Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette

TLDR

Genome engineering aims to reduce the time and labor required for custom genome modifications. The study introduces a new selection strategy for generating fluorescent protein knock‑ins via CRISPR/Cas9‑mediated homologous recombination. The approach employs a self‑excisable cassette containing a drug‑resistance gene, a visible marker, and inducible Cre recombinase, flanked by LoxP sites and inserted into a synthetic intron of an FP tag; heat shock induces Cre to excise the cassette, leaving only the FP tag. The strategy enables generation of knock‑in alleles in any genetic background without PCR screening or a second injection step, and allows simultaneous production of fluorescent protein fusions, transcriptional reporters, and loss‑of‑function alleles in a single injection.

Abstract

Abstract A central goal in the development of genome engineering technology is to reduce the time and labor required to produce custom genome modifications. Here we describe a new selection strategy for producing fluorescent protein (FP) knock-ins using CRISPR/Cas9-triggered homologous recombination. We have tested our approach in Caenorhabditis elegans. This approach has been designed to minimize hands-on labor at each step of the procedure. Central to our strategy is a newly developed self-excising cassette (SEC) for drug selection. SEC consists of three parts: a drug-resistance gene, a visible phenotypic marker, and an inducible Cre recombinase. SEC is flanked by LoxP sites and placed within a synthetic intron of a fluorescent protein tag, resulting in an FP–SEC module that can be inserted into any C. elegans gene. Upon heat shock, SEC excises itself from the genome, leaving no exogenous sequences outside the fluorescent protein tag. With our approach, one can generate knock-in alleles in any genetic background, with no PCR screening required and without the need for a second injection step to remove the selectable marker. Moreover, this strategy makes it possible to produce a fluorescent protein fusion, a transcriptional reporter and a strong loss-of-function allele for any gene of interest in a single injection step.