We would like to thank all the members of the Mitchell lab for helpful discussions. This work was supported by the Canadian Institutes of Health Research, the Canada Foundation for Innovation and the Ontario Ministry of Research and Innovation (operating and infrastructure grants held by JAM).

1. Designing and Constructing the gRNA
<ol>
	<li>To delete transcriptional enhancer regions use two gRNAs, one 5&#39; and one 3&#39; of the region of interest. Use the mouse UCSC genome browser track generated by the Zhang laboratory to identify unique gRNA sequences (http://www.genome-engineering.org15). Next check these gRNAs and their adjacent PAM for SNPs and indels using online tools provided by the Sanger Institute (www.sanger.ac.uk/sanger/Mouse_SnpViewer/rel-1211)27-28. To target both allel...

<ol>
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CRISPR/Cas9 mediated genome editing technology provides a straightforward, fast and inexpensive method for genome modification. The method detailed here to generate and analyze monoallelic enhancer deletion for functional enhancer characterization takes advantage of SNPs in F1 mouse cells. The advantages of this type of approach are: 1) monoallelic enhancer deletions do not produce confounding effects that occur when a critical enhancer is deleted from both alleles, i.e., a great reduction in the protein levels ...

Transcriptional regulatory elements are critical for spatio-temporal fine tuning of gene expression during development1 and modification of these elements can result in disease due to aberrant gene expression2. Many disease-associated regions identified by genome wide association studies are in non-coding regions and have features of transcriptional enhancers3-4. Identifying enhancers and matching them with the genes they regulate is complicated as they are often located several kilobases away from the genes they regulate and may be activated in a tissue-specific manner5-6. Enhancer predictions are commonly based on histone modification marks, mediator-cohesin complexes and binding of cell type-specific transcription factors7-10. Validation of predicted enhancers is most often done through a vector based assay in which the enhancer activates expression of a reporter gene11-12. These data provide valuable information about the regulatory potential of putative enhancer sequences but do not reveal their function in their endogenous genomic context or identify the genes they regulate. Genome editing serves as a powerful tool to study the function of transcriptional regulatory elements in their endogenous context by loss-of-function analysis.
Recent advances in genome editing, namely the CRISPR/Cas9 genome editing system, facilitate the investigation of genome function. The CRISPR/Cas9 system is easy to use and adaptable for many biological systems. The Cas9 protein is targeted to a specific site in the genome by a guide RNA (gRNA)13. The SpCas9/gRNA complex scans the genome for its target genomic sequence which must be 5&#39; to a protospacer adjacent motif (PAM) sequence, NGG14-15. Base pairing of the gRNA to its target, a 20 nucleotide (nt) sequence complementary to the gRNA, activates SpCas9 nuclease activity resulting in a double strand break (DSB) 3 bp upstream of the PAM sequence. Specificity is achieved through complete base pairing in the gRNA seed region, the 6-12 nt adjacent to the PAM; conversely, mismatches 5&#39; of the seed are usually tolerated16-17. The introduced DSB can be repaired either by the non-homologous end joining (NHEJ) DNA repair or homology directed repair (HDR) mechanisms.NHEJ DNA repair often creates insertion/deletion (indels) of a few bp at the target site that can disrupt the open reading frame (ORF) of a gene. To generate larger deletions in the genome two gRNAs, which flank the region of interest, can be used18-19. This approach is particularly useful for the study of transcriptional enhancers clustered into locus control regions or super-enhancers which are larger than conventional enhancers9,18,20-22.
Monoallelic deletions are a valuable model for studying cis-regulation of transcription. The observed change in transcript level after monoallelic deletion of an enhancer correlates to the role of that enhancer in gene regulation without the confounding effects that can occur when transcription of both alleles is affected potentially influencing cellular fitness. Evaluating reduced expression is difficult however without the ability to distinguish the deleted from the wild type allele. Furthermore, genotyping deletions at each allele without the ability to distinguish the two alleles is challenging, especially for large deletions of &#62;10 kb to 1 Mb23 in which it is difficult to amplify the entire wild type region by PCR. The use of F1 ES cells generated by crossing Mus musculus129 with Mus castaneus allows the two alleles to be differentiated by allele-specific PCR18,24. The hybrid genome in these cells facilitates allele specific deletion screening and expression analysis. On average there is a SNP every 125 bp between these two genomes, providing flexibility in primer design for expression and genotyping analyses. The presence of one SNP can influence the primer melting temperature (Tm) and target specificity in real-time quantitative PCR (qPCR) amplification allowing for discrimination of the two alleles25. Furthermore a mismatch within the 3&#39; end of the primer greatly influences the ability of DNA polymerase to extend from the primer preventing amplification of the undesired allele target26. Described in the following protocol is the use of F1 ES cells for allele specific enhancer deletions of greater than 1 kb and subsequent expression analysis using the CRISPR/Cas9 genome editing system (Figure 1).
<img alt="Figure 1" src="/files/ftp_upload/53552/53552fig1.jpg" /> 
Figure 1. Enhancer deletion using CRISPR/Cas9 to study cis-regulation of gene expression. (A) F1 ES cells generated by a cross between Mus musculus129 and Mus castaneus are used to allow for allele specific deletion. (B) Two guide RNAs (gRNA) are used to induce a large Cas9-mediated deletion of the enhancer region. (C) Primer sets are used to identify large mono- and bi-allelic deletions. The orange primers are the inside primers, the purple primers are the outside primers and the green primers are the gRNA flanking primers. (D) Changes in gene expression are monitored using allele-specific qPCR. RFU denotes relative fluorescence units. <a href="https://www.jove.com/files/ftp_upload/53552/53552fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

