scholarly journals Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression

2018 ◽  
Author(s):  
David JHF Knapp ◽  
Yale S Michaels ◽  
Max Jamilly ◽  
Quentin RV Ferry ◽  
Hector Barbosa ◽  
...  
Keyword(s):  
Rational Design ◽  
Rna Expression ◽  
Temporal Control ◽  
High Background ◽  
Pol Ii ◽  
Trna Processing ◽  
Guide Rna ◽  
Promising Strategy ◽  
Screening Platform

ABSTRACTSpatial/temporal control of Cas9 guide RNA expression could considerably expand the utility of CRISPR-based technologies. Current approaches based on tRNA processing offer a promising strategy but suffer from high background. Here we developed a variant screening platform to identify differential sequence determinants of human tRNA promoter and processing activities. Rational design based on the ensuing principles allowed us to engineer an improved tRNA scaffold that enabled highly specific guide RNA production from a Pol-II promoter.

scholarly journals Decoupling tRNA promoter and processing activities enables specific Pol-II Cas9 guide RNA expression

2019 ◽  
Vol 10 (1) ◽  
Author(s):  
David J. H. F. Knapp ◽  
Yale S. Michaels ◽  
Max Jamilly ◽  
Quentin R. V. Ferry ◽  
Hector Barbosa ◽  
...  
Keyword(s):  
Rna Expression ◽  
Pol Ii ◽  
Guide Rna

scholarly journals Catechol-Containing Compounds are a Broad Class of Protein Aggregation Inhibitors: Redox State is a Key Determinant of the Inhibitory Activities

2020 ◽  
Author(s):  
Paul Velander ◽  
Ling Wu ◽  
Sherry B. Hildreth ◽  
Nancy J. Vogelaar ◽  
Biswarup Mukhopadhyay ◽  
...  
Keyword(s):  
Protein Aggregation ◽  
Small Molecule ◽  
Rational Design ◽  
Redox State ◽  
Broad Class ◽  
Intrinsically Disordered ◽  
Amyloidogenic Proteins ◽  
Key Factor ◽  
Inhibitory Activities ◽  
Screening Platform

Abstract Background: A range of neurodegenerative and related aging diseases, such as Alzheimer’s disease, Parkinson’s disease, and type 2 diabetes, are linked to toxic protein aggregation. Yet the mechanisms of protein aggregation inhibition by small molecule inhibitors remain poorly understood, in part because most protein targets of aggregation assembly are partially unfolded or intrinsically disordered, which hinders detailed structural characterization of protein-inhibitor complexes and structural-based mechanistic elucidation. Methods: Herein we employed a small molecule screening approach to identify inhibitors against three prototype amyloidogenic proteins in neurodegeneration and related proteinopathies: amylin, Ab and tau. We further systematically investigated selected class of inhibitors under aerobic and anaerobic conditions to uncover a key determinant of the inhibitory activities.Results: One remarkable class of inhibitors identified from all three parallel screenings against different amyloidogenic proteins was catechol-containing compounds and redox-related quinones/anthraquinones. Further mechanistic studies determined that the redox state of the broad class of catechol-containing compounds is a key determinant of the amyloid inhibitor activities. Conclusion: Our small molecule library screening platform was able to identify a broad class of amyloid inhibitors. Redox was found to be a key factor not only regulating the inhibitory activities but also involving the mechanism of inhibition. The molecular insights we gained not only explain why a large number of catechol-containing natural compounds, often enriched in healthy diet, have anti-neurodegeneration and anti-aging activities, but also could guide the rational design of therapeutic or nutraceutical strategies to target a broad range of neurodegenerative and related aging diseases.

scholarly journals The cryoelectron microscopy structure of the human CDK-activating kinase

2020 ◽  
Vol 117 (37) ◽  
pp. 22849-22857 ◽  
Author(s):  
Basil J. Greber ◽  
Juan M. Perez-Bertoldi ◽  
Kif Lim ◽  
Anthony T. Iavarone ◽  
Daniel B. Toso ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Rna Polymerase Ii ◽  
Transcription Initiation ◽  
Rational Design ◽  
Control Cell ◽  
3D Structure ◽  
Structural Basis ◽  
Pol Ii ◽  
Insight Into ◽  
Cdk Activating Kinase

