Generation of a NESTIN-EGFP reporter human induced pluripotent stem cell line, KSCBi005-A-1, using CRISPR/Cas9 nuclease

Generation of a NES-mScarlet Red Fluorescent Reporter Human iPSC Line for Live Cell Imaging and Flow Cytometric Analysis and Sorting Using CRISPR-Cas9-Mediated Gene Editing

“…We chose this targeting strategy of the NES locus for several reasons. Firstly, two green fluorescent NESTIN reporter PSC lines are already available in the human context [ 17 , 18 ], but to our knowledge, a corresponding red fluorescent reporter PSC line has not been generated so far. The new NES -mScarlet hiPSC line, thus, expands the current tool set of human NESTIN reporter cell lines.…”

“…The mScarlet fluorophore is an extremely bright (high fluorescence lifetime and quantum yield) and truly monomeric (no accumulation in organelles) RFP that was selected based on these particular spectral and biochemical as well as additional properties, including its tolerance to acidic environments and very low cytotoxicity [ 23 ]. Secondly, our approach avoided the synthesis of an unphysiological NESTIN-reporter fusion protein (as in the CRISPR-Cas9-targeted green fluorescent hiPSC line KSCBi005-A-1, [ 17 ]) and allowed the stoichiometric production of independent NESTIN and mScarlet proteins in the targeted cells. Thirdly, targeting the endogenous NES locus in the hiPSCs circumvented the problems associated with the generation of transgenic hiPSC lines using the well characterized second intron of the human NES gene [ 18 , 34 ].…”

“…We can, therefore, conclude that the expression of the red fluorescent mScarlet reporter faithfully recapitulates the expression of endogenous NESTIN in our new NES -mScarlet #18 hiPSC line, thus circumventing the problems associated with other NESTIN reporter lines. These include the ectopic expression of the reporter protein in transgenic PSC lines, probably because of positional effects after random integration of the transgene [ 18 ], and the prolonged detection of the green fluorescent reporter protein because of its low turnover in the cells [ 14 , 17 ].…”

“…NESTIN is, thus, used as an NSC/NPC-specific marker protein in the context of human neural differentiation. Indeed, green fluorescent reporter PSCs for this cell population have been generated using either the endogenous NESTIN ( NES ) locus or the regulatory regions located in the second intron of this gene to direct the expression of the transgene specific to NSCs/NPCs [ 17 , 18 ].…”

Research and Therapeutic Approaches in Stem Cell Genome Editing by CRISPR Toolkit

“…Drugs with different methods (e.g., Nocodazole (G2 and M phase cell cycle arrest), RS1 (binding homologous recombination-binding protein (RAD51) to the cut DNA area facilitators), and nu7441 molecules (NHEJ inhibitor)) considerably boost HDR repair and hence KI efficiency [ 142 ]. They monitor long-term expression changes by attaching reporter genes (KI) to the c-terminus of stemness-related genes [ 143 , 144 , 145 , 146 ]. In Wilson’s disease hiPSCs, the mutation in R778L (arginine 778 lysine) in ATPase copper transporting beta (ATP7B) was induced; this has application in drug screening and finding disease model mechanisms for Wilson disease [ 147 ].…”