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. 2015 Aug;200(4):1035-49.
doi: 10.1534/genetics.115.178335. Epub 2015 Jun 3.

Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette

Daniel J Dickinson  1 , Ariel M Pani  2 , Jennifer K Heppert  2 , Christopher D Higgins  2 , Bob Goldstein  2
Affiliations

Streamlined Genome Engineering with a Self-Excising Drug Selection Cassette

Daniel J Dickinson et al. Genetics. 2015 Aug.

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.

Keywords: CRISPR/Cas9; Caenorhabditis elegans; gene tagging; homologous recombination; self-excising cassette.

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Figures

Figure 1
Figure 1
Design of an improved gene tagging workflow. (A) Design of a self-excising cassette for drug selection. SEC consists of a hygromycin resistance gene (hygR), a visible marker [sqt-1(d)], and an inducible Cre recombinase (hs::Cre). SEC is flanked by LoxP sites and placed within a synthetic intron in an FP::3xFlag tag, so that the LoxP site that remains after marker excision is within an intron. (B) Plate phenotype of animals homozygous for a sqt-1(d)::hygR selection marker (left) and appearance of wild-type animals after marker excision (right). Arrows indicate wild-type animals. See also File S2. (C) Schematic of an expedited cloning procedure for insertion of homology arms into an FP–SEC vector. The FP–SEC vector is first digested with restriction enzymes to release the ccdB markers, and 500–700 bp homology arms are inserted by Gibson assembly to generate the repair template plasmid. (D) Workflow for generation of new FP knock-ins using our strategy. The time required for each step is listed in parentheses.
Figure 2
Figure 2
Tagging of his-72 with mNG^3xFlag. (A) Illustration of the organization of the his-72 locus and the predicted transcripts from this gene before editing (top), after homologous recombination (middle), and after SEC removal (bottom). (B) Images of adult mNG::his-72 worms before (strain LP309, top) and after (strain LP310, bottom) SEC removal. Shown are maximum intensity projections of a confocal Z series through entire worms. Scale bars, 50 µm. (C) Efficiency of SEC excision following heat shock for two independent mNG^SEC^3xFlag::his-72 insertion strains. For each experiment, L1/L2 larvae were heat shocked, and the number of wild-type (WT) and Roller progeny were counted. Each data point represents an independent experiment in which all F1’s present were counted (n = 26–394 animals counted per experiment).
Figure 3
Figure 3
his-72 expression levels and spontaneous loss of SEC from a subset of cells. (A) Expression of the his-72 ORF measured by qPCR in wild-type (N2), initial insertion (LP309 and LP311), and marker excised (LP310 and LP312) strains. Results are the means of three independent experiments, and error bars indicate 95% confidence intervals. (B) Genotyping of the strains in A. The lower band in LP309 and LP311 indicates spontaneous self-excision of SEC in a population of cells in the absence of heat shock. (C) Images of L4 worms of the indicated genotypes stained with anti-Flag antibodies to label cells that have excised SEC (spontaneously in the mNG^SEC^his-72 strain LP309, or after heat shock in the mNG::his-72 strain LP310). Shown are maximum intensity projections of a confocal Z series through entire worms. Scale bars, 50 µm. Arrowheads indicate stained cells in the head and tail, and the asterisk indicates staining near the developing vulva that may be nonspecific (see text for details).
Figure 4
Figure 4
Efficiency of genome engineering using SEC. (A) Comparison of single-copy transgene insertion at the ttTi5605 locus using either unc-119(+) or SEC selection. Each data point represents a single experiment, and red lines show the means across experiments; 45–90 animals were injected for each experiment. Efficiency is defined as the fraction of injected animals yielding insertions. (B) Efficiency of SEC excision following heat shock, measured as in Figure 2C, for two transgenes at the ttTi5605 locus. Each data point represents an independent experiment in which all F1’s present were counted (n = 299–913 animals counted per experiment). (C) Efficiency of precise mNG^SEC^3xFlag insertion into eight different endogenous loci using long homology arms (500–700 bp; green bars), short homology arms (35–40 bp; purple bars) or short homology arms and 3′GG sgRNAs (blue bars). Numbers on each bar indicate the number of animals injected, and efficiency is defined as the fraction of injected animals yielding precise insertions. N/A, not applicable (3′GG sgRNAs were tested only for the genes were a 3′GG target was present near the site of insertion). See also Table 1.
Figure 5
Figure 5
Images of knock-in strains generated using SEC. mNG fluorescence was imaged in the indicated strains. Left: initial insertions, which are predicted to behave as transcriptional reporters. Right: marker-excised strains, which express mNG^3xFlag fused to the protein of interest. The follow strains are shown: mNG^SEC^3xFlag::ebp-2, LP345; mNG^3xFlag::ebp-2, LP346; mNG^SEC^3xFlag::gex-3, LP361; mNG^3xFlag::gex-3, LP362; mNG^SEC^3xFlag::mex-5, LP366; mNG^3xFlag::mex-5, LP367; mNG^SEC^3xFlag::nmy-2, LP388; mNG^3xFlag::nmy-2, LP389; mNG^SEC^3xFlag::oma-2, LP390; mNG^3xFlag::oma-2, LP391; mNG^SEC^3xFlag::rap-1, LP394; mNG^3xFlag::rap-1, LP395. Arrowheads indicate expected localization of the fusion proteins (see text for details). Scale bars, 10 µm.
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