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At least 1,063 records · Page 59

DeepGRN: prediction of transcription factor binding site across cell-types using attention-based deep neural networks

Abstract Background Due to the complexity of the biological systems, the prediction of the potential DNA binding sites for transcription factors remains a difficult problem in computational biology. Genomic DNA sequences and experimental results from parallel sequencing provide available information about the affinity and accessibility of genome and are commonly used features in binding sites prediction. The attention mechanism in deep learning has shown its capability to learn long-range dependencies from sequential data, such as sentences and voices. Until now, no study has applied this approach in binding site inference from massively parallel sequencing data. The successful applications of attention mechanism in similar input contexts motivate us to build and test new methods that can accurately determine the binding sites of transcription factors. Results In this study, we propose a novel tool (named DeepGRN) for transcription factors binding site prediction based on the combination of two components: single attention module and pairwise attention module. The performance of our methods is evaluated on the ENCODE-DREAM in vivo Transcription Factor Binding Site Prediction Challenge datasets. The results show that DeepGRN achieves higher unified scores in 6 of 13 targets than any of the top four methods in the DREAM challenge. We also demonstrate that the attention weights learned by the model are correlated with potential informative inputs, such as DNase-Seq coverage and motifs, which provide possible explanations for the predictive improvements in DeepGRN. Conclusions DeepGRN can automatically and effectively predict transcription factor binding sites from DNA sequences and DNase-Seq coverage. Furthermore, the visualization techniques we developed for the attention modules help to interpret how critical patterns from different types of input features are recognized by our model.

37 INORGANIC, ORGANIC, PHYSICAL, AND ANALYTICAL CH↗

Simulating metagenomic stable isotope probing datasets with MetaSIPSim

DNA-stable isotope probing (DNA-SIP) links microorganisms to their in-situ function in diverse environmental samples. Combining DNA-SIP and metagenomics (metagenomic-SIP) allows us to link genomes from complex communities to their specific functions and improves the assembly and binning of these targeted genomes. However, empirical development of metagenomic-SIP methods is hindered by the complexity and cost of these studies. We developed a toolkit, ‘MetaSIPSim,’ to simulate sequencing read libraries for metagenomic-SIP experiments. MetaSIPSim is intended to generate datasets for method development and testing. To this end, we used MetaSIPSim generated data to demonstrate the advantages of metagenomic-SIP over a conventional shotgun metagenomic sequencing experiment. Through simulation we show that metagenomic-SIP improves the assembly and binning of isotopically labeled genomes relative to a conventional metagenomic approach. Improvements were dependent on experimental parameters and on sequencing depth. Community level G + C content impacted the assembly of labeled genomes and subsequent binning, where high community G + C generally reduced the benefits of metagenomic-SIP. Furthermore, when a high proportion of the community is isotopically labeled, the benefits of metagenomic-SIP decline. Finally, the choice of gradient fractions to sequence greatly influences method performance. Metagenomic-SIP is a valuable method for recovering isotopically labeled genomes from complex communities. We show that metagenomic-SIP performance depends on optimization of experimental parameters. MetaSIPSim allows for simulation of metagenomic-SIP datasets which facilitates the optimization and development of metagenomic-SIP experiments and analytical approaches for dealing with these data.

59 BASIC BIOLOGICAL SCIENCES↗

Profiling the BLAST bioinformatics application for load balancing on high-performance computing clusters

