고려대학교 생명과학대학 생명과학부/ 대학원 분자생명과학과

Loss of function studies of long intergenic non-coding RNAs (lincRNAs) have remained a challenge, mainly because of incomplete gene annotations and a lack of efficient tools. Here we present a genome-wide design of guide RNA pairs for highly efficient CRISPR/Cas9-based en blocand segmental deletions of every annotated mammalian lincRNA gene. Toward this aim, we improved lincRNA annotations and established a new scoring system that predicts highly efficient guide RNAs by considering the positional nucleotide preferences, the Shannon entropy, the secondary structure, and potential off-targets of each guide RNA. Using this system, we deleted the entire XIST gene, as well as four smaller XIST segments, from K562 cells at an efficiency of 31 ？ 56%. RNA fluorescent in situ hybridization and RNA sequencing showed that both the whole and segmental deletions of XIST resulted in compatible XIST knockout phenotypes but the whole deletions with loss of regulatory DNA elements affected the expression of other genes, suggesting that segmental deletions with no loss of any regulatory element are appropriate approaches for lincRNA functional knockout. As another case study, segmental- (100 kb) and en bloc-deletions (290 kb) of a different lincRNA, RP11-307P5.1, were achieved at efficiencies of 65% and 46%.Our study provides a guideline for the efficient knockout of lincRNAs as well as useful resources for lincRNA research and genome editing.