Project description:RNA mutations are known to change mobility in native gels. What is not known is if mobility can serve as an effective tool to separate structurally similar (structural homologs) from structurally destabilized variants in a deep-mutation library of an RNA. Here we defined the proportion of a mutant in the native band from native polyacrylamide gel electrophoresis (PAGE) as a native-mobility-fitness score. The fitness scores of single and double mutants allowed a unsupervised, RNA-specific analysis to detect key secondary and tertiary base pairs through covariational signals. Subsequent amplification of these signals and their use as restraints for folding led to not only high-accuracy secondary structures with the F1-score > 0.9, but also quality tertiary-structure models between 3.6 Å and 7.7 Å RMSD from their native structures for the best in top 5 models for 6 RNAs tested including two CASP 15 difficult targets. This MobiSeq method should provide a simple and effective method for inferring 2D and 3D structures and improving mechanistic understanding of all structured RNAs.
Project description:Deep mutational scanning is a powerful method for exploring the mutational fitness landscape of proteins. Its adaptation to anti-CRISPR proteins, which are natural CRISPR-Cas inhibitors and key players in the co-evolution of microbes and phages, facilitates their characterization and optimization. Here, we developed a robust anti-CRISPR deep mutational scanning pipeline in Escherichia coli that combines synthetic gene circuits based on CRISPR interference with flow cytometry coupled sequencing and mathematical modeling. Using this pipeline, we characterized comprehensive single point mutation libraries for AcrIIA4 and AcrIIA5, two potent inhibitors of CRISPR-Cas9. The resulting mutational fitness landscapes revealed considerable mutational tolerance for both Acrs, suggesting an intrinsic redundancy with respect to Cas9 inhibitory features, and – for AcrIIA5 – indicated mutations that boost Cas9 inhibition. Subsequent in vitro characterization suggested that the observed differences in inhibitory potency between mutant inhibitors were mostly due to changes in binding affinity rather than protein expression levels. Finally, to demonstrate that our pipeline can inform Acrs-based genome editing applications, we employed a selected subset of mutant inhibitors to increase CRISPR-Cas9 target specificity by modulating Cas9 activity. Taken together, our work establishes deep mutational scanning as a powerful method for anti-CRISPR protein characterization and optimization.
Project description:This dataset provides allele counts and raw fastqs for deep mutational scanning of the HIV-1 genes tat and rev when not-overlapped with one another (placed in the nef locus) as described in Fernandes et al. Functional segregation of overlapping genes in HIV Cell 2016 (in revision). Preselection (input) and post selection (replicate 1/2) files for every possible point mutant of these two HIV proteins from the NL4-3 background are given.Tab delimited files including codon counts across the amplicons are also included and are probably the most useful thing to most researchers. The data here was used to generate Figures 3 and 4 and 7 and might be of general use for people interested in deep mutational scanning, looking for signatures of epistasis in rev or tat, or reanalyzing and mining the data. FAQ: Why do the ends of each amplicon have such variation? In order to increase diversity across the flowcell, I pooled standard primers with N, NN, and NNN extensions to throw amplicons out of phase. When aligning you should trim the ends or ignore them. This means that the overlap between PE's can vary by 3 nt. Why are the filenames not easy to deal with? The filenames are tied to separate MiSeq runs. I hope to clean up the nomenclature and update this entry in the future while preserving the run information. You can get a sense of that as different residues will vary in Q-score, and that is mostly tied to the run they were pooled on and not any interesting biology. While this is makes it a little harder to follow, I think it's good to get a sense that doing this kind of analysis in high-throughput fashion leads to a reasonable amount of failure (i.e. RNA isolation, RT, fail) that led to repetition until we had good data for every position. Can you help me deal with this dataset? Yes. Please email me at jferna10@ucsc.edu, or contact me on twitter @jdf_ev. For reagents please contact Alan Frankel at frankel@cgl.ucsf.edu. Do you have the analysis scripts you used to process the data? Yes they are on github. https://github.com/nbstrauli/allele_frequency_trajectory_sim
Project description:Deep mutational scanning of the interaction of JUN's leucine zipper domain and other human bZIPs in different experimental conditions
Project description:Despite the importance of Aβ aggregation in Alzheimer’s disease etiology, our understanding of the sequence determinants of aggregation is sparse and largely derived from in vitro studies. For example, in vitro proline and alanine scanning mutagenesis of Aβ40 proposed core regions important for aggregation. However, we lack even this limited mutagenesis data for the more disease-relevant Aβ42. Thus, to better understand the molecular determinants of Aβ42 aggregation in a cell-based system, we combined a yeast DHFR aggregation assay with deep mutational scanning. We measured the effect of 791 of the 798 possible single amino acid substitutions on the aggregation propensity of Aβ42. We found that ~75% of substitutions, largely to hydrophobic residues, maintained or increased aggregation. We identified 11 positions at which substitutions, particularly to hydrophilic and charged amino acids, disrupted Aβ aggregation. These critical positions were similar but not identical to critical positions identified in previous Aβ mutagenesis studies. Finally, we analyzed our large-scale mutagenesis data in the context of different Aβ aggregate structural models, finding that the mutagenesis data agreed best with models derived from fibrils seeded using brain-derived Aβ aggregates.