Abstract Background The Basic Local Alignment Search Tool (BLAST) is a suite of commonly used algorithms for identifying matches between biological sequences. The user supplies a database file and query file of sequences for BLAST to find identical sequences between the two. The typical millions of database and query sequences make BLAST computationally challenging but also well suited for parallelization on high-performance computing clusters. The efficacy of parallelization depends on the data partitioning, where the optimal data partitioning relies on an accurate performance model. In previous studies, a BLAST job was sped up by 27 times by partitioning the database and query among thousands of processor nodes. However, the optimality of the partitioning method was not studied. Unlike BLAST performance models proposed in the literature that usually have problem size and hardware configuration as the only variables, the execution time of a BLAST job is a function of database size, query size, and hardware capability. In this work, the nucleotide BLAST application BLASTN was profiled using three methods: shell-level profiling with the Unix “time” command, code-level profiling with the built-in “profiler” module, and system-level profiling with the Unix “gprof” program. The runtimes were measured for six node types, using six different database files and 15 query files, on a heterogeneous HPC cluster with 500+ nodes. The empirical measurement data were fitted with quadratic functions to develop performance models that were used to guide the data parallelization for BLASTN jobs. Results Profiling results showed that BLASTN contains more than 34,500 different functions, but a single function, RunMTBySplitDB, takes 99.12% of the total runtime. Among its 53 child functions, five core functions were identified to make up 92.12% of the overall BLASTN runtime. Based on the performance models, static load balancing algorithms can be applied to the BLASTN input data to minimize the runtime of the longest job on an HPC cluster. Four test cases being run on homogeneous and heterogeneous clusters were tested. Experiment results showed that the runtime can be reduced by 81% on a homogeneous cluster and by 20% on a heterogeneous cluster by re-distributing the workload. Discussion Optimal data partitioning can improve BLASTN’s overall runtime 5.4-fold in comparison with dividing the database and query into the same number of fragments. The proposed methodology can be used in the other applications in the BLAST+ suite or any other application as long as source code is available.

59 BASIC BIOLOGICAL SCIENCES↗

Improved quality metrics for association and reproducibility in chromatin accessibility data using mutual information

Correlation metrics are widely utilized in genomics analysis and often implemented with little regard to assumptions of normality, homoscedasticity, and independence of values. This is especially true when comparing values between replicated sequencing experiments that probe chromatin accessibility, such as assays for transposase-accessible chromatin via sequencing (ATAC-seq). Such data can possess several regions across the human genome with little to no sequencing depth and are thus non-normal with a large portion of zero values. Despite distributed use in the epigenomics field, few studies have evaluated and benchmarked how correlation and association statistics behave across ATAC-seq experiments with known differences or the effects of removing specific outliers from the data. Here, we developed a computational simulation of ATAC-seq data to elucidate the behavior of correlation statistics and to compare their accuracy under set conditions of reproducibility. Using these simulations, we monitored the behavior of several correlation statistics, including the Pearson’s R and Spearman’s ρ coefficients as well as Kendall’s τ and Top–Down correlation. We also test the behavior of association measures, including the coefficient of determination R 2 , Kendall’s W, and normalized mutual information. Our experiments reveal an insensitivity of most statistics, including Spear man’s ρ, Kendall’s τ, and Kendall’s W, to increasing differences between simulated ATAC-seq replicates. The removal of co-zeros (regions lacking mapped sequenced reads) between simulated experiments greatly improves the estimates of correlation and association. After removing co-zeros, the R 2 coefficient and normalized mutual information display the best performance, having a closer one-to-one relationship with the known portion of shared, enhanced loci between simulated replicates. When comparing values between experimental ATAC-seq data using a random forest model, mutual information best predicts ATAC-seq replicate relationships. Collectively, this study demonstrates how measures of correlation and association can behave in epigenomics experiments. We provide improved strategies for quantifying relationships in these increasingly prevalent and important chromatin accessibility assays.

59 BASIC BIOLOGICAL SCIENCES↗

A willow sex chromosome reveals convergent evolution of complex palindromic repeats

Background: Sex chromosomes have arisen independently in a wide variety of species, yet they share common characteristics, including the presence of suppressed recombination surrounding sex determination loci. Mammalian sex chromosomes contain multiple palindromic repeats across the non-recombining region that show sequence conservation through gene conversion and contain genes that are crucial for sexual reproduction. In plants, it is not clear if palindromic repeats play a role in maintaining sequence conservation in the absence of homologous recombination. Results: Here we present the first evidence of large palindromic structures in a plant sex chromosome, based on a highly contiguous assembly of the W chromosome of the dioecious shrub Salix purpurea . The W chromosome has an expanded number of genes due to transpositions from autosomes. It also contains two consecutive palindromes that span a region of 200 kb, with conspicuous 20-kb stretches of highly conserved sequences among the four arms that show evidence of gene conversion. Four genes in the palindrome are homologous to genes in the sex determination regions of the closely related genus Populus , which is located on a different chromosome. These genes show distinct, floral-biased expression patterns compared to paralogous copies on autosomes. Conclusion: The presence of palindromes in sex chromosomes of mammals and plants highlights the intrinsic importance of these features in adaptive evolution in the absence of recombination. Convergent evolution is driving both the independent establishment of sex chromosomes as well as their fine-scale sequence structure.

