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Genetic Engineering Publications - GEG Tech top picks
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BigField GEG Tech's insight:
Here, the authors report a novel method for targeted DNA demethylation using the widely used clustered regularly interspaced short palindromic repeat (CRISPR)-Cas system. Applications of this system would not only help us understand mechanistically how DNA methylation might regulate gene expression in specific contexts, but also enable control of gene expression and functionality with potential clinical benefits. 
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BigField GEG Tech's insight:
This review describes methods for increasing the activity and accuracy of the CRISPR-Cas9 system and discusses approaches for assessing off-target cleavage.
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CRISPR-FOCUS is freely available at http://cistrome.org/crispr-focus/
BigField GEG Tech's insight:
The recently developed CRISPR screen technology, based on the CRISPR/Cas9 genome editing system, enables genome-wide interrogation of gene functions in an efficient and cost-effective manner. Although many computational algorithms and web servers have been developed to design single-guide RNAs (sgRNAs) with high specificity and efficiency, algorithms specifically designed for conducting CRISPR screens are still lacking. Here the scientists present CRISPR-FOCUS, a web-based platform to search and prioritize sgRNAs for CRISPR screen experiments. With official gene symbols or RefSeq IDs as the only mandatory input, CRISPR-FOCUS filters and prioritizes sgRNAs based on multiple criteria, including efficiency, specificity, sequence conservation, isoform structure, as well as genomic variations including Single Nucleotide Polymorphisms and cancer somatic mutations. CRISPR-FOCUS also provides pre-defined positive and negative control sgRNAs, as well as other necessary sequences in the construct (e.g., U6 promoters to drive sgRNA transcription and RNA scaffolds of the CRISPR/Cas9). These features allow users to synthesize oligonucleotides directly based on the output of CRISPR-FOCUS. Overall, CRISPR-FOCUS provides a rational and high-throughput approach for sgRNA library design that enables users to efficiently conduct a focused screen experiment targeting up to thousands of genes.
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BigField GEG Tech's insight:
Here, the authors determine molecular structures of the catalytically active Streptococcus pyogenes Cas9 R-loop that show the displaced DNA strand located near the RuvC nuclease domain active site. These protein-DNA interactions in turn position the HNH nuclease domain adjacent to the target DNA strand cleavage site in a conformation essential for concerted DNA cutting. Cas9 bends the DNA helix by 30°, providing the structural distortion needed for R-loop formation.
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BigField GEG Tech's insight:
This review attempts to survey the potential to use traditional pharmaceuticals as opposed to biopharmaceuticals to treat single gene disorders. The available strategies include the use of antisense oligonucleotides to alter splicing or knock-down expression of a transcript, siRNAs to knock-down gene expression, and drugs for nonsense mutation read-through.
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Here the authors demonstrate that the class 2 type VI RNA-guided RNA-targeting CRISPR–Cas effector Cas13a (previously known as C2c2) can be engineered for mammalian cell RNA knockdown and binding. Their results establish CRISPR–Cas13a as a flexible platform for studying RNA in mammalian cells and therapeutic development.