Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos - PubMed (original) (raw)

Genome editing with RNA-guided Cas9 nuclease in zebrafish embryos

Nannan Chang et al. Cell Res. 2013 Apr.

Abstract

Recent advances with the type II clustered regularly interspaced short palindromic repeats (CRISPR) system promise an improved approach to genome editing. However, the applicability and efficiency of this system in model organisms, such as zebrafish, are little studied. Here, we report that RNA-guided Cas9 nuclease efficiently facilitates genome editing in both mammalian cells and zebrafish embryos in a simple and robust manner. Over 35% of site-specific somatic mutations were found when specific Cas/gRNA was used to target either etsrp, gata4 or gata5 in zebrafish embryos in vivo. The Cas9/gRNA efficiently induced biallelic conversion of etsrp or gata5 in the resulting somatic cells, recapitulating their respective vessel phenotypes in etsrp(y11) mutant embryos or cardia bifida phenotypes in fau(tm236a) mutant embryos. Finally, we successfully achieved site-specific insertion of mloxP sequence induced by Cas9/gRNA system in zebrafish embryos. These results demonstrate that the Cas9/gRNA system has the potential of becoming a simple, robust and efficient reverse genetic tool for zebrafish and other model organisms. Together with other genome-engineering technologies, the Cas9 system is promising for applications in biology, agriculture, environmental studies and medicine.

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Figures

Figure 1

Figure 1

Genome editing via the type II CRISPR system in mammalian cells. (A) Schematic diagrams showing the CRISPR system composed of Cas9 and gRNA. The expression of Cas9 protein flanked with a SV40 NLS at the N-terminus and a nucleoplasmin NLS at the C-terminus is driven by the CMV promoter, whereas the transcription of gRNA is driven by the human U6 promoter. gRNA is designed to target the genome sequence of 19-23 bp at the 5′ side of PAM (NGG). (B) A strategy of Cas9/gRNA-induced HDR used to convert a mutant eGFP (mut-eGFP) into wild-type eGFP. The mixture of Cas9/gRNA and a donor DNA fragment was delivered into a HEK293T cell line that stably expresses mutant eGFP. (C) Fluorescent image showing a successfully targeted clone of 293T cells that express the correct eGFP. Scale bar, 100 μm. (D) Sanger sequencing of the PCR amplicon confirmed the correct sequence of eGFP of the clone in C. (E) Isolating eGFP-positive cells by FACS demonstrated 2.51% HDR efficiency.

Figure 2

Figure 2

Cas9/gRNA induces indels in the etsrp locus in zebrafish. (A) Cartoon showing the position of the target site and its sequence in the etsrp locus in zebrafish. (B) The SSA recombination assay showing luciferase activity with and without cleavage of the target site by the Cas9/gRNA system in 293T cells. The luciferase activity increased by 8-fold in the group with gRNA (Cas9 + gRNA) compared with the control group (Cas9 + empty vector). Error bars indicate SD, n = 3. **P < 0.01. (C) Representative SURVEYOR assay showing 35% efficiency of Cas9-mediated cleavage in a single embryo and 20% by TALENs-mediated cleavage in 2 embryos at 50 hpf. The indels of 8 embryos were measured with a frequency of 31.2% - 38.4%. * indicates the cleavage bands. Sam, sample treated by Cas9/gRNA or TALENs; WT, wild type. (D) Representative Sanger sequencing results of the PCR amplicons from 8 individual embryos at 50 hpf showing indels (red) induced by Cas9/gRNA in the targeted etsrp locus. PCR fragments were amplified and cloned into the pEASY vector from an individual embryo for Sanger sequencing (20-30 clones for each embryo). TGG (blue) is the PAM sequence. Deletion is represented by a dashed line and insertion is highlighted in red. (E) Fluorescent images showing abnormal intersegmental vessels that were similar in the etsrpy11 mutant and Cas9/gRNA-induced mutant, compared with the wild-type transgenic embryo at 50 hpf. * indicates defects in intersegmental vessel sprouting that are enlarged in the insets. Scale bar, 100 μm.

Figure 3

Figure 3

Cas9/gRNA induces indels in the gata5 and gata4 loci in zebrafish. (A) Cartoon showing the position of the gRNA-targeting site and its sequence in the gata5 locus in zebrafish. (B) SSA recombination assay showing luciferase activity with and without cleavage of the target site by the Cas9/gRNA system in 293T cells. The luciferase activity increased by 16-fold in the group with gRNA (Cas9 + gRNA) compared with the control group (Cas9 + empty vector). Error bars indicate SD, n = 3. **P < 0.01. (C) Representative SURVEYOR assay showing the efficiency of Cas9-mediated cleavage, 26.4% in a single embryo at 50 hpf. (D) Representative Sanger sequencing results of the PCR amplicons of 8 individual embryos at 50 hpf, showing indels induced by Cas9/gRNA in the targeted gata5 locus. Twenty to thirty clones were sequenced for each embryo. Table summarizes the frequencies of site-specific indels, ranging from 50% (11/20) to 90% (18/20), in 8 individual embryos tested by Sanger sequencing. TGG (blue) is the PAM sequence. Deletion is represented by a dashed line and insertion is highlighted in red. (E) Two small hearts were formed in a Cas9/gRNA-induced mutant (right), which phenocopied that in the fautm236a genetic mutant, whereas a single heart formed in control embryos at 60 hpf. The heart was stained with phalloidin. * indicates two small hearts. Scale bar, 100 μm. (F) Representative Sanger sequencing results of the PCR amplicons of 8 individual embryos at 50 hpf, showing indels induced by Cas9/gRNA in the targeted gata4 locus as in D. AGG (blue) is the PAM sequence. AGG in 2 out of 16 sequencing results was deleted.

Figure 4

Figure 4

Targeted insertion of mloxP mediated by Cas9/gRNA in zebrafish. (A) Cartoon showing the principle of inserting mloxP into a target site in the etsrp locus. The primers for primary PCR genotyping of zebrafish embryos with the expected mloxP insertion being marked. Gel image showing 4 out of 12 randomly selected Cas9/gRNA-treated embryos at 50 hpf that had the expected mloxP insertion (arrows). (B) Sanger sequencing result of the PCR amplicon from one representative Cas9/gRNA-injected zebrafish embryo at 50 hpf, with the correct insertion of mloxP in the etsrp locus as domonstrated in A. PCR fragments were amplified from the tested embryo and cloned into the pEASY vector for sequencing (20-30 clones per embryo), and one clone was confirmed to have the correct insertion of mloxP in the etsrp locus. 1, 3, 4 or 6 represents the original sequence in the left or right arm, whereas 2 or 5 highlights unexpected sequences (red).

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