Efficient Recreation of t(11;22) EWSR1-FLI1+ in Human Stem Cells Using CRISPR/Cas9

Stem Cell Reports
Raul Torres-RuizSandra Rodriguez-Perales

Abstract

Efficient methodologies for recreating cancer-associated chromosome translocations are in high demand as tools for investigating how such events initiate cancer. The CRISPR/Cas9 system has been used to reconstruct the genetics of these complex rearrangements at native loci while maintaining the architecture and regulatory elements. However, the CRISPR system remains inefficient in human stem cells. Here, we compared three strategies aimed at enhancing the efficiency of the CRISPR-mediated t(11;22) translocation in human stem cells, including mesenchymal and induced pluripotent stem cells: (1) using end-joining DNA processing factors involved in repair mechanisms, or (2) ssODNs to guide the ligation of the double-strand break ends generated by CRISPR/Cas9; and (3) all-in-one plasmid or ribonucleoprotein complex-based approaches. We report that the generation of targeted t(11;22) is significantly increased by using a combination of ribonucleoprotein complexes and ssODNs. The CRISPR/Cas9-mediated generation of targeted t(11;22) in human stem cells opens up new avenues in modeling Ewing sarcoma.

Citations

Nov 15, 2018·Biomedicines·Marta Martinez-LageSandra Rodriguez-Perales
Jan 20, 2018·G3 : Genes - Genomes - Genetics·Emily RoggenkampGregory C Finnigan
Jun 21, 2019·Journal of Cellular Biochemistry·Vahideh AhmadzadehHabib Zarredar
May 3, 2019·BMC Medical Genetics·Raúl Torres-RuizPaloma García-Bellido
Jun 21, 2018·Frontiers in Pediatrics·Antonio Benítez-BurracoMa Salud Jiménez-Romero
Feb 16, 2019·Fungal biology and biotechnology·Megan E GoeckelGregory C Finnigan
Jun 9, 2020·Clinical Genetics·Roya RamezankhaniCatherine M Verfaillie
Oct 17, 2018·Frontiers in Immunology·Huensuk Kim, Christoph Schaniel
Aug 9, 2020·Frontiers in Genetics·Víctor Sánchez-GayaAlvaro Rada-Iglesias
May 10, 2018·Genetics in Medicine : Official Journal of the American College of Medical Genetics·Laura RemachaAlberto Cascón
Oct 10, 2020·Nature Communications·M Martinez-LageS Rodriguez-Perales
Nov 17, 2020·Frontiers in Genetics·Jennifer Redondo-AntónPilar Lauzurica
Dec 31, 2020·Cell Reports Medicine·David G P van IJzendoornValeria V Orlova
Feb 7, 2021·BMC Musculoskeletal Disorders·Wei LiuGuangrong Ji
May 8, 2021·Nature Plants·Michelle RönspiesHolger Puchta
Sep 26, 2021·Cancer Research·Troy A McEachron, Lee J Helman

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Methods Mentioned

BETA
transfection
PCR
fluorescence-activated cell sorting
FACS
targeted translocation
electrophoresis

Software Mentioned

ImageJ
Definiens Developer XD

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