Functional characterizations and molecular dissections of long noncoding RNAs (lncRNAs) are critical to understand their involvement in the cellular regulatory network. LncRNAs exert their effects through functional RNA domains that interact with other molecules, including proteins, DNA, and RNA. Here, we describe experimental procedures for generating genomic deletions in a human haploid cell line using the CRISPR/Cas9 system. This method can be applied to examine functions of lncRNAs and their domains by establishing knockout and partial deletion mutant cell lines. In addition, we describe a CRISPR-mediated knockin method for artificial tethering of partner RNA-binding proteins to lncRNAs and its use to validate lncRNA-mediated functions.
Keywords: CRISPR/Cas9; HAP1; Haploid cell; Knockin; Large genomic deletion; MS2 tethering system; lncRNA.