Coupled single-cell CRISPR screening and epigenomic profiling reveals causal gene regulatory networks, bioRxiv, 2018-09-17
SummaryHere we present Perturb-ATAC, a method which combines multiplexed CRISPR interference or knockout with genome-wide chromatin accessibility profiling in single cells, based on the simultaneous detection of CRISPR guide RNAs and open chromatin sites by assay of transposase-accessible chromatin with sequencing (ATAC-seq). We applied Perturb-ATAC to transcription factors (TFs), chromatin-modifying factors, and noncoding RNAs (ncRNAs) in ∼4,300 single cells, encompassing more than 63 unique genotype-phenotype relationships. Perturb-ATAC in human B lymphocytes uncovered regulators of chromatin accessibility, TF occupancy, and nucleosome positioning, and identified a hierarchical organization of TFs that govern B cell state, variation, and disease-associatedcis-regulatory elements. Perturb-ATAC in primary human epidermal cells revealed three sequential modules ofcis-elements that specify keratinocyte fate, orchestrated by the TFs JUNB, KLF4, ZNF750, CEBPA, and EHF. Combinatorial deletion of all pairs of these TFs uncovered their epistatic relationships and highlighted genomic co-localization as a basis for synergistic interactions. Thus, Perturb-ATAC is a powerful and general strategy to dissect gene regulatory networks in development and disease.Highlights<jatslist list-type=order><jatslist-item>A new method for simultaneous measurement of CRISPR perturbations and chromatin state in single cells.<jatslist-item><jatslist-item>Perturb-ATAC reveals regulatory factors that controlcis-element accessibility,trans-factor occupancy, and nucleosome positioning.<jatslist-item><jatslist-item>Perturb-ATAC reveals regulatory modules of coordinatedtrans-factor activity in B lymphoblasts.<jatslist-item><jatslist-item>Keratinocyte differentiation is orchestrated by synergistic activities of co-binding TFs oncis-elements.<jatslist-item>
biorxiv genomics 100-200-users 2018Optimization of T-DNA architecture for Cas9-mediated mutagenesis in Arabidopsis, bioRxiv, 2018-09-17
ABSTRACTBacterial CRISPR systems have been widely adopted to create operator-specified site-specific nucleases. Such nuclease action commonly results in loss-of-function alleles, facilitating functional analysis of genes and gene families We conducted a systematic comparison of components and T-DNA architectures for CRISPR-mediated gene editing in Arabidopsis, testing multiple promoters, terminators, sgRNA backbones and Cas9 alleles. We identified a T-DNA architecture that usually results in stable (i.e. homozygous) mutations in the first generation after transformation. Notably, the transcription of sgRNA and Cas9 in head-to-head divergent orientation usually resulted in highly active lines. Our Arabidopsis data may prove useful for optimization of CRISPR methods in other plants.
biorxiv plant-biology 100-200-users 2018Probing variability in a cognitive map using manifold inference from neural dynamics, bioRxiv, 2018-09-17
Hippocampal neurons fire selectively in local behavioral contexts such as the position in an environment or phase of a task,1-3 and are thought to form a cognitive map of task-relevant variables.1,4,5 However, their activity varies over repeated behavioral conditions,6 such as different runs through the same position or repeated trials. Although widely observed across the brain,7-10 such variability is not well understood, and could reflect noise or structure, such as the encoding of additional cognitive information.6,11-13 Here, we introduce a conceptual model to explain variability in terms of underlying, population-level structure in single-trial neural activity. To test this model, we developed a novel unsupervised learning algorithm incorporating temporal dynamics, in order to characterize population activity as a trajectory on a nonlinear manifold—a space of possible network states. The manifold’s structure captures correlations between neurons and temporal relationships between states, constraints arising from underlying network architecture and inputs. Using measurements of activity over time but no information about exogenous behavioral variables, we recovered hippocampal activity manifolds during spatial and non-spatial cognitive tasks in rats. Manifolds were low-dimensional and smoothly encoded task-related variables, but contained an extra dimension reflecting information beyond the measured behavioral variables. Consistent with our model, neurons fired as a function of overall network state, and fluctuations in their activity across trials corresponded to variation in the underlying trajectory on the manifold. In particular, the extra dimension allowed the system to take different trajectories despite repeated behavioral conditions. Furthermore, the trajectory could temporarily decouple from current behavioral conditions and traverse neighboring manifold points corresponding to past, future, or nearby behavioral states. Our results suggest that trial-to-trial variability in the hippocampus is structured, and may reflect the operation of internal cognitive processes. The manifold structure of population activity is well-suited for organizing information to support memory,1,5,14 planning,12,15,16 and reinforcement learning.17,18 In general, our approach could find broader use in probing the organization and computational role of circuit dynamics in other brain regions.
