechanism of repair; however, this pathway is utilized by the cell less frequently than NHEJ. A number of studies have ...

RT @PlantSciNews: Genome modifications in plant cells by custom-made restriction enzymes http://t.co/sxnkOrdb... | Plant Genomics

Figure 5 Phenotypic analysis of the engineered salm alleles. (A) Lethality assay of the salm-dsRed alleles as homozygous or in trans to salm[1]. (B) All tagged salm[1st intron] alleles regain homozygous viability after step 2. (C-G) Localization of the tagged Salm proteins. Untagged Salm is located in the nucleus of wild-type IFMs (C, E), whereas V5 tagged Salm is only detected in the salm[1st intron-TY1-V5] and the salm[1st intron-FRT-TY1-FRT-V5] alleles (D, G). FLAG is found in the IFM nuclei of salm[1st intron-GFP-FLAG] adults (F). The Salm-FRT-TY1-FRT-V5 protein is found in the nuclei (red arrow heads) and also located in dots in the cytoplasm (G). Note the normal fibrillar morphology of the myofibrils in all the homozygous salm[1st intron] alleles (D, F, G). Actin was stained with phalloidin and the scale bars are 10 µm. ...

The media is writing breathless stories about the recent CRISPR "embryo-editing", In Breakthrough, Scientists Edit a Dangerous Mutation From Genes in Human Embryos.. The paper is out in Nature, Correction of a pathogenic gene mutation in human embryos.. My major confusion is that this is normal science. The breakthrough was the discovery of the power of CRISPR-Cas9. Once the discovery was made there was a literally stampede to use the technique because its power and ease was so manifest. Whats happening now is that the technique is getting more powerful and effective. I think it would surprise people if it didnt get better.. A major problem for economists in modeling productivity growth is that innovation is unpredictable. But in this case the big innovation has occurred. The next few decades are likely going to see progressive and continuous improvement in the technology. Where that will lead us? Unpredictable….. ...

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Theres a good chance that we will never be able to use CRISPR in otherwise healthy human beings. Not safely and ethically, anyway. Lets assume that all the obvious technical problems are solved - that we learn which genes to modify and how, and that we learn how to deliver CRISPR-Cas complexes safely and efficiently to…

The gene editing technology CRISPR-Cas9 corrects genetic abnormalities by breaking two DNA strands and then inserting healthy genes or eliminating unhealthy ones. A Yale team has developed an alternative gene-editing technology that they say replaces CRISPRs

A federal biosafety and ethics panel on Tuesday unanimously approved the first study in patients of the genome-editing technology CRISPR-Cas9.

Scientists are using CRISPR-Cas9 technology to tag and explore specific sets of neurons in mice, in one of the first steps towards building a comprehensive atlas of brain circuitry. 0 Comments. ...

Controversy over a Chinese study that used CRISPR-Cas9 gene-editing technology shows how the West still looks at the East through the lens of Orientalism.

Scientists in China plan to use CRISPR-Cas9 in patients with lung cancer as soon as next month, marking the first use of the technology in humans.

Enhanced Green Fluorescent Protein (eGFP)-Cas9 is used in CRISPR-Cas9 genome editing, cleaves dsDNA, and allows for fluorescence-activated cell sorting.

Enhanced Green Fluorescent Protein (eGFP)-Cas9 is used in CRISPR-Cas9 genome editing, cleaves dsDNA, and allows for fluorescence-activated cell sorting.

Scientists are using CRISPR-Cas9 technology to tag and explore specific sets of neurons in mice, in one of the first steps towards building a comprehensive atlas of brain circuitry. 0 Comments. ...

In the current study, the researchers employed the CRISPR-Cas9 genome editing technology to target unique DNA sequences formed because of the gene fusion. The team used viruses to deliver the gene editing tools that cut out the mutated DNA of the fusion gene and replaced it with a gene that leads to death of the cancer cells. Because the fusion gene is present only in cancer cells, not healthy ones, the gene therapy is highly specific. Such an approach could come with significantly fewer side effects when translated to the clinic, which is a major concern with other cancer treatments such as chemotherapy ...

The gene-editing tool CRISPR-Cas9 has been used in a human for the first time. Heres how it could revolutionize medicine - and our genes.

