Journal
GENETICS
Volume 200, Issue 4, Pages 1035-+Publisher
GENETICS SOCIETY AMERICA
DOI: 10.1534/genetics.115.178335
Keywords
CRISPR/Cas9; homologous recombination; gene tagging; Caenorhabditis elegans; self-excising cassette
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Funding
- U.S. National Institutes of Health (NIH) Office of Research Infrastructure Programs [P40 OD010440]
- NIH [T32 CA009156, R01 GM083071]
- Helen Hay Whitney Foundation
- U.S. National Science Foundation (NSF) graduate research fellowship
- NSF [IOS 0917726]
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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 Caenorhabditiselegans. 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.
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