Time course analysis of the genome modifications induced by the Cas9 protein and Cas9 mRNA.

<p>Two crRNAs (spns2-crRNA1; 25 pg + tyr-crRNA; 25 pg) and tracrRNA (100 pg) were co-injected with Cas9 protein (400 pg) or Cas9 mRNA (250 pg) into zebrafish embryos and the genomic DNA was prepared from the dome stage embryos (4.3 hpf) or the shield stage embryos (6 hpf). Genome modifications induced by the Cas9 protein or Cas9 mRNA were assessed by a HMA. The positions of the expected homoduplexes are indicated by the arrowheads. Non-specific bands are indicated by the asterisks. The sizes of the DNA ladder markers are indicated by the base pairs at the left.</p

Visualization of endogenous gene expression by knock-in of the eGFP reporter using the crRNA-tracrRNA-Cas9 protein complex.

<p>(A) Whole-mount <i>in situ</i> hybridization with an <i>epdr1</i> probe. (B) An uninjected embryo. (C) The embryo injected two crRNAs (epdr1-crRNA; 25 pg + Mbait-crRNA; 25 pg), tracrRNA (100 pg) and Mbait-hs-eGFP (25 pg) with Cas9 protein (400 pg). The expression of eGFP was detected in the anterior central nervous system, including neurons.</p

Efficient Multiple Genome Modifications Induced by the crRNAs, tracrRNA and Cas9 Protein Complex in Zebrafish

<div><p>The type II clustered regularly interspaced short palindromic repeats (CRISPR) associated with Cas9 endonuclease (CRISPR/Cas9) has become a powerful genetic tool for understanding the function of a gene of interest. In zebrafish, the injection of Cas9 mRNA and guide-RNA (gRNA), which are prepared using an <i>in vitro</i> transcription system, efficiently induce DNA double-strand breaks (DSBs) at the targeted genomic locus. Because gRNA was originally constructed by fusing two short RNAs CRISPR RNA (crRNA) and <i>trans</i>-activating crRNA (tracrRNA), we examined the effect of synthetic crRNAs and tracrRNA with Cas9 mRNA or Cas9 protein on the genome editing activity. We previously reported that the disruption of <i>tyrosinase</i> (<i>tyr</i>) by tyr-gRNA/Cas9 mRNA causes a retinal pigment defect, whereas the disruption of <i>spns2</i> by spns2-gRNA1/Cas9 mRNA leads to a cardiac progenitor migration defect in zebrafish. Here, we found that the injection of spns2-crRNA1, tyr-crRNA and tracrRNA with Cas9 mRNA or Cas9 protein simultaneously caused a migration defect in cardiac progenitors and a pigment defect in retinal epithelial cells. A time course analysis demonstrated that the injection of crRNAs and tracrRNA with Cas9 protein rapidly induced genome modifications compared with the injection of crRNAs and tracrRNA with Cas9 mRNA. We further show that the crRNA-tracrRNA-Cas9 protein complex is functional for the visualization of endogenous gene expression; therefore, this is a very powerful, ready-to-use system in zebrafish.</p></div

Comparison of the crRNAs and gRNAs on the Cas9 protein-mediated genome editing activity.

<p>Three crRNAs (spns2-crRNA2; 25 pg + s1pr2-crRNA1; 25 pg + s1pr2-crRNA2; 25 pg) plus tracrRNA (100 pg) or three gRNAs (spns2-gRNA2; 25 pg + S1PR2-gRNA1; 25 pg + S1PR2-gRNA2; 25 pg) were co-injected with Cas9 protein (400 pg) into zebrafish embryos and the genomic DNA was prepared from 24 hpf embryos. The genome modifications induced by the Cas9 protein were assessed by a HMA. Target sites for S1PR2-gRNA1 and S1PR2-gRNA2 are located within the binding sites of S1PR2-specific primers. The positions of the expected homoduplexes are indicated by the asterisks. The sizes of the DNA ladder markers are indicated by the base pairs at the left.</p

Heteroduplex mobility assay in the embryos injected with two crRNAs, tracrRNA and Cas9 protein.

<p>The <i>spns2</i>- and <i>tyr</i>-targeted genomic regions were amplified from the genomic DNA of individual embryos (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128319#pone.0128319.g001" target="_blank">Fig 1</a>) by PCR using locus-specific primers. To perform the HMA, the resultant PCR amplicons were separated on a 15% polyacrylamide gel. The positions of the expected homoduplexes are indicated by the arrowheads and the multiple heteroduplexes are indicated by the white lines. The sizes of the DNA ladder markers are indicated by the base pairs at the left.</p

Detection of heteroduplexes at <i>spns2</i> and <i>tyr</i> loci by HMA in F1 embryos.