Project description:The HIV-1 genome gains access to the inside of a cell via the mechanism of the viral spike protein Env, which undergoes a series of major conformational rearrangements after binding target receptors that ultimately drive virus-cell membrane fusion. Env is expressed as a heterogenous ensemble of conformations, which can inappropriately misdirect the host immune response towards the production of non-protective, strain-specific antibodies. Potent, broadly neutralizing antibodies (bnAbs) frequently recognize a ‘closed’ Env conformation, and therefore Env has undergone significant engineering to stabilize the closed state for vaccine incorporation. Previously, we used deep mutational scanning of Env from a prototypical tier 1 clade B strain (BaL) to characterize the sequence-activity landscape for binding to PG16, a bnAb that preferentially binds the closed state. Mutations were identified that increased expression of closed Env and reduced conformational heterogeneity, but these mutations were only partially transferable to Env sequences from other strains. To generate an expanded set of mutations that may be broadly applicable to diverse HIV-1 strains, we present here the deep mutational scanning of Env from the tier 2 clade C strain DU422 for interactions with CD4 and PG16. Residues across the trimerization domain and trimer interface have low mutational tolerance for maintaining PG16 recognition. New mutations are identified that enhance presentation of the closed Env conformation, and these are applied to Env sequences spanning multiple clades and tiers.
Project description:Deep mutational scanning can provide significant insights into the function of essential genes in bacteria. Here, we developed a high-throughput method for mutating essential genes of Escherichia coli in their native genetic context. We used Cas9-mediated recombineering to introduce a library of mutations, created by error-prone PCR, within a gene fragment on the genome using a single gRNA pre-validated for high efficiency. Tracking mutation frequency through deep sequencing revealed biases in the position and the number of the introduced mutations. We overcame these biases by increasing the homology arm length and blocking mismatch repair to achieve a mutation efficiency of 85% for non-essential genes and 55% for essential genes. These experiments also improved our understanding of poorly characterized recombineering process using dsDNA donors with single nucleotide changes. Finally, we applied our technology to target rpoB, the beta subunit of RNA polymerase, to study resistance against rifampicin. In a single experiment, we validate multiple biochemical and clinical observations made in the previous decades and provide insights into resistance compensation with the study of double mutants.
Project description:The fidelity of start codon recognition by ribosomes is paramount during protein synthesis. The textbook knowledge of eukaryotic translation initiation depicts 5’→3’ unidirectional migration of the pre-initiation complex (PIC) along the 5’UTR. In probing translation initiation from ultra-short 5’UTR, we report that an AUG triplet near the 5’ end can be selected via PIC backsliding. The bi-directional ribosome scanning is supported by competitive selection of closely spaced AUG codons and recognition of two initiation sites flanking an internal ribosome entry site. Transcriptome-wide PIC profiling reveals footprints with an oscillation pattern near the 5’ end and start codons. Depleting the RNA helicase eIF4A leads to reduced PIC oscillations and impaired selection of 5’ end start codons. Enhancing the ATPase activity of eIF4A promotes nonlinear PIC scanning and stimulates upstream translation initiation. The helicase-mediated PIC conformational switch may provide an operational mechanism that unifies ribosome recruitment, scanning, and start codon selection.