Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2015 Feb;199(2):363-77.
doi: 10.1534/genetics.114.172361. Epub 2014 Dec 9.

Rapid and precise engineering of the Caenorhabditis elegans genome with lethal mutation co-conversion and inactivation of NHEJ repair

Jordan D Ward  1
Affiliations

Rapid and precise engineering of the Caenorhabditis elegans genome with lethal mutation co-conversion and inactivation of NHEJ repair

Jordan D Ward. Genetics. 2015 Feb.

Abstract

As in other organisms, CRISPR/Cas9 methods provide a powerful approach for genome editing in the nematode Caenorhabditis elegans. Oligonucleotides are excellent repair templates for introducing substitutions and short insertions, as they are cost effective, require no cloning, and appear in other organisms to target changes by homologous recombination at DNA double-strand breaks (DSBs). Here, I describe a methodology in C. elegans to efficiently knock in epitope tags in 8-9 days, using a temperature-sensitive lethal mutation in the pha-1 gene as a co-conversion marker. I demonstrate that 60mer oligos with 29 bp of homology drive efficient knock-in of point mutations, and that disabling nonhomologous end joining by RNAi inactivation of the cku-80 gene significantly improves knock-in efficiency. Homology arms of 35-80 bp are sufficient for efficient editing and DSBs up to 54 bp away from the insertion site produced knock-ins. These findings will likely be applicable for a range of genome editing approaches in C. elegans, which will improve editing efficiency and minimize screening efforts.

