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

Abstract

In principle, clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 allows genetic tags to be inserted at any locus. However, throughput is limited by the laborious construction of repair templates and guide RNA constructs and by the identification of modified strains. We have developed a reagent toolkit and plasmid assembly pipeline, called "SapTrap," that streamlines the production of targeting vectors for tag insertion, as well as the selection of modified Caenorhabditis elegans strains. SapTrap is a high-efficiency modular plasmid assembly pipeline that produces single plasmid targeting vectors, each of which encodes both a guide RNA transcript and a repair template for a particular tagging event. The plasmid is generated in a single tube by cutting modular components with the restriction enzyme SapI, which are then "trapped" in a fixed order by ligation to generate the targeting vector. A library of donor plasmids supplies a variety of protein tags, a selectable marker, and regulatory sequences that allow cellspecific tagging at either the N or the C termini. All site-specific sequences, such as guide RNA targeting sequences and homology arms, are supplied as annealed synthetic oligonucleotides, eliminating the need for PCR or molecular cloning during plasmid assembly. Each tag includes an embedded Cbr-unc-119 selectable marker that is positioned to allow concurrent expression of both the tag and the marker. We demonstrate that SapTrap targeting vectors direct insertion of 3-to 4-kb tags at six different loci in 10-37% of injected animals. Thus SapTrap vectors introduce the possibility for high-throughput generation of CRISPR/Cas9 genome modifications.KEYWORDS CRISPR/Cas9; high-throughput; gene tagging; plasmid toolkit T HE clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has revolutionized genome editing in nearly all model systems, including Caenorhabditis elegans (Frøkjaer-Jensen 2013;Doudna and Charpentier 2014;Xu 2015). The Cas9 protein cuts genomic DNA at sites that match an 20-nucleotide guide RNA sequence (Gasiunas et al. 2012;Jinek et al. 2012). Error-prone repair of the break can create point mutations, small indels, or large deletions at the cut site (Cho et al. 2013;Cong et al. 2013;Friedland et al. 2013;Jinek et al. 2013;van Schendel et al. 2015). However, the true power of CRISPR/Cas9 for genome editing lies in the insertion of exogenous DNA sequences, such as genetically encoded protein tags (Katic and Grosshans 2013;Lo et al. 2013;Mali et al. 2013;Tzur et al. 2013). To insert an exogenous sequence, one must simply supply a repair template with homology arms that flank the cut site. The cell uses homologybased repair to heal the break and copy the exogenous DNA into the cut site.Although in theory CRISPR/Cas9 makes it easy to insert exogenous sequences into a genome, practical limitations have prevented high-throughput implementation. Two critical limiting factors are (1) the time and expense required to build both the repair template a...

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