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 2018Exploiting selection at linked sites to infer the rate and strength of adaptation, bioRxiv, 2018-09-26
Genomic data encodes past evolutionary events and has the potential to reveal the strength, rate, and biological drivers of adaptation. However, robust estimation of adaptation rate (alpha) and adaptation strength remains a challenging problem because evolutionary processes such as demography, linkage, and non-neutral polymorphism can confound inference. Here, we exploit the influence of background selection to reduce the fixation rate of weakly-beneficial alleles to jointly infer the strength and rate of adaptation. We develop a novel MK-based method (ABC-MK) to infer adaptation rate and strength, and estimate alpha=0.135 in human protein-coding sequences, 72% of which is contributed by weakly adaptive variants. We show that in this adaptation regime alpha is reduced approx. 25% by linkage genome-wide. Moreover, we show that virus-interacting proteins (VIPs) undergo adaptation that is both stronger and nearly twice as frequent as the genome average (alpha=0.224, 56% due to strongly-beneficial alleles). Our results suggest that while most adaptation in human proteins is weakly-beneficial, adaptation to viruses is often strongly-beneficial. Our method provides a robust framework for estimating adaptation rate and strength across species.
biorxiv evolutionary-biology 0-100-users 2018Slow transcriptional elongation causes embryonic lethality and perturbs kinetic coupling of long neural genes, bioRxiv, 2018-09-26
The rate of RNA Polymerase II (RNAPII) elongation has an important role in the control of Alternative splicing (AS); however, the in vivo consequences of an altered elongation rate are unknown. Here, we generated mouse embryonic stem cells (ESCs) knocked-in for a slow elongating form of RNAPII. We show that a reduced transcriptional elongation rate results in early embryonic lethality in mice and impairs the differentiation of ESCs into the neural lineage. This is accompanied by changes in splicing and in gene expression in ESCs and along the pathway of neuronal differentiation. In particular, we found a crucial role for RNAPII elongation rate in transcription and splicing of long neuronal genes involved in synapse signaling. The impact of the kinetic coupling of RNAPII elongation rate with AS is more predominant in ESC-differentiated neurons than in pluripotent cells. Our results demonstrate the requirement for an appropriate transcriptional elongation rate to ensure proper gene expression and to regulate AS during development.
biorxiv molecular-biology 0-100-users 2018Stronger and higher proportion of beneficial amino acid changing mutations in humans compared to mice and flies, bioRxiv, 2018-09-26
ABSTRACTQuantifying and comparing the amount of adaptive evolution among different species is key to understanding evolutionary processes. Previous studies have shown differences in adaptive evolution across species, however their specific causes remain elusive. Here, we use improved modeling of weakly deleterious mutations and the demographic history of the outgroup species and estimate that 30–34% of nonsynonymous substitutions between humans and outgroup species have been fixed by positive selection. This estimate is much higher than previous estimates, which did not account for the population size of the outgroup species. Next, we directly estimate the proportion and selection coefficients of newly arising strongly beneficial nonsynonymous mutations in humans, mice, and D. melanogaster by examining patterns of polymorphism and divergence. We develop a novel composite likelihood framework to test whether these parameters differ across species. Overall, we reject a model with the same proportion and the same selection coefficients of beneficial mutations across species, and estimate that humans have a higher proportion of beneficial mutations compared to Drosophila and mice. We demonstrate that this result cannot be attributed to biased gene conversion. In summary, we find the proportion of beneficial mutations is higher in humans than in D. melanogaster or mice, suggesting that organismal complexity, which increases the number of steps required in adaptive walks, may be a key predictor of the amount of adaptive evolution within a species.
biorxiv evolutionary-biology 0-100-users 2018The ecological drivers of variation in global language diversity, bioRxiv, 2018-09-26
AbstractLanguage diversity is distributed unevenly over the globe. Why do some areas have so many different languages and other areas so few? Intriguingly, patterns of language diversity resemble biodiversity patterns, leading to suggestions that similar mechanisms may underlie both linguistic and biological diversification. Here we present the first global analysis of language diversity that identifies the relative importance of two key ecological mechanisms suggested to promote language diversification - isolation and ecological risk - after correcting for spatial autocorrelation and phylogenetic non-independence. We find significant effects of climate on language diversity consistent with the ecological risk hypothesis that areas of high year-round productivity lead to more languages by supporting human cultural groups with smaller distributions. Climate has a much stronger effect on language diversity than landscape features that might contribute to isolation of cultural groups, such as altitudinal variation, river density, or landscape roughness. The association between biodiversity and language diversity appears to be an incidental effect of their covariation with climate, rather than a causal link between the two. While climate and landscape provide strong explanatory signal for variation in language diversity, we identify a number of areas of high unexplained language diversity, with more languages than would be predicted from environmental features alone; notably New Guinea, the Himalayan foothills, West Africa, and Mesoamerica. Additional processes may be at play in generating higher than expected language diversity in these regions.
biorxiv ecology 0-100-users 2018Universal Light-Sheet Generation with Field Synthesis, bioRxiv, 2018-09-26
AbstractWe introduce Field Synthesis, a theorem that can be used to synthesize any scanned or dithered light-sheet, including those used in lattice light-sheet microscopy (LLSM), from an incoherent superposition of one-dimensional intensity distributions. This user-friendly and modular approach offers a drastically simplified optical design, higher light-throughput, simultaneous multicolor illumination, and a 100% spatial duty cycle, thereby providing uncompromised biological imaging with decreased rates of photobleaching.
biorxiv bioengineering 100-200-users 2018