Optimization of AsCas12a for combinatorial genetic screens in human cells, bioRxiv, 2019-08-28
ABSTRACTCas12a enzymes have attractive properties for scalable delivery of multiplexed perturbations, yet widespread usage has lagged behind Cas9-based strategies. Here we describe the optimization of Cas12a from Acidaminococcus (AsCas12a) for use in pooled genetic screens in human cells. By assaying the activity of thousands of guides, we confirm on-target design rules and extend them to an enhanced activity variant, enAsCas12a. We also develop the first comprehensive set of off-target rules for Cas12a, and demonstrate that we can predict and exclude promiscuous guides. Finally, to enable efficient higher-order multiplexing via lentiviral delivery, we screen thousands of direct repeat variants and identify 38 that outperform the wildtype sequence. We validate this optimized AsCas12a toolkit by targeting 12 synthetic lethal gene pairs with up to 400 guide pairs each, and demonstrate effective triple knockout via flow cytometry. These results establish AsCas12a as a robust system for combinatorial applications of CRISPR technology.
biorxiv genetics 100-200-users 2019Chromosome-level assemblies of multiple Arabidopsis genomes reveal hotspots of rearrangements with altered evolutionary dynamics, bioRxiv, 2019-08-23
AbstractWe report chromosome-level, reference-quality assemblies of seven Arabidopsis thaliana accessions selected across the global range of this predominately ruderal plant. Each genome revealed between 13-17 Mb rearranged and 5-6 Mb novel sequence introducing copy-number changes in ∼5,000 genes, including ∼1,900 genes which are not part of the current reference annotation. Analyzing the collinearity between the genomes revealed ∼350 regions (4.1% of the euchromatin) where accession-specific tandem duplications destroyed the syntenic gene order between the genomes. These hotspots of rearrangements were characterized by the loss of meiotic recombination in hybrids within these regions and the enrichment of genes implicated in biotic stress response. Together this suggests that hotspots of rearrangements are governed by altered evolutionary dynamics as compared to the rest of the genome, which are based on new mutations and not on the recombination of existing variation, and thereby enable a quick response to the ever-evolving challenges of biotic stress.
biorxiv genetics 100-200-users 2019Plasmodium vivaxMalaria viewed through the lens of an eradicated European strain, bioRxiv, 2019-08-16
AbstractThe protozoanPlasmodium vivaxis responsible for 42% of all cases of malaria outside Africa. The parasite is currently largely restricted to tropical and subtropical latitudes in Asia, Oceania and the Americas. Though, it was historically present in most of Europe before being finally eradicated during the second half of the 20th century. The lack of genomic information on the extinct European lineage has prevented a clear understanding of historical population structuring and past migrations ofP. vivax. We used medical microscope slides prepared in 1944 from malaria-affected patients from the Ebro Delta in Spain, one of the last footholds of malaria in Europe, to generate a genome of a EuropeanP. vivaxstrain. Population genetics and phylogenetic analyses placed this strain basal to a cluster including samples from the Americas. This genome allowed us to calibrate a genomic mutation rate forP. vivax, and to estimate the mean age of the last common ancestor between European and American strains to the 15th century. This date points to an introduction of the parasite during the European colonisation of the Americas. In addition, we found that some known variants for resistance to anti-malarial drugs, including Chloroquine and Sulfadoxine, were already present in this European strain, predating their use. Our results shed light on the evolution of an important human pathogen and illustrate the value of antique medical collections as a resource for retrieving genomic information on pathogens from the past.
biorxiv genetics 0-100-users 2019Assessment of Polygenic Architecture and Risk Prediction based on Common Variants Across Fourteen Cancers, bioRxiv, 2019-08-10
AbstractWe analyzed summary-level data from genome-wide association studies (GWAS) of European ancestry across fourteen cancer sites to estimate the number of common susceptibility variants (polygenicity) contributing to risk, as well as the distribution of their associated effect sizes. All cancers evaluated showed polygenicity, involving at a minimum thousands of independent susceptibility variants. For some malignancies, particularly chronic lymphoid leukemia (CLL) and testicular cancer, there are a larger proportion of variants with larger effect sizes than those for other cancers. In contrast, most variants for lung and breast cancers have very small associated effect sizes. For different cancer sites, we estimate a wide range of GWAS sample sizes, required to explain 80% of GWAS heritability, varying from 60,000 cases for CLL to over 1,000,000 cases for lung cancer. The maximum relative risk achievable for subjects at the 99th risk percentile of underlying polygenic risk scores, compared to average risk, ranges from 12 for testicular to 2.5 for ovarian cancer. We show that polygenic risk scores have substantial potential for risk stratification for relatively common cancers such as breast, prostate and colon, but limited potential for other cancer sites because of modest heritability and lower disease incidence.
biorxiv genetics 0-100-users 2019Negative selection on complex traits limits genetic risk prediction accuracy between populations, bioRxiv, 2019-08-02
Accurate genetic risk prediction is a key goal for medical genetics and great progress has been made toward identifying individuals with extreme risk across several traits and diseases (Collins and Varmus, 2015). However, many of these studies are done in predominantly European populations (Bustamante et al., 2011; Popejoy and Fullerton, 2016). Although GWAS effect sizes correlate across ancestries (Wojcik et al., 2019), risk scores show substantial reductions in accuracy when applied to non-European populations (Kim et al., 2018; Martin et al., 2019; Scutari et al., 2016). We use simulations to show that human demographic history and negative selection on complex traits result in population specific genetic architectures. For traits under moderate negative selection, ~50% of the heritability can be accounted for by variants in Europe that are absent from Africa. We show that this directly leads to poor performance in risk prediction when using variants discovered in Europe to predict risk in African populations, especially in the tails of the risk distribution. To evaluate the impact of this effect in genomic data, we built a Bayesian model to stratify heritability between European-specific and shared variants and applied it to 43 traits and diseases in the UK Biobank. Across these phenotypes, we find ~50% of the heritability comes from European-specific variants, setting an upper bound on the accuracy of genetic risk prediction in non-European populations using effect sizes discovered in European populations. We conclude that genetic association studies need to include more diverse populations to enable to utility of genetic risk prediction in all populations.
biorxiv genetics 100-200-users 2019