Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette

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…

• NS
  National Science Foundation

  Awards: P40 OD010440, R01 GM083071, 0917726
• HH
  Helen Hay Whitney Foundation
• NI
  National Institutes of Health

  Awards: OD010440, NIH T32, GM083071, T32 CA009156, P40 OD010440, R01 GM083071, NIH R01
• DO
  Division of Integrative Organismal Systems