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

doi:10.3791/59382

Method Article

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

7.8K views

⸱

DOI:

10.3791/59382

⸱

March 31st, 2019

Huacheng Luo¹ , Amin Sobh² , Christopher D. Vulpe² , Edmond Brewer¹ , Sinisa Dovat¹ , Yi Qiu³ , Suming Huang¹

¹Department of Pediatrics, Pennsylvania State University College of Medicine, ²Department of Physiological Sciences, University of Florida, ³Department of Anatomy and Cell Biology, University of Florida

Summary

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.

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.

Introduction

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....