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.KEYWORDS CRISPR/Cas9; homologous recombination; gene tagging; Caenorhabditis elegans; self-excising cassette A common goal in biological and biomedical research is to visualize the localization of a protein of interest within a cell or organism. This is often accomplished by fusing GFP or another fluorescent protein (FP) to the protein of interest. In the nematode Caenorhabditis elegans, GFP fusions were historically generated by injecting plasmids into the gonad of the adult hermaphrodite worm, resulting in the formation of extrachromosomal arrays (Mello et al. 1991). However, the resulting fusion proteins were typically strongly overexpressed in somatic tissues and silenced in the germline. Microparticle bombardment allowed the generation of low-copy transgenes that in some cases more closely recapitulated endogenous expression levels (Praitis et al. 2001;Sarov et al. 2012), but this technique is inefficient, time consuming, and difficult and requires expensive equipment and materials. More recently, we and others have reported CRISPR/Cas9-based approaches that together can be used to make essentially any desired change to the C. elegans genome, including insertion of GFP into endogenous loci Dickinson et al. 2013;Lo et al. 2013;Chiu et al. 2013;Cho et al. 2013;Katic and Großhans 2013;Tzur et al. 2013;Waaijers et al. 2013;Chen et al. 2013;Zhao et al. 2014;Kim et al. 2014;Arribere et al. 2014;Paix et al. 2014;Ward 2015;Farboud and Meyer 2015). The resulting GFP knock-in strains express 100% labeled protein under the control of all native regulatory elements, resulting in endogenous levels and patterns of expression in all cases reported to date (Dickinson et al. 2013;Kim et al. 2014).Our publis...

SapTrap, a Toolkit for High-Throughput CRISPR/Cas9 Gene Modification in Caenorhabditis elegans

“…A feature of the SapTrap constructs is that the unc-119 selectable marker is inserted in the reverse orientation relative to the target gene, and thus the target gene and the selectable marker can be concurrently expressed. For genes with viable mutant phenotypes concurrent expression is not crucial (Dickinson et al 2015;Norris et al 2015). However, when tagging essential genes for which loss-of-function cannot be tolerated during strain construction, concurrent expression of the target gene and tag is advantageous.…”

“…In previous studies, homology arms as short as 500 bp for plasmid repair templates and 30 bp for linear repair templates efficiently direct insertion into the C. elegans genome (Paix et al 2014;Dickinson et al 2015). To determine the effect of homology arm length on insertion frequency for SapTrap vectors, we targeted the 59 end of the snb-1 gene with a gfp cassette flanked by homology arms 0, 44, 100, or 400 bp in length ( Figure 3A and Table S3).…”

“…We observed an off-site insertion rate of 15% (7 false-positive strains/47 total positive strains). False-positive strains that appear to contain off-site insertions have been reported previously (Dickinson et al 2015;Katic et al 2015) and appear to be a general consequence of tag insertion by CRISPR/Cas9. …”

“…Insertion events among the progeny can be identified only by PCR or by screening for the expected phenotype, which creates a large screening burden. There are two strategies to enrich for the rare animals with edited DNA: either by insertion of selectable markers at the target site or by co-CRISPR events at a second site (Arribere et al 2014;Kim et al 2014;Dickinson et al 2015;Norris et al 2015;Paix et al 2015;Ward 2015).…”

“…However, the unique design and construction of each repair template is time-consuming. Recently, the Goldstein and Calarco groups have embedded selectable markers within a synthetic intron in the exogenous sequence (Dickinson et al 2015;Norris et al 2015). This arrangement simplifies construction because the selectable marker no longer needs to be positioned in a unique location within each repair template.…”