Researchers at the Francis Crick Institute have demonstrated that a genetic method called ‘pooled prime editing’ can screen hundreds of variants in a gene at once and identify which variants affect the gene’s function.
Useful terms
Variant of unknown significance: a genetic change that has been identified through genetic testing but with insufficient or conflicting evidence about whether it’s connected to a health condition.
Saturation genome editing: a technique to assess the effects of every possible variant in a gene. A library of variants is cloned into plasmids (small circular DNA molecules) and delivered to human cells. Genetic scissors cut the human DNA at the right site and introduce the desired variants. The number of cells is tracked over time, to see how many cells survive with each variant.
Base editing: a type of genome editing involving converting one base (A, C, T or G) to another at a specific site. Unlike CRISPR, base editing doesn’t break both DNA strands.
Prime editing: a newer type of genome editing, also known as ‘search-and-replace’ editing. It allows precise modifications to a sequence of bases rather than changing an individual base to another. Prime editing involves a prime editing guide RNA to find the right genomic position for a Cas9 nickase enzyme to make a break in the sequence and a reverse transcriptase enzyme to write the new piece of DNA.
Prime editing
Components of prime editing. Credit: Ldinatto.
In the future, this method could allow for genomic research on a mass scale, and help interpret the risk associated with different genetic variants for diseases like cancer.
Genetic variation happens when one or more building blocks in DNA are changed, added or removed. Genetic sequencing can reveal ‘variants of unknown significance’, which account for a large fraction of all variants seen in people. We don’t yet understand how these variants impact a person’s risk of developing a disease.
Building on their previous work using a technique called saturation genome editing to map all the variants in a cancer risk gene, the team at the Crick have now tested a new technique called pooled prime editing, outlined in research published today in Cell Genomics.
First used in 2019, prime editing, also known as ‘search-and-replace’ editing, is a genome editing tool to search for a section in the genome (the entire set of DNA), make a nick and then replace the section with an altered stretch of DNA.
The researchers optimised the technique to engineer large numbers of variants at the same time in human cells, testing this on two tumour suppressor genes, SMARCB1 and MLH1. Mutations that inactivate these genes can cause cancer.
The team first used the prime editing platform to test variants in SMARCB1, identifying specific mutations that caused ‘loss of function’ of the gene and cell death.
They then tested mutations in MLH1 – a risk gene for bowel cancer – for which over 2,000 variants of unknown significance have been recorded on a clinical database. They were able to score over 700 variants, newly identifying loss-of-function variants in both coding and non-coding regions (parts of the gene that don’t contain instructions to make proteins).
This screening identified a cluster of loss-of-function variants in an area of the gene that matched reports in the clinical database, meaning they might be linked to cancer.
The experiments suggest that pooled prime editing has potential to efficiently screen thousands of variants at once, either for basic research to assess variants, or one day for use in the clinic as a diagnostic tool.
Michael Herger, postdoctoral fellow in the Genome Function Laboratory at the Crick, and co-first author, said: “Understanding rare variants in small numbers of people is tricky but very important for doctors to make proper assessments of disease risk. We’ve optimised a pooled prime editing method which could one day screen thousands of variants in a single experiment – a very valuable tool at our disposal.”
Shedding light on which variants of unknown significance are pathogenic will bring clarity to large numbers of people who are told they have one of these variants without understanding its risk. Greg Findlay
Christina Kajba, doctoral clinical fellow in the Genome Function Laboratory at the Crick, and co-first author, said: “A classic question in biological research is what outcome genetic changes have on the function of the cell or animal, and pooled prime editing can help to answer this. We’re now trying to scale the method further to test more variants at once in the most efficient way.”
Greg Findlay, Group Leader of the Genome Function Laboratory at the Crick, and senior author, said: “Prime editing offers more precise and targeted editing than other types of genetic editing. We’ve shown that scaling up prime editing has the benefit of combining this precision with testing variants at mass scale. And we’ve also shown it works in non-coding areas of genes, which is crucial given less than 5% of the human genome codes for proteins.
“Shedding light on which variants of unknown significance are pathogenic will bring clarity to large numbers of people who are told they have one of these variants without understanding its risk.”
The researchers also worked with the Viral Vector Core and Genomics teams at the Crick.
Thumbnail image credit: ILRI/Annabel Slater.