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

(A, F) An uninjected embryo derived from Tg(cmlc2:eGFP) expressing eGFP in the cardiac cells. (B, G) Tg(cmlc2:eGFP)-derived embryos injected with spns2-crRNA1 (25 pg), tyr-crRNA (25 pg), tracrRNA (100 pg) and Cas9 protein (400 pg). (C, H) Tg(cmlc2:eGFP)-derived embryos injected with spns2-gRNA1 (25 pg), tyr-gRNA (25 pg) and Cas9 protein (400 pg). (D, I) Tg(cmlc2:eGFP)-derived embryos injected with spns2-crRNA1 (25 pg), tyr-crRNA (25 pg), tracrRNA (100 pg) and Cas9 mRNA (250 pg). (E, J) Tg(cmlc2:eGFP)-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.

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

(A) Whole-mount in situ hybridization with an epdr1 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.

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

The injection conditions used in S1 Fig were the same as those used in Fig 1. (A, F, K, P) uninjected embryos. (B, G, L, Q) Tg(cmlc2:EGFP)-derived embryos injected with spns2-crRNA1, tyr-crRNA, tracrRNA and Cas9 protein. (C, H, M, R) Tg(cmlc2:EGFP)-derived embryos injected with spns2-gRNA1, tyr-gRNA and Cas9 protein. (D, I, N, S) Tg(cmlc2:EGFP)-derived embryos injected with spns2-crRNA1, tyr-crRNA, tracrRNA and Cas9 mRNA. (E, J, O, T) Tg(cmlc2:EGFP)-derived embryos injected with spns2-gRNA1, tyr-gRNA and Cas9 mRNA. The phenotypic results between the samples in Fig 1 and S1 Fig are essentially similar. White and black arrowheads indicate the position of the developing heart and the position of the eye, respectively. The embryos in (A), (B), (C), (D), (E), (K), (L), (M), (N) and (O) correspond to the embryos in (F), (G), (H), (I), (J), (P), (Q), (R), (S) and (T), respectively. (A-E, K-O) Ventral view with anterior at the top at 1 dpf. (F-J, P-T) Lateral view with anterior to the left and dorsal at the top at 2 dpf.

Requirement of tracrRNA and crRNA for Cas9-mediated genome modifications.

(A, E) an uninjected embryo. (B, F) Tg(cmlc2:EGFP)-derived embryos injected with Cas9 protein (400 pg). (C, G) Tg(cmlc2:EGFP)-derived embryos injected with Cas9 mRNA (250 pg). (D, H) Tg(cmlc2:EGFP)-derived embryos injected with two crRNAs (spns2-crRNA; 25 pg + tyr-crRNA; 25 pg) and tracrRNA (100 pg). No abnormality in cardiac development at 1 dpf or the retinal epithelium pigmentation at 2 dpf was observed.

Phenotypic analysis of the embryos injected with three crRNAs, tracrRNA and Cas9 protein.

(A) An uninjected embryo. (B) Tg(cmlc2:EGFP)-derived embryos injected with tracrRNA (100 pg), spns2-crRNA2 (25 pg), s1pr2-crRNA1 (25 pg) s1pr2-crRNA2 (25 pg) and Cas9 protein (400 pg). (C) Tg(cmlc2:EGFP)-derived embryos injected with spns2-gRNA1 (25 pg), s1pr2-gRNA1 (25 pg), s1pr2-gRNA2 (25 pg) and Cas9 protein (400 pg). Cardia bifida was observed in the embryo injected with multiple crRNAs, tracrRNA and Cas9 protein, whereas a normal single heart was observed in an uninjected embryo and the embryo injected with multiple gRNAs and Cas9 protein. (A-C) Ventral view with anterior at the top. White arrowheads indicate the position of the developing heart.