# Protocol to rapidly screen CRISPR-Cas9 gene editing outcomes in a cell population by mutating eGFP to a blue or non-fluorescent phenotype

## TL;DR

This paper introduces a protocol to quickly assess CRISPR-Cas9 gene editing outcomes by tracking changes in fluorescent proteins in cells.

## Contribution

The protocol enables scalable and high-throughput differentiation of DNA repair outcomes using eGFP to BFP mutations.

## Key findings

- The protocol distinguishes between non-homologous end joining and homology-directed repair outcomes in eGFP-positive cells.
- It provides a scalable method for assessing gene editing techniques using fluorescence-based readouts.

## Abstract

When designing genome editing therapy, it is crucial to measure outcomes of DNA damage repair. Here, we present a protocol to distinguish the outcome of targeted DNA damage repair from the bottom up, through a previously established readout of enhanced green fluorescent protein (eGFP) to blue fluorescent protein (BFP) mutations. We describe steps for producing eGFP-positive cells and differentiating between non-homologous end joining-induced gene knockout and homology-directed repair-induced-directed mutation in these cells. This protocol has potential for high-throughput and scalable assessment of gene editing techniques.

For complete details on the use and execution of this protocol, please refer to Walther et al.1 and Wilbie et al.2

•Instructions for generation of eGFP-positive cell lines via lentiviral transduction•Steps for transfection of gene editing reagents in eGFP-positive cells•Steps for cell handling post-transfection and measuring cell fluorescence using FACS•Guidance on FACS data processing, analysis, and interpretation of gene editing outcomes

Instructions for generation of eGFP-positive cell lines via lentiviral transduction

Steps for transfection of gene editing reagents in eGFP-positive cells

Steps for cell handling post-transfection and measuring cell fluorescence using FACS

Guidance on FACS data processing, analysis, and interpretation of gene editing outcomes

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

When designing genome editing therapy, it is crucial to measure outcomes of DNA damage repair. Here, we present a protocol to distinguish the outcome of targeted DNA damage repair from the bottom up, through a previously established readout of enhanced green fluorescent protein (eGFP) to blue fluorescent protein (BFP) mutations. We describe steps for producing eGFP-positive cells and differentiating between non-homologous end joining-induced gene knockout and homology-directed repair-induced-directed mutation in these cells. This protocol has potential for high-throughput and scalable assessment of gene editing techniques.