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 2018

Stronger 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 2018

 

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