The Role of Revvity’s Cell Line in Supporting Wellcome Sanger Institute's genome variant exploration

As the biggest contributor to the Human Genome Project, the United Kingdom-based Wellcome Sanger Institute is known for embarking upon research projects that are as ambitious as they are valuable. 

One such ongoing project is the Atlas of Variant Effects (AVE) Alliance, for which the Institute is a founding member. The AVE aims to define the impacts of variants upon protein function and gene regulation in disease-related genes and functional elements. To achieve its goal, the Institute uses high-throughput, CRISPR/Cas9-mediated Saturation Genome Editing (SGE) to explore the effects of all possible single nucleotide variants (SNVs) within a gene of interest. For this technique to work at a large scale, the Institute needed a haploid cellular platform with high rates of homologous repair (HR) which could be efficiently modified with homology-directed repair (HDR). These requirements led the Institute to Revvity’s HAP1 LIG4 knockout (HAP1 LIG4(-)) cell line. 

By virtue of its mechanism, CRISPR/Cas9-mediated genome editing introduces DNA double-strand breaks (DDSBs) at precise locations within a selected gene before the break undergoes homology-directed repair (HDR; a form of HR). When cells detect DDSBs, they typically utilize either HR or non-homologous end joining (NHEJ) to repair them. NHEJ, the preferred repair pathway of both proliferating and non-dividing cells, can interfere with CRISPR/Cas9 gene editing due to its speed and mechanistic flexibility. DNA ligase 4 is a key component of NHEJ, and studies have demonstrated that by knocking out its parent gene, LIG4, the NHEJ pathway can be disabled. With an inactivated NHEJ pathway, Revvity's HAP1 LIG4 (-) cell line is well-suited for experiments that require highly efficient CRISPR-Cas9 gene editing. 

To generate their desired cellular platform, the Wellcome Sanger Institute began with the HAP1 LIG4(-) cell line and incorporated a stably expressed Cas9 nuclease (HAP1 LIG4(-) Cas9(+)). The haploid background of this line simplifies the interpretation of results, and the combined absence of LIG4 and the presence of Cas9 significantly enhances genome editing efficiency.

To learn more about the Wellcome Sanger Institute’s AVE Alliance and the role of Revvity’s HAP1 LIG4(-) cell line, see our white paper wherein we interview Dr. Sebastian Gerety, Principal Staff Scientist in the Human Genetics Program at The Wellcome Sanger Institute.