Developing a high-throughput CRISPR-based assay for saturation mutagenesis of human genes. M. L. Carpenter, C. Lee, N. Hammond, A. Li, A. Adams, C. D. Bustamante, M. C. Bassik Department of Genetics, Stanford University, Stanford, CA.

   One of the biggest challenges currently facing the clinical translation of whole-genome sequencing is our lack of knowledge about the functional impact of the majority of variants. Although many variant annotation tools have been developed to take advantage of characteristics such as conservation, amino acid properties, population frequency, and genic location (e.g., splice site, promoter), these predictions are rarely experimentally verified. Most functional testing usually occurs on a case-by-case basis, and it is often difficult to directly compare results between laboratories. In this study, we aim to develop an experimental method to simultaneously and consistently assay the impact of many mutations in a single gene. We will describe our implementation of a CRISPR/Cas9-based approach to create a population of human cells in which each cell harbors a different mutation in the same gene, TP53. These cells can be grown in bulk under an appropriate selective pressure and then sequenced to determine the abundance of each mutation. This abundance serves as a proxy for the pathogenicity of each mutation, and even has the potential to reveal unexpected effectsfor example, pathogenic synonymous mutations. As a test case, we have used CRISPR/Cas9 to install a set of 5 known pathogenic and 5 benign mutations in the human gene TP53 in the MCF7 breast cancer cell line, as well as 10 mutations categorized as variants of unknown significance (VUS). We have characterized the effects of these mutations on growth and DNA damage sensitivity in cells; these measurements will allow us to assess the sensitivity and specificity of our assay and will help set benchmarks for the future assessment of VUS. At the same time, we are developing technologies for improving the efficiency of producing single mutations in individual cells in large populations using the CRISPR system. Our experimental pathogenicity measures will eventually be incorporated into the new ClinGen resource, which is being developed by our group and others to serve as a central, curated repository for clinically relevant variant information.

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