Publications by authors named "Kevin D Dong"

2 Publications

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Robust single-cell discovery of RNA targets of RNA-binding proteins and ribosomes.

Nat Methods 2021 May 7;18(5):507-519. Epub 2021 May 7.

Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA, USA.

RNA-binding proteins (RBPs) are critical regulators of gene expression and RNA processing that are required for gene function. Yet the dynamics of RBP regulation in single cells is unknown. To address this gap in understanding, we developed STAMP (Surveying Targets by APOBEC-Mediated Profiling), which efficiently detects RBP-RNA interactions. STAMP does not rely on ultraviolet cross-linking or immunoprecipitation and, when coupled with single-cell capture, can identify RBP-specific and cell-type-specific RNA-protein interactions for multiple RBPs and cell types in single, pooled experiments. Pairing STAMP with long-read sequencing yields RBP target sites in an isoform-specific manner. Finally, Ribo-STAMP leverages small ribosomal subunits to measure transcriptome-wide ribosome association in single cells. STAMP enables the study of RBP-RNA interactomes and translational landscapes with unprecedented cellular resolution.
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http://dx.doi.org/10.1038/s41592-021-01128-0DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8148648PMC
May 2021

Evaluation of Engineered CRISPR-Cas-Mediated Systems for Site-Specific RNA Editing.

Cell Rep 2020 11;33(5):108350

Department of Cellular and Molecular Medicine, University of California San Diego, La Jolla, CA 92093, USA; Stem Cell Program, University of California San Diego, La Jolla, CA 92093, USA; Institute for Genomic Medicine, University of California at San Diego, La Jolla, CA 92093, USA. Electronic address:

Site-directed RNA editing approaches offer great potential to correct genetic mutations in somatic cells while avoiding permanent off-target genomic edits. Nuclease-dead RNA-targeting CRISPR-Cas systems recruit functional effectors to RNA molecules in a programmable fashion. Here, we demonstrate a Streptococcus pyogenes Cas9-ADAR2 fusion system that uses a 3' modified guide RNA (gRNA) to enable adenosine-to-inosine (A-to-I) editing of specific bases on reporter and endogenously expressed mRNAs. Due to the sufficient nature of the 3' gRNA extension sequence, we observe that Cas9 gRNA spacer sequences are dispensable for directed RNA editing, revealing that Cas9 can act as an RNA-aptamer-binding protein. We demonstrate that Cas9-based A-to-I editing is comparable in on-target efficiency and off-target specificity with Cas13 RNA editing versions. This study provides a systematic benchmarking of RNA-targeting CRISPR-Cas designs for reversible nucleotide-level conversion at the transcriptome level.
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http://dx.doi.org/10.1016/j.celrep.2020.108350DOI Listing
November 2020