Engineering the Caenorhabditis elegans genome using Cas9-triggered homologous recombination

Abstract

Study of the nematode Caenorhabditis elegans has provided important insights in a wide range of fields in biology. The ability to precisely modify genomes is critical to fully realize the utility of model organisms. Here we report a method to edit the C. elegans genome using the clustered, regularly interspersed, short palindromic repeats (CRISPR) RNA-guided Cas9 nuclease and homologous recombination. We demonstrate that Cas9 is able to induce DNA double-strand breaks with specificity for targeted sites and that these breaks can be repaired efficiently by homologous recombination. By supplying engineered homologous repair templates, we generated gfp knock-ins and targeted mutations. Together our results outline a flexible methodology to produce essentially any desired modification in the C. elegans genome quickly and at low cost. This technology is an important addition to the array of genetic techniques already available in this experimentally tractable model organism.

Figure 1

Adaptation of the CRISPR-Cas9 system for C. elegans. (a) Schematic of the Cas9 nuclease and sgRNA. Formation of a double-strand break requires base pairing between the sgRNA and the target DNA sequence, as well as the presence of the NGG motif (PAM) immediately adjacent to the target sequence. Cleavage occurs 3 bp 5’ of the PAM. The guanine (G) residue at the 5’ end of the sgRNA is required for transcription initiation by the U6 promoter. (b) Sequence conservation of the ten U6 RNA genes that we identified in C. elegans. The blue trace is a rolling average produced using LOWESS. The green line indicates the region of R07E5.16 that we used as the promoter in the Cas9-sgRNA construct. (c) Schematic of the Cas9-sgRNA plasmid.

Figure 2

Efficiency of Cas9-triggered homologous recombination in C. elegans. (a) Schematic for homologous recombination (HR) mediated by either Mos1 transposon excision (left) or Cas9 (right). (b) Efficiency of single-copy transgene insertion for three different transgenes using either MosSCI or Cas9. n values at the bottom of each bar indicate the number of successfully injected animals (those that yielded non-Unc progeny). Percent efficiency is the fraction of successfully injected animals that yielded integrated transgenes. See Supplementary Table 1 for raw data. (c) Images of germline GFP expression from Pmex-5::GFP::tbb-2 3’UTR transgenes generated using MosSCI or Cas9. Images were acquired, processed and displayed with identical settings. Results are representative of five animals of each strain. Scale bars represent 20 μm.

Figure 3

Tagging of endogenous nmy-2 with GFP. (a) Strategy for producing nmy-2::gfp knock-ins. Cas9 cleavage of the 3’ end of nmy-2 stimulates homologous recombination, resulting in insertion of GFP and unc-119(+) into the genome. After isolating recombinants, we excised the unc-119(+) selectable marker by expressing Cre recombinase. (b) PCR genotyping of the nmy-2 locus in the indicated strains, using primer pairs as indicated and as schematized in panel a. Results are representative of three independently isolated knock-in strains. (c) PCR genotyping of the nmy-2 locus before and after excision of the unc-119(+) marker with Cre. Results are representative of five independent Cre-mediated unc-119(+) excision experiments. (d) Western blot showing NMY-2 levels in embryonic lysates in N2 (wild type), a strain carrying zuIs45, and strains carrying three independent knock-in alleles. Coomassie staining of total protein is shown as a loading control. Results are representative of three independent experiments. (e) Stage-matched images of NMY-2–GFP localization in an nmy-2::gfp knock-in strain compared to zuIs45. The embryos shown were placed side-by-side on the same coverslip and imaged simultaneously. The images in the four left columns are maximum intensity projections of two 0.5 μm sections at a cortical focal plane and are taken from Movie S1. The far right panels are single confocal sections from a different pair of embryos at gastrulation stage. Arrows indicate apical accumulation of NMY-2–GFP in gastrulating endodermal precursors. Results are representative of 14 independent experiments. Scale bars represent 10 μm.

Figure 4

Tagging of endogenous his-72 with GFP. (a) PCR genotyping of the his-72 locus in the indicated strains using a PCR strategy similar to that outlined in Fig. 3a–b. (b) his-72 mRNA expression levels in the indicated strains, as measured by qRT-PCR. Results are the average of three independent experiments, and error bars show 95% confidence interval. N.S., not significant (p>0.05, two-tailed t-test). (c) HIS-72–GFP fluorescence in whole worms at the indicated stages. Results are representative of seven animals imaged. Scale bars represent 50 μm.

Figure 5

Targeted mutations in an endogenous gene. (a) Model for how MAPK phosphorylation affects LIN-31 function and the predicted effects of mutating these residues to either alanine or glutamic acid. (b) Strategy for simultaneous mutagenesis of four threonine residues (T145, T200, T218 and T220) in lin-31 to either alanine or glutamic acid. Cas9 is targeted to the 5’ end of the last exon of lin-31, ensuring mutation of all four threonine residues. (c) Sequence confirmation of the induced mutations in lin-31 mutant strains. Results are representative of two independently isolated lin-31(4T→A) strains and three independently isolated lin-31(4T→A) strains. (d) Vulval morphology in L4 and adult animals of the indicated genotypes. Images are representative of 15 N2 animals, 35 lin-31(4T→A) animals and 36 lin-31(4T→E) animals. Scale bars represent 20 μm. (e) Quantification of the Pvl phenotype of lin-31 adults. Scoring of phenotypes was done blindly. Results from multiple isolates of each lin-31 mutation are combined; n = 406 for N2, n = 267 for lin-31(4T→A) and n = 217 for lin-31(4T→A). See Supplementary Table 2 for the raw data.