Homology Directed Repair by Cas9Donor Co-localization in Mammalian Cells, bioRxiv, 2018-01-17
AbstractHomology directed repair (HDR) induced by site specific DNA double strand breaks (DSB) with CRISPRCas9 is a precision gene editing approach that occurs at low frequency in comparison to indel forming non homologous end joining (NHEJ). In order to obtain high HDR percentages in mammalian cells, we engineered Cas9 protein fused to a high-affinity monoavidin domain to deliver biotinylated donor DNA to a DSB site. In addition, we used the cationic polymer, polyethylenimine, to deliver Cas9 RNP-donor DNA complex into the cell. Combining these strategies improved HDR percentages of up to 90% in three tested loci (CXCR4, EMX1, and TLR) in standard HEK293 cells. Our approach offers a cost effective, simple and broadly applicable gene editing method, thereby expanding the CRISPRCas9 genome editing toolbox.SummaryPrecision gene editing occurs at a low percentage in mammalian cells using Cas9. Colocalization of donor with Cas9MAV and PEI delivery raises HDR occurrence.
biorxiv biochemistry 0-100-users 2018Resistance gene discovery and cloning by sequence capture and association genetics, bioRxiv, 2018-01-16
Genetic resistance is the most economic and environmentally sustainable approach for crop disease protection. Disease resistance (R) genes from wild relatives are a valuable resource for breeding resistant crops. However, introgression of R genes into crops is a lengthy process often associated with co-integration of deleterious linked genes1, 2 and pathogens can rapidly evolve to overcome R genes when deployed singly3. Introducing multiple cloned R genes into crops as a stack would avoid linkage drag and delay emergence of resistance-breaking pathogen races4. However, current R gene cloning methods require segregating or mutant progenies5–10, which are difficult to generate for many wild relatives due to poor agronomic traits. We exploited natural pan-genome variation in a wild diploid wheat by combining association genetics with R gene enrichment sequencing (AgRenSeq) to clone four stem rust resistance genes in <6 months. RenSeq combined with diversity panels is therefore a major advance in isolating R genes for engineering broad-spectrum resistance in crops.
biorxiv genomics 100-200-users 2018Stability of association between Arabidopsis thaliana and Pseudomonas pathogens over evolutionary time scales, bioRxiv, 2018-01-16
SummaryCrop disease outbreaks are often associated with clonal expansions of single pathogenic lineages. To determine whether similar boom-and-bust scenarios hold for wild plant pathogens, we carried out a multi-year multi-site survey of Pseudomonas in the natural host Arabidopsis thaliana. The most common Pseudomonas lineage corresponded to a pathogenic clade present in all sites. Sequencing of 1,524 Pseudomonas genomes revealed this lineage to have diversified approximately 300,000 years ago, containing dozens of genetically distinct pathogenic sublineages. These sublineages have expanded in parallel within the same populations and are differentiated both at the level of gene content and disease phenotype. Such coexistence of diverse sublineages indicates that in contrast to crop systems, no single strain has been able to overtake these A. thaliana populations in the recent past. Our results suggest that the selective pressures acting on a plant pathogen in wild hosts may be more complex than those in agricultural systems.
biorxiv microbiology 0-100-users 2018Classification of Single Particles from Human Cell Extract Reveals Distinct Structures, bioRxiv, 2018-01-15
SummaryMulti-protein complexes are necessary for nearly all cellular processes, and understanding their structure is required for elucidating their function. Current high-resolution strategies in structural biology are effective, but lag behind other fields (e.g. genomics and proteomics) due to their reliance on purified samples rather than characterizing heterogeneous mixtures. Here, we present a method combining single particle analysis by electron microscopy with protein identification by mass spectrometry to structurally characterize macromolecular complexes from extracts of human cells. We obtain three-dimensional structures of native proteasomes directly from ab initio classification of a heterogeneous mixture of protein complexes. In addition, we find an ~1 MDa size structure of unknown composition and reference our proteomics data to suggest possible identities. Our study shows the power of using a shotgun approach to electron microscopy (shotgun EM) when coupled with mass spectrometry as a tool to uncover the structures of macromolecular machines in parallel.
biorxiv biochemistry 0-100-users 2018Precise temporal regulation of alternative splicing during neural development, bioRxiv, 2018-01-15
AbstractAlternative splicing (AS) is a crucial step of gene expression that must be tightly controlled, but the precise timing of dynamic splicing switches during neural development and the underlying regulatory mechanisms are poorly understood. Here we systematically analyzed the temporal regulation of AS in a large number of transcriptome profiles of developing mouse cortices, in vivo purified neuronal subtypes, and neurons differentiated in vitro. Our analysis revealed early- and late-switch exons in genes with distinct functions, and these switches accurately define neuronal maturation stages. Integrative modeling suggests that these switches are under direct and combinatorial regulation by distinct sets of neuronal RNA-binding proteins including Nova, Rbfox, Mbnl and Ptbp. Surprisingly, various neuronal subtypes in the sensory systems lack Nova andor Rbfox expression. These neurons retain the “immature” splicing program in early-switch exons, affecting numerous synaptic genes. These results provide new insights into the organization and regulation of the neurodevelopmental transcriptome.
biorxiv molecular-biology 0-100-users 2018A systematic review of Drosophila short-term-memory genetics meta-analysis reveals robust reproducibility, bioRxiv, 2018-01-14
AbstractGeneticists use olfactory conditioning in Drosophila to identify learning genes; however, little is known about how these genes are integrated into short-term memory (STM) pathways. Here, we investigated the hypothesis that the STM evidence base is weak. We performed systematic review and meta-analysis of the field. Using metrics to quantify variation between discovery articles and follow-up studies, we found that seven genes were both highly replicated, and highly reproducible. However, ~80% of STM genes have never been replicated. While only a few studies investigated interactions, the reviewed genes could account for >1000% memory. This large summed effect size could indicate irreproducibility, many shared pathways, or that current assay protocols lack the specificity needed to identify core plasticity genes. Mechanistic theories of memory will require the convergence of evidence from system, circuit, cellular, molecular, and genetic experiments; systematic data synthesis is an essential tool for integrated neuroscience.
biorxiv animal-behavior-and-cognition 0-100-users 2018