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).

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The authors have read JoVE&#39;s policies on conflict of interest and have no conflicts to disclose.

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 of the regulated gene leading to cell lethality or altered phenotype; 2) if the frequency of monoallelic deletion is low obtaining a homozygous deletion is less likely; however, with the use of allele-specific primers in gene expression analysis one can analyze clones with a monoallelic deletion; 3) using four sets of primers to screen monoallelic deletions permits the elimination of clones containing partial or large deletions which confound the downstream analysis.
Allele-specific primer design is critical for genotyping mono/biallelic CRISPR deletions and analyzing the effect on gene expression in an allele-specific manner. This is more easily achieved when the F1 cells used contain more frequent SNPs that allow discrimination of the two alleles. Here ES cells generated from a Mus musculus129 x Mus castaneus cross are used; however, other cells could be used if SNPs between the two alleles allow for allele-specific deletion screening and expression analysis, and if sufficient data exist to predict active enhancer regions to be targeted in the chosen cell type. Therefore, this method can be adapted to any cell line where information about allelic SNPs is available. One of the limitations of the protocol is the dependence on SNPs at specific locations. Some target regions carry fewer SNPs which makes designing allele-specific outside primers that amplify a &#60;800 bp fragment challenging. In such cases a PCR approach could be used as an alternative to qPCR screening allowing for a larger amplicon. In addition there may be SNP associated differences in the phenotype of F1 ES cells; to confirm the function of specific enhancers in additional genotypes homozygous deletions can be conducted in standard ES lines. The specificity of the SpCas9 nuclease is an important issue especially in considering potential applications in clinical approaches. Investigation into SpCas9 specificity has revealed that both the 6-12 nt seed region of the gRNA recognition sequence and the adjacent PAM are important for nuclease activity13-14,17. Off target mutations can be minimized by ensuring that the seed region and adjacent PAM are unique in the genome being modified14,16.
A monoallelic deletion approach described here coupled with allele specific RNA-seq can definitively reveal the gene or genes regulated by a specific enhancer18. These experiments are important for understanding genome function as deletion of even reporter-assay validated enhancers does not always affect gene expression18. Furthermore, enhancers may not regulate the closest gene in the genome or may regulate more than one gene1,20,35. As a result, loss-of-function analysis is the most informative approach to determine the function of an enhancer region. This can be rapidly achieved using CRISPR/Cas9-mediated monoallelic deletion.

