Hello, I am going to generate knock-in zebrafish model for my gene using Bi-FoRe plasmid. the gRNA target site for my gene has 43 percent efficiency. so i did microinjection using 600ng of Cas9 mRNA, 200 ng hEMX gRNA, 300ng gene specific gRNA and 15 ng of plasmid. and after 24 hours i run PCR using junction primers 5 and 3 terminals, but no band! and also i cannot see florescence signals after 36 hpf. Has anyone done this Knock-in before? i need some guidance because i am not sure what can be the issue.