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Fig. 1
Targeted genomic integration of the Mbait-hs-eGFP reporter into the pax2a locus using the CRISPR/Cas9.
(a) A schematic representation of the pax2a locus containing the 5′ untranslated region (white box) and the pax2a-gRNA target sites (pink box), and the reporter construct consisting of the Mbait-gRNA target site (blue box), the hsp70 promoter (hs, light red box), the eGFP gene (green box) and the polyA signal (pA, purple box). The Mbait-hs-eGFP reporter was co-injected with pax2a-gRNA1, Mbait-gRNA and Cas9 mRNA into 1-cell stage zebrafish embryos. Concurrent cleavage of the targeted genomic locus and the Mbait-hs-eGFP reporter resulted in the integration of the reporter by non-homologous end joining. The scheme shows forward integration of the reporter. (b,b’,d,d’) An uninjected control embryo around 32 hpf shows no eGFP expression. (c,c’,e,e’) The expression of eGFP around 32 hpf was detected in the midbrain-hindbrain boundary (MHB)(asterisk), the optic stalk (os), the otic vesicles (ov), the hindbrain neurons (hb), the spinal cord (sc) neurons and the pronephric duct (pd) in the injected embryo at 1 dpf. (f) The integration of the reporter into the pax2a gene was determined by genomic PCR using the pax2a-specific and reporter-specific primers. The targeted positions of the primers are shown in (a). (g) The sequences of the 5′ junction and the 3′ junction at the integration site of the eGFP-positive embryo (c,c’). A 4-bp deletion and a 1-bp insertion were observed at the 5′ junction and the 3′ junction, respectively. The red letter represents the inserted nucleotide. (b–e,b’–e’) Lateral view with anterior to the left and dorsal to the top. Black letters and green letters represent pax2a sequences and the reporter sequences, respectively.