Biology subjects

Watters, K. E.

Publications and source records attributed to Watters, K. E..

5 recordsLinked to original sources

bioRxiv2019

Machine Learning Predicts New Anti-CRISPR Proteins

The increasing use of CRISPR-Cas9 in medicine, agriculture and synthetic biology has accelerated the drive to discover new CRISPR-Cas inhibitors as potential mechanisms of control for gene editing applications. Many such anti-CRISPRs have been found in mobile genetic elements that disable the CRISPR-Cas adaptive immune system. However, comparing all currently known anti-CRISPRs does not reveal a shared set of properties that can be used for facile bioinformatic identification of new anti-CRISPR families. Here, we describe AcRanker, a machine learning based method for identifying new potential anti-CRISPRs directly from proteomes using protein sequence information only. Using a training set of known anti-CRISPRs, we built a model based on XGBoost ranking and extensively benchmarked it through non-redundant cross-validation and external validation. We then applied AcRanker to predict candidate anti-CRISPRs from self-targeting bacterial genomes and discovered two previously unknown anti-CRISPRs: AcrllA16 (ML1) and AcrIIA17 (ML8). We show that AcrIIA16 strongly inhibits Streptococcus iniae Cas9 (SinCas9) and weakly inhibits Streptococcus pyogenes Cas9 (SpyCas9). We also show that AcrIIA17 inhibits both SpyCas9 and SauCas9 with low potency. The addition of AcRanker to the anti-CRISPR discovery toolkit allows researchers to directly rank potential anti-CRISPR candidate genes for increased speed in testing and validation of new anti-CRISPRs. A web server implementation for AcRanker is available online at http://acranker.pythonanywhere.com/.

biochemistry↗

bioRxiv2019

Potent CRISPR-Cas9 inhibitors from Staphylococcus genomes

Anti-CRISPRs (Acrs) are small proteins that inhibit the RNA-guided DNA targeting activity of CRISPR-Cas enzymes. Encoded by bacteriophage and phage-derived bacterial genes, Acrs prevent CRISPR-mediated inhibition of phage infection and can also block CRISPR-Cas-mediated genome editing in eukaryotic cells. To identify Acrs capable of inhibiting Staphylococcus aureus Cas9 (SauCas9), an alternative to the most commonly used genome editing protein Streptococcus pyogenes Cas9 (SpyCas9), we used both self-targeting CRISPR screening and guilt-by-association genomic search strategies. Here we describe three new potent inhibitors of SauCas9 that we name AcrIIA13, AcrIIA14 and AcrIIA15. These inhibitors share a conserved N-terminal sequence that is dispensable for anti-CRISPR function, and have divergent C-termini that are required in each case for selective inhibition of SauCas9-catalyzed DNA cleavage. In human cells, we observe robust and specific inhibition of SauCas9-induced genome editing by AcrIIA13 and moderate inhibition by AcrIIA14 and AcrIIA15. We also find that the conserved N-terminal domain of AcrIIA13-15 binds to an inverted repeat sequence in the promoter of these Acr genes, consistent with its predicted helix-turn-helix DNA binding structure. These data demonstrate an effective strategy for Acr discovery and establish AcrIIA13-15 as unique bifunctional inhibitors of SauCas9.

biochemistry↗

bioRxiv2018

Computationally Reconstructing Cotranscriptional RNA Folding Pathways from Experimental Data Reveals Rearrangement of Non-Native Folding Intermediates

The series of RNA folding events that occur during transcription, or a cotranscriptional folding pathway, can critically influence the functional roles of RNA in the cell. Here we present a method, Reconstructing RNA Dynamics from Data (R2D2), to uncover details of cotranscriptional folding pathways by predicting RNA secondary and tertiary structures from cotranscriptional SHAPE-Seq data. We applied R2D2 to the folding of the Escherichia coli Signal Recognition Particle (SRP) RNA sequence and show that this sequence undergoes folding through non-native intermediate structures that require significant structural rearrangement before reaching the functional native structure. Secondary structure folding pathway predictions and all-atom molecular dynamics simulations of folding intermediates suggest that this rearrangement can proceed through a toehold mediated strand displacement mechanism, which can be disrupted and rescued with point mutations. These results demonstrate that even RNAs with simple functional folds can undergo complex folding processes during synthesis, and that small variations in their sequence can drastically affect their cotranscriptional folding pathways.\n\nHighlights- Computational methods predict RNA structures from cotranscriptional SHAPE-Seq data\n- The E. coli SRP RNA folds into non-native structural intermediates cotranscriptionally\n- These structures rearrange dynamically to form an extended functional fold\n- Point mutations can disrupt and rescue cotranscriptional RNA folding pathways

biochemistry↗

bioRxiv2018

Controlling CRISPR-Cas9 with ligand-activated and ligand-deactivated sgRNAs

The CRISPR-Cas9 system provides the ability to edit, repress, activate, or mark any gene (or DNA element) by pairing of a programmable single guide RNA (sgRNA) with a complementary sequence on the DNA target. Here we present a new method for small-molecule control of CRISPR-Cas9 function through insertion of RNA aptamers into the sgRNA. We show that CRISPR-Cas9-based gene repression (CRISPRi) can be either activated or deactivated in a dose-dependent fashion over a >10-fold dynamic range in response to two different small-molecule ligands. Since our system acts directly on each target-specific sgRNA, it enables new applications that require differential and opposing temporal control of multiple genes.

synthetic biology↗

bioRxiv2017

Distributed Biotin-Streptavidin Transcription Roadblocks for Mapping Cotranscriptional RNA Folding

RNA molecules fold cotranscriptionally as they emerge from RNA polymerase. Cotranscriptional folding is an important process for proper RNA structure formation as the order of folding can determine an RNA molecules structure, and thus its functional properties. Despite its fundamental importance, the experimental study of RNA cotranscriptional folding has been limited by the lack of easily approachable methods that can interrogate nascent RNA structures at nucleotide resolution during transcription. We previously developed cotranscriptional selective 2-hydroxyl acylation analyzed by primer extension sequencing (SHAPE-seq) to simultaneously probe all of the intermediate structures an RNA molecule transitions through during transcription elongation. Here, we improve the broad applicability of cotranscriptional SHAPE-Seq by developing a sequence-independent streptavidin roadblocking strategy to simplify the preparation of roadblocking transcription templates. We determine the fundamental properties of streptavidin roadblocks and show that randomly distributed streptavidin roadblocks can be used in cotranscriptional SHAPE-Seq experiments to measure the Bacillus cereus crcB fluoride riboswitch folding pathway. Comparison of EcoRIE111Q and streptavidin roadblocks in cotranscriptional SHAPE-Seq data shows that both strategies identify the same RNA structural transitions related to the riboswitch decision-making process. Finally, we propose guidelines to leverage the complementary strengths of each transcription roadblock for use in studying cotranscriptional folding.

molecular biology↗