biorxiv neuroscience 0-100-users 2018Transformation of Speech Sequences in Human Sensorimotor Circuits, bioRxiv, 2018-09-17
SummaryAfter we listen to a series of words, we can silently replay them in our mind. Does this mental replay involve a re-activation of our original perceptual representations? We recorded electrocorticographic (ECoG) activity across the lateral cerebral cortex as people heard and then mentally rehearsed spoken sentences. For each region, we tested whether silent rehearsal of sentences involved reactivation of sentence-specific representations established during perception or transformation to a distinct representation. In sensorimotor and premotor cortex, we observed reliable and temporally precise responses to speech; these patterns transformed to distinct sentence-specific representations during mental rehearsal. In contrast, we observed slower and less reliable responses in prefrontal and temporoparietal cortex; these higher-order representations, which were sensitive to sentence semantics, were shared across perception and rehearsal. The mental rehearsal of natural speech involves the transformation of time-resolved speech representations in sensorimotor and premotor cortex, combined with diffuse reactivation of higher-order semantic representations.Conflict of interestThe authors declare no competing financial interests.
biorxiv neuroscience 0-100-users 2018A guide to performing Polygenic Risk Score analyses, bioRxiv, 2018-09-16
The application of polygenic risk scores (PRS) has become routine across genetic research. Among a range of applications, PRS are exploited to assess shared aetiology between phenotypes, to evaluate the predictive power of genetic data for use in clinical settings, and as part of experimental studies in which, for example, experiments are performed on individuals, or their biological samples (eg. tissues, cells), at the tails of the PRS distribution and contrasted. As GWAS sample sizes increase and PRS become more powerful, they are set to play a key role in personalised medicine. However, despite the growing application and importance of PRS, there are limited guidelines for performing PRS analyses, which can lead to inconsistency between studies and misinterpretation of results. Here we provide detailed guidelines for performing polygenic risk score analyses relevant to different methods for their calculation, outlining standard quality control steps and offering recommendations for best-practice. We also discuss different methods for the calculation of PRS, common misconceptions regarding the interpretation of results and future challenges.
biorxiv genomics 100-200-users 2018Active and repressed chromatin domains exhibit distinct nucleosome segregation during DNA replication, bioRxiv, 2018-09-16
SummaryChromatin domains and their associated structures must be faithfully inherited through cellular division to maintain cellular identity. Yet, accessing the localized strategies preserving chromatin domain inheritance, specifically the transfer of parental, pre-existing nucleosomes with their associated post-translational modifications (PTMs) during DNA replication is challenging in living cells. We devised an inducible, proximity-dependent labeling system to irreversibly mark replication-dependent H3.1 and H3.2 histone-containing nucleosomes at single desired loci in mouse embryonic stem cells such that their fate after DNA replication could be followed. Strikingly, repressed chromatin domains are preserved through the local re-deposition of parental nucleosomes. In contrast, nucleosomes decorating active chromatin domains do not exhibit such preservation. Notably, altering cell fate leads to an adjustment in the positional inheritance of parental nucleosomes that reflects the corresponding changes in chromatin structure. These findings point to important mechanisms that contribute to parental nucleosome segregation to preserve cellular identity.
biorxiv biochemistry 0-100-users 2018