# ON-Target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected

Julian Boutin, David Cappellen, Juliette Rosier, Samuel Amintas, Sandrine

Dabernat, Aurélie Bedel, François Moreau-Gaudry

► To cite this version:

Julian Boutin, David Cappellen, Juliette Rosier, Samuel Amintas, Sandrine Dabernat, et al. ON-Target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected. The Crispr Journal, 2022, 5 (1), pp.19-30. 10.1089/crispr.2021.0120. hal-05158475

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REVIEW ARTICLE

# ON-target Adverse Events of CRISPR-Cas9 Nuclease: More Chaotic than Expected

Julian Boutin,<fnref n="1" />,²,³ David Cappellen,<fnref n="1" />,²,⁴ Juliette Rosier,<fnref n="1" />,² Samuel Amintas,<fnref n="1" />,²,⁴ Sandrine Dabernat,<fnref n="1" />,²,³ Aurélie Bedel,<fnref n="1" />,²,³,*,† and François Moreau-Gaudry<fnref n="1" />,²,³,†

## Abstract

CRISPR-Cas9 is a highly promising technology for clinical development. However, this powerful tool can induce adverse genomic events. The off-target genotoxicity is well described, predictable, detectable, and resolved by the use of new generations of Cas9 nucleases with high fidelity. In contrast, the ON-target genotoxicity due to a DNA double-strand break at the targeted locus is still underestimated. Here, we review several genomic outcomes induced by CRISPR-Cas9 from the insertion/deletion of a few bases to megabase-scale rearrangements. We hope to highlight this barely detectable complex safety concern to promote further studies to understand the mechanisms better, to detect these unwanted events, and to prevent them for the safety management of future CRISPR-Cas9 clinical trials.

## Introduction

Gene therapy fascinates scientists and clinicians because it represents an opportunity to cure diseases at the genetic level. It is a smart strategy that has been developed to fill the gaps of conventional drugs in the treatment of hereditary diseases. It was previously intended to treat monogenic diseases successfully, and more recently it was tested for more frequent diseases such as heart failure and cancer with chimeric antigen receptor T cells (CAR-T) or with oncolytic viruses to destroy tumor cells.<fnref n="1" />,² Most gene therapy protocols use additive gene therapy to add a transgene and its promoter into the genome of diseased cells.

Due to semi-random integration in the genome, onco gene transactivation may occur with retroviral vectors.³,⁴ Despite the development and optimization of new vectors that have a lower risk of insertional mutagenesis, the risk/ benefit ratio of using gene therapy to treat non-lethal ge netic diseases is debated.

The holy grail of gene therapy is to correct mutated genes by precise genome editing that respects the endoge nous promoter and its physiological regulation. This is now feasible with engineered DNA endonucleases that

bind specifically to a targeted DNA sequence and induce double-stranded cleavage. The first tools developed were the I-SceI meganuclease, zinc-finger nucleases (ZFN), and transcription-activator-like effector nucleases (TALEN), followed more recently by CRISPR-Cas.⁵⁻¹⁰

The discovery of the CRISPR-Cas9 tool in 2012 led to the renaissance of gene therapy. CRISPR-Cas9 is very powerful, versatile, easy-to-use, and inexpensive. Bacterial CRISPR has enabled targeted gene correction named “genome editing” to flourish.¹¹

It is made of a ribonucleoprotein (RNP) with a guide RNA (gRNA) and an endonuclease, Cas9. gRNA is a combination of CRISPR RNA (crRNA), which is a sequence of about 20 nt, complementary to the sequence of the target gene, and of transactivating crRNA (tracrRNA), which is used for the maturation of crRNAs and the binding to Cas9.⁸ When the gRNA hybridizes to the target sequence, it brings Cas9 with it. To target the desired loci, the presence of a protospacer adjacent motif (PAM) sequence, an interacting domain between the gRNA and the DNA consisting of three NGG nucleotides for Cas9 from *Streptococcus pyogenes*, is necessary for Cas9 to recognize the DNA cleavage site.

<footnote n="1">¹Bordeaux University, Bordeaux, France; ²INSERM U1035, Biotherapy of Genetic Diseases, Inflammatory Disorders and Cancers, Bordeaux, France; ³Biochemistry Laboratory, University Hospital Bordeaux, Bordeaux, France; and ⁴Tumor Biology and Tumor Bank Laboratory, University Hospital Bordeaux, Bordeaux, France.

†These authors contributed equally to this work.</footnote>

<footnote>*Address correspondence to: Aurélie Bedel, PharmD-PhD, Université de Bordeaux, 146 rue Leo Saignat, Case10, 33076 Bordeaux, France, Email: aurelie.bedel@u-bordeaux.fr</footnote>
Cas9 then induces a DNA double-strand break (DSB) via its two nuclease domains, RuvC and HNH. DSBs can be repaired through several endogenous repair pathways, including non-homologous end joining (NHEJ), microhomology-mediated end joining (MMEJ), homologous recombination (HR), or single-strand annealing (SSA). While NHEJ and MMEJ result in random insertions/deletions (indels) and gene disruption at the target site, homology-directed repair (HDR) by HR or SSA can be harnessed to insert a specific DNA template (single stranded or double stranded) at the target site for precise gene editing.