<table border="0" cellpadding="0" cellspacing="0" width="966">
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:253px;">Phusion High-Fidelity DNA Polymerase</td>
			<td style="width:200px;">NEB</td>
			<td style="width:119px;">M0530S</td>
			<td style="width:395px;">high fidelity DNA polymerase used in gRNA assembly</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Gibson Assembly Master Mix</td>
			<td style="width:200px;">NEB</td>
			<td>E2611L</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">gRNA_Cloning Vector</td>
			<td style="width:200px;">Addgene</td>
			<td style="width:119px;">41824</td>
			<td>A target sequence is cloned into this vector to create the gRNA plasmid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">pCas9_GFP</td>
			<td style="width:200px;">Addgene</td>
			<td style="width:119px;">44719</td>
			<td>Codon-optimized SpCas9 and EGFP co-expression plasmid</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">AflII</td>
			<td style="width:200px;">NEB</td>
			<td style="width:119px;">R0520S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">EcoRI</td>
			<td style="width:200px;">NEB</td>
			<td style="width:119px;">R3101S</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Neon Transfection System 100 &#181;L Kit</td>
			<td style="width:200px;">Life Technologies</td>
			<td style="width:119px;">MPK10096</td>
			<td>Microporator transfection technology</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">prepGEM</td>
			<td style="width:200px;">ZyGEM</td>
			<td style="width:119px;">PT10500</td>
			<td>genomic DNA extraction reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Nucleo Spin Gel &#38; PCR Clean-up</td>
			<td style="width:200px;">Macherey-Nagel</td>
			<td style="width:119px;">740609.5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">High-Speed Plasmid Mini Kit</td>
			<td style="width:200px;">Geneaid</td>
			<td style="width:119px;">PD300</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Maxi Plasmid Kit Endotoxin Free&#160;</td>
			<td style="width:200px;">Geneaid</td>
			<td style="width:119px;">PME25</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">SYBR select mix for CFX</td>
			<td style="width:200px;">Life Technologies</td>
			<td style="width:119px;">4472942</td>
			<td>qPCR reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">iScript cDNA synthesis kit</td>
			<td style="width:200px;">Bio-rad</td>
			<td style="width:119px;">170-8891</td>
			<td>Reverse transcription reagent</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">0.25% Trypsin with EDTA</td>
			<td style="width:200px;">Life Technologies</td>
			<td style="width:119px;">25200072</td>
			<td></td>
		</tr>
		<tr height="25">
			<td height="25" style="height:25px;width:253px;">PBS without Ca/Mg2+</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:119px;">D8537</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">0.5M EDTA</td>
			<td style="width:200px;">Bioshop</td>
			<td style="width:119px;">EDT111.500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">HBSS</td>
			<td style="width:200px;">Life Technologies</td>
			<td style="width:119px;">14175095</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">1M HEPES</td>
			<td style="width:200px;">Life Technologies</td>
			<td style="width:119px;">13630080</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">BSA fraction V (7.5%)</td>
			<td style="width:200px;">Life Technologies</td>
			<td style="width:119px;">15260037</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Max Efficiency DH5&#945; competent cells</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:119px;">18258012</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">FBS</td>
			<td style="width:200px;">ES cell qualified</td>
			<td style="width:119px;"></td>
			<td>FBS is subjected to a prior testing in mouse ES cells for pluripotency</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">DMSO</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:119px;">D2650</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Glutamax</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:119px;">35050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">DMEM</td>
			<td style="width:200px;">Life Technologies</td>
			<td style="width:119px;">11960069</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Pencillin/Streptomycin</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:119px;">15140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Sodium pyruvate</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:119px;">11360</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Non-essential aminoacid</td>
			<td style="width:200px;">Invitrogen</td>
			<td style="width:119px;">11140</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">&#946;-mercaptoethanol</td>
			<td style="width:200px;">Sigma</td>
			<td style="width:119px;">M7522</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">96-well plate</td>
			<td style="width:200px;">Sarstedt</td>
			<td style="width:119px;">83.3924</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">Sealing tape</td>
			<td style="width:200px;">Sarstedt</td>
			<td style="width:119px;">95.1994</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">CoolCell LX</td>
			<td style="width:200px;">Biocision</td>
			<td style="width:119px;">BCS-405</td>
			<td>alcohol-free cell freezing container</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">CHIR99021</td>
			<td style="width:200px;">Biovision</td>
			<td style="width:119px;">1748-5</td>
			<td>Inhibitor for F1 ES cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">PD0325901</td>
			<td style="width:200px;">Invivogen</td>
			<td style="width:119px;">inh-pd32</td>
			<td>Inhibitor for F1 ES cell culture</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:253px;">LIF</td>
			<td style="width:200px;">Chemicon</td>
			<td style="width:119px;">ESG1107</td>
			<td>Inhibitor for F1 ES cell culture</td>
		</tr>
	</tbody>
</table>

generating crispr/cas9 mediated monoallelic deletions to study enhancer function in mouse embryonic stem cells