Like other structural biologists, Eva Nogales works in extraordinary times. The University of California, Berkeley, faculty member now has the tools to tackle important questions about cells molecular machinery that would have been impossible to answer just a few years ago. A recent project with Berkeley colleague Jennifer Doudna, the molecular biologist who co-pioneered the CRISPR-Cas9 gene-editing method, is a case in point...". Read more. ...

Like other structural biologists, Eva Nogales works in extraordinary times. The University of California, Berkeley, faculty member now has the tools to tackle important questions about cells molecular machinery that would have been impossible to answer just a few years ago. A recent project with Berkeley colleague Jennifer Doudna, the molecular biologist who co-pioneered the CRISPR-Cas9 gene-editing method, is a case in point...". Read more. ...

Using multiplex CRISPR-Cas9 editing of human hematopoietic, or blood-forming, stem cells followed by transplantation in mice, researchers designed customized mouse models for the progression of leukemia. In a number of different experiments, the animal models successfully reflected human responses to a therapeutic agent commonly used to treat blood cancers.

Scientists have created an on switch for CRISPR-Cas9 that allows it to be turned on in select cells only, specifically those that have a particular protein-cutting enzyme, or protease. Viruses produce such proteases, as.... ...

Also this week: Stem cell transplant removes active HIV from a patient, Lonza announces collaboration for point-of-care manufacturing and autologous immunotherapy, and Cellectis receives a patent for CRISPR-Cas9 edited CAR-T.

CRISPR-Cas encodes an adaptive immune system that defends prokaryotes against infectious viruses and plasmids. Immunity is mediated by Cas nucleases, which use small RNA guides (the crRNAs) to specify a cleavage site within the genome of invading nucleic acids. In type II CRISPR-Cas systems, the DNA-cleaving activity is performed by a single enzyme Cas9 guided by an RNA duplex. Using synthetic single RNA guides, Cas9 can be reprogrammed to create specific double-stranded DNA breaks in the genomes of a variety of organisms, ranging from human cells to bacteria, and thus constitutes a powerful tool for genetic engineering. Here we describe recent advancements in our understanding of type II CRISPR-Cas immunity and how these studies led to revolutionary genome editing applications.. ...

The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas (CRISPR-associated) genes are widely spread in bacteria and archaea, representing an intracellular defence system against invading viruses and plasmids. In the system, fragments from foreign DNA are captured and integrated into the host genome at the CRISPR locus. The locus is transcribed and the resulting RNAs are processed by Cas6 into small crRNAs (CRISPR RNAs) that guide a variety of effector complexes to degrade the invading genetic elements. Many bacteria and archaea have one major type of effector complex. However, Sulfolobus solfataricus strain P2 has six CRISPR loci with two families of repeats, four cas6 genes and three different types of effector complex. These features make S. solfataricus an important model for studying CRISPR-Cas systems. In the present article, we review our current understanding of crRNA biogenesis and its effector complexes, subtype I-A and subtype III-B, in S. solfataricus. We also ...

The CRISPR (clustered regularly interspaced short palindromic repeat)-Cas (CRISPR associated) system is a naturally occuring, adaptative microbial immune system for defense against invading phages and other mobile genetic elements. Type II CRISPR-Cas systems use an RNA-guided DNA endonuclease, Cas9, to generate double strand breaks in invasive DNA during an adaptative bacterial immune response. Cas9 proteins are abundant across the bacterial kingdom, but vary widely in both sequence and size. All known Cas9 enzymes contain an HNH domain that cleaves the DNA strand complementary to the guide RNA sequence (target strand), and a RuvC nuclease domain required for cleaving the noncomplementary strand (non-target strand), yielding double strand DNA breaks (DSBs) [1,2]. The Cas9-type HNH nuclease domain contains a two-stranded antiparallel β sheet flanked by two α-helices on each side (see ,PDB:4CMP,) [1,2]. The profile we developed covers the entire Cas9-type HNH domain. Last update: March 2015 / ...

