A5086851699- RNA Interference and Gene Delivery
- CRISPR and Genetic Engineering
- Advanced biosensing and bioanalysis techniques
- RNA and protein synthesis mechanisms
- Molecular Biology Techniques and Applications
- RNA regulation and disease
- Virus-based gene therapy research
- Single-cell and spatial transcriptomics
- RNA Research and Splicing
- Mass Spectrometry Techniques and Applications
- Innovation and Socioeconomic Development
- DNA and Nucleic Acid Chemistry
- CAR-T cell therapy research
- Estrogen and related hormone effects
- Diabetes and associated disorders
- Immunotherapy and Immune Responses
- Animal Genetics and Reproduction
- Cancer, Hypoxia, and Metabolism
- Pluripotent Stem Cells Research
Horizon Discovery Group (United States)
2017-2022
University of Colorado Boulder
2022
General Electric (Spain)
2015
Thermo Fisher Scientific (Israel)
2012-2014
New York State Department of Health
1978
Delivery of siRNA is a key hurdle to realizing the therapeutic promise RNAi. By targeting internalizing cell surface antigens, antibody–siRNA complexes provide possible solution. However, initial reports relied on non-specific charged interactions and have not been broadly applicable. To assess improve this delivery method, we built an industrial platform antibodies called THIOMABs, engineered enable precise covalent coupling siRNAs. We report that such generates monomeric conjugates (ARCs)...
The discovery that the bacterial clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9) acquired immune system can be utilized to create double-strand breaks (DSBs) in eukaryotic genomes has resulted ability genomic changes more easily than with other genome engineering techniques. While there is significant potential for CRISPR-Cas9 advance basic and applied research, several unknowns remain, including specificity of RNA-directed DNA cleavage...
Since its initial application in mammalian cells, CRISPR-Cas9 has rapidly become a preferred method for genome engineering experiments. The Cas9 nuclease is targeted to genomic DNA using guide RNAs (gRNA), either as the native dual RNA system consisting of DNA-targeting CRISPR (crRNA) and trans-activating crRNA (tracrRNA), or chimeric single (sgRNA). Entirely DNA-free systems protein mRNA chemically synthesized gRNAs allow transient expression components, thereby reducing potential...
MAD7 is an engineered class 2 type V-A CRISPR-Cas (Cas12a/Cpf1) system isolated from Eubacterium rectale. Analogous to Cas9, it RNA-guided nuclease with demonstrated gene editing activity in Escherichia coli and yeast cells. Here, we report that capable of generating indels fluorescent tagging endogenous genes human HCT116 U2OS cancer cell lines, respectively. In addition, highly proficient indels, small DNA insertions (23 bases), larger integrations ranging 1 14 kb size mouse rat embryos,...
Abstract The potent tumor promoter tetradecanoyl phorbol acetate (TPA) induces early changes in ion movements analogous to those induced by prostaglandins E 1 and F 2α . Among the earliest TPA is a significant increase 32 P i incorporation within 15 minutes incubation of (10 −8 −10 −6 M) with post‐confluent Swiss 3T3 mouse embryonic fibroblasts. Similarly, active ester homolog 4‐;β‐;OH didecanoate but not inactive stereoisomeric 4‐β‐OH stimulated incorporation. Also, at above concentrations...
RNAi screening using pooled shRNA libraries is a valuable tool for identifying genetic regulators of biological processes. However, successful screen, it imperative to thoroughly optimize experimental conditions obtain reproducible data. Here we performed viability screens with library ∼10 000 shRNAs at two different fold representations (100- and 500-fold transduction) report the reproducibility abundance changes between replicates determined by microarray next generation sequencing...
While the use of RNA interference (RNAi) in molecular biology and functional genomics is a well-established technology, vivo applications synthetic short interfering RNAs (siRNAs) require chemical modifications. We recently found that amides as non-ionic replacements for phosphodiesters may be useful modifications optimization siRNAs. Herein, we report comprehensive study systematic replacement single phosphate with an amide linkage throughout guide strand The results show are surprisingly...
