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Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination.

Chen C, Fenk LA, de Bono M - Nucleic Acids Res. (2013)

Bottom Line: We show that the CRISPR-CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome.The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion.The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

ABSTRACT
Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR-Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR-CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.

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CRISPR–Cas targeting in C. elegans. (A) Vectors optimized to express Cas9 and sgRNA in the C. elegans germline. NLS, nuclear localization signal. (B) Targeting sequences used for different genes, together with the 3′ PAM sequence.
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gkt805-F1: CRISPR–Cas targeting in C. elegans. (A) Vectors optimized to express Cas9 and sgRNA in the C. elegans germline. NLS, nuclear localization signal. (B) Targeting sequences used for different genes, together with the 3′ PAM sequence.

Mentions: The Cas9 open reading frame was codon-optimized for C. elegans (27), and modified by inserting a 3X FLAG tag and an SV40 nuclear localization sequence (NLS) after the ATG start codon, and a further NLS from the egl-13 gene just upstream of the stop codon (Figure 1A). This artificial gene, called Ce Cas9, was synthesized from oligonucleotides (GeneArt, Life Technologies). To drive expression of Ce Cas9 in C. elegans, we used the eft-3 promoter. We also placed the tbb-2 3′UTR after the Cas9 open reading frame (Figure 1A). The eft-3 promoter and tbb-2 3′UTR have been used previously to optimize germline expression of transposase in MosTIC protocols (24). The peft-3::Cas9::tbb-2 3′UTR construct was assembled using the multisite Gateway system (Life technologies). This plasmid will be made available via Addgene (http://www.addgene.org).Figure 1.


Efficient genome editing in Caenorhabditis elegans by CRISPR-targeted homologous recombination.

Chen C, Fenk LA, de Bono M - Nucleic Acids Res. (2013)

CRISPR–Cas targeting in C. elegans. (A) Vectors optimized to express Cas9 and sgRNA in the C. elegans germline. NLS, nuclear localization signal. (B) Targeting sequences used for different genes, together with the 3′ PAM sequence.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3814388&req=5

gkt805-F1: CRISPR–Cas targeting in C. elegans. (A) Vectors optimized to express Cas9 and sgRNA in the C. elegans germline. NLS, nuclear localization signal. (B) Targeting sequences used for different genes, together with the 3′ PAM sequence.
Mentions: The Cas9 open reading frame was codon-optimized for C. elegans (27), and modified by inserting a 3X FLAG tag and an SV40 nuclear localization sequence (NLS) after the ATG start codon, and a further NLS from the egl-13 gene just upstream of the stop codon (Figure 1A). This artificial gene, called Ce Cas9, was synthesized from oligonucleotides (GeneArt, Life Technologies). To drive expression of Ce Cas9 in C. elegans, we used the eft-3 promoter. We also placed the tbb-2 3′UTR after the Cas9 open reading frame (Figure 1A). The eft-3 promoter and tbb-2 3′UTR have been used previously to optimize germline expression of transposase in MosTIC protocols (24). The peft-3::Cas9::tbb-2 3′UTR construct was assembled using the multisite Gateway system (Life technologies). This plasmid will be made available via Addgene (http://www.addgene.org).Figure 1.

Bottom Line: We show that the CRISPR-CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome.The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion.The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.

View Article: PubMed Central - PubMed

Affiliation: Division of Cell Biology, MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cambridge CB2 0QH, UK.

ABSTRACT
Cas9 is an RNA-guided double-stranded DNA nuclease that participates in clustered regularly interspaced short palindromic repeats (CRISPR)-mediated adaptive immunity in prokaryotes. CRISPR-Cas9 has recently been used to generate insertion and deletion mutations in Caenorhabditis elegans, but not to create tailored changes (knock-ins). We show that the CRISPR-CRISPR-associated (Cas) system can be adapted for efficient and precise editing of the C. elegans genome. The targeted double-strand breaks generated by CRISPR are substrates for transgene-instructed gene conversion. This allows customized changes in the C. elegans genome by homologous recombination: sequences contained in the repair template (the transgene) are copied by gene conversion into the genome. The possibility to edit the C. elegans genome at selected locations will facilitate the systematic study of gene function in this widely used model organism.

Show MeSH