CRISPR/Cas9-mediated gene knockout in the ascidian Ciona intestinalis.
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.
Affiliation: Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, 415-0025, Japan.Show MeSH
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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.
Affiliation: Shimoda Marine Research Center, University of Tsukuba, Shimoda, Shizuoka, 415-0025, Japan.