59 BASIC BIOLOGICAL SCIENCES↗

VirSorter2: a multi-classifier, expert-guided approach to detect diverse DNA and RNA viruses

Abstract Background Viruses are a significant player in many biosphere and human ecosystems, but most signals remain “hidden” in metagenomic/metatranscriptomic sequence datasets due to the lack of universal gene markers, database representatives, and insufficiently advanced identification tools. Results Here, we introduce VirSorter2, a DNA and RNA virus identification tool that leverages genome-informed database advances across a collection of customized automatic classifiers to improve the accuracy and range of virus sequence detection. When benchmarked against genomes from both isolated and uncultivated viruses, VirSorter2 uniquely performed consistently with high accuracy (F1-score > 0.8) across viral diversity, while all other tools under-detected viruses outside of the group most represented in reference databases (i.e., those in the order Caudovirales ). Among the tools evaluated, VirSorter2 was also uniquely able to minimize errors associated with atypical cellular sequences including eukaryotic genomes and plasmids. Finally, as the virosphere exploration unravels novel viral sequences, VirSorter2’s modular design makes it inherently able to expand to new types of viruses via the design of new classifiers to maintain maximal sensitivity and specificity. Conclusion With multi-classifier and modular design, VirSorter2 demonstrates higher overall accuracy across major viral groups and will advance our knowledge of virus evolution, diversity, and virus-microbe interaction in various ecosystems. Source code of VirSorter2 is freely available ( https://bitbucket.org/MAVERICLab/virsorter2 ), and VirSorter2 is also available both on bioconda and as an iVirus app on CyVerse ( https://de.cyverse.org/de ).

59 BASIC BIOLOGICAL SCIENCES↗

Data for Comparison of Genotyping Assays for Detection of Targeted CRISPR/Cas Mutagenesis in Highly Polyploid Sugarcane

Sugarcane ( Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100–130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.

Genomics↗

Description and Use of SCALE Sampler Parametric Capability for Engineering Analysis and Optimization

The Sampler sequence was introduced into the SCALE nuclear modeling and simulation suite in SCALE 6.2 to perform uncertainty quantification via random sampling of nuclear data, material number densities, and dimensions. Sampler was expanded with the introduction of a parametric capability in SCALE 6.2.2. This paper discusses input for the Sampler parametric sequence and presents two case studies of analyses performed using the sequence. These case studies include preconceptual design of a package for transporting high assay low-enriched uranium (HALEU) oxide and scoping calculations to support subcritical limit development for a future update of the ANSI/ANS-8.1 (ANS-8.1) standard. The parametric capability within Sampler provides many benefits to analysts. For instance, parametric sweeps are frequently used to identify optimum parameter values as part of safety analysis or system design, but such sweeps can require substantial engineering time or may rely on custom-written scripts or scripts such as Write One, Run Many (or WORM) developed outside of any software quality assurance program. With the parametric capabilities in Sampler, however, a large number of inputs can be generated automatically without recourse to scripting by individual analysts. The parametric capability can also be used in lieu of the CSAS5S search sequence to identify optimum parameters more simply with straightforward inputs and outputs. Sampler can also be used to calculate input parameters from engineering specifications. For example, diameters can be converted to radii, or masses can be used to calculate number densities. Overall, the Sampler parametric capability provides a robust feature within SCALE, eliminating the need for user-developed scripting.

