This work was supported in part by National Heart, Lung, and Blood Institute Grants RO1-HL-99507, HL-114120, HL-131017, HL138023, and UO1-HL-134764 (to J. Zhang) and by American Heart Association Scientific Development Grant 17SDG33670677 (to R. Kannappan).

1. Comet Assay
<ol>
	<li>Reagent preparation
	<ol>
		<li>For low-melting-point agarose, place 500 mg of low-melting agarose in 100 mL of DNA-/RNA-free water. Heat the bottle in a microwave oven until the agarose dissolves. Place the bottle in a 37 &#176;C water bath until needed. 
		NOTE: For further reading, see Azqueta et al., who studied the effects of agarose concentration on the DNA-unwinding time and several electrophoresis factors23.</li>
		<li>Dilute SYBR green DNA dye in Tris-EDTA (TE) Buffer at a 1:10,000 ratio to obtain a 1x working stock dye solution. This dye is light-sensitive, so store it in a dark place.</li>
		<li>For the cell lysis buffer, dissolve 100 mL of lysis buffer (2.5 M NaCl, 0.1 M EDTA, 10 mM Tris, 1% Triton X-100) in deionized water (DI H2O). Then, add 10 M NaOH to adjust the pH to 10.0. Cool the buffer to 4 &#176;C.</li>
		<li>For the alkaline DNA-unwinding/electrophoresis running buffer, dissolve 1 L of alkaline solution (0.3 M NaOH, 1 mM EDTA) in DI H2O. Cool the buffer to 4 &#176;C. Make sure the pH &#62;13.</li>
		<li>For precoated comet slides, place a few drops of 0.5% agarose on a glass slide. Immediately, using the flat surface of another slide, spread the agarose to form a thin layer of agarose coating. 
		NOTE: Dry the slides before using.</li>
		<li>For iPS cell culture, briefly culture iPS cells in basement-membrane-matrix-coated (see Table of Materials) culture plates in mTESR1 culture medium until confluency is reached. 
		NOTE: Human-cardiac-fibroblast-derived iPS cells were used in this protocol. The reprogramming of iPS cell derivation and culture conditions is described in recent articles from our research group12,13,21.</li>
	</ol>
	</li>
	<li>Sample preparation
	<ol>
		<li>Discard the culture medium and wash the cells. Dissociate the cells using detachment solution (see Table of Materials). Wash the cell pellet with phosphate-buffered saline (PBS) and resuspend the cells in PBS at 1 x 105 cells/mL.</li>
	</ol>
	</li>
	<li>Sample slide preparation 
	NOTE: Perform the following steps under low-light conditions to avoid ultraviolet-light-induced cell damage.
	<ol>
		<li>Mix 10 &#956;L of cell suspension and 90 &#956;L of agarose solution (1:10 ratio) in a tube. Place the tube in a 37 &#176;C water bath to prevent the agarose from solidifying.</li>
		<li>Mix the solutions without introducing air bubbles and pipette 70 &#956;L/spot onto the precoated comet slide. Using a pipette tip, spread the cell agarose mixture to form a thin layer. Place the slide in 4 &#176;C for 15 min.</li>
		<li>In a container, completely submerge the slide in cold lysis buffer. Place the container in the dark at 4 &#176;C for 1 h.</li>
		<li>Replace the lysis buffer with cold alkaline solution. Make sure the slides are completely submerged. Place the container in the dark at 4 &#176;C for 30 min.</li>
	</ol>
	</li>
	<li>Electrophoresis
	<ol>
		<li>Place the slide in a horizontal electrophoresis tank. Fill the tank with cold alkaline electrophoresis buffer until the slides are completely submerged. Apply voltage at 1 V/cm for 15 to 30 min. 
		NOTE: Total voltage = distance between the electrodes x 1 V</li>
		<li>In a container, completely submerge the slide in cold DI H2O. After 2 min, remove the DI H2O and add fresh DI H2O. Repeat this step.</li>
		<li>Replace the DI H2O with cold 70% ethanol. Wait 5 min. Gently remove the slide, do not tilt it, and let it air-dry.</li>
		<li>Once dried, add 100 &#956;L of diluted DNA dye to each spot. Wait 15 min at room temperature. Using a FITC filter in an epifluorescence microscope, take images of 50 to 100 comets in total per sample.</li>
	</ol>
	</li>
	<li>Image analysis and calculation of the results
	<ol>
		<li>For scoring the comets, use the National Institutes of Health&#8217;s (NIH) ImageJ with comet assay plugin (download ImageJ here: https://imagej.nih.gov/ij/download.html; download the plugin here: https://www.med.unc.edu/microscopy/resources/imagej-plugins-and-macros/comet-assay/). 
		NOTE: The plugin comes with a PDF instruction which contains all details to perform the analysis. Additionally, screenshots of the comet analysis are shown in Figure 2A-C. Representative comet images of control and doxorubicin (Doxo)-treated iPS cells and the quantification of the comets are shown in Figure 3A-C.</li>
	</ol>
	</li>
</ol>
2. DNA Damage Response
<ol>
	<li>Sample and reagent preparation
	<ol>
		<li>For an iPS-CM cell culture, culture iPS-CM cells in basement-membrane-matrix-coated (see Table of Materials) culture plates in RPMI medium supplemented with B-27 (CMM) until confluency is reached. 
		NOTE: Human-iPS-cell-derived cardiomyocytes were used in this protocol. The protocol for iPS cell differentiation into cardiomyocytes can be found in recent articles from our research group12,13,21.</li>
		<li>Seed iPS-CMs in chamber slides.
		<ol>
			<li>Discard the culture medium from the iPS-CM wells. Wash the cells three times with PBS.</li>
			<li>Add 1 mL of 0.25% trypsin per well and incubate at 37 &#176;C for 5 min. Check the plate under a microscope for cell detachment. Add 1 mL of CMM to stop the trypsin.</li>
