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Heritable genome editing in C. elegans via a CRISPR-Cas9 system.

Friedland AE, Tzur YB, Esvelt KM, Colaiácovo MP, Church GM, Calarco JA - Nat. Methods (2013)

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

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.

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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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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.

Mentions: To extend our initial results and test whether we could also recover animals carrying disruptions that do not lead to visible phenotypes, we selected two additional loci (klp-12 and Y61A9LA.1) with no known loss-of-function phenotypes and generated sgRNAs to target them. We microinjected these animals with Cas9, sgRNA, and mCherry expression vectors as above, and isolated mCherry-positive F1 progeny. We allowed these F1 animals to lay eggs and then genotyped these animals by sequencing regions of genomic DNA spanning expected cleavage sites. In two replicate experiments targeting klp-12 and one targeting Y61A9LA.1, we generated disruptions in 80.3%, 77. 1%, and 18.1% of the F1s screened, respectively (Fig. 3a, 3b). Interestingly, at the klp-12 locus, 27 out of 80 F1 animals carrying a disruption were homozygous for a single disruption while other animals carried two unique disruption alleles. We speculate that these doubly-targeted mutant F1s are generated through two sequential break and repair events. The first event may occur in the haploid oocyte, where NHEJ-mediated repair introduces an indel The second event likely occurs later in the sperm-contributed chromosome, where either NHEJ introduces a second, unique indel, or through homologous recombination uses the already-disrupted chromosome as a template and copies the error, yielding a homozygous mutant. We followed the inheritance of four klp-12 alleles identified in F1 animals by genotyping single mCherry-negative F2 animals and confirmed the heritability of all of these disruptions (Supplementary Fig. 3).


Heritable genome editing in C. elegans via a CRISPR-Cas9 system.

Friedland AE, Tzur YB, Esvelt KM, Colaiácovo MP, Church GM, Calarco JA - Nat. Methods (2013)

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.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3822328&req=5

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.
Mentions: To extend our initial results and test whether we could also recover animals carrying disruptions that do not lead to visible phenotypes, we selected two additional loci (klp-12 and Y61A9LA.1) with no known loss-of-function phenotypes and generated sgRNAs to target them. We microinjected these animals with Cas9, sgRNA, and mCherry expression vectors as above, and isolated mCherry-positive F1 progeny. We allowed these F1 animals to lay eggs and then genotyped these animals by sequencing regions of genomic DNA spanning expected cleavage sites. In two replicate experiments targeting klp-12 and one targeting Y61A9LA.1, we generated disruptions in 80.3%, 77. 1%, and 18.1% of the F1s screened, respectively (Fig. 3a, 3b). Interestingly, at the klp-12 locus, 27 out of 80 F1 animals carrying a disruption were homozygous for a single disruption while other animals carried two unique disruption alleles. We speculate that these doubly-targeted mutant F1s are generated through two sequential break and repair events. The first event may occur in the haploid oocyte, where NHEJ-mediated repair introduces an indel The second event likely occurs later in the sperm-contributed chromosome, where either NHEJ introduces a second, unique indel, or through homologous recombination uses the already-disrupted chromosome as a template and copies the error, yielding a homozygous mutant. We followed the inheritance of four klp-12 alleles identified in F1 animals by genotyping single mCherry-negative F2 animals and confirmed the heritability of all of these disruptions (Supplementary Fig. 3).

Bottom Line: 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.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.

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.

Show MeSH