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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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Related in: MedlinePlus

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).
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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).

Mentions: We first generated vectors to express Cas9 and sgRNAs in the germline (Fig. 1a). An SV40 nuclear localization signal (NLS) was added to the 3′ end of the Cas9 open reading frame to ensure the enzyme would be properly localized to the nucleus8, 10. To drive expression of transcripts encoding this Cas9-SV40 NLS fusion protein, we utilized the promoter sequence from the gene eft-3, selected for its effectiveness in driving expression in the germline13. While previous studies have utilized vectors containing RNA polymerase III (pol III) promoters to transcribe small RNAs14 or sgRNAs in mammalian systems, no equivalent vector has been described in C. elegans. Studying conserved upstream and downstream regulatory sequences flanking a U6 snRNA gene in C. elegans, we derived a putative pol III promoter for sgRNA expression (Fig. 1a; Supplementary Fig. 1). It has been suggested that optimal expression from pol III promoters occurs when the first base transcribed is a purine15, 16. Combining this finding with the known sequence requirements of CRISPR-Cas guided cleavage, our sgRNA expression system enables the selection of target sequences of the form G/A(N)19NGG, where the G/A(N)19 represents a 20 nucleotide sequence that will recognize a homologous stretch of double-stranded DNA in the genome, and the 3′ NGG sequence represents the essential protospacer-associated motif (PAM)1 (Fig. 1b).


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)

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).
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3822328&req=5

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).
Mentions: We first generated vectors to express Cas9 and sgRNAs in the germline (Fig. 1a). An SV40 nuclear localization signal (NLS) was added to the 3′ end of the Cas9 open reading frame to ensure the enzyme would be properly localized to the nucleus8, 10. To drive expression of transcripts encoding this Cas9-SV40 NLS fusion protein, we utilized the promoter sequence from the gene eft-3, selected for its effectiveness in driving expression in the germline13. While previous studies have utilized vectors containing RNA polymerase III (pol III) promoters to transcribe small RNAs14 or sgRNAs in mammalian systems, no equivalent vector has been described in C. elegans. Studying conserved upstream and downstream regulatory sequences flanking a U6 snRNA gene in C. elegans, we derived a putative pol III promoter for sgRNA expression (Fig. 1a; Supplementary Fig. 1). It has been suggested that optimal expression from pol III promoters occurs when the first base transcribed is a purine15, 16. Combining this finding with the known sequence requirements of CRISPR-Cas guided cleavage, our sgRNA expression system enables the selection of target sequences of the form G/A(N)19NGG, where the G/A(N)19 represents a 20 nucleotide sequence that will recognize a homologous stretch of double-stranded DNA in the genome, and the 3′ NGG sequence represents the essential protospacer-associated motif (PAM)1 (Fig. 1b).

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
Related in: MedlinePlus