Comparison of Efficiency and Specificity of CRISPR-Associated (Cas) Nucleases in Plants An Expanded Toolkit for Precision Genome Engineering, bioRxiv, 2018-09-21

Molecular tools adapted from bacterial CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats) systems for adaptive immunity have become widely used for plant genome engineering, both to investigate gene functions and to engineer desirable traits. A number of different Cas (CRISPR-associated) nucleases are now used but, as most studies performed to date have engineered different targets using a variety of plant species and molecular tools, it has been difficult to draw conclusions about the comparative performance of different nucleases. Due to the time and effort required to regenerate engineered plants, efficiency is critical. In addition, there have been several reports of mutations at sequences with less than perfect identity to the target. While in some plant species it is possible to remove these so-called ‘off-targets’ by backcrossing to a parental line, the specificity of genome engineering tools is important when targeting specific members of closely-related gene families, especially when recent paralogues are co-located in the genome and unlikely to segregate. Specificity is also important for species that take years to reach sexual maturity or that are clonally propagated. Here, we directly compare the efficiency and specificity of Cas nucleases from different bacterial species together with engineered variants of Cas9. We find that the nucleotide content correlates with efficiency and that Cas9 from Staphylococcus aureus is comparatively most efficient at inducing mutations. We also demonstrate that ‘high-fidelity’ variants of Cas9 can reduce off-target mutations in plants. We present these molecular tools as standardised DNA parts to facilitate their re-use.

biorxiv plant-biology 0-100-users 2018

Objective versus Self-Reported Energy Intake Changes During Low-Carbohydrate and Low-Fat Diets, bioRxiv, 2018-09-21

AbstractObjectiveTo examine objective versus self-reported energy intake changes (ΔEI) during a 12-month diet intervention.MethodsWe calculated ΔEI in subjects who participated in a 1-year randomized low-carbohydrate versus low-fat diet trial using repeated body weight measurements as inputs to an objective mathematical model (ΔEIModel) and compared these values with self-reported energy intake changes assessed by repeated 24-hr recalls (ΔEI24hrRecall).ResultsΔEI24hrRecall indicated a relatively persistent state of calorie restriction ≥500 kcald throughout the year with no significant differences between diets. ΔEIModel demonstrated large early decreases in calorie intake &gt;800 kcald followed by an exponential return to approximately 100 kcald below baseline at the end of the year. The low-carbohydrate diet resulted in ΔEIModel that was 162±53 kcald lower than the low-fat diet over the first 3 months (p=0.002), but no significant diet differences were found at later times. Weight loss at 12 months was significantly related to ΔEIModel at all time intervals for both diets (p&lt;0.0001).ConclusionsSelf-reported measurements of ΔEI were inaccurate. Model-based calculations of ΔEI found that instructions to follow the low-carbohydrate diet resulted in greater calorie restriction than the low-fat diet in the early phases of the intervention, but these diet differences were not sustained.What is already known about this subject?<jatslist list-type=bullet><jatslist-item>Diet assessments that rely on self-report, such as 24hr dietary recall, are known to underestimate actual energy intake as measured by doubly labeled water. However, it is possible that repeated self-reported measurements could accurately detect changes in energy intake over time if the absolute bias of self-reported of measurements is approximately constant for each subject.<jatslist-item>What this study adds<jatslist list-type=bullet><jatslist-item>We compared energy intake changes measured using repeated 24hr dietary recall measurements collected over the course of the 1-year Diet Intervention Examining The Factors Interacting with Treatment Success (DIETFITS) trial versus energy intake changes calculated using repeated body weight measurements as inputs to a validated mathematical model.<jatslist-item><jatslist-item>Whereas self-reported measurements indicated a relatively persistent state of calorie restriction, objective model-based measurements demonstrated a large early calorie restriction followed by an exponential rise in energy intake towards the pre-intervention baseline.<jatslist-item><jatslist-item>Model-based calculations, but not self-reported measurements, found that low-carbohydrate diets led to significantly greater early decreases in energy intake compared to low-fat diets, but long-term energy intake changes were not significantly different.<jatslist-item>

biorxiv physiology 100-200-users 2018

Paleolithic 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 2018

 

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