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, 10 (8), 741-3

Heritable Genome Editing in C. Elegans via a CRISPR-Cas9 System

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Heritable Genome Editing in C. Elegans via a CRISPR-Cas9 System

Ari E Friedland et al. Nat Methods.

Abstract

We report the use of clustered, regularly interspaced, short palindromic repeats (CRISPR)-associated endonuclease Cas9 to target genomic sequences in the Caenorhabditis elegans germ line using single-guide RNAs that are expressed from a U6 small nuclear RNA promoter. Our results demonstrate that targeted, heritable genetic alterations can be achieved in C. elegans, providing a convenient and effective approach for generating loss-of-function mutants.

Figures

Figure 1
Figure 1
A set of vectors that drive expression of Cas9 and sgRNAs in C. elegans. (A) The C. elegans eft-3 promoter drives transcription of Cas9 with a 3′ SV40 nuclear localization sequence. A pol III promoter (derived from a U6 snRNA locus) drives transcription of the sgRNA, which contains a target sequence and a scaffold sequence. (B) A schematic illustration of Cas9 interacting with sgRNA and its genomic target. (C) RT-PCR results demonstrating expression of Cas9 and sgRNA transcripts. Total RNA was tested from strains carrying Cas9 vector alone (lanes 1 and 2), unc-119 sgRNA vector alone (lanes 3 and 4), and both vectors (lanes 5 and 6) with primers specific for Cas9 (top panel) or unc-119 sgRNA (bottom panel). For all samples, control reactions were run in the absence of Reverse Transcriptase (-RT; lanes 1, 3, and 5).
Figure 2
Figure 2
Heritable, targeted gene disruptions in the germline using CRISPR-Cas systems. (A) Wild type (Bristol N2) adults were injected with vectors expressing Cas9, sgRNA, and a body wall muscle-specific mCherry marker. mCherry-positive F1 animals were isolated, a small fraction of which were heterozygous for the disruption. Next, the F2 animals were screened for mutant phenotypes, reflecting homozygous disruption. All further progeny of these F2 mutants were homozygous for the disruption. (B) A table summarizing the results of the four experiments, in which 4 disruptions were found out of 402 mCherry-positive F1 animals. (C) Images of worms from our wild type background line, a disrupted unc-119 line, and a disrupted dpy-13 line. (D) Sequences of the indel mutations found in our mutant lines. Insertions are marked in blue, deletions are marked by dashes, and the PAM is marked in purple. *The third experiment targeting the unc-119 locus utilized five-fold higher concentrations of expression vectors (see Supplementary Methods for details).
Figure 3
Figure 3
Heritable, targeted gene disruptions in genes that lead to no obvious phenotypes. (A) A table summarizing the results of the three experiments, in which 93 disruptions were found out of 173 mCherry-positive F1 animals. (B) Sequences of the indel mutations found in several of our mutant lines. Insertions are marked in blue, deletions are marked by dashes, and the PAM is marked in purple. (C) Sequence at the klp-12 locus showing the target PAM site in purple and the MfeI restriction site in green. (D) An image of a 1% agarose gel showing a restriction digest of PCR amplicons spanning the klp-12 cleavage site from seven F1 animals. Wild type sequences in lanes 1 and 2 are cut into bands of 280bp and 107bp, while doubly disrupted sequences remain full length at 387bp in lanes 6 and 7. Lanes 3, 4, and 5 show all three bands, indicating worms that are singly disrupted.

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