Services

Repair of small-scale mutations in hiPSCs by CRISPR/Cas9 gene editing

Repair of nucleotide substitutions, short duplications or deletions in hiPSCs. The service includes sgRNA and ssODN design and targeting with Cas9 protein. Successfully targeted clones are identified by restriction enzyme analysis and confirmed by Sanger sequencing. Expansion of repaired sub-clones and characterisation as described (See our service Characterisation of pluripotency status, functional pluripotency and cell identity). An off-target analysis is not included. Service costs apply.

Note: This service is available for hiPSC lines at passages ≥ 10  and  ≤20 cultured in mTESR-E8 or TESR Plus that are mycoplasma-negative and have been karyotyped. For design of the sgRNAs, sequencing of the region of interest is required.

Despite the prediction of suitable sgRNAs by in silico tools, gene editing efficiencies are unpredictable and highly variable depending on the target locus and the hiPSC line. We cannot guarantee that gene-edited hiPSC clones are truly clonal, that is that they originated from a single-cell. Karyotype aberrations may occur during single cell cloning. Karyotyping is not included in the service.

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Note: This service is available for hiPSC lines at passages ≥ 10  and  ≤20 cultured in mTESR-E8 or TESR Plus that are mycoplasma-negative and have been karyotyped. For design of the sgRNAs, sequencing of the region of interest is required.

Despite the prediction of suitable sgRNAs by in silico tools, gene editing efficiencies are unpredictable and highly variable depending on the target locus and the hiPSC line. We cannot guarantee that gene-edited hiPSC clones are truly clonal, that is that they originated from a single-cell. Karyotype aberrations may occur during single cell cloning. Karyotyping is not included in the service.

Experimental strategy

sgRNAs are designed by using the CRISPOR web tool. If necessary ssODN design includes the incorporation of silent mutations facilitating screening by restriction analysis. After nucleofection of hiPSCs with ssODN, Cas9 and sgRNA clones are screened for successful gene editing by restriction enzyme analysis. Gene editing in positive clones is confirmed by Sanger sequencing. Successfully targeted sub-clones are subsequently expanded, cryopreserved and characterised as described (See our service Characterisation of pluripotency status, functional pluripotency and cell identity). Negative clones (non-targeted control) are available upon request. Analysis of potential off-target effects is not included.

Upon completion of characterisation the customer is provided with:

  • Cryovials for each characterised hiPSC line at 2 passages (3 vials each)
  • Summary of sequencing results, characterisation/ QC data (PowerPoint; raw data upon request)
  • Standard Operation Procedure (SOP) for starting up cells

Service costs upon request.