Figure 1.

Scheme of the knock-in strategy. A target site (TS), located ~500 base pairs upstream of the ATG in the non-coding region in the gene of interest were chosen. The bait plasmid was constructed by cloning 1 kb of the upstream sequence, including the target site, into a plasmid with the desired fluorescent reporter and poly A (PA) sequence. The bait plasmid, sgRNA against the target site, and Cas9 mRNA were injected at the 1-cell stage. The Cas9 protein creates double stranded breaks (DSB) at both TS, i.e., genomic locus and bait plasmid, and the linearized plasmid bait is integrated by homology independent repair. Forward integration of bait plasmid will result in expression of the fluorescent reporter that matches the expression pattern of the gene of interest. Primer pairs (A+B, C+D) can be used to verify the 5′ and 3′ junctions of the knock-in, respectively.