Abstract
Pluripotent stem cells exhibit cell cycle-regulated heterogeneity for trimethylation of histone-3 on lysine-4 (H3K4me3) on developmental gene promoters containing bivalent epigenetic domains. The heterogeneity of H3K4me3 can be attributed to Cyclin-dependent kinase-2 (CDK2) phosphorylation and activation of the histone methyltransferase, MLL2 (KMT2B), during late-G1. The deposition of H3K4me3 on developmental promoters in late-G1 establishes a permissive chromatin architecture that enables signaling cues to promote differentiation from the G1 phase. These data suggest that the inhibition of MLL2 phosphorylation and activation will prevent the initiation of differentiation. Here, we describe a method to seamlessly modify a putative CDK2 phosphorylation site on MLL2 to restrict its phosphorylation and activation. Specifically, by utilizing dimeric CRISPR RNA-guided nucleases, RFNs (commercially known as the NextGEN™ CRISPR), in combination with an excision-only piggyBac™ transposase, we demonstrate how to generate a point mutation of threonine-542, a predicted site to prevent MLL2 activation. This gene editing method enables the use of both positive and negative selection, and allows for subsequent removal of the donor cassette without leaving behind any unwanted DNA sequences or modifications. This seamless “donor-excision” approach provides clear advantages over using single stranded oligo-deoxynucleotides (ssODN) as donors to create point mutations, as the use of ssODN necessitate additional mutations in the donor PAM sequence, along with extensive cloning efforts. The method described here therefore provides the highest targeting efficiency with the lowest “off-target” mutation rates possible, while removing the labor-intensive efforts associated with screening thousands of clones. In sum, this chapter describes how seamless gene editing may be utilized to examine stem cell heterogeneity of epigenetic marks, but is also widely applicable for performing precise genetic manipulations in numerous other cell types.
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Singh, A.M., Perry, D.W., Steffey, V.V.A., Miller, K., Allison, D.W. (2016). Decoding the Epigenetic Heterogeneity of Human Pluripotent Stem Cells with Seamless Gene Editing. In: Turksen, K. (eds) Stem Cell Heterogeneity. Methods in Molecular Biology, vol 1516. Humana Press, New York, NY. https://doi.org/10.1007/7651_2016_324
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DOI: https://doi.org/10.1007/7651_2016_324
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