<p>F0 founder male 3 and female 5 were mated with wild-type fish and genomic DNA from individual F1 embryos was isolated at 1 dpf. PCR amplicons for <i>spns2</i> or <i>tyr</i> were electrophoresed on a 15% polyacrylamide gel. We observed heteroduplex bands containing mutant alleles (brackets) at <i>spns2</i> and <i>tyr</i> loci. The positions of the expected homoduplexes are indicated by the arrowheads. The sizes of the DNA ladder markers are indicated by the base pairs at the left.</p

Phenotypic analysis in the embryos injected with two crRNAs, tracrRNA and Cas9 protein.

<p>(A, F) An uninjected embryo derived from Tg(<i>cmlc2</i>:<i>eGFP</i>) expressing eGFP in the cardiac cells. (B, G) Tg(<i>cmlc2</i>:<i>eGFP</i>)-derived embryos injected with spns2-crRNA1 (25 pg), tyr-crRNA (25 pg), tracrRNA (100 pg) and Cas9 protein (400 pg). (C, H) Tg(<i>cmlc2</i>:<i>eGFP</i>)-derived embryos injected with spns2-gRNA1 (25 pg), tyr-gRNA (25 pg) and Cas9 protein (400 pg). (D, I) Tg(<i>cmlc2</i>:<i>eGFP</i>)-derived embryos injected with spns2-crRNA1 (25 pg), tyr-crRNA (25 pg), tracrRNA (100 pg) and Cas9 mRNA (250 pg). (E, J) Tg(<i>cmlc2</i>:<i>eGFP</i>)-derived embryos injected with spns2-gRNA1 (25 pg), tyr-gRNA (25 pg) and Cas9 mRNA (250 pg). (A-E) The injection of the two crRNAs and tracrRNA with Cas9 protein or Cas9 mRNA (B and C) as well as the injection of two gRNAs with Cas9 protein or Cas9 mRNA (D and E) caused the cardiac progenitor migration defects at 1 day post-fertilization (dpf), whereas an uninjected embryo had a normal heart (A). White arrowheads indicate the position of the developing heart. (F–J) The injection of the two crRNAs and tracrRNA with Cas9 protein or Cas9 mRNA (G and H) as well as the injection of two gRNAs with Cas9 protein or Cas9 mRNA (I and J) caused pigmentation defects in the retinal epithelium at 2 dpf, whereas an uninjected embryo had retinal epithelial cells with normal pigmentation (F). Black arrowheads indicate the position of the eye. The embryos in (A), (B), (C), (D) and (E) correspond to the embryos in (F), (G), (H), (I) and (J), respectively. (A-E) Ventral view with anterior at the top. (F-J) Lateral view with anterior to the left and dorsal at the top.</p

Sequence analysis of the genome modifications induced by two crRNAs, tracrRNA and Cas9 protein.

<p>PCR amplicons for the <i>spns2</i>- and <i>tyr</i>-target sites from the individual genomic DNA (as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128319#pone.0128319.g002" target="_blank">Fig 2</a>) were inserted into the pGEM-T Easy vector, and the inserted fragments derived from the individual PCR amplicons were randomly sequenced. The targeted genomic sequences and PAM sequences are indicated by the green and blue letters, respectively. The deleted and inserted nucleotides compared with the wild-type sequence (top row) are indicated by the red dashes and red letters, respectively. The numbers of nucleotides deleted (-) and inserted (+) are indicated to the right with the detection number. Slashes mean a gap in the genome sequence containing a large insertion or a large deletion.</p

Detection of Cas9 protein by western blotting using anti-Cas9 antibody.

<p>Two crRNAs (spns2-crRNA1; 25 pg + tyr-crRNA; 25 pg) and tracrRNA (100 pg) were co-injected with Cas9 protein (400 pg) or Cas9 mRNA (250 pg) into zebrafish embryos and total whole body lysate was prepared from the embryos (1 hpf or 24 hpf). Western blotting was performed using anti-Cas9 protein and anti-γ-Tubulin antibodies. Recombinant Cas9 protein (1ng) was applied at the right as a control. Essentially similar results were obtained by three independent experiments. The positions of the indicated proteins are indicated by the arrowheads. The sizes of the protein markers are indicated by kD at the left.</p