			<li>Place cell suspension in a 15 mL tube and centrifuge for 5 min at 4 &#176;C and 200 x g. Discard the medium, add 1 mL of CMM, and resuspend the cells. Count the cells and adjust the cell count to obtain 20 x 105 cells in 1.6 mL of CMM.</li>
			<li>Seed 200 &#181;L of cell suspension per well of the 8-well chamber slide (see Table of Materials). Tap the slide gently to spread the cells around the well and incubate at 37 &#176;C.</li>
		</ol>
		</li>
		<li>For the iPS-CM doxorubicin treatment, discard the culture medium from the iPS-CM wells. Wash the cells with PBS. Treat the cells with 1 &#956;M doxorubicin and CMM for 4 h. Aspirate the medium and wash the cells with PBS.</li>
		<li>For the blocking buffer, prepare 10 mL of bovine serum albumin (BSA) solution containing 3% BSA and 0.05% Triton X-100. To prepare 10 mL of blocking buffer, add 1 mL of normal donkey serum to 9 mL of prepared BSA solution.</li>
		<li>For the primary antibody solution, add 1 &#956;L of anti-rabbit phospho-histone H2A.X (Ser139) antibody to 200 &#956;L of blocking buffer.</li>
		<li>For the secondary antibody mix, add 1 &#956;L each of fluorochrome-conjugated anti-rabbit secondary antibody, DAPI, and fluorochrome-conjugated phalloidin to 200 &#956;L of blocking buffer.</li>
	</ol>
	</li>
	<li>Immunolabeling
	<ol>
		<li>Wash the cells three times with PBS and add 200 &#956;L of freshly prepared 4% paraformaldehyde (PFA) to each well. Fix the cells for 20 min in the dark at room temperature. Wash the cells with PBS.</li>
		<li>Remove the PBS and add 200 &#956;L of blocking buffer per well and block for 30 min in a humidified chamber at room temperature. Remove the blocking buffer and add 200 &#956;L of primary antibody solution. Incubate for 45 min in a dark, humidified chamber at room temperature. Then, wash the wells three times with PBS.</li>
		<li>Remove the PBS and add 200 &#956;L of secondary antibody mix. Incubate for 45 min in a dark, humidified chamber at room temperature. Then, wash the wells three times with PBS. Wash with DI H2O before mounting them with antifade. Place a coverglass and store the slides in the dark at 4 &#176;C.</li>
		<li>Using an epifluorescence microscope, take three to five images of random fields per sample, as shown in Figure 4A, and proceed to image analysis.</li>
	</ol>
	</li>
	<li>Counting of the DDR foci
	<ol>
		<li>Download and install CellProfiler24 (http://cellprofiler.org).</li>
		<li>NOTE: Image processing in this study was performed using CellProfiler version 2.1.1. Tutorials for CellProfiler that were used can be found elsewhere (https://www.youtube.com/watch?v=PvhRL9xpduk&#38;list=PL7CC87670239B4D10). If a newer version of CellProfiler is being used, the pipeline might require minor adaptations.</li>
		<li>Download the pipeline punctae_gH2AX.cpipe. Select File &#62; Import &#62; Pipeline from the file and choose punctae_gH2AX.cpipe.</li>
		<li>Drag-and-drop the folder containing the acquired images of &#947;H2A.X foci to the File list window in the Input modules/Images section for analysis (Figure 5A). Then, follow the instructions as shown in Figure 5A-L.</li>
		<li>Set the following parameters for the images:
		<ol>
			<li>Drag the desired image for analysis onto the File list. Under Input modules, select Images (see Figure 5A for module settings). In Input modules, select Metadata (see Figure 5B for module settings). In Input modules, select NamesAndTypes (see Figure 5C for module settings).</li>
			<li>For Groups, select no. In Analysis modules, select ColorToGray (see Figure 5D for module settings).</li>
		</ol>
		</li>
		<li>In Analysis modules, select Smooth (see Figure 5E for module settings). 
		NOTE: This value may need to be optimized, depending on the image resolution.</li>
		<li>In Analysis modules, select IdentifyPrimaryObjects (see Figure 5F for module settings). 
		NOTE: Optimize these values to make sure the whole nucleus is selected. After this step, the computer may lag.</li>
		<li>In Analysis modules, select Smooth (see Figure 5G for module settings). 
		NOTE: This value may need to be optimized, depending on the image resolution.</li>
		<li>In Analysis modules, select EnhanceOrSuppressFeatures (see Figure 5H for module settings).</li>
		<li>In Analysis modules, select IdentifyPrimaryObjects (see Figure 5I for module settings). 
		NOTE: Optimize these values to make sure the punctae are selected. After this step, the computer may lag again.
		<ol>
			<li>In Analysis modules, select RelateObjects (see Figure 5J for module settings). In Analysis modules, select ClassifyObjects (see Figure 5K for module settings). In Analysis modules, select ExportToSpreadsheet (see Figure 5L for module settings).</li>
			<li>Click Analyze Images at the bottom left panel to perform image analysis. At the successful completion of the analysis, several images are generated (Figure 6A-F). 
			NOTE: Users should save these images for future reference.</li>
			<li>Note the .csv file containing the quantitative data for further analysis that is generated and saved in the appropriate location on the computer. In the spreadsheet called MyExpt_Image, columns B and D will have the number of nuclei that have up to five and more punctae, respectively. Add columns B and D to get the total number of nuclei.</li>
		</ol>
		</li>
		<li>Repeat step 2.3.3 - 2.3.8 for all images (change the MyExpt_ filename before every analysis). Plots can be made for assessing DNA integrity, as shown in Figure 4C.</li>
	</ol>
	</li>
</ol>

