High Efficiency, Homology-Directed Genome Editing in Caenorhabditis elegans Using CRISPR-Cas9 Ribonucleoprotein Complexes

Abstract

Homology-directed repair (HDR) of breaks induced by the RNA-programmed nuclease Cas9 has become a popular method for genome editing in several organisms. Most HDR protocols rely on plasmid-based expression of Cas9 and the gene-specific guide RNAs. Here we report that direct injection of in vitro-assembled Cas9-CRISPR RNA (crRNA) trans-activating crRNA (tracrRNA) ribonucleoprotein complexes into the gonad of Caenorhabditis elegans yields HDR edits at a high frequency. Building on our earlier finding that PCR fragments with 35-base homology are efficient repair templates, we developed an entirely cloning-free protocol for the generation of seamless HDR edits without selection. Combined with the co-CRISPR method, this protocol is sufficiently robust for use with low-efficiency guide RNAs and to generate complex edits, including ORF replacement and simultaneous tagging of two genes with fluorescent proteins.

Keywords: C. elegans; CRISPR-Cas9; genome editing; homology-directed repair; ribonucleoprotein complexes.

Figure 1

Co-CRISPR strategy. (A) We cotargeted (1) the dpy-10 locus with a ssODN repair template to introduce a missense mutation leading to the dominant roller phenotype (Arribere et al. 2014) and (2) a second locus (gene of interest) with a PCR repair template to insert a fluorescent protein (FP) near the C-terminus. (B) Experimental outline. The gonads of 10–20 hermaphrodites are injected, and their broods are examined for the presence of rollers (dpy-10 edits) and FP⁺ animals. In typical experiments, >50% of hermaphrodites segregate rollers. Jackpot broods are the broods with the highest numbers of rollers. Edits at the gene of interest (pink) are found in both roller and nonroller worms, but only among broods that contain rollers.

Figure 2

Jackpot broods. Broods with high numbers of dpy-10 edits contain higher percentages of gtbp-1 edits compared to broods with few dpy-10 edits. For three separate experiments, we compared the frequency of FP⁺ edits (insertion of GFP or RFP) at the gtbp-1 locus among rollers (A) and nonrollers (B) derived from the top three broods with the highest numbers of rollers (jackpot broods) compared to the bottom three broods with the lowest numbers of rollers (as depicted in Figure 1). The frequency of gtbp-1 edits is higher in the jackpot broods. For experiments AP58, AP60, and AP78, the top three jackpot broods contained 70.2 (33 of 47), 44.2 (49 of 111), and 71.6% (68 of 95) of all FP⁺ edits, respectively. Numbers refer to the number of F₁ progeny screened.

Figure 3

Gene replacement strategy. (A) Replacement of the gtbp-1 ORF with GFP::H2B. Two cuts were made at either end of the gtbp-1 ORF and repaired using a PCR template containing GFP::H2B flanked by 35 bases that were homologous to the 5′ and 3′ ends of gtbp-1. (B) Replacement of GFP with RFP. Two cuts were made in GFP and repaired using a PCR template containing RFP flanked by 33 and 35 bases that were homologous to the 5′ and 3′ ends of GFP, respectively. (C) Experimental results for replacing GFP with RFP at the gtbp-1 locus. The percentages of each genotype among roller F₁ progeny are indicated.