FIGURE 3

FIGURE 3

Effect of the lssDNA strand choice on knock-in efficiency. (A) Definition of the target and non-target strands in relation to the CRISPR-Cas9 complex. The strand that is complementary to the crRNA sequence is referred to as the target strand. (B) The target and non-target strands of lssDNA used as a donor template. Each lssDNA was microinjected with 1.5 fmol of the RNP complex into the cytoplasm of one-cell stage zebrafish embryos, and genomic DNA was extracted from 20-embryo pools. (C) Schematic illustration of the sox3 knock-in allele and knock-in allele-specific PCR. (D) Agarose gel image showing the PCR amplicons of knock-in allele-specific PCR at the 3′ junction of the integration and the β-actin2 gene-specific PCR (control) to confirm DNA integrity.