<ol>
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The authors have nothing to disclose.

DNA integrity portrays cell integrity. Cells with damaged DNA are frequently in stress and eventually lose their integrity. The integrity of stem and stem-cell-derived cells that are being propagated for the purpose of transplantation is principal for the cells to perform their desired function. Transplanting cells with damaged DNA would result in a poor engraftment rate and performance of the cell8,20. Therefore, examining the DNA integrity prior to cell transpl...

Experimental and clinical evidence demonstrates that cell transplantation can moderately improve the left ventricle contractile performance of failing hearts1,2,3,4,5,6,7,8,9. Recent advances have opened further appealing opportunities for cardiovascular regeneration; these include the forced expression of reprogramming factors in somatic cells to induce pluripotency and differentiate these induced pluripotent stem cells (iPSC) into different cardiac lineages, importantly cardiomyocyte (CM)10,11,12,13. The hereditary material in every cell, including the artificially generated iPSCs and iPSC-derived CM (iPS-CM), is DNA. The genetic instructions stored in DNA dictates the growth, development, and function of cells, tissues, organs, and organisms. DNA is not inert; cell metabolites, such as reactive oxygen, carbonyl, and nitrogen species, and alkylating agents can cause DNA damage in vitro and in vivo14,15,16,17. Importantly, DNA damage occurs intuitively in every cell, with a significant frequency. If these damages are not corrected, it will lead to DNA mutation, cellular senescence, the loss of DNA and cell integrity, and possibly, diseases, including life-threatening cancers. Therefore, retaining DNA integrity is essential to any cell, especially iPSCs, that has enormous potential in the clinic.
For assessing the quantity and integrity of isolated genomic DNA, expensive equipment is available on the market. However, there are no simple and cost-effective methods to assess the DNA integrity in cells without isolating the cells. Moreover, user-induced DNA degradation during DNA isolation is one of the major drawbacks in using these methods. The single-cell gel electrophoresis (known as comet assay)18,19 and &#947;H2A.X immunolabeling8 techniques are fundamental approaches in research labs for assessing DNA damage. These two methods do not require expensive equipment or isolated genomic DNA to analyze DNA integrity8,20,21. Since, these techniques have been performed with whole cells; user-induced DNA/RNA/protein degradation during the sample preparation will not affect these protocols. Here, to assess the DNA damage and DNA damage response in stem and stem-cell-derived cells, we provide step-by-step protocols to perform both the comet assay and &#947;H2A.X immunolabeling. Moreover, combining these two approaches, we propose a naive assessment that can be used to evaluate the cell&#39;s suitability for transplantation.
The comet assay, or single-cell gel electrophoresis, measures the DNA breaks in cells. Cells embedded in low-melting agarose are lysed to form nucleoids containing supercoiled DNA. Upon electrophoresis, small pieces of fragmented DNA and broken DNA strands migrate through the agarose pores, whereas the intact DNA, due to their enormous size and their conjugation with the matrix protein, will have a restricted migration. The pattern of stained DNA under a fluorescence microscope mimics a comet. The comet head contains intact DNA and the tail is composed of fragments and broken DNA strands. The fraction of DNA damage can be measured by the fluorescence intensity of the damaged DNA (comet tail) relative to the intact DNA (comet head) intensity. The parameter tail moment can be calculated as shown in Figure 1.
DNA damage induces the phosphorylation of histone H2A.X (&#947;H2A.X) at Ser139 by ATM, ATR, and DNA-PK kinases. The phosphorylation and recruitment of H2A.X at DNA strand breaks is called DNA damage response (DDR) and happens rapidly after DNA is damaged. Following this process, checkpoint-mediated cell cycle arrest and DNA repair processes are initiated. After the successful completion of DNA repair, &#947;H2A.X is dephosphorylated and inactivated by phosphatases. Prolonged and multiple DNA strand breaks lead to the accumulation of &#947;H2A.X foci in DNA. This indicates the cell&#39;s inability to repair the DNA damage and the loss of DNA integrity. These &#947;H2A.X foci in DNA can be identified by and the number of DDR foci can be counted using the protocol in section 2.