73 NUCLEAR PHYSICS AND RADIATION PHYSICS↗

High wax ester and triacylglycerol biosynthesis potential in coastal sediments of Antarctic and Subantarctic environments

The wax ester (WE) and triacylglycerol (TAG) biosynthetic potential of marine microorganisms is poorly understood at the microbial community level. The goal of this work was to uncover the prevalence and diversity of bacteria with the potential to synthesize these neutral lipids in coastal sediments of two high latitude environments, and to characterize the gene clusters related to this process. Homolog sequences of the key enzyme, the wax ester synthase/acyl-CoA:diacylglycerol acyltransferase (WS/DGAT) were retrieved from 13 metagenomes, including subtidal and intertidal sediments of a Subantarctic environment (Ushuaia Bay, Argentina), and subtidal sediments of an Antarctic environment (Potter Cove, Antarctica). The abundance of WS/DGAT homolog sequences in the sediment metagenomes was 1.23 ± 0.42 times the abundance of 12 single-copy genes encoding ribosomal proteins, higher than in seawater (0.13 ± 0.31 times in 338 metagenomes). Homolog sequences were highly diverse, and were assigned to the Pseudomonadota, Actinomycetota, Bacteroidota and Acidobacteriota phyla. The genomic context of WS/DGAT homologs included sequences related to WE and TAG biosynthesis pathways, as well as to other related pathways such as fatty-acid metabolism, suggesting carbon recycling might drive the flux to neutral lipid synthesis. These results indicate the presence of abundant and taxonomically diverse bacterial populations with the potential to synthesize lipid storage compounds in marine sediments, relating this metabolic process to bacterial survival.

59 BASIC BIOLOGICAL SCIENCES↗

Efficacy and Delivery of Novel FAST Agents for Coronaviruses

We proposed to test and develop advanced delivery for novel agents from our collaborators Facile Accelerated Specific Therapeutics (FAST) platform to reduce coronavirus replication. Sachi Bioworks Inc., Prof. Anushree Chatterjee, and Prof. Prashant Nagpal at the University of Colorado Boulder have developed a bioinformatics and synthesis pipeline to produce sequence specific theranostic agents (agents that can be therapies and/or diagnostics) that are inherently transported into the cytoplasm of mammalian host cells and sequence-specifically interfere in nucleic acid replication. The agent comprises a small nanoparticle (2-5 nm) chosen for ideal cellular transport and/or imaging conjugated to a short, synthetic DNA analog oligomer designed for binding to one or more target viral sequences. The sequence specific binding of the FAST agent to its target prevents nucleic acid replication due to its high affinity binding. While the small nanoparticle facilitates delivery in vitro, we plan to package the FAST agents into a larger nanoparticle (80-300 nm) for future in vivo delivery applications. Our team at Sandia has expertise encapsulating biomolecules including protein, DNA, and RNA into solid lipid nanoparticles (LNP) and lipid coated mesoporous silica nanoparticles (LC-MSN) and shown successful delivery in mouse models to multiple tissues. Our team focused on formulation parameters for FAST agents into lipid nanoparticles (LNP) and lipid coated mesoporous silica nanoparticles (LC-MSN) for enhanced delivery and/or efficacy and in vivo translation. We used lipid formulas that have been shown in literature to facility in vitro and more importantly, in vivo delivery. In our work discussed below, we successfully demonstrate loading and release of FAST agents on silica core and stable LC-MSN in a reasonable size range for in vivo testing.

