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  2. Multiparametric and accurate functional analysis of genetic sequence variants using CRISPR-Select

Multiparametric and accurate functional analysis of genetic sequence variants using CRISPR-Select

  • Nat Genet. 2022 Dec;54(12):1983-1993. doi: 10.1038/s41588-022-01224-7.
Yiyuan Niu # 1 Catarina A Ferreira Azevedo # 1 Xin Li # 1 Elahe Kamali # 1 Ole Haagen Nielsen 2 Claus Storgaard Sørensen 3 Morten Frödin 4
Affiliations

Affiliations

  • 1 Biotech Research and Innovation Centre (BRIC), Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark.
  • 2 Department of Gastroenterology, Herlev Hospital, University of Copenhagen, Copenhagen, Denmark.
  • 3 Biotech Research and Innovation Centre (BRIC), Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. [email protected]
  • 4 Biotech Research and Innovation Centre (BRIC), Faculty of Health Sciences, University of Copenhagen, Copenhagen, Denmark. [email protected]
  • # Contributed equally.
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)-SelectTIME, CRISPR-SelectSPACE and CRISPR-SelectSTATE, 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.

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