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Implementation of the CRISPR-Cas9 system in fission yeast.

Jacobs JZ, Ciccaglione KM, Tournier V, Zaratiegui M - Nat Commun (2014)

Bottom Line: Application of the CRISPR-Cas9 genome editing system in the model organism Schizosaccharomyces pombe has been hampered by the lack of constructs to express RNA of arbitrary sequence.Here we present expression constructs that use the promoter/leader RNA of K RNA (rrk1) and a ribozyme to produce the targeting guide RNA.Together with constitutive expression of Cas9, this system achieves selection-free specific mutagenesis with efficiencies approaching 100%.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854, USA.

ABSTRACT
Application of the CRISPR-Cas9 genome editing system in the model organism Schizosaccharomyces pombe has been hampered by the lack of constructs to express RNA of arbitrary sequence. Here we present expression constructs that use the promoter/leader RNA of K RNA (rrk1) and a ribozyme to produce the targeting guide RNA. Together with constitutive expression of Cas9, this system achieves selection-free specific mutagenesis with efficiencies approaching 100%. The rrk1 CRISPR-Cas9 method enables rapid and efficient genome manipulation and unlocks the CRISPR toolset for use in fission yeast.

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sgRNA expression system. (a) Schema of sgRNA expression construct and processing. HHR: Hammerhead Ribozyme. (b) 5′ RACE sequence. Only 4bp of the oligodG attached to the 5′ end, resulting from the method, are shown. (c) Northern analysis of Cas9 expressing , ade6+ strains with either no sgRNA vector or a sgRNA vector targeted against ade6-M210. Marker sizes are shown to the left and hybridization probe below. (d) ade6 targeting sequences.
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Figure 1: sgRNA expression system. (a) Schema of sgRNA expression construct and processing. HHR: Hammerhead Ribozyme. (b) 5′ RACE sequence. Only 4bp of the oligodG attached to the 5′ end, resulting from the method, are shown. (c) Northern analysis of Cas9 expressing , ade6+ strains with either no sgRNA vector or a sgRNA vector targeted against ade6-M210. Marker sizes are shown to the left and hybridization probe below. (d) ade6 targeting sequences.

Mentions: We constructed an expression cassette by joining positions −1 to −358 of the rrk1 gene with the sgRNA sequence5,8, preceded by a CspCI restriction target site that serves as a placeholder for cloning of the targeting sequence (Supplementary Fig. 1). Since rrk1 is synthesized by RNA Pol II6 which has complex transcription terminators and yields polyadenylated transcripts, we cloned a Hammerhead Ribozyme9,10 immediately downstream of the construct to precisely determine the 3′ end of the mature sgRNA (Fig. 1A and Supplementary Fig. 1). Northern blotting and 5′ RACE analysis revealed the accumulation of sgRNA with correct cleavage of the 5′ and 3′ ends, as well as the presence of a larger precursor (Fig. 1B,C).


Implementation of the CRISPR-Cas9 system in fission yeast.

Jacobs JZ, Ciccaglione KM, Tournier V, Zaratiegui M - Nat Commun (2014)

sgRNA expression system. (a) Schema of sgRNA expression construct and processing. HHR: Hammerhead Ribozyme. (b) 5′ RACE sequence. Only 4bp of the oligodG attached to the 5′ end, resulting from the method, are shown. (c) Northern analysis of Cas9 expressing , ade6+ strains with either no sgRNA vector or a sgRNA vector targeted against ade6-M210. Marker sizes are shown to the left and hybridization probe below. (d) ade6 targeting sequences.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4215166&req=5

Figure 1: sgRNA expression system. (a) Schema of sgRNA expression construct and processing. HHR: Hammerhead Ribozyme. (b) 5′ RACE sequence. Only 4bp of the oligodG attached to the 5′ end, resulting from the method, are shown. (c) Northern analysis of Cas9 expressing , ade6+ strains with either no sgRNA vector or a sgRNA vector targeted against ade6-M210. Marker sizes are shown to the left and hybridization probe below. (d) ade6 targeting sequences.
Mentions: We constructed an expression cassette by joining positions −1 to −358 of the rrk1 gene with the sgRNA sequence5,8, preceded by a CspCI restriction target site that serves as a placeholder for cloning of the targeting sequence (Supplementary Fig. 1). Since rrk1 is synthesized by RNA Pol II6 which has complex transcription terminators and yields polyadenylated transcripts, we cloned a Hammerhead Ribozyme9,10 immediately downstream of the construct to precisely determine the 3′ end of the mature sgRNA (Fig. 1A and Supplementary Fig. 1). Northern blotting and 5′ RACE analysis revealed the accumulation of sgRNA with correct cleavage of the 5′ and 3′ ends, as well as the presence of a larger precursor (Fig. 1B,C).

Bottom Line: Application of the CRISPR-Cas9 genome editing system in the model organism Schizosaccharomyces pombe has been hampered by the lack of constructs to express RNA of arbitrary sequence.Here we present expression constructs that use the promoter/leader RNA of K RNA (rrk1) and a ribozyme to produce the targeting guide RNA.Together with constitutive expression of Cas9, this system achieves selection-free specific mutagenesis with efficiencies approaching 100%.

View Article: PubMed Central - PubMed

Affiliation: Department of Molecular Biology and Biochemistry, Rutgers, the State University of New Jersey, Piscataway, New Jersey 08854, USA.

ABSTRACT
Application of the CRISPR-Cas9 genome editing system in the model organism Schizosaccharomyces pombe has been hampered by the lack of constructs to express RNA of arbitrary sequence. Here we present expression constructs that use the promoter/leader RNA of K RNA (rrk1) and a ribozyme to produce the targeting guide RNA. Together with constitutive expression of Cas9, this system achieves selection-free specific mutagenesis with efficiencies approaching 100%. The rrk1 CRISPR-Cas9 method enables rapid and efficient genome manipulation and unlocks the CRISPR toolset for use in fission yeast.

Show MeSH