Functional editing of endogenous genes through rapid selection of cell pools (Rapid generation of endogenously tagged genes in Drosophila ovarian somatic sheath cells)

Abstract

The combination of genome-editing and epitope tagging provides a powerful strategy to study proteins with high affinity and specificity while preserving their physiological expression patterns. However, stably modifying endogenous genes in cells that do not allow for clonal selection has been challenging. Here, we present a simple and fast strategy to generate stable, endogenously tagged alleles in a non-transformed cell culture model. At the example of piwi in Drosophila ovarian somatic sheath cells, we show that this strategy enables the generation of an N-terminally tagged protein that emulates the expression level and subcellular localization of the wild type protein and forms functional Piwi–piRNA complexes. We further present a concise workflow to establish endogenously N-terminally and C-terminally tagged proteins, and knockout alleles through rapid selection of cell pools in fly and human models.

CRISPR‐Cas9 Genome Editing and Rapid Selection of Cell Pools

“…Our first goal was to add an epitope tag to the N‐terminus of the endogenous Piwi protein (Meng et al., 2022). Epitope tags are optimized peptides that provide a specific handle for the detection and purification of the tagged protein (Vandemoortele et al., 2019).…”

“…edit the different alleles consecutively or, if the editing efficacy is high in your cell type, simultaneously (Meng et al, 2022). 4.…”

“…After the successful tagging of your protein, we recommend performing experiments to verify that the newly tagged protein is still functional. In the example of endogenously tagged Piwi in OSCs, we performed immunoprecipitation of tagged and endogenous Piwi‐piRNA complexes and compared their associated piRNAs by next‐generation sequencing (Meng et al., 2022).…”

“…Thus, we devised a rapid strategy to select for successfully edited cell pools. We suggest generating multiple pools with the same desired edit using different sgRNAs, when possible, to minimize the contribution of sgRNA off‐target effects (Meng et al., 2022).…”

“…Here, we describe a protocol for generating endogenously tagged proteins and biallelic knockouts without the use of clonal selection in OSCs or human embryonic kidney (HEK) 293 cells (Meng et al., 2022). Our protocol enables robust genome editing in OSCs for the first time and can also be applied to other ex vivo models as a time‐saving alternative to traditional editing protocols.…”