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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-targeted knock-ins. Schematic showing targeted knock-in of an HygR cassette at the ben-1 locus. The double-strand break induced at ben-1 by CRISPR–Cas9 is repaired using the transgene containing the HygR cassette, leading to gene conversion.
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gkt805-F3: CRISPR-targeted knock-ins. Schematic showing targeted knock-in of an HygR cassette at the ben-1 locus. The double-strand break induced at ben-1 by CRISPR–Cas9 is repaired using the transgene containing the HygR cassette, leading to gene conversion.

Mentions: To confirm that gene conversion had occurred, we PCR-amplified the ben-1 region using primer pairs that flank sequences present in the transgene construct mediating repair (Figure 3). A PCR fragment of ∼9.4 kb, equivalent to the size of a single-copy hygromycin insertion, was obtained in all three lines (data not shown). We sequenced this entire fragment and found it was identical to the sequence provided on the repair transgene, confirming precise gene conversion.Figure 3.


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

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

CRISPR-targeted knock-ins. Schematic showing targeted knock-in of an HygR cassette at the ben-1 locus. The double-strand break induced at ben-1 by CRISPR–Cas9 is repaired using the transgene containing the HygR cassette, leading to gene conversion.
© Copyright Policy - creative-commons
Related In: Results  -  Collection

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

gkt805-F3: CRISPR-targeted knock-ins. Schematic showing targeted knock-in of an HygR cassette at the ben-1 locus. The double-strand break induced at ben-1 by CRISPR–Cas9 is repaired using the transgene containing the HygR cassette, leading to gene conversion.
Mentions: To confirm that gene conversion had occurred, we PCR-amplified the ben-1 region using primer pairs that flank sequences present in the transgene construct mediating repair (Figure 3). A PCR fragment of ∼9.4 kb, equivalent to the size of a single-copy hygromycin insertion, was obtained in all three lines (data not shown). We sequenced this entire fragment and found it was identical to the sequence provided on the repair transgene, confirming precise gene conversion.Figure 3.

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