CRISPR without scissors
CRISPR-Cas technology, developed in 2012, is able to eliminate defects in genes and replace them with whole elements. The newest version of CRISPR works differently. The goal is to correct the error in the genes without crossing the DNA strand and, therefore, safer gene editing.
A new version of the CRISPR enzyme was developed in the USA in 2018, it works more accurately and gives fewer errors. Researchers Maarten Geerts from the Hubrecht Institute and Eileen de Poel from the University Medical Center in Utrecht, the Netherlands, were able to use the so-called adenine base editing to repair the defective nucleotide on the spot, without cutting it out of the chain and replacing it with a healthy one.
Previous studies have shown that this method of genetic correction is safe in rice and mouse stem cells. The Utrecht study showed that adenine editing also works effectively and safely in human stem cells.
Mini intestines
The researchers used a biobank with intestinal organoids. These are a kind of mini-intestines grown from stem cells of patients with cystic fibrosis. Organoids help to investigate exactly what happens in cystic fibrosis. In addition, they are used to find the most appropriate medication for a specific patient with cystic fibrosis, as well as to test new treatments such as CRISPR-Cas. In the current study, scientists used an organoid biobank to find out whether adenine editing works in human stem cell DNA and whether it can be used to treat cystic fibrosis in the small intestine.
What goes wrong with cystic fibrosis?
Cystic fibrosis (cystic fibrosis) is caused by a mutation in the CFTR gene, leading to the inability of the corresponding protein to perform its functions. Like all genes, CFTR is a small part of DNA – a long double chain of four types of nucleotides: adenine, thymine, guanine and cytosine (A, T, G, C). Nucleotides form a long series of three-letter combinations that give the cell the information necessary to create proteins. The gene affected by cystic fibrosis consists of about 250 thousand pairs of nucleotides.
In most patients with cystic fibrosis, only one "word" of three pairs of nucleotides is missing, which leads to the absence of one amino acid and the formation of a non-functional version of the protein.
In the remaining part of patients, the replacement of just one nucleotide changes the triplet encoding the amino acid to a stop codon - a trinucleotide that signals that the gene is ending. In particular, the stop codon appears too early if the base pair of HZ is replaced by AT. This can be corrected by changing the A in the "false" stop codon back to G.
Thus, two groups of patients with cystic fibrosis have a different defect in the CFTR gene with the same result: the cell does not synthesize the normal CFTR protein, which is necessary in the body for the transport of chloride and water through the cells of the mucous membrane of many organs. If this does not happen, the mucus in the organs is not sufficiently moistened and becomes dense. As a result, various organs (lungs, pancreas, reproductive organs) do not work properly, their function gradually decreases.
Effective medications are now available for a large group of patients with cystic fibrosis who lack a trinucleotide in their DNA. But for the second group, with an erroneous stop codon, there is no treatment. Therefore, the new adenine editing technique was tested on stem cells of patients with cystic fibrosis with premature stop codon.
How does single nucleotide CRISPR editing work?
The Cas enzyme in the traditional CRISPR-Cas technique identifies a defective DNA fragment and cuts it out. In the new modification, the tool also finds a DNA error, but does not eliminate it. The purpose of adenine editing of CRISPR in cystic fibrosis is to restore the CFTR gene by replacing the wrong base A with G. For this, a protein is attached to the enzyme that destroys A in such a way that it becomes similar to G. The cell, which is able to restore defective nucleotides, now recognizes it as G and replaces it with a normal G. Thus, the trinucleotide it is no longer recognized as a stop codon, and the CFTR protein is synthesized completely.
The researchers managed to edit the CFTR genes in the intestinal organoids from the biobank: the cells in the restored mini-intestines produced the correct CFTR protein.
An important feature of adenine editing CRISPR is that it seems to be narrowly specific. It is known that the biggest drawback of the traditional CRISPR-Cas technology is that it not only eliminates the error, but also cuts off healthy DNA fragments. This leads to unintentional DNA damage. The new technology is more accurate, better recognizes the wrong DNA sequence and changes only it. This was confirmed in the study: there was no additional DNA damage in the restored mini-intestine.
Organoids of the small intestine of a patient with cystic fibrosis (left), which do not swell due to a mutation in the CFTR gene, and swollen after correcting the mutation (right). Image: Eyleen de Poel and Maarten Geurts, copyright UMC Utrecht and Hubrecht Institute.
Next steps
Demonstration of the laboratory method, however, does not mean that patients can already benefit from it. Before being introduced into clinical practice, it is necessary to make sure that the enzyme will achieve its goal in a living organism. Cystic fibrosis is a rather difficult disease to treat, as it affects many different functions of the body. Further research is needed to ensure that this new method of basic CRISPR editing can be applied in the clinic.
In addition, there are ethical aspects of genetic editing of cells. Theoretically, this method can also be used to change cells, for example, in embryos. Discussions on this topic have not yet led to any decision.
Article by M.H.Geurts et al. CRISPR-Based Adenine Editors Correct Nonsense Mutations in a Cystic Fibrosis Organoid Biobank is published in the journal Cell Stem Cell.
Aminat Adzhieva, portal "Eternal Youth" http://vechnayamolodost.ru based on the materials of the Hubrecht Institute: Curing genetic disease in human stem cells.