<table>
	<tbody>
		<tr>
			<td>0.25% Trypsin</td>
			<td>Corning</td>
			<td>25200056</td>
			<td></td>
		</tr>
		<tr>
			<td>8-well chamber slide</td>
			<td>Millicell</td>
			<td>PEZGS0816</td>
			<td></td>
		</tr>
		<tr>
			<td>Accutase</td>
			<td>Stemcell Technologies</td>
			<td>7920</td>
			<td>Cell detachment solution</td>
		</tr>
		<tr>
			<td>Alexa Fluor 488 AffiniPure 
			Donkey Anti-Rabbit IgG</td>
			<td>Jackson Immuno 
			Research Laboratories</td>
			<td>711-545-152 
			(RRID: AB_2313584)</td>
			<td></td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin</td>
			<td>Sigma-Aldrich</td>
			<td>A7906</td>
			<td></td>
		</tr>
		<tr>
			<td>Cy3 AffiniPure Donkey Anti-Rabbit IgG</td>
			<td>Jackson Immuno 
			Research Laboratories</td>
			<td>711-165-152 
			(RRID:AB_2307443)</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>D9564</td>
			<td></td>
		</tr>
		<tr>
			<td>Gibco B-27 Supplement, Serum Free,</td>
			<td>Gibco</td>
			<td>17504-044</td>
			<td></td>
		</tr>
		<tr>
			<td>Matrigel growth factor reduced</td>
			<td>Corning</td>
			<td>354230</td>
			<td></td>
		</tr>
		<tr>
			<td>mTeSR1</td>
			<td>Stemcell Technologies</td>
			<td>85850</td>
			<td></td>
		</tr>
		<tr>
			<td>PhalloidinA488</td>
			<td>Life Technology</td>
			<td>A12379</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospho-Histone H2A.X (Ser139) Antibody</td>
			<td>Cell Signaling Technology</td>
			<td>2577 (RRID: AB_2118010)</td>
			<td></td>
		</tr>
		<tr>
			<td>RPMI</td>
			<td>Gibco</td>
			<td>11875-093</td>
			<td></td>
		</tr>
		<tr>
			<td>SYBR&#160;Green I nucleic acid gel stain</td>
			<td>Sigma-Aldrich</td>
			<td>S9430</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Fisher scientific</td>
			<td>BP151-100</td>
			<td></td>
		</tr>
		<tr>
			<td>UltraPure Low melting Point Agarose</td>
			<td>Invitrogen</td>
			<td>16520050</td>
			<td></td>
		</tr>
		<tr>
			<td>Vectashield</td>
			<td>Vector Laboratories</td>
			<td>H-1000</td>
			<td>Antifade</td>
		</tr>
	</tbody>
</table>

assessing stem cell dna integrity for cardiac cell therapy

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data in all cells, including stem cells, and its integrity is fundamental to its regenerative ability. Stem cells undergo rapid propagation in labs to achieve the necessary numbers for transplantation. Accelerated cell growth leads to the loss of DNA integrity by accumulated metabolites, such as reactive oxygen, carbonyl, and alkylating agents. Transplanting these cells would result in poor engraftment and regeneration of the deteriorating organ. Moreover, transplanting DNA-damaged cells leads to mutations, DNA instability, cellular senescence, and possibly, life-threatening diseases such as cancer. Therefore, there is an immediate need for a quality control method to evaluate the cell's suitability for transplantation. Here, we provide step-by-step protocols for the assessment of the DNA integrity of stem cells prior to cell transplantation.

