Genome editing and the CRISPR

Genome editing: break and repair

In the modern era of genetics, it is simple to read the human genome, determine every single nucleotide in your cells can be done in a few days at an relatively low cost. However it is extremely challenging to change something in the DNA, e.g. to correct a disease causing mutation. This molecule is extremely stable and well-protected from any interventions, so until recently it was hardly possible to ’edit’ the genome. Editing means everything that writers or publishers do with a written text: cutting, pasting  or changing letters.  Genome editing is similarly done with the four  nucleotides (A, G, C, T) in the DNA. Genome editing is not a completely new technolgy researchers have been using other enyzmes (like the so-called zinc  finger nucleases and the TALENs) to perform genome editing. But there is a huge differnce between the these and the novel technology, CRISPR (the abbreviation is very complicated: Clustered Regularly Interspaced Short Palindromic Repeats) The former technologies use peptide decoders to find the certain part in the long DNA that has to be modified and therefore it is challenging to figure out what peptide is the one that exclusively binds to my favourite sequence. But CRISPR is a real gift from the nature: it uses a complimentary RNA, which will just pair with one strand of the DNA. As you may know, the whole genome has been already deciphered, we know the sequence of the human genome. If it was written on sheets, the tower of books would be 169 meter high!

So, if the genome sequence is known, then it is simple to design the RNA (this will be called the guide RNA, as this guides the editing enzyme to the genomic region) that is complimentary to the DNA. As you know: adenin [A] pairs with timine [T], guanine [G] with cytosine [C] and vice versa. Let’s see: here is a diseases gene sequence: AGCTGTGCTGTCGATGC. I want to edit this. So first I have to direct the editing enzyme (this is called the Cas9) to this sequence with the guide RNA, which has the sequence of: TCGACACGACAGCTACG. So look at this!

The gene to be edited: AGCTGTGCTGTCGATGC

The guide:                       TCGACACGACAGCTACG

These are pairs! So to be simple: the guide RNA can select the region of interest from 3 billion base pairs quickly and effectively.

OK, so we have something that selects a gene of interest in the genome. The next question is: how this part of the genome will be edited? Basicly, the Cas9 protein will cleave the DNA and create a double strand break (so it cuts the DNA to two parts). Cells doesn’t like this, a break in the DNA is a nightmare for them, so they want to utilize one of many repair mechanisms to rejoin the injured DNA. Among these is the so-called NHEJ – non-homologus end joining. This repair is an error-prone repair, which will create mutations (some difference compared to the original), otherwise the Cas9 enzymewill cut it again and again. So it has to be some change in the sequence so that the RNA guide will not recognize it anymore. This NHEJ can kick out a gene and inactivate it. So let’s say there is a mutation in a gene that leads to an abnormal protein that has abnormal function, like a tumor formation. We have two copies of each gene (one from the mother and one from the father), but even if we have ONE normal copy, there is that bad gene that drives tumor growth. This is called the DOMINANT effect, with one bad copy, there is still disease. But using CRISPR this bad gene coud be knocked out, so the normal copy will take over and the disease can be cured! Forever since it the DNA is modified!

But you can say, where is the editing? Where is the real correction? Fortunatley this is also possible. If you happen to deliver a repair template to the cells (from which the DNA could be repaired) you can insert anything into the genome, exactly at the cut site, where the DNA is broken. This is called the homology directed repair (HDR). So if someone has a so-called RECESSIVE disorder (when both copies of the gene is mutated), the gene can be corrected with this method.

And applications are indefinite: it is feasible to do it in plants, bacteria, or other animals to cure diseases, or to create even new lifestyles. This system can be also used as a novel technology to study the function of genes. I will tell you even more, if you come back to this site!