The emergence of technology for development of induced pluripotent stem cells (iPSCs) from somatic cells, such as skin and blood cells, has resulted in the ability of researchers to have limitless pool of iPSCs retaining the genetic make-up of the somatic cells from which they were derived. In conjunction with tools for downstream gene editing, such as clustered regularly interspaced short palindromic-repeat (CRISPR)-Cas9 nuclease systems, iPSCs can be used generate (1) knock-outs to study the impact of genes on cellular processes, or (2) knock-ins to assess the impact of reversal of point mutations on diseased states, or furthermore for generation of reporter cell lines. Briefly, CRISPR-Cas9 systems provide simple and efficient site-specific targeting that is accomplished by guiding Cas9 nuclease via a variable 20-base guide RNA sequence to the site for formation of a double stranded break. This break can then be repaired via non-homologous end joining (NHEJ) where small insertions or deletions are made in the gene of interest to knock-out its function or via homology-directed repair (HR) in which single nucleotide changes or knock-ins can be accomplished using a donor DNA template for repair. Together iPSCs and CRISPR-Cas9 systems provide researchers with effective in vitro tools for assessing gene function, disease modeling, and regenerative therapy. In this webinar, we discuss new technologies available from Thermo Fisher Scientific which support (1) efficient delivery of CRISPR Cas9 to PSCs, (2) improve cell survival following transfection while maintaining normal PSC properties, and (3) improve clonal cell survival following low density seeding of 1, 3, or 5 cells per well of 96-well plate.
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