Human induced pluripotent stem cells were cultured, and the DNA damage and the tail moment, which were used as a measure of DNA integrity, were analyzed by comet assay. iPS cells were embedded in low-melting-point agarose and placed on a glass slide. The cells were, then, treated with lysis buffer, followed by an alkaline solution, to obtain supercoiled DNA. Nucleoids were electrophoresed and comets were visualized by DNA dye (Figure 1A-D). T...

Watch this Scientific Journal Video about Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy at JoVE.com

Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Avaliar a integridade do DNA de células-tronco para terapia celular cardíaca

Here, we provide a detailed description of an experimental setup for an analysis of the assessment of DNA integrity in stem cells prior to cell transplantation.

Stem and stem-cell-derived cells have immense potential as a regenerative therapy for various degenerative diseases. DNA is the storehouse of genetic data ...

assessing-stem-cell-dna-integrity-for-cardiac-cell-therapy

Research

JoVE Journal

Genetics

7.6K visualizações.  University of Alabama at Birmingham. Para doenças neonatais como cardiomiopatia, a terapia regenerativa usando células-tronco pluripotentes induzidas tem um enorme potencial. No entanto, o crescimento acelerado das células in vitro leva a danos no DNA por metabólitos acumulados, como espécies reativas de oxigênio. Transplantar essas células danificadas pelo DNA resultaria em má engrafia e regeneração do órgão.Antes do transplante, a qualidade das células deve ser avaliada. Aqui fornecemos dois protocolos pass...

Vídeo: Assessing Stem Cell DNA Integrity for Cardiac Cell Therapy

Engineering Artificial Factors to Specifically Manipulate Alternative Splicing in Human Cells

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which specifically binds the pre-mRNA target and an effector domain. Previously, we have taken advantage of the unique RNA binding mode of the PUF domain in human Pumilio 1 to generate a programmable RNA binding scaffold, which was used to engineer various artificial RBPs to manipulate RNA metabolism. Here, a detailed protocol is described to construct Engineered Splicing Factors (ESFs) that are specifically designed to modulate the alternative splicing of target genes. The protocol includes how to design and construct a customized PUF scaffold for a specific RNA target, how to construct an ESF expression plasmid by fusing a designer PUF domain and an effector domain, and how to use ESFs to manipulate the splicing of target genes. In the representative results of this method, we have also described the common assays of ESF activities using splicing reporters, the application of ESF in cultured human cells, and the subsequent effect of splicing changes. By following the detailed protocols in this report, it is possible to design and generate ESFs for the regulation of different types of Alternative Splicing (AS), providing a new strategy to study splicing regulation and the function of different splicing isoforms. Moreover, by fusing different functional domains with a designed PUF domain, researchers can engineer artificial factors that target specific RNAs to manipulate various steps of RNA processing.

This report describes a bioengineering method to design and construct novel Artificial Splicing Factors (ASFs) that specifically modulate the splicing of target genes in mammalian cells. This method can be further expanded to engineer various artificial factors to manipulate other aspects of RNA metabolism.

Engenharia de fatores artificiais para manipular Especificamente splicing alternativo em células humanas

The processing of most eukaryotic RNAs is mediated by RNA Binding Proteins (RBPs) with modular configurations, including an RNA recognition module, which ...

Genética

Mapping RNA-RNA Interactions Globally Using Biotinylated Psoralen

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA interactions such as microRNA-mRNA interactions have been shown to regulate gene expression, the full extent to which RNA interactions occur in the cell is still unknown. Previous methods to study RNA interactions have primarily focused on subsets of RNAs that are interacting with a particular protein or RNA species. Here, we detail a method named Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH) that allows genome-wide capture of RNA interactions in vivo in an unbiased manner. SPLASH utilizes in vivo crosslinking, proximity ligation, and high throughput sequencing to identify intramolecular and intermolecular RNA base-pairing partners globally. SPLASH can be applied to different organisms including bacteria, yeast and human cells, as well as diverse cellular conditions to facilitate the understanding of the dynamics of RNA organization under diverse cellular contexts. The entire experimental SPLASH protocol takes about 5 days to complete and the computational workflow takes about 7 days to complete.

Here, we detail the method of Sequencing of Psoralen crosslinked, Ligated, and Selected Hybrids (SPLASH), which enables genome-wide mapping of intramolecular and intermolecular RNA-RNA interactions in vivo. SPLASH can be applied to study RNA interactomes of organisms including yeast, bacteria and humans.

Mapeando as Interações RNA-ARN Globalmente Usando Psoraleno Biotinilado

Knowing how RNAs interact with themselves and with others is key to understanding RNA based gene regulation in the cell. While examples of RNA-RNA ...

