Fig. 1

Experimental strategies performed in vitro and in vivo. a Experimental strategies performed in vitro. The target DNA was modified with a Cy5 group. The guide DNA was designed complementary to the target with an unpaired nucleotide at the 3′ end to form the 3′ flap structure. The SGN recognizes the 3′ flap and cleaves the target DNA. The cleaved products were analyzed by denatured polyacrylamide gel electrophoresis and fluorescence imaging. b Experimental strategies performed in vivo. Here, we used Tg(flk1 :eGFP) zebrafish embryos to investigate the SGN activity in vivo. A pair of guide DNAs were designed complementary to the target gene GFP with an unpaired nucleotide at the 3′ end to form the 3′ flap structure. We microinjected the SGN mRNA and the guide DNAs into zebrafish embryos. The expressed SGN recognized the 3′ flap and cleaved the target DNA in vivo. Genomic DNA was digested and repaired by the DNA repair pathway. To examine the DNA editing, we extracted the genomic DNA from the zebrafish embryos and then performed PCR amplification of the GFP target. The amplicons were cloned and sequenced to analyze the mutations caused by SGN. Similar experiments were performed to target the endogenous genes of wild-type zebrafish embryos