UNLABELLED: Bacterial genomes encode numerous homologs of Cas9, the effector protein of the type II CRISPR-Cas systems. The homology region includes the arginine-rich helix and the HNH nuclease domain that is inserted into the RuvC-like nuclease domain. These genes, however, are not linked to cas genes or CRISPR. Here, we show that Cas9 homologs represent a distinct group of nonautonomous transposons, which we denote ISC (insertion sequences Cas9-like). We identify many diverse families of full-length ISC transposons and demonstrate that their terminal sequences (particularly 3 termini) are similar to those of IS605 superfamily transposons that are mobilized by the Y1 tyrosine transposase encoded by the TnpA gene and often also encode the TnpB protein containing the RuvC-like endonuclease domain ...

Using a highly efficient AAV delivery platform together with potent and specific guide RNAs for CRISPR-Cas9, we and others have demonstrated efficient in vivo genome editing in mouse models (25). Following cleavage by endonuclease, HDR is generally a less efficient pathway compared to NHEJ, which creates gene-disabling indels. AAV vector has exhibited advantages as an efficient vehicle to deliver donor DNA both in vitro and in vivo. We previously demonstrated successful correction of a G-to-A mutation in 10% of OTC alleles in the liver of newborn OTC spfash mice by a CRISPR-Cas9-mediated HDR approach (25). However, this approach cannot benefit all OTC-deficient patients because disease-causing mutations and large deletions are found scattered at approximately 320 different positions throughout the OTC gene (27). The HDR-mediated gene-targeting approach described in the current study could be broadly applied to all patients carrying mutations in the same causal gene, similar to gene replacement ...

The development of CRISPR-Cas technology is revolutionizing biology. Based on machinery bacteria use to target foreign nucleic acids, these powerful techniques allow investigators to edit nucleic acids and modulate gene expression more rapidly and accurately than ever before.Featuring contributions from leading figures in the CRISPR-Cas field, this laboratory manual presents a state-of-the-art guide to the technology.

In light of this decision, the Doudna/Charpentier patent application will be returned to the Patent Examiner, who previously determined it to be allowable, thus moving the Doudna/Charpentier groups application closer to issuance as a patent covering the use of CRISPR-Cas9 in all types of cells and organisms, including bacteria, plants, animals and humans.. "The team led by UCs Jennifer Doudna and Emmanuelle Charpentier, now at the Max Planck Institute for Infection Biology, invented the CRISPR-Cas9 gene-editing technology that has been rightfully hailed as the scientific breakthrough of the century," said UC President Janet Napolitano. "We are pleased that todays ruling affirms the spectacular accomplishments of these two scientists and their research teams and highlights the incomparable value of basic research at our public research universities and scientific institutions.". The PTAB decision does, however, leave in place patents previously issued to the Broad Institute for use of ...

The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5-NGG-3 PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These ...

The CRISPR-associated protein Cas9 is an RNA-guided endonuclease that cleaves double-stranded DNA bearing sequences complementary to a 20-nucleotide segment in the guide RNA. Cas9 has emerged as a versatile molecular tool for genome editing and gene expression control. RNA-guided DNA recognition and cleavage strictly require the presence of a protospacer adjacent motif (PAM) in the target DNA. Here we report a crystal structure of Streptococcus pyogenes Cas9 in complex with a single-molecule guide RNA and a target DNA containing a canonical 5-NGG-3 PAM. The structure reveals that the PAM motif resides in a base-paired DNA duplex. The non-complementary strand GG dinucleotide is read out via major-groove interactions with conserved arginine residues from the carboxy-terminal domain of Cas9. Interactions with the minor groove of the PAM duplex and the phosphodiester group at the +1 position in the target DNA strand contribute to local strand separation immediately upstream of the PAM. These ...

First, a DNA molecule is introduced into a cell that encodes the Cas9 protein and also encodes an RNA molecule that has both the scaffold sequence and a sequence that will bind to location on the genome to be cut. Following transcription and translation the Cas9 protein binds to the scaffold section of the gRNA. This forms a gRNA-Cas9 complex causing a conformational change in the Cas9 protein enabling the RNA-protein complex to bind to double stranded DNA at loci defined by the guide RNA. This guide must contain the sequence NGG, the Protospacer Adjacent Motif (PAM) at the 3 end (see figure 1 PAM section). It is important to note that the NGG PAM sequence is not in the guide RNA molecule, but must be in the genome to allow cleavage.. Whether the PAM bound-Cas9 cleaves the DNA strands depends on base pairing between one of the genomic DNA strands and the targeting region of the gRNA (figure 1). Base pairing begins at the 3 end of the gRNA targeting region and propagates along towards the 5 ...

