FIGURE 8

Effect of lssDNA strand choice and 3′ homology arm length on composite tag knock-in into the sox11a and pax6a genes. In these knock-in designs, ∼200-nt-long composites that contain the HBH (His6-Bio-His6) tag followed by a TEV protease cleavage site and FLAG epitope tag in its trimeric form were knocked into the 5′ end of the coding sequence of the sox11a(A) and pax6a(B) genes. (a) Sequences and locations of crRNAs for DSB induction of sox11a and pax6a. (b) Target and non-target strands of lssDNA with different 3′ homology arm lengths were used as donor templates. Each lssDNA was microinjected with 1.5 fmol of the RNP complex into one-cell stage zebrafish embryos, and genomic DNA was extracted from 10-embryo pools. (c) Schematic illustration of the sox11a and pax6a knock-in alleles and knock-in allele-specific PCRs. (d) Knock-in allele-specific qPCRs for 5′ and 3′ junctions using the hydrolysis probes shown in panel (c). The vertical bars represent the means of 7–11 replicates, each of which consists of a pooled sample of 10 injected embryos and is shown as a colored circle.