13 Jul A new solution to CRISPR side effects

Article recently published by Team 08 – Biotherapies Génétique et Oncologie (BioGO) inNature communications

Authors: Cullot G*, Boutin J*, Fayet S*, Prat F*, Rosier J, Cappellen D, Lamrissi I, Pennamen P, Bouron J, Amintas S, Thibault C, Moranvillier I, Laharanne E, Merlio JP, Guyonnet-Duperat V, Blouin JM, Richard E, Dabernat S, Moreau-Gaudry F*, Bedel A* (*Contributed equally).

The CRISPR-Cas9 system is derived from bacterial immunity. It has been used to modify the genome. This discovery won Jennifer Doudna and Emmanuelle Charpentier the Nobel Prize for Chemistry in 2020. Thanks to its efficiency and ease of use, it has revolutionized our ability to precisely modify the genome, leading to gene editing for clinical applications. By targeting the genome via an RNA guide, it enables a precise double-stranded cut to be made in the DNA at a given locus. This cut enables the targeted gene to be invalidated or corrected.

Unfortunately, side effects known as genotoxicity were soon described. Off-target cuts can induce modifications to genes not initially targeted, or major changes such as chromosome translocations (“OFF-target” genotoxicity). Fortunately, this type of genotoxicity is now under control thanks to the development of “high-fidelity” CRISPR-Cas9 tools. More recently, another form of genotoxicity has been described. Double-strand breaks in targeted genes can lead to a wide range of genomic modifications, not always desired or anticipated, due to the complexity of DNA double-strand break repair mechanisms (ON-target genotoxicity).

ON-target genotoxicity has long been underestimated. Indeed, it can induce large anomalies of several thousand to several million bases that are not detectable by standard PCR-based methods. Using genomic methods (FISH, single cell CGH-array/SNP-array), our laboratory has been able to identify major chromosomal changes in 0.1 to 10% of cells [1] :

1. loss of chromosome end, called CL-LOH for loss-of heterozygosity with copy-loss, or
2. modification of the chromosomal end identical to the other allele, like “copy and paste”. These are known as CN-LOH for copy-neutral loss-of heterozygosity.

We have shown that these anomalies are induced by the double-strand break, independently of the break site and in several cell types.[2,3]. Elles peuvent par exemple être mises en évidence dans les cellules souches hématopoïétiques, cellules d’intérêt pour la thérapie génique des maladies génétiques hématologiques, en ciblant les gènes de globines [3].

These large anomalies can result, for example, in a homozygous mutation affecting a previously heterozygous tumor suppressor gene. Unexpectedly, these modifications can also induce epigenetic changes. We have highlighted methylation anomalies in genes with parental imprints upstream of the cut at the 11p15 locus, inducing transcriptional modifications [3].

Fortunately, these rare events are very difficult to identify. We have developed two sensitive detection systems based on cytometry. These two systems, called FAMReD, enable the detection, quantification and cell sorting of edited cells with chromosomal rearrangement. These tools have enabled us to show that the frequency of these events depends on p53 and proliferation. Cells deficient in p53 or proliferating at the time of CRISPR editing are more susceptible to ON-target genotoxicity. Conversely, quiescent cells appear to be protected. We have shown that the cdk4/6 inhibitor palbociclib, which temporarily blocks cells in G1 phase prior to CRISPR editing, significantly reduces these undesired events. These results have just been published in Nature communications [4]. A patent to this effect has been filed withINSERM Transfert.

CRISPR is already used in clinical trials. These results show that it would be desirable to adapt gene-editing protocols using double-strand breaks for safer use in the clinic.

Financial support : ANR, AFM Téléthon, Ligue contre cancer

Collaborations : CHU Bordeaux, TBM Core, INSERM Transfert

[1] ON-Target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected. Boutin J, Cappellen D, Rosier J, Amintas S, Dabernat S, Bedel A, Moreau-Gaudry F. CRISPR J. 2022 Feb;5(1):19-30. doi: 10.1089/crispr.2021.0120. PMID: 35099280/

[2] CRISPR-Cas9 genome editing induces megabase-scale chromosomal truncations. Cullot G*, Boutin J*, Toutain J, Prat F, Pennamen P, Rooryck C, Teichmann M, Rousseau E, Lamrissi-Garcia I, Guyonnet-Duperat V, Bibeyran A, Lalanne M, Prouzet-Mauléon V, Turcq B, Ged C, Blouin JM, Richard E, Dabernat S, Moreau-Gaudry F*, Bedel A*. Nat Commun. 2019 Mar 8;10(1):1136. doi: 10.1038/ s41467-019-09006-2. PMID: 30850590

[3] CRISPR-Cas9 globin editing can induce megabase-scale copy-neutral losses of heterozygosity in hematopoietic cells. Boutin J*, Rosier J*, Cappellen D, Prat F, Toutain J, Pennamen P, Bouron J, Rooryck C, Merlio JP, Lamrissi-Garcia I, Cullot G, Amintas S, Guyonnet-Duperat V, Ged C, Blouin JM, Richard E, Dabernat S, Moreau-Gaudry F*, Bedel A*. Nat Commun. 2021 Aug 13;12(1):4922. doi: 10.1038/s41467-021-25190-6. PMID: 34389729

[4] Cell cycle arrest and p53 prevent ON-target megabase-scale rearrangements induced by CRISPR-Cas9. Cullot G*, Boutin J*, Fayet S*, Prat F*, Rosier J, Cappellen D, Lamrissi I, Pennamen P, Bouron J, Amintas S, Thibault C, Moranvillier I, Laharanne E, Merlio JP, Guyonnet-Duperat V, Blouin JM, Richard E, Dabernat S, Moreau-Gaudry F*, Bedel A*. Nat Commun. 2023 Jul 10;14(1):4072. doi: 10.1038 /s41467-023-39632-w. PMID: 37429857