Generation of induced pluripotent cells by crispr activation
A pluripotent stem cell and inducible technology, applied in the field of generating induced pluripotent cells through CRISPR activation, can solve the problems of undetermined iPSC induction and less induction of pluripotency
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[0067] The following examples are intended to further illustrate certain embodiments of the present disclosure. Examples are presented to provide one of ordinary skill in the art and are not intended to limit the scope thereof.
example 1
[0068] Example 1. Activation of endogenous Oct4 and Sox2 with dCas9-SunTag-VP64
[0069] To determine whether and how reshaping endogenous loci triggers reprogramming toward pluripotency, the SunTag CRISPR Activation System was used to precisely reshape endogenous pluripotency loci in mouse embryonic fibroblasts (MEFs). dCas9-SunTag-VP64 was selected for its enhanced chromatin remodeling activity by recruiting multiple VP64s to a single targeting site ( figure 1 A) (Tanenbaum et al., 2014). dCas9 expression is controlled by the Tet-On promoter. The Oct4 and Sox2 loci were chosen as targets due to their important roles in the induction and maintenance of pluripotency.
[0070] MEFs were prepared from E13.5 mouse embryos. After the embryos have recovered, remove the head, limbs, and viscera, especially the gonads, under a dissecting microscope. The remaining embryoid bodies were minced finely with two razor blades and digested in 0.05% trypsin-EDTA for 15 minutes. MEF medi...
example 2
[0080] Example 2. Establishment of a pluripotency network in MEFs by gene activation with dCas9-SunTag-VP64
[0081] We next examined whether the pluripotency network could be fully reactivated and established in MEFs. The SunTag Reprogramming System is optimized in two ways. First, target more genes by adding corresponding sgRNAs. Klf4, c-Myc (Takahashi and Yamanaka, 2006), Nr5a2 (Heng et al., 2010), Glis1 (Maekawa et al., 2011) and Cebpa (DiStefano et al., 2014) were selected. For each promoter, 4-10 sgRNAs were designed and tested in differentiated ES cells ( Figure 5 E). 1-3 sgRNAs per promoter were included in the previous Oct4 / Sox2 sgRNA pool (see Table 1). Secondly, the small molecule mixture composed of Parnate, Chir99021, A83-01 and forskolin (PCAF) was added to the reprogramming medium. This chemical cocktail further increased Oct4 and Sox2 transcription by 3-4 fold on day 4 ( Figure 5 h).
[0082] Table 1. sgRNA sequences used in this disclosure*
[0083...
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