Image credit: Laura Olivares Boldú, Wellcome Connecting Science
Prime editing is the latest technique that enables scientists to alter the DNA sequence of a living cell. It is the next big step in CRISPR-Cas9 technology, with fewer errors and off-target effects. It offers precision, flexibility, and the potential to edit human cells to treat genetic diseases.
How DNA works
A genome is made of a chemical called deoxyribonucleic acid, or DNA for short. DNA contains four basic building blocks or 'letters' which are chemical bases: adenine (A), cytosine (C), guanine (G) and thymine (T). The order, or sequence, of these bases form the instructions in the genome.
DNA is a two-stranded molecule with a unique ‘double helix’ shape, like a twisted ladder.
Each strand is composed of long sequences of the four bases, A, C, G and T. The bases on one strand of the DNA molecule pair together with complementary bases on the opposite strand of DNA to form the ‘rungs’ of the DNA ladder. The bases always pair together in the same way, A with T, C with G.
DNA synthesis and replication in cells involves biological molecules called enzymes. Enzymes are usually proteins, and they are responsible for thousands of metabolic processes essential to life.
RNA is a nucleic acid similar to DNA, but with only a single, helical strand of bases. It plays a key role in turning DNA instructions into functional proteins.
DNA Structure
How DNA works
A genome is made of a chemical called deoxyribonucleic acid, or DNA for short. DNA contains four basic building blocks or 'letters' which are chemical bases: adenine (A), cytosine (C), guanine (G) and thymine (T). The order, or sequence, of these bases form the instructions in the genome.
DNA is a two-stranded molecule with a unique ‘double helix’ shape, like a twisted ladder.
Each strand is composed of long sequences of the four bases, A, C, G and T. The bases on one strand of the DNA molecule pair together with complementary bases on the opposite strand of DNA to form the ‘rungs’ of the DNA ladder. The bases always pair together in the same way, A with T, C with G.
DNA synthesis and replication in cells involves biological molecules called enzymes. Enzymes are usually proteins, and they are responsible for thousands of metabolic processes essential to life.
RNA is a nucleic acid similar to DNA, but with only a single, helical strand of bases. It plays a key role in turning DNA instructions into functional proteins.
DNA Structure
CRISPR-Cas9 genome editing
CRISPR-Cas9 Â technologies were developed in 2012. In this system, the Cas9 enzyme cuts the double-stranded DNA molecule at a specified location, determined by a guide RNA.
The cell recognises the double-stranded break, and repairs the damage. The cell’s repair process is sometimes inaccurate, meaning that mutations are introduced. If the cut is in a gene, the gene can become inactivated or altered.
DNA templates can be used to introduce sequences of bases, and multiple cuts can be made to delete stretches of DNA.
Subsequent DNA sequencing can determine the exact mutation, insertion or deletion that has occurred.
The technique has been used to understand the roles of genes in different diseases, such as cancer.
Base editors
Base editors were an innovation expanding on CRISPR-Cas9 and were called ‘molecular pencils’ for their ability to alter single bases of DNA.
Building on CRISPR-Cas9 genome editing technologies, prime editing allows DNA to be rewritten.
CRISPR-Cas9 genome editing
CRISPR-Cas9 Â technologies were developed in 2012. In this system, the Cas9 enzyme cuts the double-stranded DNA molecule at a specified location, determined by a guide RNA.
The cell recognises the double-stranded break, and repairs the damage. The cell’s repair process is sometimes inaccurate, meaning that mutations are introduced. If the cut is in a gene, the gene can become inactivated or altered.
DNA templates can be used to introduce sequences of bases, and multiple cuts can be made to delete stretches of DNA.
Subsequent DNA sequencing can determine the exact mutation, insertion or deletion that has occurred.
The technique has been used to understand the roles of genes in different diseases, such as cancer.
Base editors
Base editors were an innovation expanding on CRISPR-Cas9 and were called ‘molecular pencils’ for their ability to alter single bases of DNA.
Building on CRISPR-Cas9 genome editing technologies, prime editing allows DNA to be rewritten.
Prime editing
In the prime editing system, the Cas9 has been modified. Termed a ‘Cas9 nickase’, it doesn’t cause a double-stranded break. Instead, it cuts just one strand of the DNA molecule. The Cas9 is also fused to a reverse transcriptase enzyme, which can synthesise DNA.
