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Applications: Generation of sgRNA-edited iPS cell pools. Screening of pooled sgRNA libraries to identify pluripotency, differentiation, and drug toxicity genetic modulators.
Background: Cas9 (Streptococcus pyogenes CRISPR associated protein 9) is an endonuclease enzyme that, when recruited to a specific DNA sequence by the appropriate sgRNA, introduces a double stranded break into the DNA. This double stranded break can then be repaired through either Non-Homologous End Joining (NHEJ) or Homologous Recombination (HR). NHEJ is an error prone process and causes insertions or deletions which may result in functional inactivation of the target gene, while HR, in conjunction with a single stranded DNA repair construct, can be used to introduce mutations at specific base pair(s). Gene modifications introduced via Cas9 are now used in multiple fields of research, aimed at both understanding cellular mechanisms and developing therapeutic solutions.The discovery by Yamanaka and colleagues in 2007 that 4 factors were sufficient to reprogram terminally differentiated fibroblasts into pluripotent stem cells launched the advent of human induced pluripotent stem (iPS) cell technology. These human iPS cells are capable of both self-renewal and differentiation down all three germline lineages and provide both a tool to model human development and disease in the relevant differentiated human cell types. Together with CRISPR/Cas9 technology, gene edited iPS cells provide a unique opportunity to study disease-specific mutations or knockout genes of interest, alongside isogenic control cell pools or cell lines.
Contraindications: Maintenance of the cells requires specific reagents such as specialty culture media, Matrigel™, Accutase™, RelesR™, and Thiazovivin that are not provided with the cells. Ensure that you have all reagents on hand prior to thawing the cells. Prepare media as indicated in section "Media Required for Cell Culture" below. Thiazovivin is a Rho Kinase inhibitor used to ensure that sensitive cell types such as iPS cells survive the cell dissociation process and re-plate successfully. Thiazovivin is not stable in solution and should be added to the medium immediately before use.
Description: The Cas9 Expressing iPS Cell Pool constitutively expresses Cas9 (Streptococcus pyogenes CRISPR associated protein 9) under the control of an EF1A promoter, introduced into PBMC-derived human iPS cells via lentiviral transduction (Cas9 Lentivirus, BPS Bioscience #78066). Cas9 Expressing iPS cells can be transduced with single-guide RNA (sgRNA) targeting gene(s) of interest to generate genetically engineered cell pools or cell lines.
Mycoplasma Testing: The cell pool has been screened to confirm the absence of Mycoplasma species.
Storage Stability: Cells are shipped in dry ice and should immediately be thawed or stored in liquid nitrogen upon receipt. Do not use a -80°C freezer for long term storage. As this is a cell pool and not a cell line, BPS Bioscience cannot guarantee the stability of the genetic modifications over time. Clonal selection can be performed. We recommend freezing cell vials very early on and growing the cells for a limited number of passages. Cells should be cultured using Growth Media, which contain selection antibiotics.
Supplied As: Each vial contains 2 x 106 cells in 1 ml of iPS Cell Freezing Medium
Warnings: Avoid freeze/thaw cycles
Biosafety Level: BSL-1
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