Journal: Current protocols in human genetics

Article Title: CRISPR/Cas9 Mediated Fluorescent Tagging of Endogenous Proteins in Human Pluripotent Stem Cells

doi: 10.1002/cphg.52

Figure Lengend Snippet: Timeline for nucleofection, subcloning, and expansion of fluorescently-tagged hiPSCs. Assume a daily mTeSR1 media change for all hiPSC steps. Rho kinase inhibitor is recommended for passaging and steps where hiPSCs are plated sparsely or as single cells. A) Steps from nucleofection until initial clone picking. Begin by nucleofecting 80% confluent, low passage hiPSCs and conduct puromycin selection. After selection, allow clones to grow before replating cells in a serial dilution at very low density. Then, identify individual cells under a microscope and allow single, monoclonal colonies to grow to 100 cells in rho kinase inhibitor before picking into a 96-well plate. B) Steps from clone picking to positive HDR clone identification. After repicking monoclonal hiPSC colonies into a new 96-well plate, allow the colonies to grow in rho kinase inhibitor before redistributing them within the same well. Then, allow colonies to into 95% confluent monolayers before passaging half into a new plate while simultaneously harvesting half for HDR verification with DNA extraction and PCR/gel electrophoresis. Once positive clones are identified, proceed to C. If no positive clones are identified, re-nucleofect with the same guide RNA or order new guide RNAs. C) Once positive clones are identified, grow the clones in 96-well plates and passage positive clones into 24 and 6-well plates, consecutively. After clones are grown to 6-well plate format, freeze a subset of clones before passaging and expanding for long-term culture.

Article Snippet: Wild type, low passage hiPSCs adapted for feeder cell-free growth on Matrigel Matrigel (hESC-qualified; BD Sciences, cat. no. 354277) DMEM/F12 medium (Thermo Fisher Scientific, cat. no. 11320033) mTeSR1 medium (StemCell Technologies, cat. no. 05850) Rho kinase inhibitor (ROCKi; Tocris cat. no. 1254) Human Stem Cell Nucleofector Kit 2 (Lonza, cat. no. VPH-5022) GFP plasmid as nucleofection positive control (pMax GFP vector supplied with Human Stem Cell Nucleofector Kit 2) Cas9 plasmid DNA (from Basic Protocol 1) gRNA expression vector (from Basic Protocol 1) HDR template vector (from Basic Protocol 1) Phosphate-buffered saline (PBS; Life Technologies, cat. no. 20012-050) 0.5M EDTA (Thermo Fisher Scientific, cat. no. 15575020) 6-well tissue culture–treated plates 15- and 50-mL conical centrifuge tubes (e.g., BD Falcon) Hemocytometer for cell counting Tabletop centrifuge with plate adapter Amaxa Nucleofector II Device

Techniques: Subcloning, Passaging, Selection, Clone Assay, Serial Dilution, Microscopy, DNA Extraction, Nucleic Acid Electrophoresis