Potential in vivo applications of RNA interference (RNAi) require suppression various off-target activities. Herein, we report that replacement a single phosphate linkage between the first and second nucleosides passenger strand with an amide almost completely abolished its undesired activity restored desired guide strands had been compromised by unfavorable modifications. Molecular modeling suggested observed effect was most likely due to suppressed loading amide-modified into Ago2 caused...
The CRISPR-Cas9 system has been utilized for large-scale, loss-of-function screens mainly using lentiviral pooled formats and cell-survival phenotypic assays. Screening in an arrayed format expands the types of readouts that can be used to now include high-content, morphology-based assays, with recent availability synthetic crRNA libraries, new studies are emerging. Here, we use a cell cycle reporter line perform arrayed, crRNA:tracrRNA screen targeting 169 genes (>600 crRNAs) high content...
The CRISPR-Cas9 gene editing system requires Cas9 endonuclease and guide RNAs (either the natural dual RNA consisting of crRNA tracrRNA or a chimeric single RNA) that direct site-specific double-stranded DNA cleavage. This communication describes click ligation approach uses alkyne-azide cycloaddition to generate triazole-linked (sgRNA). conjugated sgRNA shows efficient comparable genome activity unmodified constructs.
While CRISPR interference (CRISPRi) systems have been widely implemented in pooled lentiviral screening, there has limited use with synthetic guide RNAs for the complex phenotypic readouts enabled by experiments arrayed format. Here we describe a novel deactivated Cas9 fusion protein, dCas9-SALL1-SDS3, which produces greater target gene repression than first or second generation CRISPRi when used chemically modified single (sgRNAs), while exhibiting high specificity. We show that...
RNA interference screening using pooled, short hairpin (shRNA) is a powerful, high-throughput tool for determining the biological relevance of genes phenotype. Assessing an shRNA pooled screen's performance difficult in practice; one can estimate only by reproducibility as proxy power or employing large number validated positive and negative controls. Here, we develop open-source software tool, Power Decoder simulator, generating experiments silico that be used to statistical power. Using...
Abstract Cell culture has long been essential for preclinical modeling of human development and disease. However, conventional two‐dimensional (2D) cell fails to faithfully model the complexity found in vivo, novel drug candidates that show promising results 2D models often do not translate clinic. More recently, three‐dimensional (3D) have gained popularity owing their greater physiological relevance vivo biology. In particular, 3D spheroid are becoming widely used due ability mimic solid...
While the CRISPR-Cas9 system from S. pyogenes is a powerful genome engineering tool, additional programmed nucleases would enable added flexibility in targeting space and multiplexing. Here, we characterized L. gasseri found that it has modest activity cell-free lysate assay but no mammalian cells even when altering promoter, position of tag sequences NLS, length crRNA:tracrRNA. In tested over 400 sequential crRNA target Lga Cas9 PAM NNGA/NDRA, different than NTAA predicted native bacterial...
Abstract Gene editing technologies hold promise for enabling the next generation of adoptive cellular therapies. Conventional gene platforms that rely on nuclease activity, such as Clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9 (CRISPR-Cas9), allow efficient introduction genetic modifications; however, these modifications occur via DNA double-strand breaks (DSBs) and can lead to unwanted genomic alterations genotoxicity. Here, we apply novel modular RNA...
Abstract Functional gene analysis studies have been empowered by development of CRISPR-Cas9 knockout tools, however the system has also adapted for inhibition or activation transcription. A nuclease-deactivated Cas9 (dCas9) can be fused to various effector domains produce an RNA-guided transcription factor either (CRISPRi) (CRISPRa) target genes. For overexpression studies, CRISPRa holds significant advantages over traditional vector-based expression, because genes are upregulated from their...
Functional gene analysis studies have been empowered by development of CRISPR-Cas9 knockout tools, however the system has also adapted for inhibition or activation transcription. A nuclease-deactivated Cas9 (dCas9) can be fused to various effector domains produce an RNA-guided transcription factor either (CRISPRi) (CRISPRa) target genes. For overexpression studies, CRISPRa holds significant advantages over traditional vector-based expression, because genes are upregulated from their native...