Our approach involves assembling a CRISPR-Tag within the intron region of a fluorescent protein and then integrating this cassette to N- or C-terminus of a specific gene, which enables simultaneous real-time imaging of protein and DNA of human protein-coding genes, such as HIST2H2BE, LMNA ...
We previously described a protocol for genome engineering of mammalian cultured cells with clustered regularly interspaced short palindromic repeats and associated protein 9 (CRISPR–Cas9) to generate homozygous knock-ins of fluorescent tags into endogenous genes. Here we are updating this former protocol...
Previously, researchers used zinc fingers (ZNF)156 and TALE proteins157 for targeted recruitment of fluorescent proteins to repetitive genomic regions, such as centromeres and telomeres for live cell imaging. However, the advances in the dCas9 platform technology have substantially improved both the ...
After puromycin selection, cells were transfected with a CRISPRoff plasmid expressing the red fluorescent protein mScarletI. Two days later, GFP+/TagBFP+ mScarletI+ cells were sorted and maintained without antibiotic selection for ≥10 cell divisions, allowing for the ablation of cells with silenced...
To enable HaloTag-mediated degradation of endogenous proteins, we provide protocols for HaloTag genomic insertion at the protein N or C terminus via CRISPR/Cas9 and use of HaloTag fluorescent ligands to enrich edited cells via Fluorescence-Activated Cell Sorting (FACS). Using these approaches, ...
To directly measure the rates of R-loop formation for all enzymes, we used a stopped-flow assay based on measuring fluorescence of tCo at −16 nt, a fluorescent tricyclic cytosine analog that is quenched by base stacking in dsDNA so that opening of the duplex results in a large increase ...
Fluorescent Protein Genes & Plasmids Lucigen Vectors Qiagen Vectors Yeast Plasmids Gateway® Cloning Vectors Mammalian Expression Vectors Structural Genomics Vectors The maps, notes, and annotations in the zip file on this page are copyrighted material. This material may be used without restriction by ...
Fluorescent signal was tracked with a Synergy H4 plate reader (BioTek) and data were plotted in GraphPad Prism. Plasmid cleavage assay Plasmid cleavage reactions were prepared by combining 14 nM Cas12a2 (or mutants) with 14 nM crRNA and 25 nM target RNA in NEB 3.1 buffer (50 mM ...
Finally, we expand fluorescent palette of Casilio to allow simultaneous labeling of three loci along a chromatin loop to track its folding dynamics. Results One-gRNA labeling of a nonrepetitive locus by Casilio We reasoned that the immense multimerization capability of Casilio will enable ...
To characterize the sensor in EcN, we obtained the PtlpA-tlpA temperature sensing system from Salmonella typhimurium SL1344 genomic DNA and used it to drive expression of a green fluorescent protein (GFP) reporter. The wild-type TlpA sensor induced GFP expression with a half maximal fluorescence...