CRISPR-Based Methods for Caenorhabditis elegans Genome Engineering

Abstract

The advent of genome editing techniques based on the clustered regularly interspersed short palindromic repeats (CRISPR)-Cas9 system has revolutionized research in the biological sciences. CRISPR is quickly becoming an indispensible experimental tool for researchers using genetic model organisms, including the nematode Caenorhabditis elegans. Here, we provide an overview of CRISPR-based strategies for genome editing in C. elegans. We focus on practical considerations for successful genome editing, including a discussion of which strategies are best suited to producing different kinds of targeted genome modifications.

Keywords: CRISPR/Cas9; Caenorhabditis elegans; WormBook; genome editing.

Figure 1

DNA recognition by the Cas9–sgRNA complex. Cas9 identifies its substrates by first binding to the PAM (NGG motif) and subsequently by base pairing of the sgRNA cofactor to the substrate DNA. HNH and RuvC are the two Cas9 nuclease domains that cleave the sgRNA-complementary and noncomplementary strand of the target DNA, respectively. Red and orange in the sgRNA indicate the portions derived from the bacterial crRNA and tracrRNA, respectively.

Figure 2

DNA repair approaches for CRISPR-based genome engineering. DNA double-strand breaks introduced by Cas9 can be repaired via three different mechanisms. End joining produces random insertion/deletion mutations. HDR produces error-free edits using an exogenous DNA molecule as a repair template. Although the mechanisms of HDR in C. elegans are not known, efficiency data suggest the existence of two different HDR pathways (see text). Short-range HDR is hypothesized to occur via a synthesis-dependent strand-annealing mechanism and can accommodate insertions of up to 1–2 kb, with the highest efficiency within 10 bp of the cut site. Long-range HDR is hypothesized to occur via a double-crossover mechanism and can accommodate insertions of at least 12 kb, at distances up to at least 1 kb from the cut site.

Figure 3

Genetic schemes for co-CRISPR screening. (A) dpy-10 co-CRISPR (Arribere et al. 2014) makes use of the dominant Roller phenotype conferred by the cn64 mutation to identify a desired modification at an unlinked locus. Because dpy-10(cn64)/dpy-10(o) animals have a different phenotype than dpy-10(cn64)/+, a wild-type copy of dpy-10 can be carried through the screening, eliminating the need for outcrossing to remove the marker mutation. The sqt-1(e1350) marker mutation can be used in place of dpy-10(cn64). (B) pha-1 co-CRISPR (Ward 2015) uses repair of the temperature-sensitive lethal mutation pha-1(e2123) for live/dead screening of F₁’s. No outcrossing is required because the converted pha-1 allele is wild type; however, F₂’s need to be PCR genotyped for both the desired modification and pha-1(+).

Figure 4

Gene tagging with a self-excising selection cassette. (A) Design of a self-excising cassette for drug selection. SEC consists of a drug 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) 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).

Figure 5

(A) Gibson assembly-based strategy (Dickinson et al. 2015). An FP–SEC vector is digested to release ccdB markers, and homology arms are inserted by Gibson assembly to generate the repair template plasmid. Since the ccdB-containing parent vector does not transform, correct clones make up a majority of transformants and can be identified directly by sequencing. (B) SapTrap assembly strategy (Schwartz and Jorgensen, 2016). Homology arms and the sgRNA sequence are assembled, along with pre-existing tag and selectable marker building blocks, into a destination vector. The Type II restriction enzyme SapI generates unique overhangs that ensure ligation of the various fragments in the correct order.

Figure 6

Flow chart summarizing recommended CRISPR techniques for different applications. See Recommended Strategies for Different Types of Modifications for details and rationale behind these recommendations.

Figure 7

A general strategy for structure–function analysis. (A) Illustration of the strategy. First, a null mutation is generated by inserting FP–SEC in place of the gene of interest; then, variants are reintroduced into the mutant background. Once the null mutant is made, multiple different variants (as many as desired) can be introduced using the same homology arms and sgRNA for the second HDR step. (B) Genetic scheme for executing this strategy for a nonessential gene. (C) Genetic scheme for executing this strategy for an essential gene. The only additional step is the introduction of a balancer chromosome during isolation of the initial null mutant.