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.remotexs.ntu.edu.sg/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 ideal (Figure 2). Ideally these primers have a SNP at their 3&#39; end. Primers with less allele-specificity can be used if they display a minimum of a 5 Ct value difference in their amplification of the correct vs. the incorrect genotype25. Primers that carry a SNP more 5&#39; than the 4th base from the 3&#39; end usually fail to exhibit allele specificity and amplify both genotypes with equal efficiency, revealing the importance of the SNP position in the primer34. In addition a purine/purine or pyrimidine/pyrimidine substitution has been shown to have a greater impact on the Tm difference of two primers compared to a purine/pyrimidine substitution25.
Primary screening of a 96-well plate of DNA isolated from ES cell clones is conducted with allele-specific inside primers to identify clones that carry a deletion on one or both alleles. These deleted clones are further screened with outside allele-specific primers to confirm each deletion. The example given here is from an efficient gRNA pair that resulted in 46% of clones carrying a deletion on the 129, Cast, or both alleles (Figure 3). Secondary screening is done for confirmed monoallelic deletion clones to identify indels around the gRNA target sites on the non-deleted allele as the frequency of indel occurrence at the gRNA target site is high23. Indels around the gRNA are not identifiable in the primary screening, as these clones show no deletion with inside primers and do not amplify with the outside primers (Figure 4). Monoallelic deletion clones that give amplification with both sets of left and right gRNA flanking primers have their other allele largely intact as they do not contain a deletion larger than the 5&#39; or 3&#39; gRNA flanking amplicons. Sequencing the amplicons from the gRNA flanking primers will identify indels smaller than the gRNA flanking amplicons which may not be noticeable in the qPCR. The monoallelic deletion clones that do not give amplification at both regions in the secondary screening are not included for further gene expression analysis. As gRNA target regions are chosen outside the region suspected to have enhancer function, indels smaller than the gRNA flanking amplicons are not likely to affect enhancer function and clones containing these can be included in further analysis.
To restrict a large deletion to one allele a gRNA can be chosen which overlaps a SNP in the seed region or the PAM. Described here is an example of a high-efficiency deletion (53%) on the 129 allele due to a SNP in the PAM for the 3&#39; gRNA on the Cast allele (Figure 5). This deletion removed the SCR, a recently described Sox2 specific enhancer in ES cells18,22. Although the introduction of the large deletion was greatly reduced on the Cast allele, three clones (1, 11, 75) were identified with a large deletion on the Cast allele (Figure 5). Of these three clones two (1, 11) contained 3&#39; break points within 50 bp of the 3&#39; gRNA target region. For the third clone we were not able to identify the 3&#39; break point and concluded that the deletion was greater than 11 kb18. Deletions with one or both of their break points located &#62;100 bp from either the 5&#39; of 3&#39; gRNA target region are difficult to genotype, generally account for 15-30% of all clones, and remain uncharacterized in the primary screening as the deletion is not amplified with the outside primers.
Once clones with monoallelic deletion have been identified they are analyzed for allele-specific gene expression using absolute quantification by reverse transcription qPCR. Gene expression from clones carrying a monoallelic deletion of the critical Sox2 enhancer region, the SCR, is shown here compared with expression in wild-type F1 ES cells (Figure 6). Specifically, clones carrying a deletion of the enhancer region on the 129 allele displayed a decrease in 129 transcript levels whereas clones carrying a deletion on the Cast allele displayed a decrease in Cast transcript levels. The allele-specific gene expression analysis revealed that this distal enhancer region, SCR, is a critical cis-regulator of Sox2 in ES cells18.
<img alt="Figure 2" src="/files/ftp_upload/53552/53552fig2.jpg" /> 
Figure 2. Testing the allele-specificity of 129 and Cast allele-specific primers. (A) Amplification from primers for the 129 genotype. (B) Amplification from primers for the Cast genotype. In both A and B the lines display the amplification profiles for C57BL/6 genomic DNA which has the same genotype as 129 DNA at these specific SNPs; the lines with open circles display the amplification profiles for Cast genomic DNA. Amplification resulting from the primer set with the most allele-specificity is shown in green (Allele-specific primer-1), a primer set displaying a 5 Ct difference is shown in purple (Allele-specific primer-2) and a primer set displaying a minimal difference in Ct value is represented in red (Allele-specific primer-3, primer details in Table 7). The red primer set would not be appropriate for allele-specific screening. RFU denotes relative fluorescence units. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53552/53552fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53552/53552fig3.jpg" /> 
Figure 3. Results obtained after screening a 96-well plate with allele-specific inside and outside primers. (A) qPCR results from the screen with inside primers (Enh_del_IS_F1_129, Enh_del _IS_F1C, Enh_del _IS_R1) B) qPCR results from the screen with outside primers (Enh_del_OS_F1, Enh del_OS_R1_129, Enh_del_OS_F2C, Enh_del_OS_R3, primer details in Table 7). In both grey bars represent amplification from Cast-specific primers and black bars represent amplification from 129-specific primers. Note that only clones with a deletion on one or both alleles are screened with the outside primers. The relative amplification of each allele was calculated using 2-CT to approximate the initial concentration and subsequently expressing each allele as a percentage of the sum of the two alleles. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53552/53552fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53552/53552fig4.jpg" /> 
