Title : CRISPR-Based Chromatin Remodeling of the Endogenous Oct4 or Sox2 Locus Enables
Reprogramming to Pluripotency
Journal : Cell stem cell
Impact factor : 23.394
Main techniques : CRISPR-dCas9-VP64 activation system, induced pluripotent stem cell construction, primary cell production, iPS identification
Background The process of terminally differentiated adult cells to differentiate and form a pluripotent stem cell state is called cell reprogramming. In 2006, Japanese scientist Shinya Yamanaka published a technique for the construction of induced pluripotent stem cells (iPS), using retroviruses to treat four factors of OSKM (OCT4, SOX2, KLF4 and C-myc). Overexpression in somatic cells successfully induced mouse embryonic fibroblasts to form pluripotent stem cells, opening up a new field of stem cell research. With the deepening of research, human skin fibroblasts, T lymphocytes and the like have also been successfully induced into pluripotent stem cells. These pluripotent stem cells are consistent with the donor DNA and have the potential to differentiate into a variety of cells, showing a very broad clinical application prospect. For example, when T lymphocytes in human blood are reversely differentiated to form pluripotent stem cells, when the directed differentiation technique is mature, iPS can be ordered to differentiate into desired cells, such as cardiomyocytes and nerve cells.
Epigenetic changes in important gene promoter sites on the chromosome, such as changes in DNA methylation levels, changes in histone acetylation levels, chromatin remodeling, etc., opening endogenous expression of important genes such as OCT4, SOX2, etc. , is a crucial event in the iPS induction process, directly related to whether the induction is successful.
As a powerful gene editing tool, the CRISPR-Cas9 system has been widely used in conventional cell or individual gene knockout, gene knock-in, and point mutation. When the CAS9 protein is inactivated and the domain is added, the CRISPR-dCas9-SunTag-VP64 system is obtained, which enables specific gene transcriptional activation. The CRISPR-dCas9 endogenous gene activation technology (SunTag Gene Activation) perfectly fits the iPS-inducing technology and solves the problem of activating the endogenous OSKM four genes, thus creating a new iPS construction strategy (see Figure 1). 
Figure 1. iPS induction strategy using the CRIPR-dCas9 gene activation system
Research content and results
1. Design sgRNAs targeting the promoter regions of Sox2 and Oct4 genes and the Oct4 enhancer region, and activate Sox2 and Oct4 using CRISPR-dCas9-SunTag-VP64.
In this study, we first designed the SgRNA of the corresponding gene target. The promoter position of the promoter region or enhancer region includes: sgRNA target position and binding of important transcription factors (OCT4, SOX2, NANOG) binding site, histone acetyltransferase p300 The distance between the sites, the histone acetylation distribution near the target region, and the DNA methylation distribution. Using MEF cells as materials, the activation effects of different sgRNAs on the target genes sox2 and oct4 were detected at the RNA level (Fig. 2). 
Figure 2. Verification of transcriptional activation of different sgRNAs in the promoter regions of the Sox2 and Oct4 genes and the Oct4 enhancer region in MEF cells
2. The CRISPR-dCas9-SunTag-VP64 system enables endogenous activation of important genes and successfully obtains iPS.
Subsequently, using this method, 18 important sgRNAs were screened to activate seven important genes: Oct4, Sox2, Klf4, c-Myc, Nr5a2, Glis 1, Cebpa. After the MEF was introduced into the 18 sgRNA by the virus infection method, it formed a complex with dCas9-SunTag-VP64, which activated the expression of the corresponding endogenous gene in OG2-MEF. After 17 days of induction, the iPS cells were successfully obtained and stabilized. Passage and show the same clone morphology as embryonic stem cells. By immunofluorescence and QPCR, it was shown that the obtained iPS expressed Nanog, Sox2 and SSEA-1, and the relevant stem cell gene expression levels were comparable or exceeded compared with RI embryonic stem cells. This section demonstrates successful implementation of iPS induction in the CRISPR-dCas9-SunTag-VP64 system for endogenous activation (Figure 3). 
Figure 3. The CRISPR-dCas9-SunTag-VP64 system successfully activates iPS induction when it simultaneously activates Oct4, Sox2, Klf4, c-Myc, Nr5a2, Glis 1, and Cebpa endogenous expression.
3. The CRISPR-dCas9-SunTag-VP64 system performs endogenous activation of the Sox2 gene alone, and can also effectively obtain iPS.
