A co-CRISPR strategy for efficient genome editing in Caenorhabditis elegans.
Bottom Line: Genome editing based on CRISPR (clustered regularly interspaced short palindromic repeats)-associated nuclease (Cas9) has been successfully applied in dozens of diverse plant and animal species, including the nematode Caenorhabditis elegans.The rapid life cycle and easy access to the ovary by micro-injection make C. elegans an ideal organism both for applying CRISPR-Cas9 genome editing technology and for optimizing genome-editing protocols.Our findings reveal a surprisingly high frequency of HR-mediated gene conversion, making it possible to rapidly and precisely edit the C. elegans genome both with and without the use of co-inserted marker genes.
Affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605.Show MeSH
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Mentions: To test this idea, we decided to use CRISPR-Cas9-mediated HR to introduce the gfp coding sequence immediately downstream of the start codon in the endogenous pie-1 locus (Figure 3A). The donor plasmid in this experiment contained NheI restriction sites flanking the gfp coding sequence, 1-kb homology arms, and a silent mutation that disrupts the PAM sequence at the sgRNA target site (Figure 3A). We generated three different donor constructs: gfp::pie-1(WT), gfp::pie-1(K68A), and gfp::pie-1(K68R). Each donor molecule was co-injected with vectors to express the sgRNA, Cas9, and rol-6 marker. We then directly examined the resulting F1 rolling animals for GFP::PIE-1 expression in the germline and embryos using epifluorescence microscopy (Figure 3B, see Materials and Methods). Using this approach, we obtained 9 independent gfp::pie-1(K68A) lines from 92 F1 rollers, 1 gfp::pie-1(K68R) line from 69 F1 rollers, and 1 gfp::pie-1(WT) line from 72 F1 rollers. Subsequent analyses revealed that each of these F1 animals was heterozygous for gfp::pie-1, and each strain incorporated both the gfp coding sequence and the PAM site mutation, as well as the linked K68A and K68R missense mutations. For unknown reasons, we found that one of the nine gfp::pie-1(K68A) lines could not be maintained.
Affiliation: Program in Molecular Medicine, University of Massachusetts Medical School, Worcester, Massachusetts 01605 RNA Therapeutics Institute, University of Massachusetts Medical School, Worcester, Massachusetts 01605.