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Design principles and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

ABSTRACT: Design principles and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

PROVIDER: PRJNA833456 | ENA |

REPOSITORIES: ENA

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Design principles and applications of a CRISPR activation system for facile genetic overexpression in Candida albicans

Project description:Here, we sought to assess target-specificity and detect potential off-target effects of a novel CRISPR activation (CRISPRa) system in Candida albicans. We used the CRISPRa system to target ALS1 for overexpression, and we also created a CRISPRa strain that does not target any gene for overexpression for use as a control. Differential gene expression analysis between the CRISPRa-ALS1 strain and the non-targeting strain allowed us to determine if ALS1 was the only gene overexpressed by our CRISPRa-ALS1 strain, or if the system non-specifically targeted other genes as well.

2022-05-03 | GSE201904 | GEO

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Massively parallel Cas13d screens reveal principles for guide RNA design

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Design principles for cyclin K molecular glue degraders

Project description:Molecular glue degraders are an effective therapeutic modality, but their design principles are not well understood. Recently, several unexpectedly diverse compounds were reported to deplete cyclin K by linking CDK12-cyclin K to the DDB1-CUL4-RBX1 E3 ligase. To investigate how chemically dissimilar small molecules trigger cyclin K degradation, we evaluated 91 candidate degraders in structural, biophysical, and cellular studies and reveal all compounds acquire glue activity via simultaneous CDK12 binding and engagement of DDB1 interfacial residues, in particular Arg928. While we identify multiple published kinase inhibitors as cryptic degraders, we also show that these glues do not require pronounced inhibitory properties for activity and that the relative degree of CDK12 inhibition versus cyclin K degradation is tuneable. We further demonstrate cyclin K degraders have transcriptional signatures distinct from CDK12 inhibitors, thereby offering unique therapeutic opportunities. The systematic structure-activity relationship analysis presented herein provides a conceptual framework for rational molecular glue design.

2023-10-20 | PXD041836 | Pride

Hyphomicrobium facile strain:dsm 1565

Project description:Hyphomicrobium facile subsp. facile DSM 1565 genome sequencing

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Comprehensive AAV capsid fitness landscape reveals a viral gene and enables machine-guided design

Project description:An Adeno-Associated Virus capsid fitness landscape reveals a frameshifted viral gene and in vivo design principles, enabling machine-guided engineering.

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Massively parallel Cas13d screens reveal principles for guide RNA design

Project description:Type VI CRISPR enzymes are RNA-targeting proteins with nuclease activity that enable specific and robust target gene knockdown without altering the genome. To define rules for the design of Cas13d guide RNAs, we conducted massively parallel screens targeting messenger RNAs of a green fluorescent protein transgene, and CD46, CD55 and CD71 cell-surface proteins in human cells. In total, we measured the activity of 24,460 gRNAs with and without mismatches relative to the target sequences. Knockdown efficacy is driven by gRNA-specific features and target site context. Single mismatches generally reduce knockdown to a modest degree, but spacer nucleotides 15–21 are largely intolerant of target site mismatches. We developed a computational model to identify optimal gRNAs and confirm their generalizability testing 3,979 guides targeting mRNAs of 48 endogenous genes. We show that Cas13 can be used in forward transcriptomic pooled screens and, using our model, predict optimized Cas13 gRNAs for all protein-coding transcripts in the human genome.

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Absolute quantification of protein production reveals principles underlying protein synthesis rates

Project description:A quantitative view of cellular functions requires precise measures of the rates of biomolecule production, especially proteins-the direct effectors of biological processes. Here we present a genome-wide approach, based on ribosome profiling, for measuring absolute protein synthesis rates. The resultant E. coli dataset transforms our understanding of the extent to which protein synthesis is precisely controlled to optimize function and efficiency. For example, members of multi-protein complexes are made in precise proportion to their stoichiometry, whereas components of functional modules are produced differentially according to their hierarchical role. Estimates of absolute protein abundance also reveal principles used to optimize design. These include how the level of different types of transcription factors is optimized for rapid response, and how a metabolic pathway (methionine biosynthesis) balances production cost with activity requirements. More broadly, our studies reveal how general principles, important both for understanding natural systems and for synthesizing new ones, emerge from global quantitative analyses of protein synthesis. 4 samples of E. coli ribosome profiling and mRNA-seq, including biological replicates

2014-04-24 | E-GEOD-53767 | biostudies-arrayexpress

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