Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9 - PubMed (original) (raw)

Tissue-specific genome editing in Ciona embryos by CRISPR/Cas9

Alberto Stolfi et al. Development. 2014 Nov.

Abstract

The CRISPR/Cas9 system has ushered in a new era of targeted genetic manipulations. Here, we report the use of CRISPR/Cas9 to induce double-stranded breaks in the genome of the sea squirt Ciona intestinalis. We use electroporation to deliver CRISPR/Cas9 components for tissue-specific disruption of the Ebf (Collier/Olf/EBF) gene in hundreds of synchronized Ciona embryos. Phenotyping of transfected embryos in the 'F0' generation revealed that endogenous Ebf function is required for specification of Islet-expressing motor ganglion neurons and atrial siphon muscles. We demonstrate that CRISPR/Cas9 is sufficiently effective and specific to generate large numbers of embryos carrying mutations in a targeted gene of interest, which should allow for rapid screening of gene function in Ciona.

Keywords: Ascidians; CRISPR/Cas9; Ciona; Ebf; Genome editing; Islet.

© 2014. Published by The Company of Biologists Ltd.

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Figures

Fig. 1.

Fig. 1.

CRISPR/Cas9 components in Ciona intestinalis. (A) Top: nls::dCas9::nls::eGFP protein used to assay nuclear localization in Ciona. dCas9 is Cas9 with two point mutations that render it catalytically dead. Bottom: nls::Cas9::nls used for targeted mutagenesis. (B) Tail bud-stage embryo electroporated with Mesp>nls::dCas9::nls::eGFP, confirming proper nuclear localization in B7.5 lineage cells. Scale bar: 25 μm. (C) Ebf.774 sgRNA (F+E version). The protospacer (red) is paired with its target in Ebf. ‘F’ modification is in orange, ‘E’ modification is in green. (D) In situ hybridization using eGFP probe in gastrula-stage embryo electroporated, with U6>sgRNA(F+E)::eGFP in right hemisphere, indicating successful transcription of the sgRNA in 100% of embryos (_n_=100).

Fig. 2.

Fig. 2.

CRISPR/Cas9-mediated mutagenesis of Ebf. (A) C. intestinalis Ebf gene, showing exons (solid boxes) and introns (scale bar: 1 kb). Exons are colored according to domains: green, N terminus+DNA-binding domain (DBD); orange, IPT; magenta, atypical HLH; brown, transactivation domain; gray, untranslated regions. Promoters (proximal and distal) are indicated by elbows. Alternative splicing is indicated by dotted line, giving rise to Ebf transcript variants shown below. Both contain the conserved zinc-coordinating motif of the DBD. (B) Alignment of wild-type and mutant Ebf alleles cloned from pooled embryos electroporated with 10 μg EF1α>nls::Cas9::nls, 10 μg U6>Ebf.774 and 10 μg U6>Ebf.813. Three out of seven clones had a mutation (two unique mutations). (C) Mutant Ebf alleles cloned from MACS-sorted hCD4+ cells dissociated from embryos electroporated with 10 μg EF1α>hCD4::mCherry, 25 μg EF1α>nls::Cas9::nls and 75 μg U6>Ebf.774 (see ‘Cleavage assay of genomic DNA from MACS-enriched transfected cells’ section for details). Six out of 13 clones had a mutation (all unique). Target sequences indicated in blue. Indels and substitutions are indicated in red.

Fig. 3.

Fig. 3.

Genomic cleavage assays. (A) Cleavage assay of Ebf exon 9 amplicon from pooled embryos electroporated with EF1α>nls::Cas9::nls+U6>Ebf.774 or EF1α>nls::Cas9::nls alone. Cleavage efficiency was calculated at 31.5%. Cleavage of Ebf exon 9 amplicon from control (_Cas9-_alone) embryos was not detected. Cleavage of kit control amplicon (1:1 mix of wild-type and mutant sequences) was 43.7%. (B) Assay of amplicon from MACS-sorted cells from embryos electroporated with 10 μg EF1α>nls::Cas9::nls, 10 μg U6>Ebf.774 and 10 μg EF1α>hCD4::mCherry (for MACS selection). Cleavage efficiency in sorted hCD4+ cells was 27.1%, versus 2.4% in hCD4− flow-through and 13.7% in unsorted cells from the same pool of dissociated embryos. (C) Assay of amplicon from MACS-sorted cells from embryos electroporated with 10 μg EF1α>hCD4::mCherry, 25 μg EF1α>nls::Cas9::nls and 75 μg U6>Ebf.774. Cleavage efficiency in sorted hCD4+ cells was 66.2% and 45.1% in unsorted cells from the same embryo pool (see

supplementary material Fig. S6

). (D) Assay of pooled embryos electroporated with EF1α>nls::Cas9::nls+U6>Ebf.774, collected at 4, 8 and 12 hpf. Cleavage bands are visible at 8 hpf, but not at 4 hpf. Efficiency does not increase substantially from 8 to 12 hpf.

Fig. 4.

Fig. 4.

Phenotypic assays for tissue-specific loss of Ebf in F0 embryos. (A) Larva (17.5 hpf, 22°C) electroporated with 35 μg Sox1/2/3>nls::Cas9::nls and 25 μg U6>Control sgRNA, showing normal expression of Isl>YFP reporter in A10.57 MN (observed in 68% of larvae, _n_=100). (B) CRISPR/Cas9-targeted mutagenesis of Ebf exon 9 (35 μg Sox1/2/3>nls::Cas9::nls, 25 μg U6>Ebf.774) results in only 23% of larvae showing MN-specific Isl reporter expression (_n_=100). (C) Lost Isl expression can be rescued by a CRISPR/Cas9-resistant form of Ebf (45 μg Ebf>Ebfm774), as Isl>YFP is now seen in 83% of larvae (_n_=100). (D) Larva (26 hpf, 18°C) electroporated with 50 μg Mesp>nls::Cas9::nls and 25 μg U6>Control, showing normal ASMP migration (seen in 96% of larvae, _n_=100) and _Isl_>mCherry reporter expression in ASMPs (filled arrowhead; seen in 53% of larvae, _n_=100). (E) Upon electroporation with 50 μg Mesp>nls::Cas9::nls and 25 μg U6>Ebf.774, ASMP migration is observed in only 22% of larvae (_n_=100) and _Isl_>mCherry expression in ASMPs is only seen in 3% of larvae (_n_=100; empty arrowhead). Asterisk indicates _Isl_>mCherry expression in A10.57 neuron, confirming that disruption of Ebf was B7.5 lineage specific. (F) Control larvae (22 hpf, 18°C) show normal expression of Mrf (assayed by in situ mRNA hybridization) in ASMPs (filled arrowheads; observed in 79% of larvae, _n_=100). (G) Mrf expression in ASMPs is downregulated upon CRISPR/Cas9 targeting of Ebf (50 μg Mesp>nls::Cas9::nls+25 μg U6>Ebf.774), as Mrf expression is seen in only 38% of larvae (_n_=100; empty arrowheads). (H,I) Histograms indicating the fraction of electroporated embryos displaying the phenotypes represented in A-G.

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