UCL Institute of Ophthalmology


Genome Editing

Genome editing is a method that can be used to change the DNA of cells.

What is genome editing? 

Genome editing is a method that allows researchers to make specific changes to the DNA of living cells, modifying, removing or adding to the DNA sequence. Having the ability to precisely edit the DNA in cells has great implications for inherited diseases. In theory, gene editing could allow us to fix any DNA mutation in any gene, treating or curing the disease the mutation causes. 

Image showing DNA being cut with a scalpel to represent DNA editing techniques
 Genome editing is reliant on two events 1) using an enzyme to cut DNA at a specific point and 2) a cellular repair mechanism that can fix the DNA back together, repairing or modifying the mutation site. 


Using enzymes to cut DNA 

A number of enzyme-based methods can be used to cut DNA, including Zinc-fingered nucleases (ZFNS), Transcription activator-like effector-based nucleases (TALENS), meganucleases and the CRISPR-Cas9 system, which stands for Clustered Regularly Interspaced short palindromic repeats with CRISPR associated protein 9. 

CRISPR-Cas9 is by far the most popular method to edit DNA. CRISPR-Cas9 is a simple and versatile DNA editing system, which has become an exciting prospect for developing human therapies for genetic diseases, due to its efficiency, ease of use and low cost. The CRISPR-Cas9 system was developed from a naturally occurring viral protection response present in microorganisms, like bacteria. When bacterial cells are infected by a virus, they create a DNA record of it. The bacteria then uses this DNA record as a “guide” for the Cas9 enzyme, allowing it to recognise, target and cut the foreign viral DNA, disabling the virus. 

Schematic of CRISPR-Cas9 protein complex entering a cell, binding to the target DNA and cutting at the programmed site.

Rather than using CRISPR-Cas9 to cut viral DNA, scientists have adapted the system to cut any DNA sequence, including human DNA. To do this, the Cas9 enzyme and a synthetic guide, designed to bind to the target DNA sequence, are inserted into the cell. The guide and Cas9 enzyme join together to form a complex that surveys DNA until it finds a sequence that matches the guide. Here, the CRISPR-Cas9 system acts as a pair of molecular scissors, cutting the DNA at the target point. Scientists can direct CRISPR-Cas9 to break the double strands of any region of human DNA by modifying the sequence of the guide. 


Repairing the DNA and fixing the mutation 

DNA breaks are not a rare phenomenon in cells, these breaks occur during normal cellular activities, or from exposure to ultraviolet light, ionising irradiation (e.g. x-rays) or chemicals. Because of this, cells have evolved a number of ways to repair the damaged DNA.  

These cellular repair mechanisms can be used to edit the DNA, switching off a gene or replacing the original mutated region with a healthy copy. 

End Joining 

The most common mechanism used to repair double strand DNA breaks in cells is to join the ends back together. End joining is a quick-fix method to repair DNA, however the mechanism isn’t perfect, often leading to unexpected changes in the repaired DNA sequence. Typically, end joining leads to the deletion or insertion of one or two DNA bases, altering the meaning of the subsequent DNA code once the broken DNA strands are re-joined. End joining can create “nonsensical” DNA, which means that gene no longer contains the correct instructions to make its protein. In genome editing, DNA repair using end joining is an effective way to “switch” a gene off, stopping the cell making a defective protein. 

Schematic of end join proteins binding to broken DNA in order to repair. Shows the possibility of removing DNA bases or inserting DNA bases as a by-product of repair.

Directed repair 

The cell also has more efficient DNA repair mechanisms that removes the damaged region of DNA and replaces it with a new copy, this method of DNA repair is known as recombination. To do this the cell uses a copy of the same DNA to fix the gene. Usually when damaged DNA is detected, the cell can use the sister chromosome, inherited from the other parent, to fix the broken DNA on the affected chromosome.  

For gene editing, a new copy of the DNA can be inserted with the Cas9 and guide to repair the mutated area. To do this scientists create a small copy of damaged DNA region with a “fixed” mutation site, this healthy “donor template” replaces the mutated DNA, restoring normal function in affected cells.  

Schematic showing broken DNA being repaired by the insertion of a healthy DNA template into the break site and the binding of the original DNA to either side of this template

Gene editing as a therapy for disease 

Gene editing is a relative new technology and is currently being used in the clinic to treat diseases where cells can be modified outside of the body before being replaced, such as cancer, blood disorders and AIDS. 

Gene editing could be an ideal way to treat inherited retinal diseases caused by single gene mutations. Eye researchers are using models to test the use of genome editing as a way to correct disease-causing mutations. Gene editing has been used in the lab to fix mutations causing retinal dystrophies in rats, and many research groups are using induced pluripotent cells to test whether editing works in patient cells in a dish. 

In March 2020, CRISPR-Cas9 editing was used for the first time to treat a patient with Lebers Congenital Amaurosis, an inherited disease and common cause of childhood blindness https://www.nature.com/articles/d41586-020-00655-8. The breakthrough trial, named BRILLANCE, is a major step from gene editing a cell in a dish to treating a patient by injecting the CRISPR-Cas9 therapy as a virus directly into the eye. 

However, there are still a number of challenges that need to be overcome before gene editing is commonplace in eye clinics, including delivery of the gene editing system to target cells, efficiency of the therapy and controlling the specificity of the editing mechanisms in RPE and retinal cells. 

Is genome editing available for patients with Bestrophinopathies? 

Currently genome editing is not available for patients with Bestrophinopathies however, research groups are investigating this approach to treat these diseases. 

The Carr lab at the UCL Institute of Ophthalmology is currently investigated the use of CRISPR gene editing to treat dominant forms of Bestrophinopathies. The project, funded by The Macular Society, is using RPE cells created from patient induced pluripotent stem cells to test this novel approach. 

David Gamm’s lab at the McPherson Eye Research Institute has been testing CRISPR-Cas9 genome editing using induced pluripotent stem cells from patients with Best disease. Promising results suggest that gene editing can be used to switch off the mutated copy of BEST1, however the group highlight potential concerns about the specificity of gene editing. 

David Gamm - Human iPSC modeling reveals mutation-specific responses to gene therapy in Best disease