59 BASIC BIOLOGICAL SCIENCES↗

Threat Agnostic Virulence Assessment of Pathogens

Virulence assessment of new, emerging, and engineered pathogens is critical to mounting an appropriate response to a biothreat agent. The capacity of the pathogen to colonize human and harm tissues must be characterized to understand pathogenicity pathways and optimize diagnosis and treatment of resulting disease. Respiratory pathogens are of interest because they can have high transmissibility rates, as observed with the SARS-CoV-2 virus, the causative agent of Covid-19. Current technologies are insufficient to assess threats due to their reliance on systems with only one cell type and on sequencing the pathogen. However, it is known that sequence is not an accurate predictor of function, and sequencing can be unreliable for newly emerged or engineered pathogens. An ideal system would consist of relevant epithelial cell types and an assay sensitive enough to detect changes in host responses that do not rely on DNA sequencing. We chose a system consisting of host lung epithelial cells that can be used to assess the virulence of unknown respiratory pathogens. We interrogated pathogens using this model and assess features of pathogenicity. Our objective is to leverage PNNLs strengths in tissue engineering and proteomics capabilities to build a multiple reaction monitoring (MRM) or parallel reaction monitoring (PRM) liquid chromatography-tandem mass spectrometry assay for human host cell proteins whose abundance is influenced by infection. These responses can were then assessed for relative virulence using pathogen agnostic signatures. When confronted with a pathogen, cells activate dedicated signaling pathways, typically through phosphorylation of regulatory proteins and downstream activation of host cell networks.

59 BASIC BIOLOGICAL SCIENCES↗

Determination of the spent fuel decay heat with the VERA core simulator

VERA core simulator capabilities to predict spent fuel decay heat have been explored. The comparison with the Serpent2 and NEWT/TRITON sequence from the SCALE package, performed on the NPP Krško fuel test cases, has enabled independent verification of the successful implementation of the VERA depletion/decay calculation sequence applied on the extended nuclide set. Since the VERA is a 3-D core simulator, the focus in the rest of the paper was on the investigation of the 3-D effects and non-linearity of the soluble boron concentration averaging process, which are difficult to assess with most of available computational tools. A difference in the decay heat prediction between the 3-D and 2-D model approaches was analyzed. In addition, the impact of the Inconel grids on the decay heat was determined. Averaging the concentration of soluble boron over the fuel irradiation period has been found to be a viable approach, since it results in slight decay heat overprediction, which is considered conservative for most applications. Finally, demonstration on the Watts Bar Unit 1 cycle 1 depletion has shown formidable VERA capabilities to accurately predict spent fuel decay heat.

Kromar, Marjan↗

Comparison of genotyping assays for detection of targeted CRISPR/Cas mutagenesis in highly polyploid sugarcane

Sugarcane (Saccharum spp.) is an important biofuel feedstock and a leading source of global table sugar. Saccharum hybrid cultivars are highly polyploid (2n = 100–130), containing large numbers of functionally redundant hom(e)ologs in their genomes. Genome editing with sequence-specific nucleases holds tremendous promise for sugarcane breeding. However, identification of plants with the desired level of co-editing within a pool of primary transformants can be difficult. While DNA sequencing provides direct evidence of targeted mutagenesis, it is cost-prohibitive as a primary screening method in sugarcane and most other methods of identifying mutant lines have not been optimized for use in highly polyploid species. In this study, non-sequencing methods of mutant screening, including capillary electrophoresis (CE), Cas9 RNP assay, and high-resolution melt analysis (HRMA), were compared to assess their potential for CRISPR/Cas9-mediated mutant screening in sugarcane. These assays were used to analyze sugarcane lines containing mutations at one or more of six sgRNA target sites. All three methods distinguished edited lines from wild type, with co-mutation frequencies ranging from 2% to 100%. Cas9 RNP assays were able to identify mutant sugarcane lines with as low as 3.2% co-mutation frequency, and samples could be scored based on undigested band intensity. CE was highlighted as the most comprehensive assay, delivering precise information on both mutagenesis frequency and indel size to a 1 bp resolution across all six targets. This represents an economical and comprehensive alternative to sequencing-based genotyping methods which could be applied in other polyploid species.

60 APPLIED LIFE SCIENCES↗

The molecular basis of the neutralization breadth of the RBD-specific antibody CoV11

