Name: hox loci focused crispr/sgrna library screening identifying critical ctcf boundaries
Uploaded: 2019-03-31T14:00:00
Description: Watch this Scientific Journal Video about HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries at JoVE.com

The authors also thank Nicholas Cesari for editing the manuscript. The work was supported by grants from National Institute of Health (S.H., R01DK110108, R01CA204044).

&#8203;1. CTCF sgRNALibrary Design Using an Online Tool
<ol>
	<li>Design the sgRNA targeting CTCF binding sites in the human HOX loci using the genetic perturbation platform (GPP) designer tool (https://portals.broadinstitute.org/gpp/public/analysis-tools/sgrna-design).</li>
	<li>Synthesize a total of 1,070 sgRNAs consisting of sgRNAs targeting 303 random targeting genes, 60 positive controls, 500 non-Human-targeting controls, and 207 CTCF elements or lncRNA targeting genes (Figure 1</stro...

<ol>
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Protein-coding gene related sgRNA libraries have been applied in a functional screening system to identifying genes and networks regulating specific cellular functions through sgRNA enrichment24,25,26,27,28. Several non-coding region related sgRNA libraries were also shown in gene-specific functional screens for distal and proximal regulating elements, includi...

Recent genome interaction studies revealed that the human nuclear genome forms stable topologically associating domains (TADs) that are conserved across cell types and species. The organization of the genome into separate domains facilitates and restricts interactions between regulatory elements (e.g., enhancers and promoters). The CCCTC-binding factor (CTCF) binds to TAD boundaries and plays a critical role in constraining interactions of DNA elements that are located in neighboring TADs1. However, genome wide CTCF binding data revealed that although CTCF mostly interacts with the same DNA-sites in different cell types, it often functions as a chromatin barrier at a specific site in one cell type but not in the other, suggesting that CTCF functions together with other activities in the formation of chromatin boundaries2. What remains unknown is whether the boundary elements (CTCF-binding sites) are directly linked to the biological function of CTCF, and how these links occur. Therefore, we hypothesize that specific CTCF binding sites in the genome directly regulate the formation of TADs and control promoter/enhancer interactions within these domains or between neighboring domains. The completion of the human and mouse genome sequencing projects and subsequent epigenetic analyses have uncovered new molecular and genetic signatures of the genome. However, the role of specific signatures/modifications in gene regulation and cellular function, as well as their molecular mechanism(s), have yet to be fully understood.
Multiple lines of evidence support that the CTCF-mediated TADs represent functional chromatin domains3,4,5. Although CTCF mostly interacts with the same DNA-sites in different cell types, genome wide CTCF ChIP-seq data revealed that CTCF often functions as a chromatin barrier in one cell type but not in the other2. CTCF plays an essential role during development by mediating genome organization4,6,7. Disruption of CTCF boundaries impaired enhancer/promoter interactions and gene expression, leading to developmental blockage. This suggests that CTCF mediated TADs are not only structural components, but also regulatory units required for proper enhancer action and gene transcription5,8,9.
HOX genes play critical roles during embryonic development and they are temporally and spatially restricted in their expression patterns. The HOXA locus forms two stable TADs separating anterior and posterior genes by a CTCF-associated boundary element in both hESCs and IMR90 cells1. Recent reports demonstrated that HoxBlinc, a HoxB locus associated lncRNA, mediates the formation of CTCF directed TADs and enhancer/promoter interactions in the HOXB locus. This leads to anterior HOXB gene activation during ESC commitment and differentiation10. Furthermore, at specific gene loci including the HOXA locus, alteration of CTCF mediated TAD domains changed lineage specific gene expression profiles and was associated with the development of disease states11,12. The evidence supports a primary function for CTCF in coordinating gene transcription and determining cell identity by organizing the genome into functional domains.
Despite its role in the embryonic development, during hematopoiesis, HOX genes regulate hematopoietic stem and progenitor cell (HS/PC) function. This is done by controlling the balance between proliferation and differentiation10,13,14,15. The expression of HOX genes is tightly regulated throughout the specification and differentiation of hematopoietic cells, with highest expression in HS/PCs. HOX gene expression gradually decreases during maturation, with its lowest levels occurring in differentiated hematopoietic cells16. HOX gene dysregulation is a dominant mechanism of leukemic transformation by dysregulating self-renewal and differentiation properties of HS/PCs leading to leukemic transformation17,18. However, the mechanism of establishing and maintaining normal vs. oncogenic expression patterns of HOX genes as well as associated regulatory networks remains unclear.
CRISPR-Cas9 sgRNA library screening has been widely used to interrogate protein-coding genes19 as well as non-coding genes, such as lncRNA20 and miRNA21 in different species. However, the cost to use the CRISPR-Cas9 sgRNA library to identify new genomic targets remains high, because high-throughput genome sequencing is often applied to verify the sgRNA library screening. Our sgRNA screening system is focused on the specific genome loci and evaluates the targeting sgRNAs through one-step RT-PCR according to the marker gene expression, such as HOXA9. Additionally, Sanger sequencing confirmed that the sgRNA was integrated into the genome, and Indel mutations can be detected to identify the sgRNA targeting site. Through the loci-specific CRISPR-Cas9 genetic screening, the CBS7/9 chromatin boundary has been identified as a critical regulator for establishing oncogenic chromatin domain and maintaining ectopic HOX gene expression patterns in AML pathogenesis12. The method can be widely applied to identify not only specific function of CTCF boundary in embryonic development, hematopoiesis, leukemogenesis, but also CTCF boundary as potential therapeutic targets for future epigenetic therapy.

