An Introduction to CRISPR/Cas9

DNA is usually depicted as a book in which the information contained within - given as a combination of triplets of A, T, G or C – provides the instruction for the development and the function of a living organism. For a long while, DNA has been deeply studied but the information contained within was still considered inaccessible to human manipulation. However, for the past 20-years, this is not true anymore as the newest discoveries in molecular biology have granted us the tools to interact with the DNA like never before.

Gene editing in a nutshell

Gene editing (or genetic engineering) entails the ability to manipulate the genetic information contained within the DNA by using molecular biology tools also known as “molecular scissors”. As the name suggests, molecular scissors are DNA nucleases that can be programmed to cut the DNA at the desired location. The cut usually occurs on both strands of the DNA double helix and therefore it is also defined as Double-Strand-Break(DSB). Gene editing takes advantage of the endogenous DNA repair mechanisms which are triggered when a DSB is detected by the cells. This complex cascade of events is also known as DNA Damage Response(DDR). A DSB damage can be generally repaired via two main mechanisms: the Non-Homologous End-Joining (NHEJ) and the Homology-Directed Repair (HDR). The NHEJ is regarded as an error-prone mechanism meaning that when the DSB is repaired some errors may occur because of which DNA bases may be mistakenly added (Insertions) or they can be lost (Deletions), shortened to indels. Addition or deletion of bases within the portion of a gene codifying for the respective mRNA – defined also as coding sequence – may alter the so-called reading frame, meaning how the triplets are read by the transcriptional machinery. This can result in mutations generating a premature stop codon (nonsense mutation) or the protein produced contains the wrong amino acid (missense mutation). Gene editing relies particularly on generating nonsense mutations to inactivate a gene, this is also defined as Knock-Out (KO). On the other hand, HDR is an error-free mechanism and the DSB repair is guided by the presence of a homology sequence to identify a DNA portion to be used as a blueprint to repair the DSB without introducing any indel. Usually, the “natural blueprint” is contained within the homologous chromosome. However, for gene editing applications it is possible to design an “artificial blueprint” that is made of the same homology arms of the homologous chromosome but in between the two homology arms (left and right), it can be inserted a desired DNA sequence. This way it is possible to integrate an exogenous DNA sequence where the cut has been generated by the molecular scissors. This practice is also known as targeted integration or Knock-In (KI) [1].

Molecular scissors and CRISPR/Cas

As we saw, gene editing relies on the natural DNA repair mechanisms already present in the cells to repair DNA damage. It is possible to trigger these mechanisms by using these novel tools named molecular scissors.

Although they have been around for almost 20 years, only in the past 8 years or so we have started hearing about gene editing more often.

As you may have already guessed, we are speaking about Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR). In nature, CRISPR is a bacteria defense system that is activated to protect the bacteria from the invasion of exogenous infections such as bacteriophages and plasmid DNA (Fig.1).