Protocol for permanent gene repression by CRISPR-adenine base editing of promoter CCAAT motifs

Here, we present a protocol to achieve permanent downregulation of gene expression by editing the CCAAT box in promoter regions using CRISPR-adenine base editors (ABEs). We outline steps for guide RNA (gRNA) design, transfection, genomic DNA extraction, Sanger sequencing, and gene expression quantification. The protocol is optimized for mammalian cell lines (e.g., NIH3T3). It allows for precise disruption of transcription factor binding site without double-strand breaks and offers a novel alternative to RNAi or CRISPR interference (CRISPRi). For complete details on the use and execution of this protocol, please refer to Daliri et al.¹.