User:Pml2p/sandbox

Introduction
To survive in a variety of challenging, inhospitable habitats that are filled with bacteriophages, bacteria and archea have evolved methods to evade and fend off predatory viruses. This includes the CRISPR system of adaptive immunity. In practice, CRISPR acts as a self-programmable restriction enzyme. CRISPR loci are composed of short, palindromic repeats that occur at regular intervals composed of alternate CRISPR repeats and variable CRISPR spacers between 24-48 nucleotides long. These CRISPR loci are usually accompanied by adjacent CRISPR-associated (cas) genes. In 2005, it was discovered by three separate groups that the spacer regions were homologous to foreign DNA elements, including plasmids and viruses. These reports provided the first biological evidence that CRISPRs might function as an immune system.

Genomic Editing in Eukaryotic Cells
While genomic editing in eukaryotic cells has been possible using various methods since the 1980's the methods employed had proved to be inefficient and impractical to implement on a larger scale. Genomic editing leads to irreversible changes to the gene. Working like genetic scissors, the Cas9 nuclease opens both strands of the targeted sequence of DNA to introduce the modification by one of two methods. Knock-in mutations, facilitated via Homology Directed Repair, is the traditional pathway of targeted genomic editing approaches. This allows for the introduction of targeted DNA damage and repair. HDR employs the use of similar DNA sequences to drive the repair of the break via the incorporation of exogenous DNA to function as the repair template. This method relies on the periodic and isolated occurrence of DNA damage at the target site in order for a repair to commence. Knock-out mutations caused by Cas9/CRISPR results in the repair of the double-strand break by means of NHEJ (Non-Homologous End Joining). NHEJ can often result random deletions or insertions at the repair site disrupting or altering gene functionality. Therefore, genomic engineering by CRISPR/Cas9 gives researches the ability to generated targeted random gene disruption. Because of this, the precision of genomic editing is a great concern. With the discovery of CRISPR and specifically the Cas9 nuclease molecule, efficient and highly selective editing is now a reality. Cas9 allows for a reliable method of creating a targeted break at a specific location as designated by the crRNA and tracrRna guide strands. Cas9 derived from S. pyogenes has facilitated the targeted genomic modification in eukaryotic cells. The ease by which researches can insert Cas9 and template RNA in order to silence or cause point mutations on specific loci has proved invaluable to the quick and efficient mapping of genomic models and biological processes associated with various genes in a variety of eukaryotes. New engineered variant of the Cas9 nuclease have been developed that significantly reduce off-target manipulation. Called spCas9-HF1 (Streptococcus pyogenes Cas9 High Fidelity 1), has a success rate of modification in vivo of 85% and undetectable off-target manipulations as measured by genome wide break capture and targeted sequencing methods used to measure total genomic changes.

Cellular Modeling
Cas9 genomic modification has allowed for the quick and efficient generation of transgenic models within the field of genetics. Cas9 can be easily introduced into the target cells via plasmid transfection along with sgRNA in order to model the spread of diseases and the cell's response and defense to infection. The ability of Cas9 to be introduced in vivo allows for the creation of more accurate models of gene function, mutation effects, all while avoiding the of off-target mutations typically observed with older methods of genetic engineering. The CRISPR and Cas9 revolution in genomic modeling doesn’t only extent to mammals. Traditional genomic models such as Drosophila Melanogaster, one of the first model species, have seen further refinement in their resolution with the use of Cas9.