Samuel Hasson (SH): With CRISPR, we can really get into the endogenous realm in R&D. We can endogenously tag a protein instead of having to overexpress it, and that can make a huge difference with respect to the results of our experiments. As John noted, we remove a layer of artifacts, and so we can make cleaner observations of phenotypic changes, and the results are clearer.. How are you using CRISPR-Cas9 in your own work?. JD: I love technology development. My job is to work with experts in infectious diseases, cancer biology, neurobiology and other areas who want to solve specific biological problems. I apply the latest genome perturbation tools, such as CRISPR, to their model system and, working together, we learn which genes are involved in their particular disease processes. For example, Ive collaborated with researchers here at the Broad on various components of the immune system to learn about factors involved in influenza and norovirus infection. Weve also used CRISPR technology in ...

Functional elucidation of causal genetic variations and genetic elements requires precise genome manipulation technologies. We have recently developed a new class of eukaryotic genome engineering technology based on the bacterial CRISPR (clustered regularly interspaced short palindromic repeats) adaptive immune system. We reconstituted the CRISPR crRNA processing and interference system in mammalian cells and demonstrate that the Cas9 nuclease can be targeted to specific genomic loci by short crRNA guides to induce DNA double strand breaks. In a variety of cell types and species, Cas9 mediates editing of endogenous chromatin. Here we describe most recent advances for the Cas9 technology through interrogation and enhancement of targeting specificity, conversation of Cas9 into a modular DNA targeting domain, as well as application of the Cas9 system to probe gene function and genetic variations. Our results demonstrate the versatility of the RNA-guided CRISPR Cas9 nuclease system and open the ...

CRISPR-Cas RNA-guided nucleases are derived from an adaptive immune system that evolved in bacteria to defend against invading plasmids and viruses. Decades of work investigating CRISPR systems in various microbial species has elucidated a mechanism by which short sequences of invading nucleic acids are incorporated into CRISPR loci. They are then transcribed and processed into CRISPR RNAs (crRNAs) which, together with a trans-activating crRNAs (tracrRNAs), complex with CRISPR-associated (Cas) proteins to dictate specificity of DNA cleavage by Cas nucleases through Watson-Crick base pairing between nucleic acids. Building off of two studies showing that the three components required for the type II CRISPR nuclease system are the Cas9 protein, the mature crRNA and the tracrRNA, Doudna, Charpentier and colleagues showed through in vitro DNA cleavage experiments that this system could be reduced to two components by fusion of the crRNA and tracrRNA into a single guide RNA (gRNA). Furthermore, they ...

An innovative workflow to guide the insertion of 10-12 nucleotides into a gene of interest. Genome engineering using CRISPR-Cas9 requires expression of the Cas9 nuclease with the crRNA and tracrRNA. This can be achieved by co-transfection of a plasmid expressing Cas9 and crRNA:tracrRNA, or by creation of a cell line in which the Cas9 cassette is delivered using lentiviral particles and stably integrated and expressed prior to transfection with crRNA:tracrRNA. The Dharmacon Edit-R™ CRISPR-Cas9 product line represents the most comprehensive portfolio of tools for gene editing available, but tools are useless without an understanding how to use them. The Edit-R CRISPR-Cas9 genome engineering platform employing chemically synthesized RNAs is a relatively quick and easy method to test multiple guide sequences for optimizing % indel formation through non homologous end joining (NHEJ) and achieving functional gene knockouts.. One topic that comes up frequently when speaking with researchers and ...

Although the two primary forces driving the overall genome evolution of S. thermophilus consist of genome reduction by iterative gene losses in combination with occasional acquisition of beneficial genes through horizontal gene transfer for adaptation to a rich environment (primarily milk) [6,53,58,59], we show in the present article that CRISPR plays a major role in genome evolution following exposure to phages. Indeed, regressive genome evolution by extensive gene loss has been a key driving force shaping the adaptation of S. thermophilus to the rich milk environment, illustrated by the loss of virulence genes widely distributed in most streptococci. Overall, the DGCC7710 genome shares a high degree of synteny with other S. thermophilus genomes, with a few unique genomic islands and hypervariable loci that include the eps operon, the gp operon and CRISPR-Cas systems. Focusing on genome interplay within host-virus dynamics, we propose that the impact of the virus on host genome evolution is ...