The human CDK-activating kinase (CAK), a complex composed of cyclin-dependent kinase (CDK) 7, cyclin H, and MAT1, is a critical regulator of transcription initiation and the cell cycle. It acts by phosphorylating the C-terminal heptapeptide repeat domain of the RNA polymerase II (Pol II) subunit RPB1, which is an important regulatory event in transcription initiation by Pol II, and it phosphorylates the regulatory T-loop of CDKs that control cell cycle progression. Here, we have determined the three-dimensional (3D) structure of the catalytic module of human CAK, revealing the structural basis of its assembly and providing insight into CDK7 activation in this context. The unique third component of the complex, MAT1, substantially extends the interaction interface between CDK7 and cyclin H, explaining its role as a CAK assembly factor, and it forms interactions with the CDK7 T-loop, which may contribute to enhancing CAK activity. We have also determined the structure of the CAK in complex with the covalently bound inhibitor THZ1 in order to provide insight into the binding of inhibitors at the CDK7 active site and to aid in the rational design of therapeutic compounds.

scholarly journals CRISPR/Cas9 with single guide RNA expression driven by small tRNA promoters showed reduced editing efficiency compared to a U6 promoter

RNA ◽  
2016 ◽  
Vol 23 (1) ◽  
pp. 1-5 ◽  
Author(s):  
Yuda Wei ◽  
Yan Qiu ◽  
Yanhao Chen ◽  
Gaigai Liu ◽  
Yongxian Zhang ◽  
...  
Keyword(s):  
Rna Expression ◽  
Editing Efficiency ◽  
Guide Rna ◽  
U6 Promoter

scholarly journals Highly multiplex guide RNA expression units of CRISPR/Cas9 were completely stable using cosmid amplification in a novel polygonal structure

2019 ◽  
Vol 21 (11) ◽  
Author(s):  
Tomoko Nakanishi ◽  
Aya Maekawa ◽  
Hirotaka Tabata ◽  
Takashi Yoshioka ◽  
Zheng Pei ◽  
...  
Keyword(s):  
Rna Expression ◽  
Guide Rna ◽  
Polygonal Structure

Rational design of minimum CRISPR guide RNA by site-specific Cas9–RNA conjugation

2020 ◽  
Vol 56 (54) ◽  
pp. 7515-7518
Author(s):  
Xinyu Ling ◽  
Xiaoqin Gao ◽  
Liying Chang ◽  
Heqi Chen ◽  
Xiaomeng Shi ◽  
...  
Keyword(s):  
Rational Design ◽  
Site Specific ◽  
Guide Rna

Site-specific Cas9–crRNA conjugation reduces the synthetic burden of customized guide RNA.

scholarly journals Rational design of a split-Cas9 enzyme complex

2015 ◽  
Vol 112 (10) ◽  
pp. 2984-2989 ◽  
Author(s):  
Addison V. Wright ◽  
Samuel H. Sternberg ◽  
David W. Taylor ◽  
Brett T. Staahl ◽  
Jorge A. Bardales ◽  
...  
Keyword(s):  
Conformational Changes ◽  
Dna Cleavage ◽  
Rational Design ◽  
Genome Engineering ◽  
Guide Rna ◽  
Protein Architecture ◽  
Target Dna ◽  
Molecular Determinants ◽  
Versatile Tool ◽  
Bacterial Immune Systems

Cas9, an RNA-guided DNA endonuclease found in clustered regularly interspaced short palindromic repeats (CRISPR) bacterial immune systems, is a versatile tool for genome editing, transcriptional regulation, and cellular imaging applications. Structures of Streptococcus pyogenes Cas9 alone or bound to single-guide RNA (sgRNA) and target DNA revealed a bilobed protein architecture that undergoes major conformational changes upon guide RNA and DNA binding. To investigate the molecular determinants and relevance of the interlobe rearrangement for target recognition and cleavage, we designed a split-Cas9 enzyme in which the nuclease lobe and α-helical lobe are expressed as separate polypeptides. Although the lobes do not interact on their own, the sgRNA recruits them into a ternary complex that recapitulates the activity of full-length Cas9 and catalyzes site-specific DNA cleavage. The use of a modified sgRNA abrogates split-Cas9 activity by preventing dimerization, allowing for the development of an inducible dimerization system. We propose that split-Cas9 can act as a highly regulatable platform for genome-engineering applications.

