Comprehensive Protocols for CRISPR/Cas9‐based Gene Editing in Human Pluripotent Stem Cells

Abstract

Application of the CRISPR/Cas9 system to edit the genomes of human pluripotent stem cells (hPSCs) has the potential to revolutionize hPSC-based disease modeling, drug screening, and transplantation therapy. Here, we aim to provide a single resource to enable groups, even those with limited experience with hPSC culture or the CRISPR/Cas9 system, to successfully perform genome editing. The methods are presented in detail and are supported by a theoretical framework to allow for the incorporation of inevitable improvements in the rapidly evolving gene-editing field. We describe protocols to generate hPSC lines with gene-specific knock-outs, small targeted mutations, or knock-in reporters.

GenEditID: an open-access platform for the high-throughput identification of CRISPR edited cell clones

“…After cells had reached approximately 70% confluence, we duplicated plates to allow one plate of clones to be frozen down for later use and the other to provide genomic DNA for our sequencing pipeline. This gene editing workflow is described in detail elsewhere (47).…”

“…For the production of gRNAs, a 120 nucleotide oligo (Integrated DNA Technologies Inc.) including the SP6 promoter, gRNA sequences, and scaffold region were used as a template for synthesis by in vitro transcription using the MEGAscript SP6 kit (Thermo Fisher, AM1330) as previously described [19]. The resulting sgRNAs were purified using the E.Z.N.A miRNA purification kit (Omega Bio-tek, R7034-01), eluted in RNase-free water, and stored at −80°C.Since gRNAs vay in their efficacy, we designed at least four gRNAs per gene of interest and tested their relative cutting efficiencies in in vitro cleavage assays as previously described (47). We selected the gRNAs that showed activity at the lowest Cas9 concentration at each target gene for transfection in the hPSC cells.…”

Generation and Characterization of Functional Human Hypothalamic Neurons

“…Do not attempt hypothalamic differentiation if there is evidence of cells with a morphology different from that of hPSCs. Eliminate unwanted differentiated cells by aspirating them at each feeding or by manually picking hPSC colonies as described elsewhere (International Stem Cell Initiative Consortium et al, 2010;Ludwig & Thomson, 2007;Santos et al, 2016). b. Wash cells gently and briefly with room temperature DPBS --. c. Add warm EDTA solution to cultures, 1 ml per well in 6-well plate, 5 ml per 10-cm plate.…”

“…The presence of spontaneously differentiated cells in hPSC cultures is likely to interfere with efficient hypothalamic differentiation, and the presence of unwanted mutations will complicate downstream analyses. Detailed methods for hPSC culture are provided elsewhere (International Stem Cell Initiative Consortium et al, 2010;Ludwig & Thomson, 2007;Santos et al, 2016).…”

“…If desired, cultures can be frozen at this point for later thawing as progenitors/immature neurons using the same procedure used for freezing hPSCs (Santos et al, 2016). k. Plate cells onto Geltrex-coated plates in maturation medium with 10 µM Y-27632 at a concentration of 1 × 10 5 cells per cm 2 (corresponding to 9.5 × 10 5 cells per well of a 6-well plate, or 5.5 × 10 6 cells per 10-cm plate). This usually corresponds to a minimum of a 1:3 passage, but passaging ratio will vary between differentiations.…”