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often located distally from the regulated gene in intergenic regions or even within the body of another gene. The position independent nature of enhancer activity makes it difficult to match enhancers with the genes they regulate. Deletion of an enhancer region provides direct evidence for enhancer activity and is the gold standard to reveal an enhancer's role in endogenous gene transcription. Conventional homologous recombination based deletion methods have been surpassed by recent advances in genome editing technology which enable rapid and precisely located changes to the genomes of numerous model organisms. CRISPR/Cas9 mediated genome editing can be used to manipulate the genome in many cell types and organisms rapidly and cost effectively, due to the ease with which Cas9 can be targeted to the genome by a guide RNA from a bespoke expression plasmid. Homozygous deletion of essential gene regulatory elements might lead to lethality or alter cellular phenotype whereas monoallelic deletion of transcriptional enhancers allows for the study of cis-regulation of gene expression without this confounding issue. Presented here is a protocol for CRISPR/Cas9 mediated deletion in F1 mouse embryonic stem (ES) cells (Mus musculus129 x Mus castaneus). Monoallelic deletion, screening and expression analysis is facilitated by single nucleotide polymorphisms (SNP) between the two alleles which occur on average every 125 bp in these cells.

The protocol described here uses F1 ES cells to study cis-regulation of gene expression in monoallelic enhancer deleted cells generated using CRISPR/Cas9 genome editing (Figure 1). The gRNA and allele-specific primer design for genotyping and gene expression are the key factors in this approach. Each allele-specific primer set must be validated by qPCR to confirm allele specificity. Allele-specific primers that amplify only their respective genomic DNA target are...

Watch this Scientific Journal Video about Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells at JoVE.com

Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus castaneus).

Enhancers control cell identity by regulating tissue-specific gene expression in a position and orientation independent manner. These enhancers are often ...

