Fig. 1

A schematic pipeline for the prediction of genomic safe harbor loci in the chicken genome. Seven steps were followed to predict the chicken genomic safe harbor loci. Step 1) A few validated GSH loci in the human, murine, and porcine genomes were nominated (A). Step 2) Flanking genes surrounding the validated GSH loci were analyzed in the chicken genome using the genome data viewer of NCBI (B). Step 3) Gene arrangement around the validated GSH loci was compared to the potential GSH locus in the chicken genome (C). Step 4) If one flanking gene was similar, the pairwise alignment was excluded and the process was followed from step 5 (C-a). If two flanking genes were similar, pairwise alignment was performed (C-b). And if there was no similarity, the region was not considered a potential GSH (C–c). Step 5) The presence of possible annotated coding or non-coding genes in the predicted GSH locus was evaluated by GDV (Gallus gallus genome assembly bGalGal1.GRCg7b/w) and the UCSC Genome Browser (D). Step 6) The coordinates of predicted GSH loci were evaluated by Hi-C data to ensure that the insertion site was contained within an individual TAD (E). Step 7) The expression levels of the genes flanking the potential GSH locus were accessed from Gene Expression Atlas to determine whether these genes are highly-transcribed (F). A sgRNA-binding site in close proximity of the highly-transcribed gene will be selected by a valid online tool (G)