Figure 1
From: Generation of knock-in lampreys by CRISPR-Cas9-mediated genome engineering
CRISPR-Cas9-mediated knock-in strategy using LcHsp70A promoter. (A) For the generation of knock-in lamprey, sgRNA1 (for genome digestion), sgRNA2 (for plasmid digestion), the donor plasmids having a bait sequence, and Cas9 mRNA in distilled water containing Fast Green to aid visualization of the spread of the injection are co-injected into lamprey zygotes. (B) After injection, the CRISPR-Cas9-mediated concurrent cleavage occurs in the genome at the site upstream (approximately, 200–600 bp) of the target gene and in the donor plasmid at the bait sequence. This leads to a homology independent DNA repair, resulting in the integration of the donor plasmid into the targeted locus. Here, both forward and reverse integrations can occur. Cis-regulatory DNA sequences for the target gene expression act on the LcHsp70A promoter (enhancer-trapping), resulting in the expression of the reporter gene in cells that express the target gene.