By Kevin Holden, PhD, Head of Synthetic Biology at Synthego. This is the fourth part in the CRISPR 101 series by Synthego, providing a crash course on CRISPR-Cas9 and its applications in a wide range of life science disciplines.. The CRISPR revolution helped to trigger some fast-paced developments in biomedical research in the past year. Some of these included the first report of editing disease causing gene mutations in a human embryo and the development of transgenic pigs that are free of integrated viral genes, which could make them useful for future organ transplants in humans (1, 2).. However, in the past year, weve also seen some incredible advances to the CRISPR technology itself. The development of new tools and applications for CRISPR-Cas9 will undoubtedly serve to enable even greater advances in the field of biomedical research in the years to come. Some of the latest advancements in CRISPR technology are elaborated below.. Editing a single nucleotide using CRISPR One of the important ...

...CRISPR a system of genes that bacteria use to fend off viruses is in...The results are scheduled for publication in PNAS Early Edition/e...The CRISPR system has attracted considerable attention for its potenti...Bacteria incorporate small bits of DNA from phages into their CRISPR r...,CRISPR,system,can,promote,antibiotic,resistance,biological,biology news articles,biology news today,latest biology news,current biology news,biology newsletters

Probing gene function in the mammalian brain can be greatly assisted with methods to manipulate the genome of neurons in vivo. The clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease (Cas)9 from Streptococcus pyogenes (SpCas9) can be used to edit single or multiple genes in replicating eukaryotic cells, resulting in frame-shifting insertion/deletion (indel) mutations and subsequent protein depletion. Here, we delivered SpCas9 and guide RNAs using adeno-associated viral (AAV) vectors to target single (Mecp2) as well as multiple genes (Dnmt1, Dnmt3a and Dnmt3b) in the adult mouse brain in vivo. We characterized the effects of genome modifications in postmitotic neurons using biochemical, genetic, electrophysiological and behavioral readouts. Our results demonstrate that AAV-mediated SpCas9 genome editing can enable reverse genetic studies of gene function in the brain ...

An article recently published in Nature (Gaudelli et al., 2017) reports an approach of altering DNA sequences without cleaving the DNA strands. This method of gene editing, exploits a modified version of the CRISPR-Cas9 system and an RNA-based deamination enzyme.INTRODUCTION: GENOME-EDITING BIOTECHNOLOGIESThe clustered regularly interspaced short palindromic repeat (CRISPR) technology is widely used to mediate genome-editing in a variety of species (Sander and Joung, 2014; Barrangou and Doudna, 2016). CRISPR, a microbial cellular immunity system (Barrangou et al., 2007), allows the precise editing of DNA sequences and interrogation of regulatory elements, gene function, and protein networks (Doudna and Charpentier, 2014; Zhang et al., 2014; Amitai and Sorek, 2016). This function requires the presence of a set of CRISPR-associated (Cas) genes, which usually are found adjacent to the CRISPR locus. The wild-type Cas9 endonuclease and its different variants (Jinek et al., 2012; Cong et al., 2013; Qi et al.,

Broad-spectrum antiviral drugs targeting host processes could potentially treat a wide range of viruses while reducing the likelihood of emergent resistance. Despite great promise as therapeutics, such drugs remain largely elusive. Here we used parallel genome-wide high-coverage short hairpin RNA (shRNA) and clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 screens to identify the cellular target and mechanism of action of GSK983, a potent broad-spectrum antiviral with unexplained cytotoxicity. We found that GSK983 blocked cell proliferation and dengue virus replication by inhibiting the pyrimidine biosynthesis enzyme dihydroorotate dehydrogenase (DHODH). Guided by mechanistic insights from both genomic screens, we found that exogenous deoxycytidine markedly reduced GSK983 cytotoxicity but not antiviral activity, providing an attractive new approach to improve the therapeutic window of DHODH inhibitors against RNA viruses. Our results highlight the distinct advantages and ...

