ASXL1 mutation correction by CRISPR/Cas9 restores gene function in leukemia cells and increases survival in mouse xenografts.

Simona Valletta,Swagata Roy,Amit Verma,Matthias Bartenstein,Laura Scifo,Jacqueline Boultwood,Shanisha Gordon,Andrea Pellagatti,Jacqueline Shaw,Erica Bello,Anna Schuh,Yiting Yu,Bon Ham Yip,Tudor A Fulga,Hamid Dolatshad

doi:10.18632/oncotarget.6392

Abstract

Recurrent somatic mutations of the epigenetic modifier and tumor suppressor ASXL1 are common in myeloid malignancies, including chronic myeloid leukemia (CML), and are associated with poor clinical outcome. CRISPR/Cas9 has recently emerged as a powerful and versatile genome editing tool for genome engineering in various species. We have used the CRISPR/Cas9 system to correct the ASXL1 homozygous nonsense mutation present in the CML cell line KBM5, which lacks ASXL1 protein expression. CRISPR/Cas9-mediated ASXL1 homozygous correction resulted in protein re-expression with restored normal function, including down-regulation of Polycomb repressive complex 2 target genes. Significantly reduced cell growth and increased myeloid differentiation were observed in ASXL1 mutation-corrected cells, providing new insights into the role of ASXL1 in human myeloid cell differentiation. Mice xenografted with mutation-corrected KBM5 cells showed significantly longer survival than uncorrected xenografts. These results show that the sole correction of a driver mutation in leukemia cells increases survival in vivo in mice. This study provides proof-of-concept for driver gene mutation correction via CRISPR/Cas9 technology in human leukemia cells and presents a strategy to illuminate the impact of oncogenic mutations on cellular function and survival.

Highlights

The clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) is a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements
The CRISPR/Cas9 system has recently emerged as a powerful genome editing tool that has been used for gene correction in cells derived from patients with inherited disorders
We sought to explore the potential of CRISPR/Cas9 genome editing to correct acquired mutations in human leukemia cells and to determine the impact of mutation correction on cellular function

Summary

Introduction

The clustered regularly interspaced short palindromic repeats (CRISPR)–CRISPR-associated protein 9 (Cas9) (CRISPR/Cas9) is a microbial adaptive immune system that uses RNA-guided nucleases to cleave foreign genetic elements. This system has recently emerged as a powerful and versatile tool for genome engineering in various species, and can be used to correct gene mutations in cells via genome editing [1,2,3,4]. It has been recently demonstrated that the CRISPR/Cas system can be used for rapid genome editing in mouse embryos and human stem cells in culture [3, 10,11,12]. Expansion of Fah-positive hepatocytes rescued the body weight loss phenotype [12]

Methods

Results

Conclusion