To manoeuvre the nickase to the desired location on the genome, the Cas9 binds to a guide RNA sequence
The RNA sequence several parts. One is the guide sequence, which can be made to compliment any area of the genome, and so directs the Cas9 into place. It also has the desired sequence ‘template’, which can be used to add to, or alter, the genome. The guide RNA also includes a primer sequence. This RNA molecule is the prime editing guide or 'pegRNA'.
Once the DNA has been cut by the nickase, the RNA primer in the pegRNA binds to the resulting DNA flap.
The reverse transcriptase then synthesises DNA, based on the RNA template. The edited sequence can be incorporated into the genome by the cell’s natural machinery.
The other, unedited, strand of DNA is repaired by the cell to match the new sequence. Alternatively, the unedited sequence is incorporated back into the genome leaving the site in its original state and the editing process can start again.
The system can be used to change a base of DNA to any other. This is an improvement on previous technologies, which could only make four of the potential 12 base substitutions. Or, it can be used to insert or delete a specific sequence of DNA, of varying length.
Unwanted and off-target effects have been shown to be relatively rare.
Dubbed ‘molecular word processors’, the technology was developed by a team of researchers at the Broad Institute of MIT and Harvard, led by Dr David Liu, who has been working on developing gene-editing technologies for over a decade.
Prime editing
In the prime editing system, the Cas9 has been modified. Termed a ‘Cas9 nickase’, it doesn’t cause a double-stranded break. Instead, it cuts just one strand of the DNA molecule. The Cas9 is also fused to a reverse transcriptase enzyme, which can synthesise DNA.
To manoeuvre the nickase to the desired location on the genome, the Cas9 binds to a guide RNA sequence
The RNA sequence several parts. One is the guide sequence, which can be made to compliment any area of the genome, and so directs the Cas9 into place. It also has the desired sequence ‘template’, which can be used to add to, or alter, the genome. The guide RNA also includes a primer sequence. This RNA molecule is the prime editing guide or 'pegRNA'.
Once the DNA has been cut by the nickase, the RNA primer in the pegRNA binds to the resulting DNA flap.
The reverse transcriptase then synthesises DNA, based on the RNA template. The edited sequence can be incorporated into the genome by the cell’s natural machinery.
The other, unedited, strand of DNA is repaired by the cell to match the new sequence. Alternatively, the unedited sequence is incorporated back into the genome leaving the site in its original state and the editing process can start again.
The system can be used to change a base of DNA to any other. This is an improvement on previous technologies, which could only make four of the potential 12 base substitutions. Or, it can be used to insert or delete a specific sequence of DNA, of varying length.
Unwanted and off-target effects have been shown to be relatively rare.
Dubbed ‘molecular word processors’, the technology was developed by a team of researchers at the Broad Institute of MIT and Harvard, led by Dr David Liu, who has been working on developing gene-editing technologies for over a decade.
How are scientists using it?
Because prime editing is more accurate than previous CRISPR-Cas9 technologies, and a single letter of DNA can be edited to any other letter, as well as additional letters added, the hope is that diseases caused by genetic mutations can be targeted.
The team that developed the technology has used it to alter the genome sequence of human cell lines, grown in the laboratory, to correct the genetic mistakes responsible for sickle cell disease and Tay-Sachs disease, a rare inherited condition.
The applications, in the future, could be huge. Over 16,000 small deletion variants – where a small number of DNA bases have been removed from the genome – have been causally linked to disease. This includes cystic fibrosis, where 70 per cent of cases are caused by the deletion of just three DNA bases. In 2022, base-edited T-cells were successfully used to treat a patient’s leukaemia, where chemotherapy and bone marrow transplant had failed.
However, much more research is needed to understand and improve prime editing in a broad range of cell types. Off-target effects need to be assessed across the whole genome. Long-term effects (if any) also need to be measured.
At the Wellcome Sanger Institute, several teams are using prime editing to alter cells’ genomes and determine the effects. In a 2023 study, scientists designed 3,604 DNA sequences of between one and 69 DNA bases in length. These were inserted into three different human cell lines, using different prime editor delivery systems in various DNA repair contexts. The cells were genome sequenced to see if the edits had been successful or not.
The insertion efficiency, or success rate, of each sequence was assessed to determine common factors in the success of each edit. The length of sequence was found to be a key factor, as was the type of DNA repair mechanism involved. To help make sense of these data, the researchers turned to machine learning to detect patterns that determine insertion success, such as length and the type of DNA repair involved. Once trained on the existing data, the algorithm was tested on new data and was found to accurately predict insertion success.
Find out more
- Find out more about genomics at yourgenome.org.
- For more information about how Sanger Institute researchers are using prime editing, see Dr Leopold Parts research page.
