Crispr / Cas9
Introduction
Changing the genome of cell lines in culture has been historically limited to very simple processes such as those supported by gene transfections, with the introduction of sequences of DNA that, for example, express modified proteins or enhance the activity of promoters. Modification of the genome of cells within a living organism is a much more complicated process that uses very well characterized models, such as D. melanogaster or C. elegans, requiring an extensive knowledge on the genetics of those animal models, or very lengthy protocols (months to years) when using higher organisms such as M. musculus (mouse), involving DNA gene replacement by homology recombination.
Recent studies have however shown that it is possible to shorten dramatically the time to specifically change the genome of animals, plants or cell lines by using the CRISPR/Cas9 technology, being this a step forward into a world full of opportunities that will change the approach to biotechnology and, for the time being, gene therapy and enhancement in animal biology.
Definition and Origin
CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) is a natural method with bacterial origin; it is an innovative and recent technique which can alter a cell's genome by a much more direct and simple process. Bacteria use this technique in order to protect themselves from viruses.
With the detection of a virus' DNA, the bacteria produce two types of short RNA, one of which is a gRNA (guide RNA) that contains the sequence that matches that of the invading virus, in order to recognize the sequence where it will attach itself. These two strands of RNA form a complex with a protein called Cas9, which is an endonuclease capable of unzipping and cutting DNA. This complex (including the gRNA, Cas9 and the other portion of RNA) will lock itself onto a sequence of the viral DNA, proceeding to the unzipping of the two DNA strands. The gRNA will then find its target and Cas9 will then cut the DNA directly, in order to disable the VIRUS.
This procedure was then explored and found to be possible to use in other situations, namely to fit our needs in DNA manipulation. That said, we can manually recreate those mechanisms.
State of the Art
For CRISPR to perform as expected, an intensive study of the cell and its genome is mandatory. The CRISPR/Cas9 complex may consist of various components that, using directed means of targeting to the desired cells, will infiltrate them and alter the genome at defined specific sites. For example, if a mutation has occurred and we intend to correct that mutation, that sequence will be a target site. The strategy is to replace individual amino acids or substitute a larger DNA sequence for one of our choosing.
The essential components of the complex are the RNA of the Cas9 and the gRNA. The RNA of the protein will be transcribed inside the cell, which finalizes the job. The gRNA is the complementary RNA sequence to the RNA you wish to modify; being complementary, it will attach itself to the mutated portion of the genome and guide the rest of the components to the desired place.
In order to replace that part of the genome and not just cut and let it correct itself, it will be needed to insert, along with the other components, the corrected sequence - manually designed and purchased or synthesized - having to include both the beginning and the end of the original RNA sequence. These parts ensure that the swap is done with the right placement and orientation.
With the administration of the complex into the cell, the Cas9 RNA will transcribe itself into the protein, the gRNA will guide the complex to the mutated portion of DNA, Cas9 will unzip and perform the cut and the mutated sequence of the gene will be swapped with the "healthy" RNA sequence.