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| 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!
A recent work from the Porteus lab (Stanford, California) presents the use of chemically altered gRNAs to improve low efficiency genome editing rates in primary human T cells and CD34+ hemopoietic stem cells. Modified sugar-phosphate backbone renders the gRNA more stable in cells leading to enhanced gene engineering, including more efficient non-homologous end joining and homology directed repair rates. These gRNAs have a particular advantage over unmodified gRNAs when the system is delivered as a ribonucleoprotein particle (RNP, i.e. Cas9 protein and gRNA) or as pure RNAs (Cas9 mRNA and gRNA), advantages over plasmid DNA expressed Cas9 are less clear. The authors claim there is somewhat better on/off target ratio with modified gRNAs, but we have to be cautious with this: the authors modified the terminal bases on the gRNA, perhaps functionally truncating them. Truncated gRNAs have significantly higher specificity according to the paper of from the K. Joung lab. Nevertheless, this system could be highly advantageous when working with primary cells, where genome editing rates are relatively low.
