Multiparametric and accurate functional analysis of genetic sequence variants using CRISPR-Select
Yiyuan Niu, Catarina A. Ferreira Azevedo, Xin Li, Elahe Kamali, Ole Haagen Nielsen, Claus Storgaard Sørensen, Morten Frödin
Abstract
Abstract Determining the functional role of thousands of genetic sequence variants (mutations) associated with genetic diseases is a major challenge. Here we present clustered regularly interspaced short palindromic repeat (CRISPR)-Select TIME , CRISPR-Select SPACE and CRISPR-Select STATE , a set of flexible knock-in assays that introduce a genetic variant in a cell population and track its absolute frequencies relative to an internal, neutral control mutation as a function of time, space or a cell state measurable by flow cytometry. Phenotypically, CRISPR-Select can thereby determine, for example, pathogenicity, drug responsiveness/resistance or in vivo tumor promotion by a specific variant. Mechanistically, CRISPR-Select can dissect how the variant elicits the phenotype by causally linking the variant to motility/invasiveness or any cell state or biochemical process with a flow cytometry marker. The method is applicable to organoids, nontransformed or cancer cell lines. It is accurate, quantitative, fast and simple and works in single-well or 96-well higher throughput format. CRISPR-Select provides a versatile functional variant assay for research, diagnostics and drug development for genetic disorders.