The overall goal of this procedure is to use allele specific CRISPR/Cas9 mediated deletion to study regulation of gene transcription in mouse embryonic stem cells. The main advantage of this technique, which uses a hybrid genome to generate and analyze monoallelic deletions. Instead, it eliminates the need to analyze monoallelic enhanced deletions that may not be viable.This method can help answer key questions in the functional genomics field. Such as, which gene does a specific enhancer regulate and how dependent is that gene on any one enhancer for its activity. Those this method can provide insight into stem cell pluripotency, it can also be applied to other systems.For example, it can help us to understand how stem cells change their fate to become neurons, blood cells, or muscle. After designing, constructing, and confirming the sequence of the guide RNA, or gRNA, to be used, prepare to transfect the endotoxin-free plasmids into ES cells. Using a centrifuge, pellet one times ten to the sixth F1 embryonic stem cells generated by crossing Mus musculus 129 with Mus castaneus in a 1.5 milliliter tube at 300 times g'for five minutes.After the spin, discard the supernatant and add 100 microliters of the resuspension buffer supplied by the kit manufacturer. For deletion of the target DNA region, add five micrograms each of P Cas9 GFP 5-prime and 3-prime gRNA plasmids. Using a pipette, gently mix.Avoid introducing any bubbles. Next, use the electronic pipette tip of the microporator to aspirate 100 microliters of the electroporation mix, taking care to avoid introducing air into the tip. Then, plug the pipette into the transfection device.Select the appropriate protocol and press start. While, the electroporation is running, observe the tip. With the correct set-up, tiny bubbles should emerge from the tube electrode.A spark indicates the presence of an air bubble and will interfere with the transfection. If this occurs, the electroporation must be repeated with a new batch of cells. Following the electroporation, eject the transfected cells into a ten centimeter gelatin coated dish containing ten milliliters of ES cell medium.Incubate the transfected cells at 37 degrees Celsius, 5%carbon dioxide. After 48 hours, use a flow cytometer to sort and isolate the cast nine GFP positive ES cells. Then, feed to 1 to 1.5 times 10 to the fourth GFP positive cells in a 10 centimeter, gelatin-coated dish containing 10 milliliters of ES cell medium.Plating at this low density will facilitate picking up individual ES cell colonies. Four to five days later, use a microscope to identify individual ball-shaped, single ES cell colonies. Using a pipette transfer each colony to one well of a 96-well plate pretreated with gelatin and containing 30 microliters of tripsom.Once all of the colonies have been picked and dissociated into medium, grow the cells at 37 degrees Celsius in the carbon dioxide incubator until most of the wells are over 70%confluent. This usually takes about two days. When the cells are ready for splitting, remove the medium and add 30 microliters of tripsom.Incubate the cells for five minutes. Then, to each well add 180 microliters of ES cell medium to neutralize the tripsom. Pipette up and down to achieve a single cell suspension.From each well, transfer 70 microliters to each of three gelatin-coated 96-well plates containing 130 microliters of ES cell medium in each well. Incubate at 37 degrees Celsius. When the plates reach 70 to 85%confluency, use them to perform genotyping and expression analysis of each clone, as described in the sections that follow.Freeze the cell stocks as described in the accompanying document. Here, four sets of primers will be designed to screen the clones for the desired deletion. These include:inside primers, outside primers, and gRNA flanking primers for both 5-prime and 3-prime gRNA target sites.Begin by visiting the website shown here to obtain the single nucleotide polymorphism or snip track corresponding to the 129 and Cas genotypes. This directs to the Mitchell Lab CRISPR webpage. There, click on the given link.The site will redirect to the UCSC genome browser containing the track displaying snips between the 129 and Cas genomes in the MM-9 mouse genome assembly. Next, enter the coordinates of the region to be deleted. and click on go"Zoom in on a region of about 500 base pairs in the middle of the desired deletion containing more than three snips.Allele-specific primer design can be complicated if snips are scarce. If it's not possible to design absolute allele-specific primers, moderate allele-specific primers may be used. Just remember to compare the CT values between the deleted and non-deleted clones during analysis of the qPCR results.Now, click on View"in the top panel. From the drop-down menu, select DNA. The Get DNA in Window"page will appear.In the sequence formatting options, choose all uppercase, then click on Get DNA"to display the target sequence in all uppercase format. Copy this sequence and past it into a Word document file. Then, compare the sequence with the 129 cast snip track and highlight the snip positions and mark the 129 snips with the lowercase.When you are finished with the 129 sequence, copy it to create two fast day formatted sequences. One for 129 and one for Cas. In the cast sequence, substitute the correct base at each snip position, marking the snip with a lowercase.Next, label the sequence with its corresponding genome, 129 or Cas. Then, in the browser, type in the address for Primer3Plus. In the appropriate area, paste the snip substituted 129 sequence.Use the default settings to design the primers. To design the inside allele-specific primers, click on the drop-down menu to the right of Task"and select Primer List"the click on Pick Primers. A new page will open displaying a list of forward and reverse primers.Check the select box to choose a forward or reverse primer that has snip marked by a lowercase either at the 3-prime end or within four bases of the 3-prime end. Note the primers that have a snip at the 3-prime end display increased allele specificity during qPCR. Click Send to Primer3 Manager"at the bottom of the screen.A new page will open displaying the selected primer. To select the second primer, return to the primer list page, and click the back button to return to the main page. In the task drop-down menu, select Detection.The main page will refresh and a left and right primer sequence box will appear at the bottom. Paste the first primer sequence in the appropriate box. Next, navigate to the General Settings tab located next to the Main tab and change the setting for the product size range to 80 to 200 base pairs.Click Pick Primers"A page will open displaying five possible primer pairs for the first primer sequence. Choose a primer set from the list of primer pairs by checking the boxes to the left of the primers and click on Send to Primer3 Manager"at the bottom of the screen. These will be the 129 inside allele-specific primers.Repeat these steps to design primers for the cast allele. Then, apply this technique to the design of outside allele-specific primers and non-allele-specific primers. Finally, test the primers for allele specificity using qPCR as described in the accompanying document.Extract the genomic DNA from the 96-well plate prepared earlier. Centrifuge the plate briefly to settle any condensation to the bottom of the well. This DNA will serve as the template for deletion screening.Next, on a 384-well plate, preform qPCR and duplicate for each clone. Use a two step PCR followed by melt curve analysis with detection. Following the run, first check each allele for amplification with the inside allele-specific primers.No amplification of one allele or CT value differences of more than five cycles between alleles, suggests these clones carry a heterozygous deletion. No amplification of both alleles suggests that they carry a homozygous deletion. Next, check each allele for amplification with the outside allele-specific primers.If the target deletion is larger than one KB, amplification with outside primers will only occur if a deletion is present. A CT value of 22 to 28 confirms the deletion. For target deletions smaller than one KB, confirm the amplicon size by eletrophoresis.A 96-well plate of deletion clones was screened by qPCR using allele-specific inside primers. Gray bars represent from Cas-specific primers and black bars represent amplification from 129-specific primers. A low CT value or no amplification with inside primers indicates target deletion.Clones with a deletion on one or both alleles are then screened with the outside primers. With outside primers, amplification confirms the deletion. Clones with a monoallelic deletion are screened using primers flanking the gRNA target regions to confirm the absence of insertions or deletions, called indels, on the non-deleted allele.Only monoallelic clones without large indels at the target sites on the non-deleted allele are used in the subsequent expression analysis. Shown here are representative results from 129 or Cas SCR deleted clones. The SCR is a recently described Sox2 specific enhancer in ES cells.Red bars represent expression of the Sox2 Cas allele, and blue bars represent amplification of the Sox2 129 allele. As can be seen in this figure, deletion of the SCR on the 129 or Cas allele reduced expression of Sox2 in CIS. After watching this video, you should have a good understanding of how to generate a large deletion using CRISPR technology and use allele-specific primers to genotype your deleted clones.

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Developmental Biology

13.9K ビュー.  University of Toronto. The overall goal of this procedure is to use allele specific CRISPR/Cas9 mediated deletion to study regulation of gene transcription in mouse embryonic stem cells. The main advantage of this technique, which uses a hybrid genome to generate and analyze monoallelic deletions. Instead, it eliminates the need to analyze monoallelic enhanced deletions that may not be viable.This method can help answer key questions in the functional genomics field. Such as, which gene does a specific enhan...

