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The CRISPR–Cas9 system provides a versatile toolkit for genome engineering that can introduce various DNA lesions at specific genomic locations. However, a better understanding of the nature of these lesions and the repair pathways engaged is critical to realizing the full potential of this technology. Here we characterize the different lesions arising from each Cas9 variant and the resulting repair pathway engagement. We demonstrate that the presence and polarity of the overhang structure is a critical determinant of double-strand break repair pathway choice. Similarly, single nicks deriving from different Cas9 variants differentially activate repair: D10A but not N863A-induced nicks are repaired by homologous recombination. Finally, we demonstrate that homologous recombination is required for repairing lesions using double-stranded, but not single-stranded DNA as a template. This detailed characterization of repair pathway choice in response to CRISPR–Cas9 enables a more deterministic approach for designing research and therapeutic genome engineering strategies. CRISPR-Cas9 has rapidly become a common molecular biology tool for modifying genomes and has been modified to generate single-strand nicks as well as double-strand breaks. Here the authors explore the DNA repair pathways activated by the different variants of Cas9.

Defects in epigenetic regulation play a fundamental role in the development of cancer, and epigenetic regulators have recently emerged as promising therapeutic candidates. We therefore set out to systematically interrogate epigenetic cancer dependencies by screening an epigenome-focused deep-coverage design shRNA (DECODER) library across 58 cancer cell lines. This screen identified BRM/SMARCA2, a DNA-dependent ATPase of the mammalian SWI/SNF (mSWI/SNF) chromatin remodeling complex, as being essential for the growth of tumor cells that harbor loss of function mutations in BRG1/SMARCA4. Depletion of BRM in BRG1-deficient cancer cells leads to a cell cycle arrest, induction of senescence, and increased levels of global H3K9me3. We further demonstrate the selective dependency of BRG1 -mutant tumors on BRM in vivo. Genetic alterations of the mSWI/SNF chromatin remodeling complexes are the most frequent among chromatin regulators in cancers, with BRG1/SMARCA4 mutations occurring in ∼10–15% of lung adenocarcinomas. Our findings position BRM as an attractive therapeutic target for BRG1 mutated cancers. Because BRG1 and BRM function as mutually exclusive catalytic subunits of the mSWI/SNF complex, we propose that such synthetic lethality may be explained by paralog insufficiency, in which loss of one family member unveils critical dependence on paralogous subunits. This concept of “cancer-selective paralog dependency” may provide a more general strategy for targeting other tumor suppressor lesions/complexes with paralogous subunits.

Macrophages are key cell types of the innate immune system regulating host defense, inflammation, tissue homeostasis and cancer. Within this functional spectrum diverse and often opposing phenotypes are displayed which are dictated by environmental clues and depend on highly plastic transcriptional programs. Among these the 'classical' (M1) and 'alternative' (M2) macrophage polarization phenotypes are the best characterized. Understanding macrophage polarization in humans may reveal novel therapeutic intervention possibilities for chronic inflammation, wound healing and cancer. Systematic loss of function screening in human primary macrophages is limited due to lack of robust gene delivery methods and limited sample availability. To overcome these hurdles we developed cell-autonomous assays using the THP-1 cell line allowing genetic screens for human macrophage phenotypes. We screened 648 chromatin and signaling regulators with a pooled shRNA library for M1 and M2 polarization modulators. Validation experiments confirmed the primary screening results and identified OGT (O-linked N-acetylglucosamine (GlcNAc) transferase) as a novel mediator of M2 polarization in human macrophages. Our approach offers a possible avenue to utilize comprehensive genetic tools to identify novel candidate genes regulating macrophage polarization in humans.

Social media provides the means by which extremist social movements, such as white supremacy and anti LGBTQ, thrive online. Yet, we know little about the roles played by the participants of such movements. In this paper, we investigate these participants to characterize their roles, their role dynamics, and their influence in spreading online extremism. Our participants, online extremist accounts, are 4,876 public Facebook pages or groups that have shared information from the websites of 289 Southern Poverty Law Center designated extremist groups. By clustering the quantitative features followed by qualitative expert validation, we identify five roles surrounding extremist activism: educators, solicitors, flamers, motivators, sympathizers. For example, solicitors use links from extremist websites to attract donations and participation in extremist issues, whereas flamers share inflammatory extremist content inciting anger. We further investigate role dynamics such as, how stable these roles are over time and how likely will extremist accounts transition from one role into another. We find that roles core to the movement, educators and solicitors, are more stable, while flamers and motivators can transition to sympathizers with high probability. We further find that educators and solicitors exert the most influence in triggering extremist link posts, whereas flamers are influential in triggering the spread of information from fake news sources. Our results help in situating various roles on the trajectory of deeper engagement into the extremist movements and understanding the potential effect of various counter extremism interventions. Our findings have implications for understanding how online extremist movements flourish through participatory activism and how they gain a spectrum of allies for mobilizing extremism online.

The disruptive offline mobilization of participants in online conspiracy theory (CT) discussions has highlighted the importance of understanding how online users may form radicalized conspiracy beliefs. While prior work researched the factors leading up to joining online CT discussions and provided theories of how conspiracy beliefs form, we have little understanding of how conspiracy radicalization evolves after users join CT discussion communities. In this paper, we provide the empirical modeling of various radicalization phases in online CT discussion participants. To unpack how conspiracy engagement is related to radicalization, we first characterize the users' journey through CT discussions via conspiracy engagement pathways. Specifically, by studying 36K Reddit users through their 169M contributions, we uncover four distinct pathways of conspiracy engagement: steady high, increasing, decreasing, and steady low. We further model three successive stages of radicalization guided by prior theoretical works. Specific sub-populations of users, namely those on steady high and increasing conspiracy engagement pathways, progress successively through various radicalization stages. In contrast, users on the decreasing engagement pathway show distinct behavior: they limit their CT discussions to specialized topics, participate in diverse discussion groups, and show reduced conformity with conspiracy subreddits. By examining users who disengage from online CT discussions, this paper provides promising insights about conspiracy recovery process.

Online discussion platforms offer a forum to strengthen and propagate belief in misinformed conspiracy theories. Yet, they also offer avenues for conspiracy theorists to express their doubts and experiences of cognitive dissonance. Such expressions of dissonance may shed light on who abandons misguided beliefs and under which circumstances. This paper characterizes self-disclosures of dissonance about QAnon, a conspiracy theory initiated by a mysterious leader Q and popularized by their followers, anons in conspiracy theory subreddits. To understand what dissonance and disbelief mean within conspiracy communities, we first characterize their social imaginaries, a broad understanding of how people collectively imagine their social existence. Focusing on 2K posts from two image boards, 4chan and 8chan, and 1.2 M comments and posts from 12 subreddits dedicated to QAnon, we adopt a mixed methods approach to uncover the symbolic language representing the movement, expectations, practices, heroes and foes of the QAnon community. We use these social imaginaries to create a computational framework for distinguishing belief and dissonance from general discussion about QAnon. Further, analyzing user engagement with QAnon conspiracy subreddits, we find that self-disclosures of dissonance correlate with a significant decrease in user contributions and ultimately with their departure from the community. We contribute a computational framework for identifying dissonance self-disclosures and measuring the changes in user engagement surrounding dissonance. Our work can provide insights into designing dissonance-based interventions that can potentially dissuade conspiracists from online conspiracy discussion communities.