CRISPR/Cas9 alter NFYA binding site on CD44-regulating cis-element and control CD44 expression in breast cancer cells

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Abstract
Background CRISPR/Cas9 is used for editing of non-coding sequence. This study tested the involvement of downstream cis-element in regulating CD44 expression and the possibility of using CRISPR/Cas9 system to manipulate the transcription factor binding site. Bioinformatic tools predicted two binding sites (P1 and P2) for NFYA transcription factors. CRISPR/Cas9 was used to knockout NFYA gene and alter the NFYA binding site (P2). Results The data revealed decrease of CD44 gene expression and CD44+ CD24− sub-population after editing of P2 sequence more than the decrease resulted from editing of NFYA gene itself, confirming the involvement of NFYA in regulating CD44 gene. Both editing inhibited the migration ability of MDA-MB-231 cells. Unlike editing of NFYA gene, altering P2 sequence induced apoptosis. CHIP assay revealed that NFY have the ability to bind both P1 and P2 sequences, with higher enrichment in case of P2 than P1. After performing site-directed mutagenesis and luciferase assay we confirmed the involvement of both P1 and P2 in gene regulation, with higher potential in case of P2 than P1. Conclusion The findings confirmed the regulation of CD44 by NFYA and the efficacy of using CRISPR/Cas9 in altering the binding site, and downregulation of CD44.

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