ビデオ: Generating CRISPR/Cas9 Mediated Monoallelic Deletions to Study Enhancer Function in Mouse Embryonic Stem Cells

Experimental validation of enhancer activity is best approached by loss-of-function analysis. Presented here is an efficient protocol that uses CRISPR/Cas9 mediated deletion to study allele-specific regulation of gene transcription in F1 ES cells which contain a hybrid genome (Mus musculus129 x Mus...

crispr/cas9-mediated highly efficient gene targeting in embryonic stem cells for developing gene-manipulated mouse models

CRISPR/Cas9-Mediated Highly Efficient Gene Targeting in Embryonic Stem Cells for Developing Gene-Manipulated Mouse Models

Here we present a protocol for developing genetically modified mouse models using embryonic stem cells, especially for large DNA knock-in (KI). This protocol is tuned up using CRISPR/Cas9 genome editing, resulting in significantly improved KI efficiency compared with the conventional homologous recombination-mediated linearized DNA targeting...

generation of genomic deletions in mammalian cell lines via crispr/cas9

Generation of Genomic Deletions in Mammalian Cell Lines via CRISPR/Cas9

CRISPR/Cas9 is a robust system to produce disruption of genes and genetic elements. Here we describe a protocol for the efficient creation of genomic deletions in mammalian cell lines using...

structure-function studies in mouse embryonic stem cells using recombinase-mediated cassette exchange

Structure-function Studies in Mouse Embryonic Stem Cells Using Recombinase-mediated Cassette Exchange

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a...

lentiviral crispr/cas9-mediated genome editing for the study of hematopoietic cells in disease models

Lentiviral CRISPR/Cas9-Mediated Genome Editing for the Study of Hematopoietic Cells in Disease Models

Described are protocols for the highly efficient genome editing of murine hematopoietic stem and progenitor cells (HSPC) by the CRISPR/Cas9 system to rapidly develop mouse model systems with hematopoietic system-specific gene modifications.

crispr-cas9 mediated gene deletion in human pluripotent stem cells cultured under feeder-free conditions

Lentiviral Infections and Single-Cell Clonal Propagation for Gene Targeting in Human Embryonic Stem Cells

The presented method describes the generation of a CRISPR-mediated gene knockout in the human embryonic stem cell (hESC) line H9, which stably expresses sgRNAs targeting the L2HGDH gene using a highly efficient lentiviral-mediated gene delivery...

CRISPR-Cas9 Mediated Gene Deletion in Human Pluripotent Stem Cells Cultured Under Feeder-Free Conditions

dissection of enhancer function using multiplex crispr-based enhancer interference in cell lines

Dissection of Enhancer Function Using Multiplex CRISPR-based Enhancer Interference in Cell Lines

This protocol describes the steps needed to design and perform multiplexed targeting of enhancers with the deactivating fusion protein SID4X-dCas9-KRAB, also known as enhancer interference (Enhancer-i). This protocol enables the identification of enhancers that regulate gene expression and facilitates the dissection of relationships between enhancers regulating a common target...

in vivo crispr/cas9 screening to simultaneously evaluate gene function in mouse skin and oral cavity

In Vivo CRISPR/Cas9 Screening to Simultaneously Evaluate Gene Function in Mouse Skin and Oral Cavity

Here we describe a rapid and direct in vivo CRISPR/Cas9 screening methodology using ultrasound-guided in utero embryonic lentiviral injections to simultaneously assess functions of several genes in the skin and oral cavity of immunocompetent...

endogenous protein tagging in human induced pluripotent stem cells using crispr/cas9

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

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...

generating cortical interneuron precursors from mouse embryonic stem cells

This video presents a method for generating cortical interneuron precursors from mouse embryonic stem cells. The protocol entails culturing the stem cells in media containing specific small molecule inhibitors, which facilitate stem cell proliferation and aggregation into embryoid bodies (EBs). Subsequently, the EBs are dissociated with enzymes and plated on culture plate wells coated with adhesion proteins to promote their differentiation into cortical interneuron...

Generating Cortical Interneuron Precursors From Mouse Embryonic Stem Cells

crispr-cas9-mediated precise knock-in edits in zebrafish hearts

CRISPR-Cas9-Mediated Precise Knock-In Edits in Zebrafish Hearts

This protocol describes an approach to facilitate precise knock-in edits in zebrafish embryos using CRISPR-Cas9 technology. A phenotyping pipeline is presented to demonstrate the applicability of these techniques to model a Long QT Syndrome-associated gene...

selection-dependent and independent generation of crispr/cas9-mediated gene knockouts in mammalian cells

Selection-dependent and Independent Generation of CRISPR/Cas9-mediated Gene Knockouts in Mammalian Cells

Recent advances in the ability to genetically manipulate somatic cell lines hold great potential for basic and applied research. Here, we present two approaches for CRISPR/Cas9 generated knockout production and screening in mammalian cell lines, with and without the use of selectable...

retroviral crispr/cas9-mediated gene targeting for the study of th17 differentiation in vitro

Author Spotlight: Studying Th17 Cell Differentiation Using CRISPR/Cas9 and Retroviral Transduction

We present a protocol for retroviral transduction of guide RNA into primary T cells from Cas9 transgenic mice, providing an efficient alternative for gene editing in studying Th17...

