title: Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette.
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.
authors.affiliation: Department of Biology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-3280 ddickins@live.unc.edu.
authors.forename: Daniel J
authors.initials: DJ
authors.lastname: Dickinson
authors.affiliation: Department of Biology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-3280.
authors.forename: Ariel M
authors.initials: AM
authors.lastname: Pani
authors.affiliation: Department of Biology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-3280.
authors.forename: Jennifer K
authors.initials: JK
authors.lastname: Heppert
authors.affiliation: Department of Biology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-3280.
authors.forename: Christopher D
authors.initials: CD
authors.lastname: Higgins
authors.affiliation: Department of Biology and Lineberger Comprehensive Cancer Center, University of North Carolina, Chapel Hill, North Carolina 27599-3280.
authors.forename: Bob
authors.initials: B
authors.lastname: Goldstein
mesh_terms: D000483:Alleles; D000818:Animals; D017173:Caenorhabditis elegans / Q000235:genetics; D050939:Gene Fusion; D055879:Gene Knock-In Techniques; D056426:Genetic Loci / Q000235:genetics; D023281:Genomics; D020071:Interspersed Repetitive Sequences / Q000235:genetics*; D008164:Luminescent Proteins / Q000235:genetics; D016254:Mutagenesis, Insertional / Q000379:methods*; D011401:Promoter Regions, Genetic / Q000235:genetics; D012689:Sequence Homology, Nucleic Acid
chemical_list: D008164:Luminescent Proteins
keywords: CRISPR/Cas9; Caenorhabditis elegans; gene tagging; homologous recombination; self-excising cassette