CRISPR interference (CRISPRi) technology repurposes the CRISPR machinery to induce gene silencing by guiding a nuclease deficient Cas9 protein to target gene transcription start site (TSS) and thus impairing transcription.
CRISPRi can be used to conduct genome-wide "pooled" screens enabling tens of thousands of genes to be simultaneously silenced in a heterogeneous cell population which is then submitted to a filtering step (such as proliferation or reporter-based GFP signaling). Candidate genes, that is those affecting the process of interest, are then identified based on sgRNA representation in the initial cell population compared to the filtered one. While pooled screening is an extremely powerful target identification strategy, candidates are investigated relative to a sole phenotype and thus requires prior knowledge of said phenotype (or pathway) of interest. As a result of this limitation, pooled screening is not compatible to investigate the function of targets in an phenotypically unbiased manner. A growing body of evidence demonstrate the roles of long non-coding RNAs (lncRNAs) in most cellular process and diseases including cancer. However their systematic characterization and integration into the molecular networks supporting specific cancer types or immune-tumor cell interactions, is hindered by the lack of insights on their putative function. The OncoRNA lab focuses on lncRNAs contribution to various cancer types and other diseases. We thus developed an arrayed CRISPR interference workflow to best take advantage of the CRISPRi technology while accommodating our throughput needs. In this workflow, sgRNAs are delivered to CRISPRi competent cells in 96-well plates. Real-time cellular monitoring is used for cellular phenotyping and RNA-sequencing for each well is used to characterize the molecular phenotype of target silencing. This approaches enables us to identify the pathways in which targeted lncRNAs are involved and thus gain insights into their functions without the need for prior knowledge of said functions. Moreover, as lncRNAs are typically less abundant than mRNAs, coverage can be too low to assess. Therefore, we have established a capture sequencing approach that results in a 100- to 1000-fold coverage increase. We are currently applying our platform to investigate lncRNAs capable of modulating immune cell evasion and believe our approach can benefit other investigations focused or not on lncRNAs.
