Scalpel leaves the DNA alone
CRISPR changes DNA permanently, and any careless errors will remain for the rest of your life. A new method avoids the problem by editing at a later stage, without touching the original DNA.
scientist to create humans that had been genetically edited through and through. His aim was to create babies resistant to HIV virus. He intended to use CRISPR to cause a specific mutation of the CCR5 gene in fertilised egg cells from a donor. This mutation exists naturally in some people – primarily Europeans – and it confers total or partial protection against HIV. He set to work in early 2018, and in October of the same year the world’s first genetically-edited babies were born: a pair of identical twin girls.
Scientists throughout the world were shocked. He Jiankui was sentenced to three years in prison for unethical research, and a detailed report of his experiments was never published. Leaked information indicates that the scientist may never have reached his goal of making the twins resistant to HIV. The CRISPR tool did not cut quite as expected, and instead of the desired mutation, the girls got other CCR5 mutations, the effects of which scientists have not yet mapped out. And further, the gene
technology caused at least one other extra mutation in a totally different position in the DNA. According to some scientists, He Jiankui has probably overlooked a series of other extra mutations in the twins’ DNA. So He Jiankui may have introduced a series of harmful mutations by mistake, errors that could now be present anywhere in the girls’ bodies – including in the reproductive cells that might one day form the basis of the girls’ own children.
Hope for fortunate mutation
The case of the twins emphasised CRISPR’s weaknesses, and moderated enthusiasm for the new technology.
But scientists do have a pretty good idea of where the editing goes wrong. The most widespread version of CRISPR uses an enzyme by the name of Cas9, which comes from the bacterium Streptococcus pyogenes. Cas9 slices DNA in two, producing two loose ends with completely clean cuts. When the DNA has been cut in this way, the cell will try to repair the damage, but the clean cuts provide a challenge. The cell may combine the loose ends with entirely different DNA that has suitable clean cuts, causing new and unexpected DNA sequences and combinations. The cell will also often remove DNA bases from the loose ends, or add new bases to the ends to be able to more efficiently glue the two ends together. The result is changes in the DNA over which scientists have no control; the scientists can only hope that the required gene mutation results. The success rate depends on the changes they want to make, but typically they only get the intended result in a few per cent of the cells that undergo the CRISPR treatment.
A low success rate is acceptable when scientists treat cells in the lab, where they can carefully select the few cells with the right mutation. It is more problematic when CRISPR is to be injected directly into patients with a genetic disease. In those cases a low success rate could have unintended and serious consequences. With the further risk of unintended mutations in other places of the DNA, such treatment could prove more harmful than beneficial.
1303 patent applications including CRISPR were recorded globally in 2017.
New CRISPR makes cautious cuts
So several scientists are working to fix the most severe weaknesses of the method. One of the major breakthroughs has been made by chemist David Liu, who developed a new and more accurate variant of CRISPR:
called prime editing. Liu’s method uses a new version of the Cas9 enzyme that cuts only halfway through, so that only one of the two DNA strands is cut. Unlike traditional CRISPR, such prime editing does not produce two loose ends that can be combined with different DNA by mistake.
The Cas9 enzyme also carries a blueprint within it for the new DNA sequence that the scientists would like to insert into the gene, along with an additional enzyme which can build the new DNA sequence based on this blueprint.
Finally, Cas9 cuts the other strand of the DNA in two, and the required change is also inserted there.
In 2019, David Liu showed how he could use prime editing to exchange one single DNA base in a gene with a success rate of up to 55%. The chemist also designed a system to remove or insert entire sequences of DNA bases – and this performed with a success rate of up to 78%.
Liu’s method is already employed by other scientists. In 2020, Dutch scientists used the method to correct gene errors in small fragments of sick tissue that they had extracted from patients and cultivated in the lab. The scientists were able to insert three extra DNA bases in the DGAT1 gene and thereby remove the genetic reason for a severe hereditary type of diarrhoea that makes it almost impossible for the carrier to absorb nutrients from food.
Gene editing that avoids genes
The risk that CRISPR creates permanent damage to the DNA can also be lowered in a completely different way: by creating genetic changes without changing the gene itself.
Genes function as blueprints for the formation of proteins, and usually the ultimate aim of editing a gene is to change the protein. In 2017, biochemist Feng Zhan developed a CRISPR variant that can alter the protein by interfering with a step in between the reading of the gene and the making of the protein. When the cell produces a protein, it first translates the gene in question into an RNA sequence that then carries the gene’s instructions to the cell protein factory. Zhang’s new method corrects the RNA sequence, rather than the gene itself. The result is the same, but with the advantage that unlike DNA, RNA is continuously broken down in the cells. When scientists stop the treatment, all their changes will disappear, and the cell will not have any permanent damage to its hereditary material.
The method seems obviously applicable to treatment of diseases that require a change of the proteins of cells only for specific periods of time – for headaches, say, or other types of pain in which proteins involved in nerve cell pain signals can be temporarily altered or invalidated.
A third new CRISPR variant can affect the formation of proteins without edits to either DNA or RNA sequences. Instead, it removes methyl groups – small molecules that stick to the DNA and prevent a gene being translated into RNA and protein. The method has already been used in the lab to clear the FMR1 gene of methyl groups. When this gene is blocked, it can result in fragile X syndrome – a condition that can cause intellectual challenges.
But the third method has still greater potential. Methyl groups play an important role in many conditions from autism to cancer, and a method for removing them could lead to treatments that are unimaginable using traditional CRISPR.
CRISPR in your eye
The race to improve CRISPR needs to be a quick one, given that the method is already being increasingly tested on people. In March 2020, American ophthalmologist Mark Pennesi initiated an experiment by which CRISPR is injected directly under the retina of the eye. This technique was used on a series of patients who suffer from a type of blindness known as LCA10, or Leber congenital amaurosis 10. The condition is caused by a congenital mutation of the CEP290 gene. The CRISPR treatment aims to cut this mutation out of the gene in the patients’ retinas, so the patients might be able to regain their eyesight. The method has proved promising in mice, but there are not yet any positive results from the experiment in humans. Hopefully it works as planned, but there remains some risk that it could cause new and unwanted mutations in the patients, just as in He Jinankui’s Chinese twins.