Late last month, Bayer and CRISPR Therapeutics announced their intention to form a joint venture combining the CRISPR-Cas9 gene editing tool created by its namesake company with Bayers knowledge of disease treatment. The team plans to set their sights on developing potential treatments and cures for genetic diseases, including congenital heart disease, blindness and certain blood disorders.. The joint venture is being described by the companies as, "the first long-term strategic partnership of its kind to make a substantial investment in the development of target delivery systems in an effort to bring systemic in vivo CRISPR-Cas9 gene editing technology applications to patients." In collaboration with CRISPR Therapeutics, Bayers new LifeScience Center (BLSC) division will work towards making its mandate a reality by partnering with innovative biotech companies to speed the development of medical breakthroughs.. "The new Bayer LifeScience Center and the partnership with CRISPR Therapeutics are ...

CRISPR/Cas9 is becoming a most popular genome editing tool due to its simplicity, with guide RNA (gRNA or crRNA and tracrRNA) and cas9 recognizing specific target, cas9 will cut the DNA and make DSB (double strand break) just near the PAM site.

Cas9/Csn1 | CRISPR-associated endonuclease, anti-Cas9/Csn1, anti-Cas9, anti-Csn1 | CRISPR-associated endonuclease antibody, AS16 3690

Magic scissors. The CRISPR scissor technique has transformed research in just five years. It has made it possible for hundreds of teams of scientists to snip out portions of chromosome that are mutated, or to see what happens when a certain gene isnt there. To understand CRISPR in simple terms, imagine a pair of scissors that have one side of a zipper attached to the tip of the blades. In order to cut a stretch of DNA at exactly the right spot, the zipper has to match up exactly with a stretch of DNA leading up to that spot - forming a tight bond that positions the scissors in just the right place. In CRISPR, the zipper is made of specially designed RNA, and the scissor effect comes from the harnessing the natural cutting action of a protein, or enzyme, called Cas9. The CRISPR revolution has made it possible to design unique RNA zippers that can attach to specific genes that play a role in a disease, and cut them out. The first human clinical trials using CRISPR to cut a flawed section of ...

Review on history and future of the CRISPR-Cas9 system published by HZI researcher. Genome engineering with the RNA-guided CRISPR-Cas9 system in animals and plants is changing biology. It is easier to use and more efficient than other genetic …

Co-lead author Benjamin Kleinstiver, PhD, of the MGH Molecular Pathology Unit and Michelle Prew, a research technician in Joungs lab, then tested all 15 possible variants in which any combination of one, two, three or four of those amino acid side-chains were altered and found that one three-substitution and one four-substitution variant appeared to show the greatest promise in discriminating against mismatched target sites while retaining full on-target activities in human cells. The researchers then more fully characterized the four-substitution variant, which they called SpCas9-HF1 (Sp for the Streptococcus pyogenes bacteria, which is the source of this widely used Cas9, and HF for high-fidelity). They found that this variant induced on-target effects comparable to those observed with the original unaltered SpCas9 when used with more than 85 percent of 37 different guide RNAs they tested. Using GUIDE-Seq, a highly sensitive system Joungs lab developed in 2014 to detect off-target ...

The Art/Sci Salon in Toronto, Canada is offering a workshop and a panel discussion (I think) on the topic of CRISPR( (clustered regularly interspaced short palindromic repeats)/Cas9. CRISPR Cas9 Workshop with Marta De Menezes From its Art/Sci Salon event page (on Eventbrite), This is a two day intensive workshop on Jan. 24 5:00-9:00 pm and Jan. 25 5:00-9:00 pm This workshop will address issues pertaining to the uses, ethics, and representations of CRISPR-cas9 genome editing… Continue reading ...

The Art/Sci Salon in Toronto, Canada is offering a workshop and a panel discussion (I think) on the topic of CRISPR( (clustered regularly interspaced short palindromic repeats)/Cas9. CRISPR Cas9 Workshop with Marta De Menezes From its Art/Sci Salon event page (on Eventbrite), This is a two day intensive workshop on Jan. 24 5:00-9:00 pm and Jan. 25 5:00-9:00 pm This workshop will address issues pertaining to the uses, ethics, and representations of CRISPR-cas9 genome editing… Continue reading ...

