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Fig. 2 Design the sgRNAs targeting the last intron of pdgfrb (A) Screenshot of the CRISPOR website showing the process of identifying sgRNAs with minimal off-target potential for zebrafish genes. (B) Screenshot showing the list of sgRNAs with lower off-off-target potential (high MIT/CFD Specificity Score) as identified by CRISPOR website for zebrafish pdgfrb. (C) The sites of three sgRNAs chosen for targeting pdgfrb to create knockin line. (D) Schematic representation of the microinjection of Cas9/sgRNA into one-cell stage zebrafish eggs. The eggs are positioned on an agarose plate with the animal pole facing upwards. Cas9 is labeled in red, sgRNA in green, and the solution in the needle in chartreuse. (E) Sequencing result for the larvae injected with Cas9 protein and pdgfrb sgRNA-3. The pdgfrb sgRNA-3 displays high efficiency in inducing indels at the target genomic site, as indicated by significant mixed peaks in the chromatogram. pdgfrb sgRNA-3 binding site, red; PAM area, green.