Common computational tools for analyzing CRISPR screens

CRISPR–Cas technology offers a versatile toolbox for genome editing, with applications in various cancer-related fields such as functional genomics, immunotherapy, synthetic lethality and drug resistance, metastasis, genome regulation, chromatic accessibility and RNA-targeting. The variety of screening platforms and questions in which they are used have caused the development of a wide array of analytical methods for CRISPR analysis. In this review, we focus on the algorithms and frameworks used in the computational analysis of pooled CRISPR knockout (KO) screens and highlight some of the most significant target discoveries made using these methods. Lastly, we offer perspectives on the design and analysis of state-of-art multiplex screening for genetic interactions.

Caspase-mediated nuclear pore complex trimming in cell differentiation and endoplasmic reticulum stress

Introductory ParagraphDuring programmed cell death, caspases degrade 7 out of ∼30 nucleoporins (Nups) to irreversibly demolish the nuclear pore complex (NPC)1. However, for poorly understood reasons, caspases are also activated in differentiating cells in a non-apoptotic manner2,3. Here, we describe reversible, caspase-mediated NPC “trimming” during early myogenesis. We find that sublethal levels of caspases selectively proteolyze 4 peripheral Nups, Nup358, Nup214, Nup153, and Tpr, resulting in the transient block of nuclear export pathways. Several nuclear export signal (NES)-containing focal adhesion proteins concomitantly accumulate in the nucleus where they function as transcription cofactors4. We show that one such protein, FAK (focal adhesion kinase), drives a global reconfiguration of MBD2 (methyl CpG binding domain protein 2)-mediated genome regulation. We also observe caspase-mediated NPC trimming during neurogenesis and endoplasmic reticulum (ER) stress. Our results illustrate that the NPC can be proteolytically regulated in response to non-apoptotic cues, and call for a reassessment of the death-centric view of caspases.

Deciphering Plant Chromatin Regulation via CRISPR/dCas9-Based Epigenome Engineering

CRISPR-based epigenome editing uses dCas9 as a platform to recruit transcription or chromatin regulators at chosen loci. Despite recent and ongoing advances, the full potential of these approaches to studying chromatin functions in vivo remains challenging to exploit. In this review we discuss how recent progress in plants and animals provides new routes to investigate the function of chromatin regulators and address the complexity of associated regulations that are often interconnected. While efficient transcriptional engineering methodologies have been developed and can be used as tools to alter the chromatin state of a locus, examples of direct manipulation of chromatin regulators remain scarce in plants. These reports also reveal pitfalls and limitations of epigenome engineering approaches that are nevertheless informative as they are often associated with locus- and context-dependent features, which include DNA accessibility, initial chromatin and transcriptional state or cellular dynamics. Strategies implemented in different organisms to overcome and even take advantage of these limitations are highlighted, which will further improve our ability to establish the causality and hierarchy of chromatin dynamics on genome regulation.

R-loop resolution promotes co-transcriptional chromatin silencing

RNA-mediated chromatin silencing is central to genome regulation in many organisms. However, how nascent non-coding transcripts regulate chromatin is poorly understood. Here, through analysis of Arabidopsis FLC, we show that resolution of a nascent-transcript-induced R-loop promotes chromatin silencing. Stabilization of an antisense-induced R-loop at the 3′ end of FLC enables an RNA binding protein FCA, with its direct partner FY/WDR33 and other 3′-end processing factors, to polyadenylate the nascent antisense transcript. This clears the R-loop and recruits the chromatin modifiers demethylating H3K4me1. FCA immunoprecipitates with components of the m6A writer complex, and m6A modification affects dynamics of FCA nuclear condensates, and promotes FLC chromatin silencing. This mechanism also targets other loci in the Arabidopsis genome, and consistent with this fca and fy are hypersensitive to a DNA damage-inducing drug. These results show how modulation of R-loop stability by co-transcriptional RNA processing can trigger chromatin silencing.