Keywords: CRISPR/Cas9; co-conversion; nonhomologous end joining; oligonucleotide-mediated homologous recombination; pha-1.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Selection for pha-1(ts) oligo-mediated repair enriches for nhr-23::2×FLAG knock-in. (A) Sequence of the pha-1(e2123) genomic locus targeted, with the PAM (red text), e2123 G-to-A mutation (boldface text and underline), sgRNA target sequence, and position of the DSB indicated. The 80mer repair oligo also contains a silent C-to-A mutation to inactivate the PAM. (B) Sequence of the nhr-23 genomic locus targeted. The stop codon (blue text), PAM (no. 1, red text) and sgRNA target, the DSB position, and an alternate PAM (no. 2, red text) are indicated. The 200mer repair oligo was designed to insert a 2×FLAG epitope with a flexible GSGGGG linker sequence, which also contains a BamHI site. The oligo contains silent C-to-A and G-to-A mutations to inactivate the two indicated PAMs. (C) (i) pha-1(ts) mutants propagated at the permissive temperature were injected with 60 ng/µl each of CRISPR/Cas9 plasmids targeting the PAM in pha-1(ts) and PAM no. 1 in nhr-23, 50 ng/µl of an oligo designed to correct the pha-1(ts) allele, and 50 ng/µl of the 200mer nhr-23::2×FLAG repair oligo. (ii) P0 animals were singled onto individual plates and shifted to the restrictive temperature (25°). (iii) Three to four days later, the plates were screened for the presence of viable progeny (L3s to adults); the e2123 embryonic lethality phenotype is completely penetrant at 25°, and only rescued animals develop. Rescued F1 progeny were singled onto individual plates, allowed to lay eggs (2–3 days), and the parental F1 was genotyped by PCR followed by BamHI digestion. Correct insertion of the epitope is confirmed by sequencing-purified PCR products with knock-in-specific primers. (iv) Homozygotes were recovered by plating 12–24 progeny from candidate nhr-23:2×FLAG knock-in F1’s, allowing them to lay eggs (2–3 days), and genotyping the parental F2 animal by PCR and BamHI digestion. Marker size in kilobases is provided. (D) Summary of pha-1(ts) co-conversion experiments. Viable P0 are the number of injected animals that produced eggs; a variable number of animals are sterile in each experiment. The length and polarity (with respect to the coding strand) of the pha-1(ts) repair and nhr-23::2×FLAG oligos are provided. P0 were propagated on OP50 E. coli for these injections.
Figure 2
Figure 2
Detection of NHR-23::2×FLAG in precise knock-ins. Four micrograms of protein from synchronized gravid adults of the indicated strains was analyzed by immunoblotting with anti-FLAG. KRY48 contains a frameshift in the 2×FLAG tag and thus does not express the epitope. Stain-free (Bio-Rad) analysis of total protein on the blot is provided as a loading control. Marker size (in kilodaltons) is provided.
Figure 3
Figure 3
PCR generated sgRNA templates can be used for pha-1(ts) co-conversion. (A) Method for generating PU6::sgRNA templates by PCR. The U6 promoter and sgRNA template were separately subcloned to generate pJW1310 and 1311, respectively. The U6 promoter is amplified with oligos 1 and 2, and the sgRNA template is amplified with oligos 3 and 4. Oligo 3 contains 20 bp of homology to the sgRNA template, the new 20 bp sgRNA targeting sequence, and 20 bp of homology to the U6 promoter. The resulting PU6 and sgRNA template PCR products are then mixed and used as template in a second PCR reaction using oligos 1 + 4. (B) Animals were injected with 60 ng/µl of the Cas9 plasmid, 25 ng/µl each of PCR generated PU6::sgRNA templates targeting pha-1 and nhr-23 (same targeting sequence as in Figure 2), and 50 ng/µl each of the 200mer sense pha-1(ts) repair oligo and nhr-23::2×FLAG oligo. Viable P0 are the number of injected animals that produced eggs; a variable number of animals are sterile in each experiment. P0 were propagated on HB101 E. coli for these injections. Oligo polarity (sense) is with respect to the coding strand.
Figure 4
Figure 4
DSBs up to 54 bp from an insertion site, and 35-bp homology arms can be used for oligo-templated repair in nhr-23. (A) Schematic of the nhr-23 3′ end, indicating the stop codon (blue text), four PAMs tested (red text), and position of the DSBs (scissor). Mutations used to inactivate PAMs in repair templates are provided in the nhr-23(PAM MUT) sequence and indicated by the vertical lines between the (+) and (PAM MUT) sequence. (B) Testing effect of DSB position on nhr-23::2×FLAG epitope knock-in efficiency. The PAMs for the sgRNAs used to generate the DSB and mutations used to inactivate the PAMs in the repair templates are provided in A. Animals were co-injected with 50 ng/µl of pha-1 targeting CRISPR/Cas9 plasmid, 50 ng/µl each of the pha-1(ts) repair oligo and a 200mer sense nhr-23::2×FLAG repair oligo, and either 50 ng/µl of nhr-23 targeting CRISPR/Cas9 plasmid (PAMs no. 1 or no. 2), or 25 ng/µl of PU6::sgRNA template PCR product (PAMs no. 3 and no. 4). For the PAM no. 1 and no. 2 sgRNA experiments, the repair template carried mutations in both PAMs. For the PAM no. 3 and no. 4 experiments, the repair template carried mutations in PAMs no. 1, no. 2, and no. 3. PAM no. 3 had a single PCR hit with a 2×FLAG insertion carrying a 1-bp deletion. For the PAM no. 1 row, all experiments using nhr-23::2×FLAG 200mers were pooled (Figure 1, Figure 3, Table 2); only animals that displayed a knock-in signature in the diagnostic BamHI digest from these experiments were sequenced. For the PAM no. 2, no. 3, and no. 4 experiments, all pha-1(ts) F1 rescued animals were sequenced. (C) Testing homology arm length on nhr-23::2×FLAG knock-in efficiency. Homology lengths in basepairs for 5′ and 3′ arms are provided. 5′ arm homology numbering starts from the middle G in PAM no. 1; 3′ homology numbering starts from the first basepair of the stop codon. Animals were co-injected with 50 ng/µl each of pha-1 targeting and nhr-23 PAM no. 1 targeting CRISPR/Cas9 plasmids, 50 ng/µl each of the pha-1(ts) repair oligo and nhr-23::2×FLAG repair oligo. For the 76/54-bp homology arm row in the table, data were pooled from all non-RNAi experiments using pha-1 sense repair oligos and nhr-23::2×FLAG sense 200mers (Figure 1 and Figure 3). With the exception of the pooled data, all injected P0 animals were grown on HB101 E. coli in B and C. Oligo polarity (sense) is with respect to the coding strand.
Figure 5
Figure 5
Detection of FLAG epitope expression in nhr-23-, nhr-25-, and smo-1-tagged lines. Anti-FLAG immunoblot analyses of lysates are from mixed stage animals of the indicated genotypes. The 2x and 3xFLAG tagged nhr-23 (A) and nhr-25 (B) lines and a 2×FLAG::smo-1 tagged line (C) were assayed. Stain-free (Bio-Rad) analysis of total protein on each blot is provided as a loading control. Marker size (in kilodaltons) is provided. The same exposure time was used to image all anti-FLAG blots. For the NHR-23 blot (A), the background band observed in Figure 2 was not detected, likely because a more potent ECL substrate was used for that experiment.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Adamo A., Collis S. J., Adelman C. A., Silva N., Horejsi Z., et al. , 2010. Preventing nonhomologous end joining suppresses DNA repair defects of Fanconi anemia. Mol. Cell 39: 25–35. - PubMed
    1. Arribere J. A., Bell R. T., Fu B. X. H., Artiles K. L., Hartman P. S., et al. , 2014. Efficient marker-free recovery of custom genetic modifications with CRISPR/Cas9 in Caenorhabditis elegans. Genetics 198: 837–846. - PMC - PubMed
    1. Asahina M., Ishihara T., Jindra M., Kohara Y., Katsura I., et al. , 2000. The conserved nuclear receptor Ftz-F1 is required for embryogenesis, moulting and reproduction in Caenorhabditis elegans. Genes Cells 5: 711–723. - PubMed
    1. Asahina M., Valenta T., Silhánková M., Korinek V., Jindra M., 2006. Crosstalk between a nuclear receptor and beta-catenin signaling decides cell fates in the C. elegans somatic gonad. Dev. Cell 11: 203–211. - PubMed
    1. Bassik M. C., Lebbink R. J., Churchman L. S., Ingolia N. T., Patena W., et al. , 2009. Rapid creation and quantitative monitoring of high coverage shRNA libraries. Nat. Methods 6: 443–445. - PMC - PubMed

Publication types