Esther Lau

Non-coding RNA: Zooming in on lncRNA functions

PhotoDisc/Getty Images Long non-coding RNAs (lncRNAs) are emerging as important regulators of gene expression; however, in contrast to transcription factors, their functional domain architecture remains poorly understood. Now, a new method has been developed to simultaneously map RNA–RNA, RNA–DNA and RNA–protein interactions at the level of individual RNA domains with increased sensitivity. Quinn et al. developed a method called domain-specific chromatin isolation by RNA purification (dChIRP), in which several antisense oligonucleotide pools are used to target specific domains of lncRNAs. In this method, cells are subjected to fixation, crosslinking and sonication, and the resultant sheared chromatin is hybridized to the biotinylated oligonucleotide pools to recover chromatin fragments containing specific lncRNA domains of interest. The RNA, DNA and protein components associated with the lncRNA domains can then be analysed separately. The authors successfully applied dChIRP to study putative functional domains in the roX1 lncRNA of Drosophila melanogaster. roX1 is essential for dosage compensation, in which expression from the single X chromosome in male flies is upregulated by twofold to match expression from the pairs of autosomal chromosomes. They also showed that the three D domains of roX1 bind directly to male-specific lethal (MSL) proteins to mediate X chromosome upregulation and that the three intervening U domains exhibit minimal binding to MSL proteins but instead associate with each other. In addition, quantitative PCR and high-throughout DNA sequencing were used to identify genome-wide roX1-binding sites. Importantly, by focusing only on the domains that strongly associate with DNA, N O N C O D I N G R N A

Genetic screens: CRISPR screening from both ways.

The CRISPR–Cas9 (clustered regularly interspaced short palindromic repeat–CRISPR-associated protein 9) system, in which the Cas9 endonuclease is guided by short guide RNAs (sgRNAs) to specific genomic locations, is a widely used genome editing tool and has been used in loss-of-function genetic screens. Now, Gilbert and Horlbeck et al. report the development of two related tools called CRISPRi and CRISPRa to repress or induce, respectively, individual transcripts with minimal off-target effects, which provide complementary biological insights through both lossand gain-of-function genetic screens. In CRISPRi, a catalytically inactive Cas9 (dCas9) can either directly block RNA polymerase activity or be fused to a silencing effector domain to repress transcription. By contrast, the dCas9 fusion protein in CRISPRa was engineered to recruit multiple copies of an activating effector domain. The researchers used a pooled high-throughput tiling screen to assess CRISPRi and CRISPRa activity across the genome in 10-kb windows surrounding the transcription start sites (TSSs) of 49 endogenous genes that are involved in modulating cellular susceptibility to the AB toxin ricin. These screens defined sequence features of highly active sgRNAs and revealed the locations (relative to TSSs) where CRISPRi and CRISPRa complexes should be targeted to maximally repress or activate transcription. These ‘rules’ enabled the authors to design two genome-scale libraries (using 10 sgRNAs to target each gene), which were validated in genetic screens in human myeloid leukaemia cells. The complementary CRISPRi and CRISPRa approaches identified both positive and negative regulators of cell growth. Moreover, using these tools to reversibly G E N E T I C S C R E E N S

Complex disease: Piecing together the puzzle of coronary artery disease

Subject terms: nDisease genetics• nFunctional genomics• nGenome-wide association studies• nHeritable quantitative trait