SARS-CoV-2, the virus behind the COVID-19 pandemic, has changed over time to the extent that the current virus is substantially different from what originally led to the pandemic in 2019–2020. Viral variants have modified the severity and transmissibility of the disease and continue do so. How much of this change is due to viral fitness versus a response to immune pressure is hard to define. One class of antibodies that continues to afford some level of protection from emerging variants are those that closely overlap the binding site for angiotensin-converting enzyme 2 (ACE2) on the receptor binding domain (RBD). Some members of this class that were identified early in the course of the pandemic arose from the VH 3-53 germline gene (IGHV3-53*01) and had short heavy chain complementarity-determining region 3s (CDR H3s). Here, we describe the molecular basis of the SARS-CoV-2 RBD recognition by the anti-RBD monoclonal antibody CoV11 isolated early in the COVID-19 pandemic and show how its unique mode of binding the RBD determines its neutralization breadth. CoV11 utilizes a heavy chain VH 3-53 and a light chain VK 3-20 germline sequence to bind to the RBD. Two of CoV11’s four heavy chain changes from the VH 3-53 germline sequence, $Thr^{28}_{FWRH1}$ to Ile and $Ser^{31}_{CDRH1}$ to Arg, and some unique features in its CDR H3 increase its affinity to the RBD, while the four light chain changes from the VK 3-20 germline sequence sit outside of the RBD binding site. Antibodies of this type can retain significant affinity and neutralization potency against variants of concern (VOCs) that have diverged significantly from original virus lineage such as the prevalent omicron variant. We also discuss the mechanism by which VH 3-53 encoded antibodies recognize spike antigen and show how minimal changes to their sequence, their choice of light chain, and their mode of binding influence their affinity and impact their neutralization breadth.

60 APPLIED LIFE SCIENCES↗

Molecular evolution of human coronavirus-NL63, -229E, -HKU1 and -OC43 in hospitalized children in China

Human coronaviruses (HCoVs) HCoV-NL63, HCoV-229E, HCoV-HKU1 and HCoV-OC43 have been circulated in the human population worldwide, and they are associated with a broad range of respiratory diseases with varying severity. However, there are neither effective therapeutic drugs nor licensed vaccines available for the treatment and prevention of infections by the four HCoVs. In this study, we collected nasopharyngeal aspirates of children hospitalized for respiratory tract infection in China during 2014–2018 and conducted next-generation sequencing. Sequences of four HCoVs were then selected for an in-depth analysis. Genome sequences of 2 HCoV-NL63, 8 HCoV-229E, 2 HCoV-HKU1, and 6 HCoV-OC43 were obtained. Based on the full-length S gene, a strong temporal signal was found in HCoV-229E and the molecular evolutionary rate was 6 × 10 −4 substitutions/site/year. Based on the maximum-likelihood (ML) phylogenetic tree of complete S gene, we designated H78 as a new sub-genotype C2 of HCoV-HKU1, and the obtained P43 sequence was grouped into the reported novel genotype K of HCoV-OC43 circulating in Guangzhou, China. Based on the complete genome, potential recombination events were found to occur as two phenomena, namely intraspecies and interspecies. Moreover, we observed two amino acid substitutions in the S1 subunit of obtained HCoV-NL63 (G534V) and HCoV-HKU1 (H512R), while residues 534 and 512 are important for the binding of angiotensin-converting enzyme 2 and neutralizing antibodies, respectively. Our findings might provide a clue for the molecular evolution of the four HCoVs and help in the early diagnosis, treatment and prevention of broad-spectrum HCoV infection.

Shao, Nan↗

Evaluating the Impact of gRNA SNPs in CasRx Activity for Reducing Viral RNA in HCoV-OC43

Viruses within a given family often share common essential genes that are highly conserved due to their critical role for the virus’s replication and survival. In this work, we developed a proof-of-concept for a pan-coronavirus CRISPR effector system by designing CRISPR targets that are cross-reactive among essential genes of different human coronaviruses (HCoV). We designed CRISPR targets for both the RNA-dependent RNA polymerase (RdRp) gene as well as the nucleocapsid (N) gene in coronaviruses. Using sequencing alignment, we determined the most highly conserved regions of these genes to design guide RNA (gRNA) sequences. In regions that were not completely homologous among HCoV species, we introduced mismatches into the gRNA sequence and tested the efficacy of CasRx, a Cas13d type CRISPR effector, using reverse transcription quantitative polymerase chain reaction (RT-qPCR) in HCoV-OC43. We evaluated the effect that mismatches in gRNA sequences has on the cleavage activity of CasRx and found that this CRISPR effector can tolerate up to three mismatches while still maintaining its nuclease activity in HCoV-OC43 viral RNA. This work highlights the need to evaluate off-target effects of CasRx with gRNAs containing up to three mismatches in order to design safe and effective CRISPR experiments.

