CRISPR/Cas is a state-of-the-art genome editing technology
At AST, we use CRISPR to generate fluorescent reporter lines and disease lines harboring mutations associated with inherited disease.
In 2012, Jennifer Doudna and Emmanuelle Charpentier made the groundbreaking discovery that the Cas enzyme can be programmed to a genetic scissor able to modify the genome at any genomic location in any organism.
To act as an adaptable genetic scissor, the naturally occurring CRISPR needs to be replaced by a single RNA molecule, the so-called guide RNA. With this guide RNA (sgRNA), the Cas cutting enzyme can be directed to target any specific gene sequence.
The guide sgRNA-Cas complex binds to the target sequence and induces a double strand break in the DNA double helix. A DNA break is an emergency for the cell and thus the cell will immediately attempt to repair it. In a very simple way, the DNA strands will be glued together. While this way to repair the break is very efficient, it is also error-prone and often changes the genetic sequence. Any change in frame of the genetic code often leads to knockout of the gene.
When using a designed repair template that can be introduced into the cell together with the guide RNA and Cas enzyme, the sequence of the DNA can be changed to either introduce or repair a gene mutation or add an additional sequence.