Gene editing innovation to improve genome variant exploration

The haploid cell line HAP1 LIG4 KO has been modified to be highly efficient for CRISPR-based experiments by researchers at the Wellcome Sanger Institute. Available via Revvity to both academic and industry researchers, the cell line has been used to greatly increase gene editing rates in Saturation Genome Editing (SGE) experiments to assess variant effects at scale, with many other applications possible.

The Sanger Institute is renowned for its unique capabilities in building high-throughput platforms, such as the ones built during the pandemic to sequence 20 per cent of all the world’s SARS-COV-2 viruses, or those that enable sequencing thousands of species’ genomes for the first time in the Darwin Tree of Life project.?

A specific cell line called HAP1 LIG4 KO is now at the centre of one of those pipelines - built to explore the effects of all possible single nucleotide variants (SNVs) in genes using Saturation Genome Editing (SGE). SGE uses multiplexed CRISPR/Cas9 assays to introduce each and every mutation across a stretch of DNA to assess its function and implications.

Saturation Genome Editing is a recently developed technique. It was proposed in 2018 by Dr Greg Findlay, at the time at the University of Washington, now at The Francis Crick Institute, to investigate changes in the DNA bases that form each of our genes. This new technique enables researchers to look at how all changes at the individual DNA base level can affect our health.

Talking about it with Dr Andrew Waters , staff scientist in Dr David Adams ’ Lab within the Cancer, Ageing and Somatic Mutations Programme at the Sanger Institute, he explains that as more genomes are being sequenced, researchers are seeing ever-more gene variants. Often, researchers don’t understand whether these variants are disease-causing so they call them Variants of Uncertain Significance (VUS).?

These are modifications of our genes whose effect on our health and well-being is unknown. “These have a big clinical burden,” Andrew says. “Doctors are unsure what those variants do. When one is detected in a patient’s genome, there are complications in terms of patient and family management. Most changes are benign natural variations, but some can lead to serious disease. We need to be able to detect them and ensure clinicians are equipped with the best knowledge to manage their patient’s treatment effectively.”?

The solution is to systematically assess all potential variants in genes before they’re seen in the clinic, as well as those we already know the existence of. According to Andrew, the best way to do this is to work with this new technique, Saturation Genome Editing (SGE). Ambitious alliances such as the Atlas of Variant Effects Alliance are exploring genome variants in depth. They are looking to map the effects of all mutations in our genes to predict what a certain mutation - even one no one has ever seen - can do to our health.?

As with all new techniques, the pipelines built around them needed to be tailored. Andrew and his colleagues at the Sanger Institute are constantly innovating to enable these complex experiments. They have now engineered an existing cell line, HAP1 LIG4 KO - originally available from the commercial company Revvity - to create an enhanced cellular platform for ambitious projects such as the Atlas of Variant Effects. The cell line is available and distributed by Horizon Discovery to both academic and industry researchers.?

SGE relies on a process called Homology Directed Repair (HDR) to incorporate variants into the genome. Revvity HAP1 cells with a knockout of the gene LIG4 have higher rates of HDR, making HAP1 LIG4 KO-cells ideal for saturation genome editing. Sanger researchers edited these cells further by incorporating stably expressed Cas9 nuclease into the genome of the cell line, creating a successfully engineered cell that can now be used by other researchers working in the field of Saturation Genome Editing and other CRISPR-based approaches.?

They used Revvity’s HAP1 LIG4 KO cell line due to it being haploid, where cells contain a single set of chromosomes rather than the usual two. This allows experiments to be more precise as variant effects are not masked by any potentially unedited/wild-type gene copies.?

Saturation Genome Editing projects are key to diagnosing rare diseases in particular.? Developing this enhanced platform for gene variation exploration has allowed Andrew and his colleagues within the Adams Lab to study the effects of more than 18,000 variants in the BAP1 gene . Pathogenic variants in this gene can lead to a rare inherited disorder called BAP1 tumour predisposition syndrome, which increases the risk of developing cancerous tumours in the skin, brain, kidney, eye or mesothelium, the layer of tissue surrounding the organs in our chest. In very rare cases BAP1 variants cause a neurodevelopmental syndrome in children.?

When asked why this technique is an improvement on previous approaches, Andrew explains that deep mutational scanning allows for the examination of all possible variants within a gene, a goal that is not achievable using other techniques.

“Previous studies examined single variants in isolation, which is a tremendous amount of work when you consider the scale of conceivable variation in human genes. Deep mutational scanning allows all possible mutations to be studied in one experiment, and with SGE, we can also measure variants at very high resolution,” Andrew says. “With this new technology, we can look at the variant in its native genomic context, which means we get a much more accurate representation of how these variants behave in people, not just in a petri dish.”

In his latest papers, Andrew and the Adams Lab have analysed specific genes and correlated their findings with patient data from the UK BioBank repository. Finding that BAP1 variants measured by SGE as deleterious do indeed correlate with cancer diagnoses, and further that this may be due to higher levels of a growth factor in the carriers’ blood, which with more research, could be targeted in new therapies against BAP1-related disease.?

This work wouldn’t have been possible without the team’s innovative approach to their experiments. By tweaking an existing cell line, they have developed a pipeline that will allow for SGE to be scaled to cover hundreds of genes active in cancer and/or neurodevelopmental disorders. The cell line is now available for other researchers in the field to use via Revvity’s platform.?

Sebastian Gerety, principal scientist at the Sanger Institute, who has worked with Andrew on these projects in addition to his own team’s work on the neurodevelopmental disorder gene DDX3X , says: “At the Sanger Institute we are keen to share the cell lines that we generate with the scientific community. We hope that the collective efforts in studying disease-associated genes and functional elements will lead to advances in diagnosing and treating human disease and understanding genes, gene products, and their regulation.”

Agnieszka Wabik , Business Development Manager at the Sanger Institute, says: “Partnering with Revvity allows us to extend the reach and properly distribute this cell line. Our scientists obtained the HAP1 LIG4 KO cell line from Revvity with a set of conditions that allowed us to further modify the cell line, but not distribute it. With this innovation, our scientists have created a resource which we hope will accelerate the understanding of Variants of Uncertain Significance. Partnering with commercial providers is often a more efficient way to create a stable distribution of our resources for academics and industry researchers alike to make use of our innovation.”