Genome-wide screening for functional long noncoding RNAs in human cells by Cas9 targeting of splice sites

The functions of many long noncoding RNAs (lncRNAs) in the human genome remain unknown owing to the lack of scalable loss-of-function screening tools. We previously used pairs of CRISPR-Cas9 (refs. 1, 2, 3) single guide RNAs (sgRNAs) for small-scale functional screening of lncRNAs. Here we demonstrate genome-wide screening of lncRNA function using sgRNAs to target splice sites and achieve exon skipping or intron retention. Splice-site targeting outperformed a conventional CRISPR library in a negative selection screen targeting 79 ribosomal genes. Using a genome-scale library of splicing-targeting sgRNAs, we performed a screen covering 10,996 lncRNAs and identified 230 that are essential for cellular growth of chronic myeloid leukemia K562 cells. Screening GM12878 lymphoblastoid cells and HeLa cells with the same library identified cell-type-specific differences in lncRNA essentiality. Extensive validation confirmed the robustness of our approach.