Figure 4. Clones with a monoallelic deletion are screened to identify large indels at the gRNA target sites. Primers flanking the gRNA target regions are used to confirm that the non-deleted allele is intact. Only monoallelic clones without large indels at the target sites on the non-deleted allele are used in the subsequent expression analysis. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53552/53552fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 5" src="/files/ftp_upload/53552/53552fig5.jpg" /> 
Figure 5. Clones obtained from the Sox2 SCR deletion. Shown are the qPCR results from a screen with inside primers (pr111R, pr111F_129, pr111F_Cast, details in Table 7). Grey bars represent amplification from Cast-specific primers and black bars represent amplification from 129-specific primers. Note that the deletion is heavily skewed towards the 129 allele due to the presence of a SNP in the PAM of the 3&#39; gRNA target region on the Cast allele. The relative amplification of each allele was calculated using 2-CT to approximate the initial concentration and subsequently expressing each allele as a percentage of the sum of the two alleles. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53552/53552fig5large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 6" src="/files/ftp_upload/53552/53552fig6.jpg" /> 
Figure 6. SCR deletion dramatically reduces the expression of Sox2. Representative results from 129 or Cast SCR deleted clones. Red bars represent expression of the Sox2 Cast allele and blue bar represents amplification of the Sox2 129 allele. Deletion of the SCR on the 129 allele reduced expression of Sox2 (129) whereas deletion of the SCR on the Cast allele reduced expression of Sox2 (Cast). Data displayed are an average of three technical replicates, error bars not shown. Primers used for Sox2 expression analysis [Sox2_F, Sox2(129)_R, Sox2(Cast)_R] are listed in Table 7. <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/53552/53552fig6large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Stock concentration&#160;</td>
			<td>Volume</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>200 mM</td>
			<td>6 ml</td>
			<td>2 mM&#160;</td>
		</tr>
		<tr>
			<td>2-Mercaptoethanol</td>
			<td>10 mM</td>
			<td>6 ml</td>
			<td>0.1 mM</td>
		</tr>
		<tr>
			<td>MEM Non-essential aminoacids (NEAA)</td>
			<td>10 mM</td>
			<td>6 ml</td>
			<td>0.1 mM</td>
		</tr>
		<tr>
			<td>Sodium Pyruvate</td>
			<td>100 mM</td>
			<td>6 ml</td>
			<td>1 mM</td>
		</tr>
		<tr>
			<td>Pencillin/Streptomycin</td>
			<td>10,000 units</td>
			<td>3 ml</td>
			<td>50 U/ml</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td></td>
			<td>90 ml</td>
			<td>15%</td>
		</tr>
		<tr>
			<td>CHIR99021*</td>
			<td>10 mM</td>
			<td></td>
			<td>3 &#181;M</td>
		</tr>
		<tr>
			<td>PD0325901*</td>
			<td>10 mM</td>
			<td></td>
			<td>1 &#181;M</td>
		</tr>
		<tr>
			<td>LIF*</td>
			<td>107 units/ml</td>
			<td></td>
			<td>1000 U/ml</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4"># to prepare ES cell media, add the above components to 500 ml of high glucose DMEM. The ES cell media should not be stored for more than 4 weeks and with inhibitors* not more than 2 weeks.</td>
		</tr>
	</tbody>
</table>
Table 1. ES cell media.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Stock concentration&#160;</td>
			<td>Volume</td>
			<td>Final concentration</td>
		</tr>
		<tr>
			<td>GlutaMAX</td>
			<td>200 mM</td>
			<td>5 ml</td>
			<td>2 mM&#160;</td>
		</tr>
		<tr>
			<td>Pencillin/Streptomycin</td>
			<td>10,000 units</td>
			<td>5 ml</td>
			<td>100 U/ml</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td></td>
			<td>50 ml</td>
			<td>10%</td>
		</tr>
		<tr>
			<td colspan="4"></td>
		</tr>
		<tr>
			<td colspan="4"># to prepare Spin media, add the above components to 500 ml of high glucose DMEM.&#160;</td>
		</tr>
	</tbody>
</table>
Table 2. Spin media.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Final concentration</td>
			<td>Volume (50 ml)</td>
		</tr>
		<tr>
			<td>1x PBS without Ca/Mg2+</td>
			<td></td>
			<td>42.0 ml</td>
		</tr>
		<tr>
			<td>BSA Fraction V (7.5 %)</td>
			<td>15% (v/v)</td>
			<td>7.5 ml</td>
		</tr>
		<tr>
			<td>0.5 M EDTA</td>
			<td>5 mM</td>
			<td>0.5 ml</td>
		</tr>
	</tbody>
</table>
Table 3. Collection buffer.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Reagent</td>
			<td>Final concentration</td>
			<td>Volume (50 ml)</td>
		</tr>
		<tr>
			<td>1x HBSS</td>
			<td></td>
			<td>47.25 ml</td>
		</tr>
		<tr>
			<td>1M HEPES</td>
			<td>25 mM</td>
			<td>1.25 ml</td>
		</tr>
		<tr>
			<td>0.5 M EDTA</td>
			<td>5 mM</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>BSA Fraction V (7.5 %)</td>
			<td>1% (v/v)</td>
			<td>0.5 ml</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>1% (v/v)</td>
			<td>0.5 ml</td>
		</tr>
	</tbody>
</table>
Table 4. Sorting buffer.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ES cell media</td>
			<td>60%</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>40%</td>
		</tr>
	</tbody>
</table>
Table 5. Recovery media.
<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>ES cell media</td>
			<td>60%</td>
		</tr>
		<tr>
			<td>FBS</td>
			<td>20%</td>
		</tr>
		<tr>
			<td>DMSO</td>
			<td>20%</td>
		</tr>
	</tbody>
</table>
Table 6. 2x freezing media.
<img alt="Table 7a" src="/files/ftp_upload/53552/53552table7a.jpg" /> 
<img alt="Table 7b" src="/files/ftp_upload/53552/53552table7b.jpg" /> 
Table 7. List of primers.