Using a Fluorescent PCR-capillary Gel Electrophoresis Technique to Genotype CRISPR/Cas9-mediated Knockout Mutants in a High-throughput Format

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play in the functioning of cells and whole organisms. This cause has been tremendously aided by the recent introduction of the CRISPR/Cas9 system-a versatile tool that allows researchers to manipulate the genome and transcriptome in order to, among other things, knock out, knock down, or knock in genes in a targeted manner. For the purpose of knocking out a gene, CRISPR/Cas9-mediated double-strand breaks recruit the non-homologous end-joining DNA repair pathway to introduce the frameshift-causing insertion or deletion of nucleotides at the break site. However, an individual guide RNA may cause undesirable off-target effects, and to rule these out, the use of multiple guide RNAs is necessary. This multiplicity of targets also means that a high-volume screening of clones is required, which in turn begs the use of an efficient high-throughput technique to genotype the knockout clones. Current genotyping techniques either suffer from inherent limitations or incur high cost, hence rendering them unsuitable for high-throughput purposes. Here, we detail the protocol for using fluorescent PCR, which uses genomic DNA from crude cell lysate as a template, and then resolving the PCR fragments via capillary gel electrophoresis. This technique is accurate enough to differentiate one base-pair difference between fragments and hence is adequate in indicating the presence or absence of a frameshift in the coding sequence of the targeted gene. This precise knowledge effectively precludes the need for a confirmatory sequencing step and allows users to save time and cost in the process. Moreover, this technique has proven to be versatile in genotyping various mammalian cells of various tissue origins targeted by guide RNAs against numerous genes, as shown here and elsewhere.

The genotyping technique described here, which couples fluorescent polymerase chain reaction (PCR) to capillary gel electrophoresis, allows for high-throughput genotyping of nuclease-mediated knockout clones. It circumvents limitations faced by other genotyping techniques and is more cost effective than sequencing methods.

Usando uma técnica de PCR fluorescente-capilar Electroforese em Gel de genótipo CRISPR / cas9 mediada KO mutantes em um formato de elevado rendimento

The development of programmable genome-editing tools has facilitated the use of reverse genetics to understand the roles specific genomic sequences play ...

A Universal Protocol for Large-scale gRNA Library Production from any DNA Source

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 nuclease or a nuclease-dead dCas9 fusion protein) is targeted to a specific site in the genome by a small synthetic RNA known as the guide RNA, or gRNA. The bipartite nature of the CRISPR system enables its use in screening approaches since plasmid libraries containing expression cassettes of thousands of individual gRNAs can be used to interrogate many different sites in a single experiment. 
To date, gRNA sequences for the construction of libraries have been almost exclusively generated by oligonucleotide synthesis, which limits the achievable complexity of sequences in the library and is relatively cost-intensive. Here, a detailed protocol for CORALINA (comprehensive gRNA library generation through controlled nuclease activity), a simple and cost-effective method for the generation of highly complex gRNA libraries based on enzymatic digestion of input DNA, is described. Since CORALINA libraries can be generated from any source of DNA, plenty of options for customization exist, enabling a large variety of CRISPR-based screens.

Methods for generating large-scale gRNA libraries should be simple, efficient and cost-effective. We describe a protocol for the production of gRNA libraries based on enzymatic digestion of target DNA. This method, CORALINA (comprehensive gRNA library generation through controlled nuclease activity) presents an alternative to costly custom oligonucleotide synthesis.

Um protocolo Universal para grande escala gRNA biblioteca de produção a partir de qualquer fonte de DNA

The popularity of the CRISPR/Cas9 system for both genome and epigenome engineering stems from its simplicity and adaptability. An effector (the Cas9 ...

Semi-quantitative Detection of RNA-dependent RNA Polymerase Activity of Human Telomerase Reverse Transcriptase Protein

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase activity. Although TERT is named as a reverse transcriptase, structural and phylogenetic analyses of TERT demonstrate that TERT is a member of right-handed polymerases, and relates to viral RNA-dependent RNA polymerases (RdRPs) as well as viral reverse transcriptase. We firstly identified RdRP activity of human TERT that generates complementary RNA stand to a template non-coding RNA and contributes to RNA silencing in cancer cells. To analyze this non-canonical enzymatic activity, we developed RdRP assay with recombinant TERT in 2009, thereafter established in vitro RdRP assay for endogenous TERT. In this manuscript, we describe the latter method. Briefly, TERT immune complexes are isolated from cells, and incubated with template RNA and rNTPs including radioactive rNTP for RdRP reaction. To eliminate single-stranded RNA, reaction products are treated with RNase I, and the final products are analyzed with polyacrylamide gel electrophoresis. Radiolabeled RdRP products can be detected by autoradiography after overnight exposure.

