# Generating CRISPR-edited clonal lines of cultured Drosophila S2 cells

## TL;DR

This paper introduces a new CRISPR method to create gene-edited clonal lines in Drosophila S2 cells, overcoming challenges in generating stable cell lines.

## Contribution

A streamlined CRISPR/Cas9 method with selectable markers for efficient generation of clonal Drosophila S2 cell lines.

## Key findings

- A CRISPR method using antibiotic resistance genes improves clonal line generation in Drosophila S2 cells.
- The first acentrosomal S2 cell lines were created by knocking out centriole genes Polo-like Kinase 4/Plk4 or Ana2.
- The method enhances practicality and efficiency of CRISPR genome editing in Drosophila cultured cells.

## Abstract

CRISPR/Cas9 genome editing is a pervasive research tool due to its relative ease of use. However, some systems are not amenable to generating edited clones due to genomic complexity and/or difficulty in establishing clonal lines. For example, Drosophila Schneider 2 (S2) cells possess a segmental aneuploid genome and are challenging to single-cell select. Here, we describe a streamlined CRISPR/Cas9 methodology for knock-in and knock-out experiments in S2 cells, whereby an antibiotic resistance gene is inserted in-frame with the coding region of a gene-of-interest. By using selectable markers, we have improved the ease and efficiency for the positive selection of null cells using antibiotic selection in feeder layers followed by cell expansion to generate clonal lines. Using this method, we generated the first acentrosomal S2 cell lines by knocking-out centriole genes Polo-like Kinase 4/Plk4 or Ana2 as proof of concept. These strategies for generating gene-edited clonal lines will add to the collection of CRISPR tools available for cultured Drosophila cells by making CRISPR more practical and therefore improving gene function studies.