A High-throughput CRISPR-interference Screening Platform to Study lncRNA Functions Through Serial Cellular and Molecular Phenotyping

A High-throughput CRISPR-interference Screening Platform to Study lncRNA Functions Through Serial Cellular and Molecular Phenotyping

Abstract
Technological advances in RNA-sequencing revealed that the human genome contains thousands of non-protein coding genes giving rise to long non-coding RNAs (lncRNAs). While the matter of their functionality remains debated, their sheer numbers beg the question if and how they are involved in human development and disease and may potentially serve as candidate therapeutic targets. To date, studies aimed at unravelling lncRNA functionality have mainly focussed on individual lncRNAs that are prioritized based on a number of selection criteria. These studies have been extremely valuable as they clearly demonstrate that lncRNAs are not per definition transcriptional noise as many have claimed, but are actively involved in regulating various cellular processes in development and disease. On the other hand, CRISPR-based library screens have more recently been used to perturb lncRNA expression on a much larger scale. While these pooled library screens enable a more systematic evaluation by increasing the number of lncRNAs investigated, they require a selection process that is based on a single phenotype or molecular reporter construct. This restriction limits the fraction of functional lncRNAs identified. To enable a more comprehensive and unbiased analysis of lncRNA function, we established a unique high-throughput parallel CRISPRi screening platform that combines live cell imaging with a scalable RNA-sequencing workflow. For each lncRNA target a pool of sgRNAs is delivered to cells with stable dCas9-KRAB-MeCP2 expression grown in 96-well plates. Cells are subsequently monitored in real time using an Incucyte device to quantify cell growth, proliferation, and apoptosis. Finally, cells are lysed followed by RNA-sequencing library preparation directly on crude cell lysates allowing accurate quantification of the expression of 7000-8000 genes per sample at ultra-low cost and high throughput. This unique platform enables the analysis of hundreds of lncRNAs in a single experiment, generating a cellular and molecular readout for each of them. Of note, this platform can also be deployed for the characterization of mRNAs and support therapeutic target identification and characterization in general.