The researchers independently selected the SgRNA that activates Sox2 for iPS induction, and found that in the case of efficient activation of endogenous Sox2 gene expression, induced pluripotent stem cells can be successfully obtained, and proliferation and passage can be stably achieved. IPS was obtained through in vitro and in vivo experiments, such as stem cell gene expression detection experiments (immunofluorescence, qPCR, etc.), blastocyst injection experiments, etc. After the blastocyst injection experiment, chimeric mice were also obtained, and stable reproductive transmission was possible. Mice of the OG2 mouse strain were obtained in the offspring. These experiments demonstrate the complete dryness of the iPS cell line: ability to stabilize self-renewal and multi-directional differentiation (Figure 4). 
Figure 4. The CRISPR-dCas9-SunTag-VP64 system successfully activates iPS induction when it activates Sox2 endogenous expression alone.
4. The CRISPR-dCas9-SunTag-VP64 system simultaneously performs endogenous activation of the promoter region and enhancer region of Oct4 gene, and can effectively obtain iPS.
Next, the investigator selected the sgRNA with the target position of the promoter region of the Oct4 or the enhancer region for induction experiments. It was found that when the sgRNA or the enhancer region sgRNA was used to activate the Oct4 gene alone, although the expression was activated, The source strength could not be compared to the embryonic stem cell line R1. During the induction process, iPS was not obtained by sgRNA alone. When the sgRNA of the Oct4 promoter region or the sgRNA of the enhancer region was combined, the expression level of endogenous Oct4 was improved, and iPS was finally obtained, and the proliferation and passage were stably achieved. IPS was obtained through in vitro and in vivo experiments, such as stem cell gene expression detection experiments (immunofluorescence, qPCR, etc.), blastocyst injection experiments, etc. After the blastocyst injection experiment, chimeric mice were also obtained, and stable reproductive transmission was possible. Mice of the OG2 mouse strain were obtained in the offspring. These experiments demonstrate the complete dryness of the iPS cell line: ability to stabilize self-renewal and multi-directional differentiation (Figure 5).
Figure 5. The CRISPR-dCas9-SunTag-VP64 system simultaneously gains iPS for endogenous activation of the promoter region and enhancer region of Oct4 gene.
Summary of the article As an iPS build using the CRISPR-dCas9 activation system, the authors strongly demonstrate the effectiveness of the CRISPR-dCas9-SunTag-VP64 system in building iPS. It is foreseeable that in subsequent studies in which MEF directly induces the formation of cardiomyocytes and nerve cells, this system is likely to have a place. This article has not only explored relevant technical methods, but also answered a very good scientific question: the epigenetic changes of related endogenous genes are intuitively important in the iPS induction process.
In epigenetic studies, the application of CRIPSR-dCas9 to activate endogenous gene expression will be relatively broad. The CRISPR-dCas9-SunTag-VP64 system has been slightly improved and is likely to be a powerful tool for directly altering the epigenetic features of related genes.
Analytical literature
P. Liu, M. Chen, Y. Liu, LS Qi, S. Ding. CRISPR-Based Chromatin Remodeling of the Endogenous Oct4 or Sox2 Locus Enables Reprogramming to Pluripotency, Cell stem cell, 22 (2018) 252-261.
references
1. Black, JB, Adler, AF, et al. (2016) Targeted Epigenetic Remodeling of Endogenous Loci by CRISPR/Cas9-Based Transcriptional Activators Directly Converts Fibroblasts to Neuronal Cells. Cell stem cell 19, 406-414
2Chakraborty, S., Ji, H., Kabadi, AM, et al. (2014) A CRISPR/Cas9-based system for reprogramming cell lineage specification. Stem cell reports 3, 940-947
3. Chavez, A., Scheiman, J., et al. (2015) Highly efficient Cas9-mediated transcriptional programming. Nature methods 12, 326-328
4. Thakore, PI, D'Ippolito, AM, et al. (2015) Highly specific epigenome editing by CRISPR-Cas9 repressors for silencing of distal regulatory elements. Nature methods 12, 1143-1149
5. Buganim, Y., Faddah, DA, et al. (2012) Single-cell expression analyses during cellular reprogramming reveal an early stochastic and a late hierarchic phase. Cell 150, 1209-1222
6.Takahashi, K., and Yamanaka, S. (2006) Induction of pluripotent stem cells from mouse embryonic and adult fibroblast cultures by defined factors. Cell 126, 663-676
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