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 ...

generating-crisprcas9-mediated-monoallelic-deletions-to-study

Nanyang Technological University

Research

JoVE Journal

Developmental Biology

13.9K Views.  University of Toronto. 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).

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

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, ...

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

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 ...

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give rise to multiple cell types, including the myotome (MYO), sclerotome (SCL), dermatome (D), and syndetome (SYN), which in turn develop into skeletal muscle, axial skeleton, dorsal dermis, and axial tendon/ligament, respectively. Therefore, the generation of SMs and their derivatives from human induced pluripotent stem cells (iPSCs) is critical to obtain pluripotent stem cells (PSCs) for application in regenerative medicine and for disease research in the field of orthopedic surgery. Although the induction protocols for MYO and SCL from PSCs have been previously reported by several researchers, no study has yet demonstrated the induction of SYN and D from iPSCs. Therefore, efficient induction of fully competent SMs remains a major challenge. Here, we recapitulate human SM patterning with human iPSCs in vitro by mimicking the signaling environment during chick/mouse SM development, and report on methods of systematic induction of SM derivatives (MYO, SCL, D, and SYN) from human iPSCs under chemically defined conditions through the presomitic mesoderm (PSM) and SM states. Knowledge regarding chick/mouse&#160;SM development was successfully applied to the induction of SMs with human iPSCs. This method could be a novel tool for studying human somitogenesis and patterning without the use of embryos and for cell-based therapy and disease modeling.

We present here a protocol for the differentiation of human induced pluripotent stem cells into each somite derivative (myotome, sclerotome, dermatome, and syndetome) in chemically defined conditions, which has applications in future disease modeling and cell-based therapies in orthopedic surgery.

In response to signals such as WNTs, bone morphogenetic proteins (BMPs), and sonic hedgehog (SHH) secreted from surrounding tissues, somites (SMs) give ...

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 ...

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs during early development are technically challenging due to the limiting cell number. Moreover, many approaches require a miRNA of interest to be defined in assays such as northern blotting, microarray, and qPCR. Therefore, the expression of many miRNAs and their isoforms have not been studied during early development. Here, we demonstrate a protocol for small RNA sequencing of sorted cells from early mouse embryos to enable relatively unbiased profiling of miRNAs in early populations of neural crest cells. We overcome the challenges of low cell input and size selection during library preparation using amplification and gel-based purification. We identify embryonic age as a variable accounting for variation between replicates and stage-matched mouse embryos must be used to accurately profile miRNAs in biological replicates. Our results suggest that this method can be broadly applied to profile the expression of miRNAs from other lineages of cells. In summary, this protocol can be used to study how miRNAs regulate developmental programs in different cell lineages of the early mouse embryo.

We describe a technique for profiling microRNAs in early mouse embryos. This protocol overcomes the challenge of low cell input and small RNA enrichment. This assay can be used to analyze changes in miRNA expression over time in different cell lineages of the early mouse embryo.

MicroRNAs (miRNAs) are important for the complex regulation of cell fate decisions and developmental timing. In vivo studies of the contribution of miRNAs ...

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

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Chapters in this video

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

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

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

Single-cell Photoconversion in Living Intact Zebrafish

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

In Vitro Generation of Somite Derivatives from Human Induced Pluripotent Stem Cells

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

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

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

Preparation of Small RNA Libraries for Sequencing from Early Mouse Embryos