Researchers from the Broad Institute and MIT have created a new mouse model to simplify application of the CRISPR-Cas9 system for genome-editing experiments in living animals.. The researchers successfully used the new "Cas9 mouse" model to edit multiple genes in a variety of cell types, and to model lung adenocarcinoma, one of the most lethal human cancers. The mouse has already been made available to the scientific community and is being used by researchers at more than a dozen institutions.. A paper describing this new model and its initial applications in oncology appears today in the journal Cell.. In recent years, genetic studies have found thousands of links between genes and various diseases. But in order to prove that a specific gene is playing a role in the development of the disease, researchers need a way to perturb it - that is, turn the gene off, turn it on, or otherwise alter it - and study the effects.. A convenient genome-editing system. The CRISPR-Cas9 genome-editing system is ...

Sequence-directed genetic interference pathways control gene expression and preserve genome integrity in all kingdoms of life. In many bacteria and most archaea, clustered, regularly interspaced, short palindromic repeats (CRISPRs) specify a recently discovered genetic interference pathway that protects cells from viruses (phages) and conjugative plasmids. Within CRISPR sequences, the repeats are separated by short spacer sequences that match phage or plasmid genomes and specify the targets of interference. Spacer sequences are transcribed into CRISPR RNAs (crRNAs) - small RNAs that, through base-pairing interactions with the target sequence, guide Cas nucleases to the invasive nucleic acid. Upon infection, CRISPR arrays can acquire new repeat-spacer units that match the infecting phage or plasmid. Therefore CRISPR-Cas systems provide adaptive and inheritable immunity to the bacterial cell. The spacer content of CRISPR arrays reflects the many different invaders encountered by the host and can ...

In this study, we developed a cloning-free CRISPR/Cas-mediated genome editing system for highly efficient and convenient one-step generation of knock-in mice carrying a functional gene cassette. This system has several advantages. First, the CRISPR/Cas vector construction and in vitro RNA transcription can be omitted by using commercially available Cas9 protein and chemically synthesized crRNA and tracrRNA, leading to a cloning-free CRISPR/Cas system. Although chemical synthesis of sgRNA might also be possible and convenient, technical limitations for the synthesis of long sgRNAs (more than 100 mer) must be considered. In contrast, shorter crRNAs and tracrRNAs can be chemically synthesized easily in a cost-effective manner. Furthermore, tracrRNAs can be commonly used independently of target sequences as well as Cas9 protein. The targeting vectors are already chemically synthesizable. Second, the efficiency of CRISPR/Cas-mediated digestion can be evaluated with a cell-free IDA system using target ...

Sangal, Vartul, Blom, Jochen, Sutcliffe, Iain, von Hunolstein, Christina, Burkovski, Andreas and Hoskisson, Paul (2015) Adherence and invasive properties of Corynebacterium diphtheriae strains correlates with the predicted membrane-associated and secreted proteome. BMC Genomics, 16 (1). p. 765. ISSN 1471-2164 Sangal, Vartul, Burkovski, Andreas, Hunt, Alison, Edwards, Becky, Blom, Jochen and Hoskisson, Paul (2014) A lack of genetic basis for biovar differentiation in clinically important Corynebacterium diphtheriae from whole genome sequencing. Infection, Genetics and Evolution, 21. pp. 54-57. ISSN 1567-1348 Sangal, Vartul, Fineran, Peter and Hoskisson, Paul (2013) Novel configurations of type I and II CRISPR-Cas systems in Corynebacterium diphtheriae. Microbiology, 159 (Pt 10). pp. 2118-26. ISSN 1465-2080 Sangal, Vartul, Girvan, Kirsty, Jadhav, Sagar, Lawes, Timothy, Robb, Andrew, Vali, Leila, Edwards, Giles, Yu, Jun and Gould, Ian (2012) Impacts of a long-term programme of active surveillance ...

Clustered regularly interspaced short palindromic repeats (CRISPR) consist of highly conserved direct repeats interspersed with variable spacer sequences. They can protect bacteria against invasion by foreign DNA elements. The genome sequence of Streptococcus mutans strain UA159 contains two CRISPR loci, designated CRISPR1 and CRISPR2. The aims of this study were to analyse the organization of CRISPR in further S. mutans strains and to investigate the importance of CRISPR in acquired immunity to M102-like phages. The sequences of CRISPR1 and CRISPR2 arrays were determined for 29 S. mutans strains from different persons. More than half of the CRISPR1 spacers and about 35 % of the CRISPR2 spacers showed sequence similarity with the genome sequence of M102, a virulent siphophage specific for S. mutans. Although only a few spacers matched the phage sequence completely, most of the mismatches had no effect on the amino acid sequences of the phage-encoded proteins. The results suggest that S. mutans ...

