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Updated: Oct 3, 2025

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Genome Editing in Mammalian Cell Lines using CRISPR-Cas

Published on: April 11, 2019

Saturation variant interpretation using CRISPR prime editing.

Steven Erwood^1,2, Teija M I Bily², Jason Lequyer^1,3

¹Department of Molecular Genetics, University of Toronto, Toronto, ON, Canada.

Nature Biotechnology

|February 22, 2022

Summary

This summary is machine-generated.

Saturation prime editing (SPE) enables high-throughput functional characterization of genetic variants. This method accurately classifies mutations, advancing understanding of genetic disorders like Niemann-Pick disease.

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Published on: December 11, 2020

Area of Science:

Genetics
Molecular Biology
Biotechnology

Background:

Functional characterization of genetic variants is crucial for understanding disease.
Existing methods using homology-directed repair have limitations in editing efficiency.
High-throughput analysis of variants in their endogenous locus is challenging.

Purpose of the Study:

To adapt CRISPR prime editing for high-throughput variant classification.
To develop a strategy for locus haploidization to simplify variant interpretation.
To establish an efficient and accurate method for functional variant characterization.

Main Methods:

Adapted CRISPR prime editing for high-throughput variant classification.
Combined prime editing with a locus haploidization strategy.
Applied saturation prime editing (SPE) to NPC1 and BRCA2 genes.

Main Results:

Demonstrated the utility of SPE for variant classification in the NPC1 gene.
Identified 410 out of 706 missense mutations in NPC1 as deleterious.
Showed that SPE is translatable to other genes, exemplified by BRCA2 functional characterization.
Derived variant function scores reflecting diverse molecular defects.

Conclusions:

SPE provides an efficient and accurate method for high-throughput functional characterization of genetic variants.
This approach simplifies variant interpretation through locus haploidization.
SPE is a versatile tool applicable to various genes and disease contexts, including Niemann-Pick disease and potentially cancer-related genes like BRCA2.