CRISPR interference at the FAAH-OUT genomic region reduces FAAH expression

FAAH-OUT encodes a putative long non-coding RNA which is located next to the FAAH gene on human chromosome 1. Recently an ~8 kb microdeletion, that removes upstream regulatory elements and the first 2 exons of FAAH-OUT, was reported in a pain insensitive patient (PFS) with additional clinical symptoms including a happy, non-anxious disposition, fast wound healing, fear and memory deficits, and significant post-operative nausea and vomiting induced by morphine. PFS also carries a hypomorphic SNP in FAAH that significantly reduces the activity of the encoded fatty-acid amide hydrolase enzyme. The FAAH and FAAH-OUT mutations identified in PFS result in elevated levels of anandamide (AEA), palmitoylethanolamide (PEA) and oleoylethanolamine (OEA) measured in peripheral blood. These bioactive lipids, which are normally degraded by FAAH, have diverse biological functions including known roles in pain pathways.Here we report the first mechanistic insights into how the FAAH-OUT microdeletion affects FAAH function. Gene editing in a human cell line to mimic the FAAH-OUT microdeletion observed in PFS results in reduced expression of FAAH. Furthermore, CRISPRi experiments targeting the promoter region of FAAH-OUT or a short highly evolutionarily conserved element in the first intron of FAAH-OUT also result in reduced expression of FAAH. These experiments confirm the importance of FAAH-OUT and specific genomic elements within the ~8 kb microdeleted sequence to normal FAAH expression. Our results also highlight the potential of CRISPRi and gene editing strategies that target the FAAH-OUT region for the development of novel FAAH-based analgesic and anxiolytic therapies.