In this review, we introduce the three central methods, zinc finger nucleases (ZFNs), transcription activator-like effectors (TALENs), and CRISPR, that have been widely used to manipulate cells or organisms. Focusing on CRISPR technology, we give an overview on homology-directed repair (HDR)-,...
Endogenous fluorescence tagging by CRISPR. Trends Cell Biol. 29, 912–928 (2019). Article CAS PubMed Google Scholar Kamiyama, R. et al. Cell-type-specific, multicolor labeling of endogenous proteins with split fluorescent protein tags in Drosophila. Proc. Natl Acad. Sci. USA 118, e202...
This work was supported by NIH grants R01 NS107558 and R21 NS128750.References (22) K.C. Bui et al. CRISPR/Cas9-mediated knock-in in ebony gene using a PCR product donor template in Drosophila Gene and Genome Editing (2023) H. Bukhari et al. Endogenous fluorescence tagging by CRISPR ...
At 24 h post-transduction, the lentivirus was replaced by fresh culture medium. After 48 h of transduction, the HEK 293 T cells were sorted using fluorescence-activated cell sorting (FACSAria Fusion SORP, BD). Live cell imaging COS-7 cells were cultured on 35-mm glass bottom dishes...
All Cas-Repressor cell lines were generated through piggyBac transposition, antibiotic selection with blasticidin and FACS based on tBFP fluorescence levels (see below). mESCs culture All mESC lines were grown without feeder cells on gelatin-coated flasks (Millipore, 0.1%). mESCs were passaged every...
We applied this approach to mNG tagging of HNRNPA1 using Cas12a- RNP (Fig. 4a). After electroporation and subsequent cell expansion for two to three weeks, we collected mNG-positive cells by fluorescence-activated cell sort- ing (FACS) to enrich knock-in cells, mimicking the selec- ...
Twenty days after electroporation, live parasites appeared and were collected to check the tagging by PCR, DNA sequencing, Western blotting and immuno-fluorescence assays. The results showed that the tags were successfully integrated into the C-terminus of these three proteins. We have improved the ...
Here, we show that by changing the gating used to select for fluorescence during flow cytometry sorting, namely utilizing the width of the signal as opposed to the area, we can highly enrich for positively integrated cells. Reproducible gates were created to select even minuscule percentages of ...
CRISPR Before you begin Endogenously (i.e., “endo”)-tagging genes to create fusion proteins expressed from native genomic loci and detectable by fluorescence or affinity reagents is critical for examining protein functions at close to physiological levels, especially when high-quality antibodies are...
Although the current data demonstrate absolute quantification, this is of luciferase-tagged CXCL12 under endogenous promotion rather than endogenous CXCL12, and CRISPR/Cas9 tagging of native proteins may alter expression (Khan et al., 2019; White et al., 2020). Although no such effects were seen...