Retroviral CRISPR/Cas9-Mediated Gene Targeting for the Study of Th17 Differentiation in Vitro

On DD2, aspirate the poly-L-lysine and coat the plates with laminin overnight at 37 degrees Celsius. On DD3, before beginning EB dissociation, aspirate laminin, and allow the plates to completely dry in a tissue culture...

generation of defined genomic modifications using crispr-cas9 in human pluripotent stem cells

Generation of Defined Genomic Modifications Using CRISPR-CAS9 in Human Pluripotent Stem Cells

This protocol provides a method to facilitate the generation of defined heterozygous or homozygous nucleotide changes using CRISPR-CAS9 in human pluripotent stem...

a technique for generating neural progenitor cells from mouse embryonic stem cells

This video demonstrates a method for the in vitro generation of neural progenitor cells (NPCs) from mouse embryonic stem cells (mESCs). The mESCs are cultured in suspension to form embryoid bodies (EBs), which are then guided to differentiate into NPCs via retinoic acid treatment in an adherent...

A Technique for Generating Neural Progenitor Cells from Mouse Embryonic Stem Cells

crispr-cas9-mediated genome editing in the filamentous ascomycete huntiella omanensis

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

The CRISPR-Cas9 genome editing system is an easy-to-use genome editor that has been used in model and non-model species. Here we present a protein-based version of this system that was used to introduce a premature stop codon into a mating gene of a non-model filamentous ascomycete...

CRISPR-Cas9-Mediated Genome Editing in the Filamentous Ascomycete Huntiella omanensis

After two days, transfer the cell aggregates into 15-milliliter centrifuge tubes, and let them settle by gravity. Remove the supernatant and add 10 milliliters of fresh basal differentiation medium to resuspend the embryoid bodies. Then, replant them into a new non-adhesive dish and incubate them for another two...

generating recombinant avian herpesvirus vectors with crispr/cas9 gene editing

Generating Recombinant Avian Herpesvirus Vectors with CRISPR/Cas9 Gene Editing

Herpesvirus of turkeys (HVT) is widely used as a vector platform for the generation of recombinant vaccines against a number of avian diseases. This article describes a simple and rapid approach for the generation of recombinant HVT-vectored vaccines using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox...

Neural Differentiation of Mouse Embryonic Stem Cells in Serum-free Monolayer Culture

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as well as the generation of mature neural cell types for further study. In this protocol we describe a method for the differentiation of ESC to neural progenitors using serum-free, monolayer culture. The method is scalable, efficient and results in production of ~70% neural progenitor cells within 4 - 6 days. It can be applied to ESC from various strains grown under a variety of conditions. Neural progenitors can be allowed to differentiate further into functional neurons and glia or analyzed by microscopy, flow cytometry or molecular techniques. The differentiation process is amenable to time-lapse microscopy and can be combined with the use of reporter lines to monitor the neural specification process. We provide detailed instructions on media preparation and cell density optimization to allow the process to be applied to most ESC lines and a variety of cell culture vessels.

This protocol describes in detail the method for generating neural progenitors from embryonic stem cells using a serum-free monolayer method. These progenitors can be used to derive mature neural cell types or to study the process of neural specification and is amenable to multiwell format scaling for compound screening.

The ability to differentiate mouse embryonic stem cells (ESC) to neural progenitors allows the study of the mechanisms controlling neural specification as ...

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, cells from the roof of the blastocoel are pluripotent. These cells can be isolated, and programmed to generate various tissues through manipulation of genes expression or induction by morphogens. In this manuscript protocols are described for the use of Xenopus laevis blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development. These protocols allow the investigation of fate acquisition, cell migration behaviors, and cell autonomous and non-autonomous properties. The blastocoel roof explants can be cultured in a serum-free defined medium and grafted into host embryos. This transplantation into an embryo allows the investigation of the long-term lineage commitment, the inductive properties, and the behavior of transplanted cells in vivo. These assays can be exploited to investigate molecular mechanisms, cellular processes and gene regulatory networks underlying neural development. In the context of regenerative medicine, these assays provide a means to generate neural-derived cell types in vitro that could be used in drug screening.

Stem cell-like Xenopus Embryonic Explants to Study Early Neural Developmental Features In Vitro and In Vivo

In Xenopus embryos, cells from the roof of the blastocoel are pluripotent and can be programmed to generate various tissues. Here, we describe protocols to use amphibian blastocoel roof explants as an assay system to investigate key in vivo and in vitro features of early neural development.

Understanding the genetic programs underlying neural development is an important goal of developmental and stem cell biology. In the amphibian blastula, ...

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of the cell and often consist of multiple functional domains, which can be influenced by posttranslational modifications. The depletion of the entire protein in conditional or constitutive knock-out (KO) mice does not take into account this functional diversity and regulation. An mESC line and a derived mouse model, in which a docking site for FLPe recombination-mediated cassette exchange (RMCE) was inserted within the ROSA26 (R26) locus, was previously reported. Here, we report on a structure-function approach that allows for molecular dissection of the different functionalities of a multidomain protein. To this end, RMCE-compatible mice must be crossed with KO mice and then RMCE-compatible KO mESCs must be isolated. Next, a panel of putative rescue constructs can be introduced into the R26 locus via RMCE targeting. The candidate rescue cDNAs can be easily inserted between RMCE sites of the targeting vector using recombination cloning. Next, KO mESCs are transfected with the targeting vector in combination with an FLPe recombinase expression plasmid. RMCE reactivates the promoter-less neomycin-resistance gene in the ROSA26 docking sites and allows for the selection of the correct targeting event. In this way, high targeting efficiencies close to 100% are obtained, allowing for insertion of multiple putative rescue constructs in a semi-high throughput manner. Finally, a multitude of R26-driven rescue constructs can be tested for their ability to rescue the phenotype that was observed in parental KO mESCs. We present a proof-of-principle structure-function study in p120 catenin (p120ctn) KO mESCs using endoderm differentiation in embryoid bodies (EBs) as the phenotypic readout. This approach enables the identification of important domains, putative downstream pathways, and disease-relevant point mutations that underlie KO phenotypes for a given protein.

