Deletion of Large Intergenic Chromosomal Fragments in Human Induced Pluripotent Stem Cells (hiPSC) by CRISPR/Cas9 Genome Editing

Genome-wide mapping interactions between the proximal/distal enhancers and gene promoters display landscapes of the spatiotemporal gene expression in the context of tissue complexity. Increasing data demonstrated that distal enhancers located in the intergenic regions play critical roles in controlling the cognate promoter activities even at great genomic distance from targeted genes. Mutations in these regions contribute to the disease susceptibility and developmental malformations. The CRISPR/Cas9 system has been extensively used for gene editing in biomedical studies. In particular, knockout of functional genes with CRISPR/Cas9 has been widely achieved by creating small insertion or deletion (indels) in the open-reading frame. However, it remains challenging to delete large chromosomal fragments due to the very low efficiency of CRISPR/Cas9-mediated genome editing. In this chapter, we present an efficient protocol to delete an 81.1 kb noncoding intergenic region (Chr8: 11759913-11841048) in human inducible pluripotent stem cells (hiPSCs), which may lead to the disease development of human bicuspid aortic valve (BAV).