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A delicate equilibrium of WNT agonists and antagonists in the intestinal stem cell (ISC) niche is critical to maintaining the ISC compartment, as it accommodates the rapid renewal of the gut lining. Disruption of this balance by mutations in the tumour suppressor gene APC, which are found in approximately 80% of all human colon cancers, leads to unrestrained activation of the WNT pathway1,2. It has previously been established that Apc-mutant cells have a competitive advantage over wild-type ISCs3. Consequently, Apc-mutant ISCs frequently outcompete all wild-type stem cells within a crypt, thereby reaching clonal fixation in the tissue and initiating cancer formation. However, whether the increased relative fitness of Apc-mutant ISCs involves only cell-intrinsic features or whether Apcmutants are actively involved in the elimination of their wild-type neighbours remains unresolved. Here we show that Apc-mutant ISCs function as bona fide supercompetitors by secreting WNT antagonists, thereby inducing differentiation of neighbouring wild-type ISCs. Lithium chloride prevented the expansion of Apc-mutant clones and the formation of adenomas by rendering wild-type ISCs insensitive to WNT antagonists through downstream activation of WNT by inhibition of GSK3β. Our work suggests that boosting the fitness of healthy cells to limit the expansion of pre-malignant clones may be a powerful strategy to limit the formation of cancers in high-risk individuals.

Long non-coding RNAs (lncRNAs) are emerging as key regulators in the initiation, growth, and progression of cancer. High-throughput CRISPR-based techniques systematically assess the function of genes or regulatory elements present in the human genome. Here, we present a protocol for identifying essential lncRNAs in cancer using CRISPRi-based dropout screens. We describe steps to select target sites, design guide RNAs, and generate CRISPRi cell lines. We then detail the execution and analysis of CRISPRi-based dropout screens.

Introduction:Regulation of gene expression involves a variety of mechanisms driven by a complex regulatory network of factors. Control of transcription is an important step in gene expression regulation, which integrates the function of cis-acting and trans-acting elements. Among cis-regulatory elements, enhancer RNA (eRNA)-producing domains recently emerged as widespread and potent regulators of transcription and cell fate decision. Thus, manipulation of eRNA levels becomes a novel and appealing avenue for the design of new therapeutic treatments.Areas covered:In this review, we focus on eRNA-producing domains. We describe mechanisms involved in their cell-type specific selection and activation as well as their epigenetic features. In addition, we present their function and the growing evidences of their deregulation in human diseases. Finally, we discuss eRNAs as potential therapeutic targets.Expert opinion:As key factors in the control of transcription, eRNAs appear to possess a great potential for the establishment of new therapy options. However, thorough testing as well as providing the genetic toolbox to target eRNAs will be needed to fully assess the practical and clinical possibilities.