scholarly journals A multiplex guide RNA expression system and its efficacy for plant genome engineering

Plant Methods ◽  
2020 ◽  
Vol 16 (1) ◽  
Author(s):  
Youngbin Oh ◽  
Bora Lee ◽  
Hyeonjin Kim ◽  
Sang-Gyu Kim
Keyword(s):  
Genome Engineering ◽  
Expression System ◽  
Plant Genome ◽  
Rna Expression ◽  
Guide Rna

scholarly journals Image-based pooled whole genome CRISPR screening for Parkin and TFEB subcellular localization

2020 ◽  
Author(s):  
Gil Kanfer ◽  
Shireen A. Sarraf ◽  
Yaakov Maman ◽  
Heather Baldwin ◽  
Kory R. Johnson ◽  
...  
Keyword(s):  
Neural Network ◽  
Subcellular Localization ◽  
Fluorescent Protein ◽  
Network Models ◽  
Neural Network Models ◽  
Guide Rna ◽  
Genome Wide ◽  
A Genome ◽  
Screening Platform ◽  
Cellular Phenotypes

AbstractGenome-wide CRISPR screens have transformed our ability to systematically interrogate human gene function, but are currently limited to a subset of cellular phenotypes. We report a novel pooled screening approach for a wider range of cellular and subtle subcellular phenotypes. Machine learning and convolutional neural network models are trained on the subcellular phenotype to be queried. Genome-wide screening then utilizes cells stably expressing dCas9 (CRISPRi), photoactivatable fluorescent protein (PA-mCherry), and a lentiviral guide RNA (gRNA) pool. Cells are screened by microscopy and classified by artificial intelligence (AI) algorithms, which precisely identify the genetically altered phenotype. Cells with the phenotype of interest are photoactivated, isolated via flow cytometry, and the gRNAs are identified by sequencing. A proof-of-concept screen accurately identified PINK1 as essential for Parkin recruitment to mitochondria. A genome-wide screen identified factors mediating TFEB relocation from the nucleus to the cytosol upon prolonged starvation. Twenty of the sixty-four hits called by the neural network model were independently validated, revealing new effectors of TFEB subcellular localization. This approach, AI-Photoswitchable Screening (AI-PS) offers a novel screening platform capable of classifying a broad range of mammalian subcellular morphologies, an approach largely unattainable with current methodologies at genome-wide scale.

scholarly journals Rational design of a compact CRISPR-Cas9 activator for AAV-mediated delivery

2018 ◽  
Author(s):  
Suhani Vora ◽  
Jenny Cheng ◽  
Ru Xiao ◽  
Nathan J. VanDusen ◽  
Luis Quintino ◽  
...  
Keyword(s):  
Transcriptional Activation ◽  
Rational Design ◽  
Somatic Tissue ◽  
Transcription Activator ◽  
Adeno Associated Virus ◽  
Guide Rna ◽  
Delivery Vector ◽  
Transcriptional Modulators ◽  
Rna Complex

AbstractAkin to Zinc Finger and Transcription Activator Like Effector based transcriptional modulators, nuclease-null CRISPR-Cas9 provides a groundbreaking programmable DNA binding platform, begetting an arsenal of targetable regulators for transcriptional and epigenetic perturbation, by either directly tethering, or recruiting, transcription enhancing effectors to either component of the Cas9/guide RNA complex. Application of these programmable regulators is now gaining traction for the modulation of disease-causing genes or activation of therapeutic genes, in vivo. Adeno-Associated Virus (AAV) is an optimal delivery vehicle for in vivo delivery of such regulators to adult somatic tissue, due to the efficacy of viral delivery with minimal concerns about immunogenicity or integration. However, present Cas9 activator systems are notably beyond the packaging capacity of a single AAV delivery vector capsid. Here, we engineer a compact CRISPR-Cas9 activator for convenient AAV-mediated delivery. We validate efficacy of the CRISPR-Cas9 transcriptional activation using AAV delivery in several cell lines.

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