The pace of technical developments allowing the direct manipulation of genome sequences has seen a marked acceleration in recent years with the emergence of RNA-targeted nucleases derived from bacterial immune systems (Doudna and Charpentier 2014; Zetsche et al. 2015). In particular, the binary system relying on the Streptococcus pyogenes Cas9 endonuclease targeted by CRISPR (clustered, regularly interspaced, short, palindromic repeat) RNAs has been successfully used to generate point mutations, deletion, or DNA insertions in an ever-growing number of experimental systems. S. pyogenes CRISPR/Cas9 has been adapted early on in the model nematode Caenorhabditis elegans (Friedland et al. 2013; Dickinson et al. 2013; Chen et al. 2013; Frøkjær-Jensen 2013; Dickinson and Goldstein 2016). Previously, heritable genome engineering could only be achieved in C. elegans by remobilizing a Drosophila Mos1 transposon, which could be inserted and excised in the germline (Robert and Bessereau 2007; ...

CRISPR/Cas9 has become a powerful genome-editing tool for introducing genetic changes into crop species. In order to develop capacity for CRISPR/Cas9 technology in the tropical staple cassava (Manihot esculenta), the Phytoene desaturase (MePDS) gene was targeted in two cultivars using constructs carrying gRNAs targeting two sequences within MePDS exon 13. After Agrobacterium-mediated delivery of CRISPR/Cas9 reagents into cassava cells, both constructs induced visible albino phenotypes within cotyledon-stage somatic embryos regenerating on selection medium and the plants regenerated therefrom. A total of 58 (cv. 60444) and 25 (cv. TME 204) plant lines were recovered, of which 38 plant lines (19 from each cultivar) were analysed for mutagenesis. The frequency of plant lines showing albino phenotype was high, ranging from 90% to 100% in cv. TME 204. Observed albino phenotypes were comprised of full albinos devoid of green tissue and chimeras containing a mixture of white and green tissues. Sequence

Arahal, D. R., Dewhirst, F. E., Paster, B. J., Volcani, B. E., & Ventosa, A. (1996). Phylogenetic analyses of some extremely halophilic archaea isolated from Dead Sea water, determined on the basis of their 16S rRNA sequences. Applied and Environmental Microbiology, 62(10), 3779-3786. Ding, J. Y., Chiang, P. W., Hong, M. J., Dyall-Smith, M., & Tang, S. L. (2014). Complete genome sequence of the extremely halophilic archaeon Haloarcula hispanica strain N601. Genome announcements, 2(2), e00178-14. Juez, G., Rodriguez-Valera, F., Ventosa, A., & Kushner, D. J. (1986). Haloarcula hispanica spec. nov. and Haloferax gibbonsii spec, nov., two new species of extremely halophilic archaebacteria. Systematic and Applied Microbiology, 8(1), 75-79. Li, M., Wang, R., Zhao, D., & Xiang, H. (2014). Adaptation of the Haloarcula hispanica CRISPR-Cas system to a purified virus strictly requires a priming process. Nucleic Acids Research, 42(4), 2483-2492. Liu, H., Wu, Z., Li, M., Zhang, F., Zheng, H., Han, J., & ...

Genetically modified food would include almost all the food we eat. Several different way plant genomes are altered "conventionally" and via genetic engineering are described here. Genetically engineering is the direct manipulation of an organisms genome using biotechnology. Although many people think this means moving genes from one species to another, that is not always the case. There are several biotechnological methods of manipulating genes. Sometime this is done by actually moving genes within a species or from a closely related species. This resulting organism is referred to as cisgenic. Gene editing is another method of manipulating DNA. There are several techniques available for gene editing including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspersed short palindromic repeats (CRISPR)/Cas systems. Gene editing may involve deletion, insertion, silencing or repression. The resulting organism from gene editing ...