CRISPR/Cas9-mediated gene knockout in the ascidian Ciona intestinalis - PubMed (original) (raw)

. 2014 Sep;56(7):499-510.

doi: 10.1111/dgd.12149. Epub 2014 Sep 12.

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

Haruka Sasaki et al. Dev Growth Differ. 2014 Sep.

Abstract

Knockout of genes with CRISPR/Cas9 is a newly emerged approach to investigate functions of genes in various organisms. 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. Short guide RNA (sgRNA) and Cas9 mRNA, when they are expressed in Ciona embryos by means of microinjection or electroporation of their expression vectors, introduced mutations in the target genes. 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.

Keywords: CRISPR/Cas9; Ciona intestinalis; ascidian; knockout; mutagenesis.

© 2014 The Authors Development, Growth & Differentiation © 2014 Japanese Society of Developmental Biologists.

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Figures

Figure 1

Figure 1

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.

Figure 2

Figure 2

Insertions and/or deletions induced by microinjection of CRISPR/Cas9 RNAs. (A) Examples of mutations induced by simultaneous injection of Hox3-sg3 RNA and Cas9 mRNA. The quantity of injected RNAs was 3.0 pg of Hox3-sg3 RNA and 15 pg of Cas9 mRNA. “Wild type” indicates the un-mutated sequence. The recognition site of Hox3-sg3 was shown in red. Deleted nucleotides were shown by “-”. Inserted nucleotides were shown in blue. (B) Examples of mutations induced by simultaneous injection of Hox5-sg1 RNA and Cas9 mRNA. The quantity of injected RNAs was 3.0 pg of Hox5-sg1 RNA and 15 pg of Cas9 mRNA.

Figure 3

Figure 3

CRISPR/Cas9 system does not have a strong side-effect on embryogenesis. (A–C) Morphology of larvae. (A) An un-treated control larva, lateral view. Anterior is toward right. (B) A larvae into which 3.0 pg of Hox3-sg3 and 30 pg of Cas9 mRNA was microinjected. (C) A larvae into which 3.0 pg of Hox5-sg1 and 30 pg of Cas9 mRNA was microinjected. (D) Relative rate of normally developed larvae. In each experiment, the % of normally developed larvae in the population injected with 3.0 pg of sgRNA and 30 pg of Cas9 mRNA was normalized with the score of the uninjected control populations. Therefore, “100%” indicates the rates of normal larvae were identical between two populations. formula image, Experiment 1; formula image, Experiment 2.

Figure 4

Figure 4

Mutations induced by electroporation of expression vectors of Cas9 and sgRNA. (A) Schematic illustrations of expression vectors for Cas9 and sgRNA. pA, poly adenylation sequence. (B) Cel-I assay of polymerase chain reaction (PCR) amplifications that contained the target site of Hox5-sg1. “Not electroporated” lane indicates the PCR band from untreated control larvae. “Hox5-sg1+Cas9” lane indicates the PCR band from larvae electroporated with pSPCiEF1α>Cas9 and pSPU6>Hox5-sg1 vectors. Cel-I cleaved bands were shown by arrows. M, marker lane. (C) Examples of mutations induced by expression vectors of Cas9 and Hox3-sg3. “Wild type” indicates the un-mutated sequence. The recognition site of Hox3-sg3 was shown in red. Deleted nucleotides were shown by “–”. (D) Examples of mutations induced by expression vectors of Cas9 and Hox5-sg1. Inserted nucleotides were shown in blue.

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