Proteins often contain multiple domains that can exert different cellular functions. Gene knock-outs (KO) do not consider this functional diversity. Here, we report a recombination-mediated cassette exchange (RMCE)-based structure-function approach in KO embryonic stem cells that allows for the molecular dissection of various functional domains or variants of a protein.

Gene engineering in mouse embryos or embryonic stem cells (mESCs) allows for the study of the function of a given protein. Proteins are the workhorses of ...

Single-cell Photoconversion in Living Intact Zebrafish

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause disease when disrupted. Scientists have developed clever techniques to investigate characteristics and natural dynamics of these cells within intact tissue by expressing fluorescent proteins in subsets of cells. However, at times, experiments require more selected visualization of cells within the tissue, sometimes at the single-cell or population-of-cells manner. To achieve this and visualize single cells within a population of cells, scientists have utilized single-cell photoconversion of fluorescent proteins. To demonstrate this technique, we show here how to direct UV light to an Eos-expressing cell of interest in an intact, living zebrafish. We then image those photoconverted Eos+ cells 24 h later to determine how they changed in the tissue. We describe two techniques: single cell photoconversion and photoconversions of populations of cell. These techniques can be used to visualize cell-cell interactions, cell-fate and differentiation, and cell migrations, making it a technique that is applicable in numerous biological questions.

Here, we present a protocol to show how cell photoconversion is achieved through UV exposure to specific areas expressing the fluorescent protein, Eos, in living animals.

Animal and plant tissue is composed of distinct populations of cells. These cells interact over time to build and maintain the tissue and can cause ...

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and chromosome dynamics. While the germline is an excellent model, the analysis is often two dimensional due to the time and labor required for three-dimensional analysis. Major readouts in such studies are the number/position of nuclei and protein distribution within the germline. Here, we present a method to perform automated analysis of the germline using confocal microscopy and computational approaches to determine the number and position of nuclei in each region of the germline. Our method also analyzes germline protein distribution that enables the three-dimensional examination of protein expression in different genetic backgrounds. Further, our study shows variations in cytoskeletal architecture in distinct regions of the germline that may accommodate specific spatial developmental requirements. Finally, our method enables automated counting of the sperm in the spermatheca of each germline. Taken together, our method enables rapid and reproducible phenotypic analysis of the C. elegans germline.

Computational Analysis of the Caenorhabditis elegans Germline to Study the Distribution of Nuclei, Proteins, and the Cytoskeleton

We present an automated method for three-dimensional reconstruction of the Caenorhabditis elegans germline. Our method determines the number and position of each nucleus within the germline and analyses germline protein distribution and cytoskeletal structure.

The Caenorhabditis elegans (C. elegans) germline is used to study several biologically important processes including stem cell development, apoptosis, and ...

CRISPR/Cas9 Technology in Restoring Dystrophin Expression in iPSC-Derived Muscle Progenitors

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the exhaustion of muscle progenitor cells. Clustered regularly interspaced short palindromic repeats/CRISPR-associated 9 (CRISPR/Cas9) gene editing has the potential to restore the expression of the dystrophin gene. Autologous induced pluripotent stem cells (iPSCs)-derived muscle progenitor cells (MPC) can replenish the stem/progenitor cell pool, repair damage, and prevent further complications in DMD without causing an immune response. In this study, we introduce a combination of CRISPR/Cas9 and non-integrated iPSC technologies to obtain muscle progenitors with recovered dystrophin protein expression. Briefly, we use a non-integrating Sendai vector to establish an iPSC line from dermal fibroblasts of Dmdmdx mice. We then use the CRISPR/Cas9 deletion strategy to restore dystrophin expression through a non-homologous end joining of the reframed dystrophin gene. After PCR validation of exon23 depletion in three colonies from 94 picked iPSC colonies, we differentiate iPSC into MPC by doxycycline (Dox)-induced expression of MyoD, a key transcription factor playing a significant role in regulating muscle differentiation. Our results show the feasibility of using CRISPR/Cas9 deletion strategy to restore dystrophin expression in iPSC-derived MPC, which has significant potential for developing future therapies for the treatment of DMD.

Here, we present a Cas9-based exon23 deletion protocol to restore dystrophin expression in iPSC from Dmdmdx mouse-derived skin fibroblasts and directly differentiate iPSCs into myogenic progenitor cells (MPC) using the Tet-on MyoD activation system.

Duchenne muscular dystrophy (DMD) is a severe progressive muscle disease caused by mutations in the dystrophin gene, which ultimately leads to the ...

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C (UV-C) Damage

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational health hazard. Here, we demonstrate the exposure of primary stem cells from the mouse ocular surface to UV-C radiation. Reactive oxygen species (ROS) formation is the readout of the extent of oxidative stress/damage. In an experimental in vitro setting, it is also essential to assess the percentage of dead cells generated due to oxidative stress. In this article, we will demonstrate the 2&#39;,7&#39;-Dichlorofluoresceindiacetate (DCFDA) staining of UV-C exposed mouse primary ocular surface stem cells and their quantification based on the fluorescent images of DCFDA staining. DCFDA staining directly corresponds to ROS generation. We also demonstrate the quantification of dead and live cells by simultaneous staining with propidium iodide (PI) and Hoechst 3332 respectively and the percentage of DCFDA (ROS positive) and PI positive cells.