59 BASIC BIOLOGICAL SCIENCES↗

Comparison of Auxenochlorella protothecoides and Chlorella spp. Chloroplast Genomes: Evidence for Endosymbiosis and Horizontal Virus-like Gene Transfer

Resequencing of the chloroplast genome (cpDNA) of Auxenochlorella protothecoides UTEX 25 was completed (GenBank Accession no. KC631634.1), revealing a genome size of 84,576 base pairs and 30.8% GC content, consistent with features reported for the previously sequenced A. protothecoides 0710, (GenBank Accession no. KC843975). The A. protothecoides UTEX 25 cpDNA encoded 78 predicted open reading frames, 32 tRNAs, and 4 rRNAs, making it smaller and more compact than the cpDNA genome of C. variabilis (124,579 bp) and C. vulgaris (150,613 bp). By comparison, the compact genome size of A. protothecoides was attributable primarily to a lower intergenic sequence content. The cpDNA coding regions of all known Chlorella species were found to be organized in conserved colinear blocks, with some rearrangements. The Auxenochlorella and Chlorella species genome structure and composition were similar, and of particular interest were genes influencing photosynthetic efficiency, i.e., chlorophyll synthesis and photosystem subunit I and II genes, consistent with other biofuel species of interest. Phylogenetic analysis revealed that Prototheca cutis is the closest known A. protothecoides relative, followed by members of the genus Chlorella. The cpDNA of A. protothecoides encodes 37 genes that are highly homologous to representative cyanobacteria species, including rrn16, rrn23, and psbA, corroborating a well-recognized symbiosis. Several putative coding regions were identified that shared high nucleotide sequence identity with virus-like sequences, suggestive of horizontal gene transfer. Despite these predictions, no corresponding transcripts were obtained by RT-PCR amplification, indicating they are unlikely to be expressed in the extant lineage.

59 BASIC BIOLOGICAL SCIENCES↗

YPK9 and WHI2 Negatively Interact during Oxidative Stress

Yeast PARK9 (YPK9) shares homology with human ATP13A2, which encodes a polyamine transporter implicated in juvenile forms of Parkinson’s disease. We used YPK9 to gain insight into how ATP13A2 affects cell growth and sensitivity to oxidative stress. Surprisingly, the YPK9 deletion strain from the Saccharomyces cerevisiae deletion collection (YKO) in wildtype BY4741 (mating type a) grew faster and was more resistant to hydrogen peroxide than a commercial, putative parental BY4741 wildtype strain (BY4741 COM ). In contrast, deleting YPK9 from BY4741 COM rendered it very sensitive to hydrogen peroxide, suggesting its background is different from that of the deletion collection. Whole-genome sequencing revealed that BY4741 COM and BY4741 COM ypk9Δ contain a novel premature stop codon near the 3' end of WHI2 (WHI2 G1324T ), whereas the collection’s YPK9 deletion strain contains WHI2, which encodes a 486 amino acid protein, Whi2p. Replacing full-length WHI2 with the sequence coding for the predicted truncation (Whi2p E442* ) rendered strains more sensitive to hydrogen peroxide, whereas the converse replacement rendered them more resistant. The sequences of WHI2 in 20 randomly chosen strains from the collection encode the full-length protein, indicating that the putative parental BY4741 WHI2 G1324T strain’s genetic background differs from that of the deletion collection. Examination of WHI2 sequences in several commonly used wildtype S. cerevisiae strains and isolates revealed other Whi2p truncations that might yield altered phenotypes. Together, these results demonstrate a novel premature stop codon in WHI2 that renders yeast sensitive to hydrogen peroxide; they also reveal a negative genetic interaction between WHI2 and YPK9 in the presence of hydrogen peroxide in the BY4741 background.

59 BASIC BIOLOGICAL SCIENCES↗