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Genetic Engineering Publications - GEG Tech top picks
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BigField GEG Tech's insight:
The authors describe an efficient method to induce specific chromosomal rearrangements in vivo using viral-mediated delivery of the CRISPR/Cas9 system to somatic cells of adult animals. They apply it to generate a mouse model of Eml4–Alk-driven lung cancer. 
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Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1.
BigField GEG Tech's insight:
Cas9/CRISPR-mediated Fan1 disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrum of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1.
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BigField GEG Tech's insight:
The authors show that Cas9 binds with high affinity to single-stranded RNA (ssRNA) targets matching the Cas9-associated guide RNA sequence when the PAM is presented in trans as a separate DNA oligonucleotide. Furthermore, PAM-presenting oligonucleotides (PAMmers) stimulate site-specific endonucleolytic cleavage of ssRNA targets, similar to PAM-mediated stimulation of Cas9-catalysed DNA cleavage. These results reveal a fundamental connection between PAM binding and substrate selection by Cas9, and highlight the utility of Cas9 for programmable transcript recognition without the need for tags.
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BigField GEG Tech's insight:
The authors use CRISPR-Cas technology to create antimicrobials whose spectrum of activity is chosen by design.
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BigField GEG Tech's insight:
The authors provide an online tool for the design of highly active sgRNAs for any gene of interest. Indeed, they discovered sequence features that improved activity, including a further optimization of the protospacer-adjacent motif (PAM) of Streptococcus pyogenes Cas9. The results from 1,841 sgRNAs were used to construct a predictive model of sgRNA activity to improve sgRNA design for gene editing and genetic screens.
http://www.broadinstitute.org/rnai/public/analysis-tools/sgrna-design
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BigField GEG Tech's insight:
The authors used CRISPR/Cas9–mediated genome editing to correct the dystrophin gene (Dmd) mutation in the germline of mdx mice, a model for DMD, and then monitored muscle structure and function. Genome editing produced genetically mosaic animals containing 2 to 100% correction of the Dmd gene. The degree of muscle phenotypic rescue in mosaic mice exceeded the efficiency of gene correction, likely reflecting an advantage of the corrected cells and their contribution to regenerating muscle.
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BigField GEG Tech's insight:
The authors describe a new method of cancer model generation using the CRISPR/Cas system delivered in vivo by hydrodynamic injection in wild-type mice. This strategy open a new way for rapid development of liver cancer models and functional genomics.
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BigField GEG Tech's insight:
The authors' observations suggest a mechanism for a Protospacer Adjacent Motif (PAM)-dependent target DNA melting and RNA–DNA hybrid formation. Furthermore, this study establishes a framework for the rational engineering of Cas9 enzymes with novel PAM specificities.
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BigField GEG Tech's insight:
The fusion Cas9/FokI enhances specificity of CRISPR system without reduce the efficiency. In human cells, the authors shown that this fusion allows the modification of target DNA sites with a 140-fold higher specificity than wild-type Cas9 and with an efficiency similar to that of paired Cas9 'nickases'.
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BigField GEG Tech's insight:
The first high resolution three-dimensional structure of the DNA modifying enzyme Cas9. Nice!
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BigField GEG Tech's insight:
This study establishes guide RNA features that drive DNA targeting by Cas9 and open new design and engineering avenues for CRISPR technologies.
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BigField GEG Tech's insight:
The authors identify rules for specific targeting of transcriptional repressors (CRISPRi), and activators (CRISPRa) to endogenous genes via endonuclease-deficient Cas9. Using these rules, they construct genome-scale CRISPRi and CRISPRa libraries. Growth-based screens identify essential genes, tumor suppressors, and regulators of differentiation. These results establish CRISPRi and CRISPRa as powerful tools that provide information for mapping complex pathways.
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Coupling the specificity of CRISPR-Cas nucleases and bacteriophage delivery enables exquisitely precise bacterial killing.
BigField GEG Tech's insight:
The authors show that Cas9, delivered by a bacteriophage and reprogrammed to target virulence genes, kills virulent, but not avirulent, Staphylococcus aureus. Reprogramming the nuclease to target antibiotic resistance genes destroys staphylococcal plasmids that harbor antibiotic resistance genes and immunizes avirulent staphylococci to prevent the spread of plasmid-borne resistance genes. The authors also show that CRISPR-Cas9 antimicrobials function in vivo to kill S. aureus in a mouse skin colonization model. This technology creates opportunities to manipulate complex bacterial populations in a sequence-specific manner.
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BigField GEG Tech's insight:
The authors demonstrated in vivo as well as ex vivo genome editing using adeno-associated virus (AAV)-, lentivirus-, or particle-mediated delivery of guide RNA in neurons, immune cells, and endothelial cells using Cre-dependent Cas9 knockin mouse.
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BigField GEG Tech's insight:
The authors has developed a CRISPR/Cas9 nuclease specific for the bovine Nanog locus They showed that this genome editing tool can be used for homologous recombination targeting in bovine pluripotent cells is an efficient gene editing method that can be used to generate transgenic livestock in the future.
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From BioPortfolio: Type I CRISPR-Cas systems require a target-searching Cascade complex and the Cas3 degradation machine to drive prokaryotic adaptation to alien nucleic acids. Ca...
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An international, peer-reviewed genome sciences journal featuring outstanding original research that offers novel insights into the biology of all organisms
BigField GEG Tech's insight:
The authors combined CRISPR/Cas9 technology with the piggyBac transposon to efficiently correct the HBB mutations in patient-derived iPSCs without leaving any residual footprint. No off-target effects were detected in the corrected iPSCs, and the cells retain full pluripotency and exhibit normal karyotypes. When differentiated into erythroblasts gene-corrected iPSCs restored expression of HBB compared to the parental iPSCs line.
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BigField GEG Tech's insight:
The CRISPR system has attracted considerable attention for its potential uses in genetic engineering and biotechnology, but its roles in bacterial gene regulation are still surprising scientists. The authors find that when the gene encoding Cas9 is mutated in F. novicida bacteria, they become more vulnerable to polymyxin B as well as standard antibiotic treatments such as streptomycin and kanamycin. They were able to trace the effects of the mutation back to a defect in "envelope integrity." Cas9 regulates production of a lipoprotein, which appears to alter membrane permeability.
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BigField GEG Tech's insight:
Genome manipulation in the malaria parasite Plasmodium falciparum remains largely intractable and improved genomic tools are needed to further understand pathogenesis and drug resistance. We demonstrated the CRISPR-Cas9 system for use in P. falciparum by disrupting chromosomal loci and generating marker-free, single-nucleotide substitutions with high efficiency. Additionally, an artemisinin-resistant strain was generated by introducing a previously implicated polymorphism, thus illustrating the value of efficient genome editing in malaria research.
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PubMed comprises more than 23 million citations for biomedical literature from MEDLINE, life science journals, and online books. Citations may include links to full-text content from PubMed Central and publisher web sites.
BigField GEG Tech's insight:
CRISPR to modify HPSC by homologous recombination, and one use of more for this technology!
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Scientists designed a new approach which provides a synthetic biology platform for targeting and controlling bladder cancer cells in vitro.
http://geg-tech.com/