The regulation of target genes by distal enhancers usually determines the fate and function of cells. Active enhancers in specific regions of chromatin may transcribe bidirectionally to produce long non-coding enhancer RNA (eRNA) to regulate gene expression. We recently found that an antisense enhancer eRNA PEARL (Pcdh eRNA associated with R-loop formation) regulates gene expression of members of the Pcdhα cluster via R-loop formation. To further explore the biological function of eRNA, we performed additional genetic and molecular experiments such as CRISPR (clustered regularly interspaced short palindromic repeats) DNA-fragment editing, RT-PCR, and qPCR. First, we performed expression analyses of the HS5-1 eRNA PEARL and found that it was expressed in a tissue-specific manner. In addition, upon CRISPR DNA-fragment deletion or inversion of the CTCF sites in the HS5-1 enhancer region, the expression of eRNA PEARL was reduced to 2%-10% and the expression of Pcdhα gene cluster was also reduced to 13%-68% of the original levels. Finally, deletion of the bidirectional transcription start site (TSS) of HS5-1 eRNA or inversion of TSS of the eRNA PEARL resulted in approximately 60% or 40% decrease of levels of Pcdhα gene expression. In summary, these data suggested a functional role of the HS5-1 eRNA in gene regulation of the Pcdhα cluster, providing a new direction for future researches on the regulatory mechanisms of clustered Pcdh gene expression in the brain.