Project description:We develop a CRISPR-Assisted RNA-Protein Interaction Detection method (CARPID), which leverages CRISPR/CasRx-based RNA targeting and proximity labeling to identify binding proteins of specific lncRNA in the native cellular context.
Project description:Delineating the protein network associated with long non-coding RNAs (lncRNAs) is fundamental to understanding the functional mechanisms of lncRNAs. Current methods to identify lncRNA binding proteins either rely on crosslinking mediated complex co-precipitation or require extensive molecular engineering, leading to drawbacks such as loss of cellular context and low capture efficiency, and limiting their broad application. We develop a CRISPR-Assisted RNA-Protein Interaction Detection method (CARPID), which leverages CRISPR/CasRx-based RNA targeting and proximity labeling to identify binding proteins of specific lncRNA in the native cellular context. Applied to the nuclear lncRNA XIST, CARPID captured a list of known interacting proteins and multiple previously uncharacterized binding proteins. We generalize CARPID to explore binders of lncRNA DANCR and MALAT1, revealing its wide applicability in identifying RNA binding proteins.
Project description:We report Proximity Ligation Assisted ChIP-sequencing (PLAC-seq), a method for comprehensive detection of long-range interactions associated with proteins of interest. PLAC-seq requires up to 500-fold less starting material compared to ChIA-PET and using experimentally determined input as control precisely reveals protein associated interaction upto single-element resolution. Application of PLAC-seq to mouse embryonic stem cells revealed a comprehensive map of regulatory interactions.
Project description:This method, termed Repair Assisted Damage Detection sequencing (RADD-seq), uses repair enzymes to excise the DNA lesions leaving a single-strand gap. The damage sites are then repaired in-vitro with biotinylated nucleotides, followed by DNA fragmentation, immunoprecipitation and sequencing. Reads are mapped back to the reference genome, indicating the locations of damage sites.
Project description:We applied chemical-assisted, m6A-SEAL method to profile m6A distributions in the transcriptomes of human and plant cells and compared its results against MeRIPm6A-seq.
Project description:G-quadruplex (G4) structures formed by guanine-rich nucleic acids are implicated in essential physiological and pathological processes and serve as important drug targets. The genome-wide detection of G4s in living cells is important for exploring the functional role of G4s but has not yet been achieved due to the lack of a suitable G4 probe. Here we report an artificial 6.7 kDa G4 probe (G4P) protein that binds G4s with high affinity and specificity. We used it to capture G4s in living human, mouse, and chicken cells with the ChIP-Seq technique, yielding genome-wide landscape as well as details on the positions, frequencies, and sequence identities of G4 formation in these cells. Our results indicate that transcription is accompanied by a robust formation of G4s in genes. In human cells, we detected up to >123,000 G4P peaks, of which >1/3 had a fold increase of ≥5 and were present in >60% promoters and ~70% genes. Being much smaller than a scFv antibody (27 kDa) or even a nanobody (12-15 kDa), we expect that the G4P may find diverse applications in biology, medicine, and molecular devices as a G4 affinity agent.