Human telomerase reverse transcriptase (TERT) synthesizes not only telomeric DNA but also double-stranded RNA through RNA-dependent RNA polymerase activity. Here, we describe a newly established assay to detect RNA-dependent RNA polymerase activity of endogenous TERT.

Detecção semi-quantitativa de atividade RNA-dependente do RNA polimerase humana Telomerase Transcriptase reversa proteína

Human telomerase reverse transcriptase (TERT) is the catalytic subunit of telomerase, and it elongates telomere through RNA-dependent DNA polymerase ...

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA mutation rates. Changes in replication timing occur during development and in cancer, but the role replication timing plays in development and disease is not known. Zebrafish were recently established as an in vivo model system to study replication timing. Here is detailed the protocols for using the zebrafish to determine DNA replication timing. After sorting cells from embryos and adult zebrafish, high-resolution genome-wide DNA replication timing patterns can be constructed by determining changes in DNA copy number through analysis of next generation sequencing data. The zebrafish model system allows for evaluation of the replication timing changes that occur in vivo throughout development, and can also be used to assess changes in individual cell types, disease models, or mutant lines. These methods will enable studies investigating the mechanisms and determinants of replication timing establishment and maintenance during development, the role replication timing plays in mutations and tumorigenesis, and the effects of perturbing replication timing on development and disease.

Profiling DNA Replication Timing Using Zebrafish as an In Vivo Model System

Zebrafish were recently used as an in vivo model system to study DNA replication timing during development. Here is detailed the protocols for using zebrafish embryos to profile replication timing. This protocol can be easily adapted to study replication timing in mutants, individual cell types, disease models, and other species.

Sincronismo de replicação de DNA usando Zebrafish como um sistema modelo In Vivo de criação de perfil

DNA replication timing is an important cellular characteristic, exhibiting significant relationships with chromatin structure, transcription, and DNA ...

Highly Efficient Gene Disruption of Murine and Human Hematopoietic Progenitor Cells by CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. Complete gene ablation in HSCs required the generation of knockout mice from which HSCs could be isolated, and gene ablation in primary human HSCs was not possible. Viral transduction could be used for knock-down approaches, but these suffered from variable efficacy. In general, genetic manipulation of human and mouse hematopoietic cells was hampered by low efficiencies and extensive time and cost commitments. Recently, CRISPR/Cas9 has dramatically expanded the ability to engineer the DNA of mammalian cells. However, the application of CRISPR/Cas9 to hematopoietic cells has been challenging, mainly due to their low transfection efficiencies, the toxicity of plasmid-based approaches and the slow turnaround time of virus-based protocols.
A rapid method to perform CRISPR/Cas9-mediated gene editing in murine and human hematopoietic stem and progenitor cells with knockout efficiencies of up to 90% is provided in this article. This approach utilizes a ribonucleoprotein (RNP) delivery strategy with a streamlined three-day workflow. The use of Cas9-sgRNA RNP allows for a hit-and-run approach, introducing no exogenous DNA sequences in the genome of edited cells and reducing off-target effects. The RNP-based method is fast and straightforward: it does not require cloning of sgRNAs, virus preparation or specific sgRNA chemical modification. With this protocol, scientists should be able to successfully generate knockouts of a gene of interest in primary hematopoietic cells within a week, including downtimes for oligonucleotide synthesis. This approach will allow a much broader group of users to adapt this protocol for their needs.

A protocol for fast CRISPR/Cas9-mediated gene disruption in mouse and human primary hematopoietic cells is described in this article. Cas9-sgRNA ribonucleoproteins are introduced via electroporation with sgRNAs generated through in vitro transcription and commercial Cas9. High editing efficiencies are achieved with limited time and financial cost.

Disrupção do Gene altamente eficiente de células progenitoras hematopoiéticas murino e humano por CRISPR/Cas9

Advances in the hematopoietic stem cell (HSCs) field have been aided by methods to genetically engineer primary progenitor cells as well as animal models. ...

A Combinatorial Single-cell Approach to Characterize the Molecular and Immunophenotypic Heterogeneity of Human Stem and Progenitor Populations

