Genome-scale deletion screening of human long non-coding RNAs using a paired-guide RNA CRISPR-Cas9 library

Nat Biotechnol. 2016 Dec;34(12):1279-1286. doi: 10.1038/nbt.3715. Epub 2016 Oct 31.


CRISPR-Cas9 screens have been widely adopted to analyze coding-gene functions, but high-throughput screening of non-coding elements using this method is more challenging because indels caused by a single cut in non-coding regions are unlikely to produce a functional knockout. A high-throughput method to produce deletions of non-coding DNA is needed. We report a high-throughput genomic deletion strategy to screen for functional long non-coding RNAs (lncRNAs) that is based on a lentiviral paired-guide RNA (pgRNA) library. Applying our screening method, we identified 51 lncRNAs that can positively or negatively regulate human cancer cell growth. We validated 9 of 51 lncRNA hits using CRISPR-Cas9-mediated genomic deletion, functional rescue, CRISPR activation or inhibition and gene-expression profiling. Our high-throughput pgRNA genome deletion method will enable rapid identification of functional mammalian non-coding elements.

MeSH terms

  • CRISPR-Cas Systems / genetics*
  • Chromosome Mapping / methods*
  • Clustered Regularly Interspaced Short Palindromic Repeats / genetics*
  • Gene Deletion*
  • Genetic Testing / methods
  • Genome, Human / genetics*
  • High-Throughput Nucleotide Sequencing / methods
  • Humans
  • RNA, Long Noncoding / genetics*
  • Sequence Analysis, RNA / methods


  • RNA, Long Noncoding