CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations

Grégoire Cullot,Béatrice Turcq,Aurélie Bedel,Cécile Ged,Jérôme Toutain,François Moreau‐Gaudry ,Valérie Prouzet‐Mauléon ,Caroline Rooryck,Julian Boutin,Emmanuel Richard,Alice Bibeyran,Isabelle Lamrissi-Garcia,Véronique Guyonnet‐Duperat ,Emilie Rousseau,Florence Prat,Martin Teichmann,Sandrine Dabernat,Perrine Pennamen,Magalie Lalanne,Jean-Marc Blouin

doi:10.1038/s41467-019-09006-2

Abstract

CRISPR-Cas9 is a promising technology for genome editing. Here we use Cas9 nuclease-induced double-strand break DNA (DSB) at the UROS locus to model and correct congenital erythropoietic porphyria. We demonstrate that homology-directed repair is rare compared with NHEJ pathway leading to on-target indels and causing unwanted dysfunctional protein. Moreover, we describe unexpected chromosomal truncations resulting from only one Cas9 nuclease-induced DSB in cell lines and primary cells by a p53-dependent mechanism. Altogether, these side effects may limit the promising perspectives of the CRISPR-Cas9 nuclease system for disease modeling and gene therapy. We show that the single nickase approach could be safer since it prevents on- and off-target indels and chromosomal truncations. These results demonstrate that the single nickase and not the nuclease approach is preferable, not only for modeling disease but also and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies.

Highlights

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not
uroporphyrinogen III synthase (UROS) gene was edited in HEK293T cells by transient expression of Cas9-nuclease, a single-guide RNA (sgRNA) and 181nt-single-stranded oligodeoxynucleotide template
T > C in UROS exon 4, chromosome 10), we designed a sgRNA inducing a double-strand break (DSB) near the c.217 position and devised a 181nt-single-stranded oligodeoxynucleotide (ssODN) carrying a silent SacI restriction site close to the c

Summary

Introduction

HAL is a multi-disciplinary open access archive for the deposit and dissemination of scientific research documents, whether they are published or not. We show that the single nickase approach could be safer since it prevents on- and off-target indels and chromosomal truncations These results demonstrate that the single nickase and not the nuclease approach is preferable, for modeling disease and and more importantly for the safe management of future CRISPR-Cas9-mediated gene therapies. Nuclease is the standard approach for genome editing, it is still unknown whether a part or an entire chromosome could be eliminated after only one DSB in human cells Another goal of using CRISPR-Cas[9] is the possibility to perform homology-directed repair (HDR) to make precise genome editing (PGE) to insert or correct point mutations. It is still unclear whether large amounts of unwanted on-target indels could ablate the residual function of the original protein, thereby modifying the phenotype of cells and leading to iatrogenic effects

Methods

Results

Conclusion