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

Huacheng Luo, Amin Sobh, Christopher D. Vulpe, Edmond Brewer, Sinisa Dovat, Yi Qiu, Suming Huang

Research output: Contribution to journalArticle

Abstract

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.

Original languageEnglish (US)
JournalJournal of visualized experiments : JoVE
Issue number145
DOIs
StatePublished - Mar 31 2019

Fingerprint

Clustered Regularly Interspaced Short Palindromic Repeats
Chromatin
Screening
Genes
Gene Expression
Gene expression
Body Patterning
Binding Sites
Human Genome Project
Biological Phenomena
Binding sites
Hematopoiesis
Genetic Testing
Acute Myeloid Leukemia
Libraries
Embryonic Development
Genome
DNA

All Science Journal Classification (ASJC) codes

  • Neuroscience(all)
  • Chemical Engineering(all)
  • Biochemistry, Genetics and Molecular Biology(all)
  • Immunology and Microbiology(all)

Cite this

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title = "HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries",
abstract = "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.",
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HOX Loci Focused CRISPR/sgRNA Library Screening Identifying Critical CTCF Boundaries. / Luo, Huacheng; Sobh, Amin; Vulpe, Christopher D.; Brewer, Edmond; Dovat, Sinisa; Qiu, Yi; Huang, Suming.

In: Journal of visualized experiments : JoVE, No. 145, 31.03.2019.

Research output: Contribution to journalArticle

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AU - Qiu, Yi

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