Genome Editing Solutions to Develop the Right Biological Model
Functional genomics applications and more
Background and history
The use of CRISPR has started as a revolution in the field of genome editing and has now become increasingly a normality and a widely used technology for screening applications in Drug Discovery, especially in functional genomics.
CRISPRs (Clustered Regularly Interspaced Palindromic Repeats) were first discovered in bacteria in the 80’s1 and the use of CRISPR-Cas9 gained traction when the potential for side-directed gene knockout was recognized and proposed by Marraffini and Sontheimer in Science 20082. The principle of CRISPR-Cas9 is fairly simple: Cas9 (Cas stands for CRISPR associated) is a dual RNA-guided DNA endonuclease with the ability to generate site-specific DNA doublestrand (ds) breaks. In 2012, Doudna and Charpentier3 achieved a major breakthrough for this methodology and its implications for genome editing. They combined the before used precursor CRISPR RNAs (pre-crRNAs) and trans-activating CRISPR RNA (tracrRNA) into a single guide-RNA (sgRNA). With this simplification it was then possible to have a two component-system consisting of only CRISPR-Cas9 and sgRNA that could introduce double-strand breaks in virtually any gene of the genome. An even easier approach is used when pre-complexing Cas9 with the sgRNA resulting in a ribonucleoprotein (RNP) as a single component for site-directed gene modification.
The ability of CRISPR to introduce specific modifications into the genome allows tremendous flexibility in the choices of targets and assays and allows a precise determination of the drug target as part of functional genomics. In general, CRISPR-Cas9 allows for DNA modifications such as:
- DNA deletion
- DNA insertion
- DNA modification
- Gene expression activation
- Gene expression repression
More and more applications are developed and used as time progresses, leading to a plethora of methods for the use in functional genomics.