Assessment of Oxidative Damage in the Primary Mouse Ocular Surface Cells/Stem Cells in Response to Ultraviolet-C UV-C Damage

This protocol demonstrates the simultaneous detection of reactive oxygen species (ROS), live cells, and dead cells in live primary cultures from mouse ocular surface cells. 2&#39;,7&#39;-Dichlorofluoresceindiacetate, propidium iodide, and Hoechst staining are used to assess the ROS, dead cells, and live cells, respectively, followed by imaging and analysis.

The ocular surface is subjected to regular wear and tear due to various environmental factors. Exposure to UV-C radiation constitutes an occupational ...

A Static Self-Directed Method for Generating Brain Organoids from Human Embryonic Stem Cells

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a three-dimensional system. Here we present a relatively straightforward and inexpensive method that yields brain organoids. In this protocol human pluripotent stem cells are broken into small clusters instead of single cells and grown in basic media without a heterologous basement membrane matrix or exogenous growth factors, allowing the intrinsic developmental cues to shape the organoid&#39;s growth. This simple system produces a diversity of brain cell types including glial and microglial cells, stem cells, and neurons of the forebrain, midbrain, and hindbrain. Organoids generated from this protocol also display hallmarks of appropriate temporal and spatial organization demonstrated by brightfield images, histology, immunofluorescence and real-time quantitative reverse transcription polymerase chain reaction (qRT-PCR). Because these organoids contain cell types from various parts of the brain, they can be utilized for studying a multitude of diseases. For example, in a recent paper we demonstrated the use of organoids generated from this protocol for studying the effects of hypoxia on the human brain. This approach can be used to investigate an array of otherwise difficult to study conditions such as neurodevelopmental handicaps, genetic disorders, and neurologic diseases.

This protocol was generated as a means to produce brain organoids in a simplified, low cost manner without exogenous growth factors or basement membrane matrix while still maintaining the diversity of brain cell types and many features of cellular organization.

Human brain organoids differentiated from embryonic stem cells offer the unique opportunity to study complicated interactions of multiple cell types in a ...

Differentiation and Characterization of Neural Progenitors and Neurons from Mouse Embryonic Stem Cells

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays to characterize the differentiated cells. The E14 mouse embryonic stem cells were used to form embryoid bodies through the hanging drop method, and then induced to differentiate into neural progenitor cells by retinoic acid, and finally differentiated into neurons. Quantitative&#160;reverse transcription&#160;polymerase chain reaction (RT-qPCR) and immunofluorescence experiments revealed that the neural progenitors and neurons exhibit corresponding markers (nestin for neural progenitors and neurofilament for neurons) at day 8 and 12 post-differentiation, respectively. Flow cytometry experiments on an E14 line expressing a Sox1 promoter-driven GFP reporter showed that about 60% of cells at day 8 are GFP positive, indicating the successful differentiation of neural progenitor cells at this stage. Finally, RNA-seq analysis was used to profile the global transcriptomic changes. These methods are useful for analyzing the involvement of specific genes and pathways in regulating the cell identity transition during neuronal differentiation.

We describe the procedure for the in vitro differentiation of mouse embryonic stem cells into neuronal cells using the hanging drop method. Furthermore, we perform a comprehensive phenotypic analysis through RT-qPCR, immunofluorescence, RNA-seq, and flow cytometry.

We describe the step-by-step procedure for culturing and differentiating mouse embryonic stem cells into neuronal lineages, followed by a series of assays ...

Accurate Follicle Enumeration in Adult Mouse Ovaries

Sexually reproducing female mammals are born with their entire lifetime supply of oocytes. Immature, quiescent oocytes are found within primordial follicles, the storage unit of the female germline. They are non-renewable, thus their number at birth and subsequent rate of loss largely dictates the female fertile lifespan. Accurate quantification of primordial follicle numbers in women and animals is essential for determining the impact of medicines and toxicants on the ovarian reserve. It is also necessary for evaluating the need for, and success of, existing and emerging fertility preservation techniques. Currently, no methods exist to accurately measure the number of primordial follicles comprising the ovarian reserve in women. Furthermore, obtaining ovarian tissue from large animals or endangered species for experimentation is often not feasible. Thus, mice have become an essential model for such studies, and the ability to evaluate primordial follicle numbers in whole mouse ovaries is a critical tool. However, reports of absolute follicle numbers in mouse ovaries in the literature are highly variable, making it difficult to compare and/or replicate data. This is due to a number of factors including strain, age, treatment groups, as well as technical differences in the methods of counting employed. In this article, we provide a step-by-step instructional guide for preparing histological sections and counting primordial follicles in mouse ovaries using two different methods: [1] stereology, which relies on the fractionator/optical dissector technique; and [2] the direct count technique. Some of the key advantages and drawbacks of each method will be highlighted so that reproducibility can be improved in the field and to enable researchers to select the most appropriate method for their studies.

Here, we describe, compare, and contrast two different techniques for accurate follicle counting of fixed mouse ovarian tissues.