Efficient generation of endogenous fluorescent reporters by Nested CRISPR in Caenorhabditis elegans, bioRxiv, 2018-09-26
AbstractCRISPR-based genome editing methods in model organisms are evolving at an extraordinary speed. Whereas the generation of deletion or missense mutants is quite straightforward, the production of endogenous fluorescent reporters is still inefficient. The use of plasmids with selection markers is an effective methodology, but often requires laborious and complicated cloning steps. We have established a cloning-free ribonucleoprotein-driven Nested CRISPR method that robustly produces endogenous fluorescent reporters. This methodology is based on the division of the GFP and mCherry sequences in three fragments. In the first step we use ssDNA donors (≤200 bp) to insert 5’ and 3’ fragments in the place of interest. In the second step, we use these sequences as homology regions for Homology Directed Repair (HDR) with a dsDNA donor (PCR product, ≈700 bp) including the middle fragment, thus completing the fluorescent protein sequence. This method is advantageous because the first step with ssDNA donors is known to be very efficient, and the second step, uses universal reagents, including validated PCR products and crRNAs, to create fluorescent reporters reaching reliable editing efficiencies as high as 40%. We have also used Nested CRISPR in a non-essential gene to produce a deletion mutant in the first step and a transcriptional reporter in the second step.In the search of modifications to optimize the method, we tested synthetic sgRNAs, but we did not observe a significant increase in the efficacy compared to independently adding tracrRNA and crRNA to the injection mix. Conveniently, we also found that both steps of Nested CRISPR could be performed in a single injection. Finally, we discuss the utility of Nested CRISPR for targeted insertion of long DNA fragments in other systems and prospects of this method in the future.
biorxiv genetics 0-100-users 2018The genetics of university success, Scientific Reports, 2018-09-25
University success, which includes enrolment in and achievement at university, as well as quality of the university, have all been linked to later earnings, health and wellbeing. However, little is known about the causes and correlates of differences in university-level outcomes. Capitalizing on both quantitative and molecular genetic data, we perform the first genetically sensitive investigation of university success with a UK-representative sample of 3,000 genotyped individuals and 3,000 twin pairs. Twin analyses indicate substantial additive genetic influence on university entrance exam achievement (57%), university enrolment (51%), university quality (57%) and university achievement (46%). We find that environmental effects tend to be non-shared, although the shared environment is substantial for university enrolment. Furthermore, using multivariate twin analysis, we show moderate to high genetic correlations between university success variables (0.27–0.76). Analyses using DNA alone also support genetic influence on university success. Indeed, a genome-wide polygenic score, derived from a 2016 genome-wide association study of years of education, predicts up to 5% of the variance in each university success variable. These findings suggest young adults select and modify their educational experiences in part based on their genetic propensities and highlight the potential for DNA-based predictions of real-world outcomes, which will continue to increase in predictive power.
scientific reports genetics 500+-users 2018Trans effects on gene expression can drive omnigenic inheritance, bioRxiv, 2018-09-25
Early genome-wide association studies (GWAS) led to the surprising discovery that, for typical complex traits, the most significant genetic variants contribute only a small fraction of the estimated heritability. Instead, it has become clear that a huge number of common variants, each with tiny effects, explain most of the heritability. Previously, we argued that these patterns conflict with standard conceptual models, and that new models are needed. Here we provide a formal model in which genetic contributions to complex traits can be partitioned into direct effects from core genes, and indirect effects from peripheral genes acting as trans-regulators. We argue that the central importance of peripheral genes is a direct consequence of the large contribution of trans-acting variation to gene expression variation. In particular, we propose that if the core genes for a trait are co-regulated – as seems likely – then the effects of peripheral variation can be amplified by these co-regulated networks such that nearly all of the genetic variance is driven by peripheral genes. Thus our model proposes a framework for understanding key features of the architecture of complex traits.
biorxiv genetics 200-500-users 2018Paleolithic DNA from the Caucasus reveals core of West Eurasian ancestry, bioRxiv, 2018-09-21
AbstractThe earliest ancient DNA data of modern humans from Europe dates to ∼40 thousand years ago1-4, but that from the Caucasus and the Near East to only ∼14 thousand years ago5,6, from populations who lived long after the Last Glacial Maximum (LGM) ∼26.5-19 thousand years ago7. To address this imbalance and to better understand the relationship of Europeans and Near Easterners, we report genome-wide data from two ∼26 thousand year old individuals from Dzudzuana Cave in Georgia in the Caucasus from around the beginning of the LGM. Surprisingly, the Dzudzuana population was more closely related to early agriculturalists from western Anatolia ∼8 thousand years ago8 than to the hunter-gatherers of the Caucasus from the same region of western Georgia of ∼13-10 thousand years ago5. Most of the Dzudzuana population’s ancestry was deeply related to the post-glacial western European hunter-gatherers of the ‘Villabruna cluster’3, but it also had ancestry from a lineage that had separated from the great majority of non-African populations before they separated from each other, proving that such ‘Basal Eurasians’6,9 were present in West Eurasia twice as early as previously recorded5,6. We document major population turnover in the Near East after the time of Dzudzuana, showing that the highly differentiated Holocene populations of the region6 were formed by ‘Ancient North Eurasian’3,9,10 admixture into the Caucasus and Iran and North African11,12 admixture into the Natufians of the Levant. We finally show that the Dzudzuana population contributed the majority of the ancestry of post-Ice Age people in the Near East, North Africa, and even parts of Europe, thereby becoming the largest single contributor of ancestry of all present-day West Eurasians.
biorxiv genetics 100-200-users 2018Polygenicity of complex traits is explained by negative selection, bioRxiv, 2018-09-19
Complex traits and common disease are highly polygenic thousands of common variants are causal, and their effect sizes are almost always small. Polygenicity could be explained by negative selection, which constrains common-variant effect sizes and may reshape their distribution across the genome. We refer to this phenomenon as flattening, as genetic signal is flattened relative to the underlying biology. We introduce a mathematical definition of polygenicity, the effective number of associated SNPs, and a robust statistical method to estimate it. This definition of polygenicity differs from the number of causal SNPs, a standard definition; it depends strongly on SNPs with large effects. In analyses of 33 complex traits (average N=361k), we determined that common variants are ∼4x more polygenic than low-frequency variants, consistent with pervasive flattening. Moreover, functionally important regions of the genome have increased polygenicity in proportion to their increased heritability, implying that heritability enrichment reflects differences in the number of associations rather than their magnitude (which is constrained by selection). We conclude that negative selection constrains the genetic signal of biologically important regions and genes, reshaping genetic architecture.
biorxiv genetics 100-200-users 2018Maternal gut and breast milk microbiota affect infant gut antibiotic resistome and mobile genetic elements, Nature Communications, 2018-09-18
The infant gut microbiota has a high abundance of antibiotic resistance genes (ARGs) compared to adults, even in the absence of antibiotic exposure. Here we study potential sources of infant gut ARGs by performing metagenomic sequencing of breast milk, as well as infant and maternal gut microbiomes. We find that fecal ARG and mobile genetic element (MGE) profiles of infants are more similar to those of their own mothers than to those of unrelated mothers. MGEs in mothers’ breast milk are also shared with their own infants. Termination of breastfeeding and intrapartum antibiotic prophylaxis of mothers, which have the potential to affect microbial community composition, are associated with higher abundances of specific ARGs, the composition of which is largely shaped by bacterial phylogeny in the infant gut. Our results suggest that infants inherit the legacy of past antibiotic consumption of their mothers via transmission of genes, but microbiota composition still strongly impacts the overall resistance load.
nature communications genetics 200-500-users 2018