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is inherently a single-cell assay, however prior to molecular analysis, the target cells are often prospectively isolated in bulk, thereby losing single-cell resolution. Single-cell gene expression analysis provides a means to understand molecular differences between individual cells in heterogeneous cell populations. In bulk cell analysis an overrepresentation of a distinct cell type results in biases and occlusions of signals from rare cells with biological importance. By utilizing FACS index sorting coupled to single-cell gene expression analysis, populations can be investigated without the loss of single-cell resolution while cells with intermediate cell surface marker expression are also captured, enabling evaluation of the relevance of continuous surface marker expression. Here, we describe an approach that combines single-cell reverse transcription quantitative PCR (RT-qPCR) and FACS index sorting to simultaneously characterize the molecular and immunophenotypic heterogeneity within cell populations.
In contrast to single-cell RNA sequencing methods, the use of qPCR with specific target amplification allows for robust measurements of low-abundance transcripts with fewer dropouts, while it is not confounded by issues related to cell-to-cell variations in read depth. Moreover, by directly index-sorting single-cells into lysis buffer this method, allows for cDNA synthesis and specific target pre-amplification to be performed in one step as well as for correlation of subsequently derived molecular signatures with cell surface marker expression. The described approach has been developed to investigate hematopoietic single-cells, but have also been used successfully on other cell types.
In conclusion, the approach described herein allows for sensitive measurement of mRNA expression for a panel of pre-selected genes with the possibility to develop protocols for subsequent prospective isolation of molecularly distinct subpopulations.

Bulk gene expression measurements cloud individual cell differences in heterogeneous cell populations. Here, we describe a protocol for how single-cell gene expression analysis and index sorting by Florescence Activated Cell Sorting (FACS) can be combined to delineate heterogeneity and immunophenotypically characterize molecularly distinct cell populations.

Uma combinatória célula única abordagem para caracterizar o Molecular e imunofenotípica heterogeneidade de tronco humano e populações de Progenitor

Immunophenotypic characterization and molecular analysis have long been used to delineate heterogeneity and define distinct cell populations. FACS is ...

Isolation, Characterization and MicroRNA-based Genetic Modification of Human Dental Follicle Stem Cells

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such as initial massive cell death and low therapeutic effects, impaired their broad clinical translation. Genetic engineering of stem cells prior to transplantation emerged as a promising method to optimize therapeutic stem cell effects. However, safe and efficient gene delivery systems are still lacking. Therefore, the development of suitable methods may provide an approach to resolve current challenges in stem cell-based therapies.
The present protocol describes the extraction and characterization of human dental follicle stem cells (hDFSCs) as well as their non-viral genetic modification. The postnatal dental follicle unveiled as a promising and easily accessible source for harvesting adult multipotent stem cells possessing high proliferation potential. The described isolation procedure presents a simple and reliable method to harvest hDFSCs from impacted wisdom teeth. Also this protocol comprises methods to define stem cell characteristics of isolated cells. For genetic engineering of hDFSCs, an optimized cationic lipid-based transfection strategy is presented enabling highly efficient microRNA introduction without causing cytotoxic effects. MicroRNAs are suitable candidates for transient cell manipulation, as these small translational regulators control the fate and behavior of stem cells without the hazard of stable genome integration. Thus, this protocol represents a safe and efficient procedure for engineering of hDFSCs that may become important for optimizing their therapeutic efficacy.

This protocol describes the transient genetic engineering of dental stem cells extracted from the human dental follicle. The applied non-viral modification strategy may become a basis for the improvement of therapeutic stem cell products.

Isolamento, caracterização e baseada em MicroRNA modificação genética de células-tronco humanas folículo Dental

To date, several stem cell types at different developmental stages are in the focus for the treatment of degenerative diseases. Yet, certain aspects, such ...

Endogenous Protein Tagging in Human Induced Pluripotent Stem Cells Using CRISPR/Cas9

A protocol is presented for generating human induced pluripotent stem cells (hiPSCs) that express endogenous proteins fused to in-frame&#160;N- or C-terminal fluorescent tags. The prokaryotic CRISPR/Cas9 system (clustered regularly interspaced short palindromic repeats/CRISPR-associated 9) may be used to introduce large exogenous sequences into genomic loci via homology directed repair (HDR). To achieve the desired knock-in, this protocol employs the ribonucleoprotein (RNP)-based approach where wild type Streptococcus pyogenes Cas9 protein, synthetic 2-part guide RNA (gRNA), and a donor template plasmid are delivered to the cells via electroporation. Putatively edited cells expressing the fluorescently tagged proteins are enriched by fluorescence activated cell sorting (FACS). Clonal lines are then generated and can be analyzed for precise editing outcomes. By introducing the fluorescent tag at the genomic locus of the gene of interest, the resulting subcellular localization and dynamics of the fusion protein can be studied under endogenous regulatory control, a key improvement over conventional overexpression systems. The use of hiPSCs as a model system for gene tagging provides the opportunity to study the tagged proteins in diploid, nontransformed cells. Since hiPSCs can be differentiated into multiple cell types, this approach provides the opportunity to create and study tagged proteins in a variety of isogenic cellular contexts.

Described here is a protocol for tagging endogenously expressed proteins with fluorescent tags in human induced pluripotent stem cells using CRISPR/Cas9. Putatively edited cells are enriched by fluorescence activated cell sorting and clonal cell lines are generated.