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CRISPR/Cas9-mediated gene knockout in the ascidian Ciona intestinalis.

Sasaki H, Yoshida K, Hozumi A, Sasakura Y - Dev. Growth Differ. (2014)

Bottom Line: We demonstrate that CRISPR/Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan.The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on-target site.CRISPR/Cas9-mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases.

View Article: PubMed Central - PubMed

Affiliation: Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, 415-0025, Japan.

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CRISPR/Cas9-mediated mutations of Hox genes in Ciona intestinalis. (A) Cel-I assay of polymerase chain reaction (PCR) amplifications that contained the target site of Hox3-sg3. PCR bands were treated with Cel-I prior to electrophoresis. The “Hox3-sg3+Cas9” lane indicates the PCR product derived from larvae into which 3.0 pg of Hox3-sg3 RNA and 15 pg of Cas9 mRNA were microinjected. “Uninjected” lane indicates the PCR product derived from uninjected control larvae. M, marker lane. The arrow indicates the position of Cel-I cleaved band. (B) Cel-I assay of PCR amplifications that contained the target site of Hox5-sg1. 3.0 pg of Hox5-sg1 RNA and 15 pg of Cas9 mRNA were microinjected. (C) Cel-I assays of PCR amplifications that contained the target sites of Hox12-sg2 and Hox12-sg3. 3.0 pg of a sgRNA and 15 pg of Cas9 mRNA were microinjected. In both cases, Cel-I sensitive bands were not detected.
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fig01: CRISPR/Cas9-mediated mutations of Hox genes in Ciona intestinalis. (A) Cel-I assay of polymerase chain reaction (PCR) amplifications that contained the target site of Hox3-sg3. PCR bands were treated with Cel-I prior to electrophoresis. The “Hox3-sg3+Cas9” lane indicates the PCR product derived from larvae into which 3.0 pg of Hox3-sg3 RNA and 15 pg of Cas9 mRNA were microinjected. “Uninjected” lane indicates the PCR product derived from uninjected control larvae. M, marker lane. The arrow indicates the position of Cel-I cleaved band. (B) Cel-I assay of PCR amplifications that contained the target site of Hox5-sg1. 3.0 pg of Hox5-sg1 RNA and 15 pg of Cas9 mRNA were microinjected. (C) Cel-I assays of PCR amplifications that contained the target sites of Hox12-sg2 and Hox12-sg3. 3.0 pg of a sgRNA and 15 pg of Cas9 mRNA were microinjected. In both cases, Cel-I sensitive bands were not detected.

Mentions: As shown in Figure1A, Cel-I cleaved Hox3 PCR product from larvae into which about 3.0 pg of a sgRNA for Hox3 (Hox3-sg3 in Table1) was introduced with 15 pg of Cas9 mRNA. The presence of Cel-I cleaved bands suggests that the PCR fragment contained nucleotide sequence variations, probably caused by mutations induced by the CRISPR/Cas9 system. Likewise, Cel-I cleaved Hox5 the PCR product derived from larvae into which 3.0 pg of the sgRNA for Hox5 (Hox5-sg1) was introduced with 15 pg of Cas9 mRNA (Fig.1B). The other six sgRNAs designed to target Hox3 or Hox12 were not able to yield a Cel-I sensitive band (Fig.1C), suggesting that appearance of Cel-I sensitive bands are dependent on the sequence of sgRNAs.


CRISPR/Cas9-mediated gene knockout in the ascidian Ciona intestinalis.

Sasaki H, Yoshida K, Hozumi A, Sasakura Y - Dev. Growth Differ. (2014)

CRISPR/Cas9-mediated mutations of Hox genes in Ciona intestinalis. (A) Cel-I assay of polymerase chain reaction (PCR) amplifications that contained the target site of Hox3-sg3. PCR bands were treated with Cel-I prior to electrophoresis. The “Hox3-sg3+Cas9” lane indicates the PCR product derived from larvae into which 3.0 pg of Hox3-sg3 RNA and 15 pg of Cas9 mRNA were microinjected. “Uninjected” lane indicates the PCR product derived from uninjected control larvae. M, marker lane. The arrow indicates the position of Cel-I cleaved band. (B) Cel-I assay of PCR amplifications that contained the target site of Hox5-sg1. 3.0 pg of Hox5-sg1 RNA and 15 pg of Cas9 mRNA were microinjected. (C) Cel-I assays of PCR amplifications that contained the target sites of Hox12-sg2 and Hox12-sg3. 3.0 pg of a sgRNA and 15 pg of Cas9 mRNA were microinjected. In both cases, Cel-I sensitive bands were not detected.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4231237&req=5

fig01: CRISPR/Cas9-mediated mutations of Hox genes in Ciona intestinalis. (A) Cel-I assay of polymerase chain reaction (PCR) amplifications that contained the target site of Hox3-sg3. PCR bands were treated with Cel-I prior to electrophoresis. The “Hox3-sg3+Cas9” lane indicates the PCR product derived from larvae into which 3.0 pg of Hox3-sg3 RNA and 15 pg of Cas9 mRNA were microinjected. “Uninjected” lane indicates the PCR product derived from uninjected control larvae. M, marker lane. The arrow indicates the position of Cel-I cleaved band. (B) Cel-I assay of PCR amplifications that contained the target site of Hox5-sg1. 3.0 pg of Hox5-sg1 RNA and 15 pg of Cas9 mRNA were microinjected. (C) Cel-I assays of PCR amplifications that contained the target sites of Hox12-sg2 and Hox12-sg3. 3.0 pg of a sgRNA and 15 pg of Cas9 mRNA were microinjected. In both cases, Cel-I sensitive bands were not detected.
Mentions: As shown in Figure1A, Cel-I cleaved Hox3 PCR product from larvae into which about 3.0 pg of a sgRNA for Hox3 (Hox3-sg3 in Table1) was introduced with 15 pg of Cas9 mRNA. The presence of Cel-I cleaved bands suggests that the PCR fragment contained nucleotide sequence variations, probably caused by mutations induced by the CRISPR/Cas9 system. Likewise, Cel-I cleaved Hox5 the PCR product derived from larvae into which 3.0 pg of the sgRNA for Hox5 (Hox5-sg1) was introduced with 15 pg of Cas9 mRNA (Fig.1B). The other six sgRNAs designed to target Hox3 or Hox12 were not able to yield a Cel-I sensitive band (Fig.1C), suggesting that appearance of Cel-I sensitive bands are dependent on the sequence of sgRNAs.

Bottom Line: We demonstrate that CRISPR/Cas9 can mutate endogenous genes of the ascidian Ciona intestinalis, a splendid model for elucidating molecular mechanisms for constructing the chordate body plan.The specificity of target choice by sgRNA is relatively high compared to the reports from some other organisms, and a single nucleotide mutation at the sgRNA dramatically reduced mutation efficiency at the on-target site.CRISPR/Cas9-mediated mutagenesis will be a powerful method to study gene functions in Ciona along with another genome editing approach using TALE nucleases.

View Article: PubMed Central - PubMed

Affiliation: Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, 415-0025, Japan.

Show MeSH
Related in: MedlinePlus