Generation of 3′-UTR knockout cell lines by CRISPR/Cas9-mediated genome editing

In addition to the protein code, messenger RNAs (mRNAs) also contain untranslated regions (UTRs). 3'UTRs span the region between the translational stop codon and the poly(A) tail. Sequence elements located in 3' UTRs are essential for pre-mRNA processing. 3'UTRs also contain elements that can regulate protein abundance, localization, and function. At least half of all human genes use alternative cleavage and polyadenylation (APA) to further diversify the regulatory potential of protein functions. Traditional gene editing approaches are designed to disrupt the production of functional proteins. Here, we describe a method that allows investigators to manipulate 3' UTR sequences of endogenous genes for both single- 3'UTR and multi-3'UTR genes. As 3'UTRs can regulate individual functions of proteins, techniques to manipulate 3' UTRs at endogenous gene loci will help to disentangle multi-functionality of proteins. Furthermore, the ability to directly examine the impact of gene regulatory elements in 3'UTRs will provide further insights into their functional significance.