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			<td height="21" style="height:21px;width:263px;">Lipofectamine 3000 reagent</td>
			<td style="width:155px;">Thermo Fisher Scientific</td>
			<td style="width:100px;">L3000-008</td>
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			<td>Thermo Fisher Scientific</td>
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			<td height="21" style="height:21px;">Puromycin</td>
			<td>Thermo Fisher Scientific</td>
			<td>A1113802</td>
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			<td height="21" style="height:21px;">Stbl3 cells&#160;</td>
			<td>Life Technologies</td>
			<td>&#160;C737303</td>
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			<td>ATCC</td>
			<td>CRL-3216</td>
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			<td height="21" style="height:21px;">MOLM-13</td>
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			<td>ACC 554</td>
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			<td height="21" style="height:21px;">lentiCRISPRv2</td>
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			<td height="21" style="height:21px;">pMD2.G</td>
			<td>Addgene</td>
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			<td>Addgene</td>
			<td>12260</td>
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			<td height="21" style="height:21px;">pGEM&#174;-T Easy Vector Systems&#160;</td>
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			<td>A137A</td>
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			<td height="21" style="height:21px;">T4 ligase&#160;</td>
			<td>New England Biolabs</td>
			<td>&#160;M0202S</td>
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			<td height="21" style="height:21px;">QIAquick Gel Extract kit</td>
			<td>QIAGEN</td>
			<td>28706</td>
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			<td height="21" style="height:21px;">QIAuick PCR purification kit</td>
			<td>QIAGEN</td>
			<td>28106</td>
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			<td height="21" style="height:21px;">SingleShot&#8482; SYBR&#174;&#160;Green One-Step Kit</td>
			<td>Bio-Rad Laboratories</td>
			<td>1725095</td>
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			<td height="21" style="height:21px;width:263px;">QIAGEN Plasmid Maxi Kit</td>
			<td>QIAGEN</td>
			<td style="width:100px;">12163</td>
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			<td height="21" style="height:21px;">Dulbecco&#8217;s Modified Eagle Medium&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:100px;">&#160;11965084</td>
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			<td height="21" style="height:21px;">RPMI 1640&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:100px;">11875093</td>
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			<td height="21" style="height:21px;">Fetal bovine serum (FBS)&#160;</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:100px;">10-082-147</td>
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			<td height="21" style="height:21px;">Penicillin/streptomycin/L-glutamine&#160;</td>
			<td>Life Technologies&#160;</td>
			<td style="width:100px;">10378016</td>
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			<td style="width:155px;">Clontech</td>
			<td style="width:100px;">631232</td>
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			<td height="21" style="height:21px;width:263px;">Trypan Blue Solution</td>
			<td>Thermo Fisher Scientific</td>
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			<td height="21" style="height:21px;width:263px;">Polybrene</td>
			<td style="width:155px;">Santa Cruz Biotechnology</td>
			<td>sc-134220</td>
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hox loci focused crispr/sgrna library screening identifying critical ctcf boundaries

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements that are located in neighboring TADs. CTCF plays an important role in regulating the spatial and temporal expression of HOX genes that control embryonic development, body patterning, hematopoiesis, and leukemogenesis. However, it remains largely unknown whether and how HOX loci associated CTCF boundaries regulate chromatin organization and HOX gene expression. In the current protocol, a specific sgRNA pooled library targeting all CTCF binding sites in the HOXA/B/C/D loci has been generated to examine the effects of disrupting CTCF-associated chromatin boundaries on TAD formation and HOX gene expression. Through CRISPR-Cas9 genetic screening, the CTCF binding site located between HOXA7/HOXA9 genes (CBS7/9) has been identified as a critical regulator of oncogenic chromatin domain, as well as being important for maintaining ectopic HOX gene expression patterns in MLL-rearranged acute myeloid leukemia (AML). Thus, this sgRNA library screening approach provides novel insights into CTCF mediated genome organization in specific gene loci and also provides a basis for the functional characterization of the annotated genetic regulatory elements, both coding and noncoding, during normal biological processes in the post-human genome project era.

CRISPR-Cas9 technology is a powerful research tool for functional genomic studies. It is rapidly replacing conventional gene editing techniques and has high utility for both genome-wide and individual gene-focused applications. Here, the first individually cloned loci-specific CRISPR-Cas9-arrayed sgRNA library contains 1,070 sgRNAs consisting of sgRNAs targeting 303 random targeting genes, 60 positive controls, 500 non-Human-targeting controls, and 207 CTCF elements or lncRNA targeting ge...

Watch this Scientific Journal Video about HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries at JoVE.com

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

A CRISPR/sgRNA library has been applied to interrogating protein-coding genes.However, the feasibility of a sgRNA library to uncover the function of a CTCF boundary in gene regulation remains unexplored. Here, we describe a HOX loci specific sgRNA library to elucidate the function of CTCF boundaries in HOX loci.

HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries

CCCTC-binding factor (CTCF)-mediated stable topologically associating domains (TADs) play a critical role in constraining interactions of DNA elements ...

Research

JoVE Journal

Genetics

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

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

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