Deletion of transcription factor binding motifs using the CRISPR/spCas9 system in the β-globin LCR

Abstract

Transcription factors play roles in gene transcription through direct binding to their motifs in genome, and inhibiting this binding provides an effective strategy for studying their roles. Here, we applied the CRISPR (clustered, regularly interspaced, short palindromic repeat)/spCas9 (CRISPR-associated protein 9) system to mutate the binding motifs of transcription factors. Binding motifs for erythroid-specific transcription factors were mutated in the locus control region (LCR) hypersensitive sites (HSs) of the human β-globin locus. Guide RNAs targetting binding motifs were cloned into lentiviral CRISPR vector containing the spCas9 gene, and transduced into MEL/ch11 cells carrying human chromosome 11. DNA mutations in clonal cells were initially screened by quantitative PCR (qPCR) in genomic DNA and then clarified by sequencing. Mutations in binding motifs reduced occupancy by transcription factors in a chromatin environment. Characterization of mutations revealed that the CRISPR/spCas9 system mainly induced deletions in short regions of <20 bp and preferentially deleted nucleotides around the fifth nucleotide upstream of Protospacer adjacent motifs (PAMs). These results indicate that the CRISPR/Cas9 system is suitable for mutating the binding motifs of transcription factors, and, consequently, would contribute to elucidate the direct roles of transcription factors.