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

Area of Science:

• Genetics
• Molecular Biology
• Biotechnology

Background:

• Thousands of genetic variants are linked to diseases, but their functional roles remain largely unknown.
• Accurate assessment of variant pathogenicity and functional impact is crucial for genetic disease research and therapeutic development.

Purpose of the Study:

• To develop a versatile and quantitative assay for determining the functional consequences of genetic variants.
• To enable mechanistic dissection of how specific variants influence cellular phenotypes and processes.

Main Methods:

• Introduction of CRISPR-Select (clustered regularly interspaced short palindromic repeat-Select) assays, including CRISPR-Select^TIME, CRISPR-Select^SPACE, and CRISPR-Select^STATE.
• These assays track the absolute frequencies of knock-in genetic variants relative to a neutral control within cell populations.
• Utilizing flow cytometry to measure variant frequencies as a function of time, space, or specific cell states.

Main Results:

• CRISPR-Select assays accurately quantify variant pathogenicity, drug responsiveness, and in vivo tumor promotion.
• The method causally links variants to phenotypes like motility and invasiveness by correlating them with flow cytometry markers.
• Demonstrated applicability across organoids, nontransformed, and cancer cell lines in both single-well and 96-well formats.

Conclusions:

• CRISPR-Select offers a fast, simple, and quantitative method for functional variant analysis.
• This versatile assay supports research, diagnostics, and drug development for genetic disorders.
• Enables high-throughput functional characterization of genetic variants in diverse biological systems.