The CAS9 DNA-cutter is a unique enzyme that is the primary core of an intrinsic DNA editing system found in bacteria. This primitive immune system is used by bacteria to kill and neutralize attacking viruses and confer resistance to bacteriophages. There exist distinct features within most bacterial genomes commonly known as clustered regularly interspaced short palindromic repeats (CRISPR) that dictate the resistance specificity. This RNA-guided editing requires only 75-100 nucleotides of RNA for targeting. The powerful ability of CAS9 to drive parallel targeted DNA editing has groundbreaking implications for a huge range of biotechnology applications from gene therapy and agriculture. Compared to current brute force sequence-specific endonucleases, CAS9 is a fine-tuned system that can be easily customized and promises to be one of the most hugely valuable and versatile genome engineering breakthroughs of this decade. It has potential not only in modifying - but also in regulating and marking - genomic loci across a wide variety of cells and organisms.
Immunoprecipitation: Cas9 Antibody (7A9-3A3) [NBP2-36440] - HEK293T expressing N-terminally Flag-tagged S.pyogenes Cas9 were lysed 72h post transfection by resuspending the cells in Hunt buffer and subjecting to 3 freeze-thaw cycles in liquid nitrogen/ice.
Doudna et al share a comprehensive timeline review of CRISPR history and its role in genome manipulation and engineering in their 2014 Science review (1). Researchers from Horizon Discovery in England share their thoughts on the impact of CAS9-based editing in streamlining the establishment of cell-line and animal models, as well as genome-wide knock out screens (2). Peng’s group from the Mayo Clinic provides a broad overview of recent advances in targeted genome editing that include not only CAS9 but also the zinc-finger nucleases (ZFNs) and TALEN systems (3). They are keen to see these novel processes be used in less well established experimental organisms and systems. The application of CAS9 in plant genome editing is reviewed by the Belhaj et al in their Current Opinions in Biotechnology 2014 publication where they examine the remarkable ability of CAS9 to produce homozygous knockout mutants within a single plant generation (4). In their 2015 review, St. Jude immunology researchers discuss the advantages of CAS9 over other conventional methods with a focus on immune system challenges and questions (5).
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