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Genome Editing - Generation of Reporter Stem Cells


Human pluripotent stem cells (PSCs) such as induced pluripotent stem cells (iPSC) and embryonic stem cells (hESC)...

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Genome Editing - Generation of Reporter Stem Cells

Human pluripotent stem cells (PSCs) such as induced pluripotent stem cells (iPSC) and embryonic stem cells (hESC) hold great promise in the regenerative medicine. PSCs and their ability to form any cell type in the human body has allowed researchers to use them as research tools for applications in basic developmental research, drug screening and potential therapeutic implementations. An emerging need for stem cell biology is efficient and homogeneous differentiation of stem cells into mature, specialized, functional cells. Reporter stem cell lines are valuable models which enable noninvasive, live monitoring of marker onset and expression in a cell-specific manner. Some methods have been used to derive such cell lines based on lineage promoter-driven reporter expression. A more regulated expression achieved with a reporter knock-in into the endogenous promoter loci was less utilized due to the associated technical difficulty. While new advances in genome-editing technologies have lowered these barriers for creating knock-in reporter lines.

Gene reporter pluripotent lines

Gene reporter is a useful in vitro tool that facilitates live monitoring and tracking a cell type of interest. Lineage reporters are created using minimal lineage-specific promoter-driven reporter systems or via knock-in of the reporter into the endogenous promoter region. Choice of reporter is critical based on intended application. Although there is a wide choice of reporters that can be used, each system has its own advantages and disadvantages. Green fluorescent protein (GFP) and variants, such as yellow fluorescent protein (YFP), cyan fluorescent protein (CFP) and DsRed, have been widely used to visualize cells.

There are two main methods to create reporter lines:

Minimal promoter-driven reporter expression

Exogenous lineage-specific promoter and the regulatory fragments can be cloned upstream of a reporter gene to generate cell-specific promoter reporters. In most instances, DNA sequences upstream of the gene-encoding regions containing cis-acting elements are designated as the promoter region; the represents a very restrictive representation of the regulatory elements as in some cases gene regulation is known to be complex, requiring the combination of distal and proximal elements. Therefore, the primary concern about the use of promoter- reporter systems is that the reporter may fail to faithfully recapitulate the activity of the endogenous target gene. And it is essential to carry out extensive validation of the promoter-reporter constructs to confirm context-specific expression of the reporter. Expression level is also much dependent on how the construct is delivered for stable expression into host cells. The promoter-reporter construct can either be maintained episomally without integration, or integrated either randomly or site-specifically into the host genome (Figure. 1).

Methods of lineage reporter lines generation using lineage promoter-driven reporter constructs.Figure 1. Methods of lineage reporter lines generation using lineage promoter-driven reporter constructs.

Reporter knock-in into endogenous promoter site

Reporter genes can be inserted into specific genomic sites of interest by homologous recombination. The targeting construct composed of a core region carrying the reporter cassette is flanked by homology arms which recombine with the target genomic loci, leading to site-specific insertion of the reporter. The process is very inefficient, generally occurring at a rate of one in a million cells which can be significantly enhanced in the presence of double-strand breaks. New gene- targeting technologies aim to precisely cleave genomic loci to facilitate insertion or deletion of genes at the specific cleavage site (Figure. 2). Some reporter lines in ESC and iPSC have been generated using these methods.

Gene-editing methods for reporter knock-in into endogenous promoter sites generation.Figure 2. Gene-editing methods for reporter knock-in into endogenous promoter sites generation.

The most recent study in genome editing has been the development of clustered regularly interspaced palindromic repeat (CRISPR)-guided Cas9 nuclease-mediated cleavage. There is an important aspect of CRISPR/Cas9 that makes it unique. The Cas9 nuclease remains independent of the guide sequence until the two are introduced upon performing an experiment. The flexibility means that targeting multiple sites is relatively simple. Because the CRISPR/Cas9 method only requires the synthesis of only a new guide RNA, instead of the production of a new target-specific nuclease from scratch. The guide RNA is a single transcript containing two wild-type parts: target-specific crRNA and tracrRNA which helps to properly dock the Cas9 nuclease. The biggest advantage of CRISPR-Cas9 is the efficiency, specificity and ease with which the system can be utilized to target multiple sites, individually or together.

Applies of reporter stem cells

Reporters can be expressed in the pluripotent state and turned off with differentiation or vice versa. Reporter lines enable isolation of a homogeneous population of cells that is vital for characterization and use in downstream applications. And a visual reporter offers the opportunity of tracking cell fate progression in live cells. In addition, lineage reporters rely on onset of reporter expression as the pluripotent cell undergoes differentiation into lineage of choice. This has been best showcased in cardiomyocyte differentiation to help and define cardiac cell lineages. In brief, lineage reporters are valuable cell models for tracing and enriching target cell types of interest. With the progress in gene-editing methods, one or more reporters can be engineered in the same pluripotent stem cell line and can provide visual cues for the progression from stem cell to progenitor to highly functional mature cell types.

References:

  1. Avagyan S, et al. Developments in the Generation of Reporter Stem Cells// Genome Editing. Springer International Publishing, 2016.
  2. Hentze H, et al. Cell therapy and the safety of embryonic stem cell-derived grafts. Trends in Biotechnology, 2007, 25(1):24-32.
  3. van Weerd J H, et al. A large permissive regulatory domain exclusively controls Tbx3 expression in the cardiac conduction system. Circulation Research, 2014, 115(4):432.
  4. Fontes A, Lakshmipathy U. Advances in genetic modification of pluripotent stem cells. Biotechnology Advances, 2013, 31(7):994.
  5. Harrison M M, et al. A CRISPR view of development. Genes & Development, 2014, 28(17):1859-72.
  6. Ran F A, et al. Genome engineering using the CRISPR-Cas9 system. Trends in Genetics Tig, 2013, 32(12):815.
  7. Veres A, et al. Low Incidence of Off-Target Mutations in Individual CRISPR-Cas9 and TALEN Targeted Human Stem Cell Clones Detected by Whole-Genome Sequencing. Cell Stem Cell, 2014, 15(1):27.

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