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. 2017 May 15:121-122:86-93.
doi: 10.1016/j.ymeth.2017.03.023. Epub 2017 Apr 7.

Precision genome editing using CRISPR-Cas9 and linear repair templates in C. elegans

Alexandre Paix  1 , Andrew Folkmann  2 , Geraldine Seydoux  3
Affiliations

Precision genome editing using CRISPR-Cas9 and linear repair templates in C. elegans

Alexandre Paix et al. Methods. .

Abstract

The ability to introduce targeted edits in the genome of model organisms is revolutionizing the field of genetics. State-of-the-art methods for precision genome editing use RNA-guided endonucleases to create double-strand breaks (DSBs) and DNA templates containing the edits to repair the DSBs. Following this strategy, we have developed a protocol to create precise edits in the C. elegans genome. The protocol takes advantage of two innovations to improve editing efficiency: direct injection of CRISPR-Cas9 ribonucleoprotein complexes and use of linear DNAs with short homology arms as repair templates. The protocol requires no cloning or selection, and can be used to generate base and gene-size edits in just 4days. Point mutations, insertions, deletions and gene replacements can all be created using the same experimental pipeline.

Keywords: C. elegans; CRISPR; Cas9 RNP; Gene conversion; Genome editing; Homology-dependent repair (HDR); Linear repair templates; Short homology.

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Figures

Fig. 1.
Fig. 1.
Protocol overview. See Section 1.1 for details.
Fig. 2.
Fig. 2.
Template design. Sequence of locus and repair templates are shown for two insertional edits, one right at the Cas9-induced double-strand break (DSB) (A) and one away from the DSB (B). Homology arms are in blue, inserts in green, crRNA sequence is bolded and underlined, PAM is in capital letters. In (A), the insert disrupts the Cas9 targeting sequence and so no recoding is needed to prevent recutting. In (B), the insert is away from the Cas9 targeting sequence and so silent mutations are required to prevent re-cutting (red) and to prevent premature template switching (orange) during copying of the template. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
Fig. 3.
Fig. 3.
Editing strategies. Schematic representation of chromosome (grey), DSB (scissors) and DNA repair templates above chromosome (ssODNs: single line or PCR fragments: two lines). Homology arms are highlighted in red (left arm) and blue (right arm). Inserts are in black. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
See this image and copyright information in PMC

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References

    1. Doudna JA, Charpentier E, Genome editing. The new frontier of genome engineering with CRISPR-Cas9, Science 346 (6213) (2014) 1258096. - PubMed
    1. Jasin M, Haber JE, The democratization of gene editing: Insights from site-specific cleavage and double-strand break repair, DNA Repair 44 (2016) 6–16. - PMC - PubMed
    1. Jiang W, Marraffini LA, CRISPR-Cas: new tools for genetic manipulations from bacterial immunity systems, Annu. Rev. Microbiol 69 (2015) 209–228. - PubMed
    1. Paix A, Wang Y, Smith HE, Lee CY, Calidas D, Lu T, Smith J, Schmidt H, Krause MW, Seydoux G, Scalable and versatile genome editing using linear DNAs with microhomology to Cas9 Sites in Caenorhabditis elegans, Genetics 198 (4) (2014) 1347–1356. - PMC - PubMed
    1. Paix A, Schmidt H, Seydoux G, Cas9-assisted recombineering in C. elegans: genome editing using in vivo assembly of linear DNAs, Nucleic Acids Res (2016). - PMC - PubMed

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