This robust tool is revolutionary because it can target any gene in any species, thereby making gene editing easier and faster than ever. The tremendous potential of CRISPR for treating diseases is already clear with the first ongoing clinical trials. Moreover, it opens up possibilities well beyond human health, especially for agriculture and the food industry. The effectiveness of editing by CRISPR-Cas9 is widely documented in the literature.

However, its rapid development is not without some undesired side effects. First, off-target (OFF-T) genotoxic risks of the CRISPR-Cas9 system have become evident. They are due to unintentional cleavage(s) in non-targeted genomic loci because gRNA can tolerate mismatches.⁹,¹⁰,¹²⁻¹⁴ The ON-target (ON-T) risks, which are unwanted genomic rearrangements due to DSBs at the targeted locus, have received less attention than the OFF-T risks.

Here, we briefly review the development of engineered nucleases and CRISPR-based successful gene therapy applications. Then, we describe the genotoxicity associated with CRISPR-Cas9 technology, with particular emphasis on ON-T risks.

## Success Story of Nucleases: From Laboratory Research to Clinical Applications

The ability to create artificial DNA endonucleases that bind specifically to a chosen DNA sequence and induce DSBs has opened a new era in genome editing. Engineered nucleases achieve long-standing goals such as disrupting somatic genes, targeting transgenic integration, and *in situ* gene correction for gene therapy. Interestingly, transient expression of the nuclease complex and, in some cases, concomitant presence of a repair template is sufficient to obtain a permanently modified genome. Briefly, gene editing is a one-off “hit-and-run” process.

The first clinical use of gene-edited cell therapies began roughly a decade ago. The first gene-editing platform to enter clinical trials was ZFNs. For example, they were used to create a DSB in the *CCR5* gene, the major co-receptor used by human immunodeficiency

virus type 1 (HIV-1) in T lymphocytes.¹⁵ DSBs are rapidly repaired by a NHEJ non-conservative mechanism, leading to *CCR5* disruption in HIV-infected lymphocytes. This system has demonstrated its safety and the long-term persistence of the modified cells. Hematopoietic stem-cell (HSC) editing is currently being tested to provide a more effective and long-term antiviral effect (NCT02500849).¹⁶,¹⁷ For almost a decade, Sangamo's SB-728 ZFN for HIV patients was the only ZFN gene-edited cell therapy entering clinical trials.

More recently, gene targeting was used to inactivate a repressor transcription factor and thereby lift inhibition. For example, the disruption of the transcriptional repressor gene *BCL11A* in erythroid progenitors makes it possible to reactivate the expression of fetal γ-globins to compensate the dysfunction of the beta-globin chains in beta-thalassemia (NCT03432364) or in sickle cell anemia (NCT03653247).¹⁸

Gene correction has become more ambitious in attempting to replace a targeted sequence with an exogenous version of choice using the HDR pathway instead of adding it into the genome of the targeted cells. To achieve this, an artificial nuclease is used with an exogenous double-stranded DNA template having homologies to the targeted site, so that the new sequence is delivered to the cell at the desired site. This approach has great potential in *ex vivo* gene therapy because the targeted integration of an expression cassette into a safe, preselected site of the genome and the *in situ* repair of a mutant gene will ensure robust and physiological expression without the risk of insertional oncogenic mutagenesis. For example, adeno-associated vectors can deliver ZFN and a template in hepatocytes to insert the factor IX transgene into the endogenous albumin locus (NCT02695160).

Alternatively, another easier-to-use and more efficient tool has been developed for gene correction: TALEN, which have a straightforward design.¹⁹ To date, TALEN have been mainly used to modify CAR-T cells. For example, to bypass the immune system, they are used for TCRα constant gene *ex vivo* inactivation to develop a universal allogeneic approach that is termed “universal CAR-T cells” (NCT02808442, NCT02746952).²⁰ It is intended to overcome limitations of the autologous approach and aims to offer a standardized therapy with greater consistency, improved quality control, and immediate availability to patients.

Overall, like ZFNs, TALEN therapies are still in early clinical development, but confidence in their design and manufacturing has developed rapidly. However, ZFN technology is very tedious, since it requires finding efficient DNA binding blocks—a procedure that requires
considerable experience—and TALENs are time-
consuming for building DNA binding domains.

The most recent gene editing system is the CRISPR sys-
tem. Thanks to its simplicity and its versatility, CRISPR
gene editing clinical studies are rapidly emerging.

Like TALEN, its first application was cancer im-
munotherapy, with programmed cell death-1 (PD-1) dis-
ruption in patients with non-small cell lung cancer
(NCT02793856).<sup>21</sup> Recently, Stadtmauer *et al*. disrupted
PD-1 in T lymphocytes in three patients with advanced
refractory cancers. Autologous T cells were edited *ex
vivo* using CRISPR-Cas9 to knock out the immune
checkpoint inhibitor PD-1 prior to reinfusion back into
the patient (NCT03399448).<sup>22</sup> Other clinical trials
used CD19-specific CAR-T cell therapy for hematologi-
cal malignancies (NCT03398967, NCT03166878, and
NCT04438083).<sup>23</sup>

Two clinical trials reported good results in beta-
hemoglobinopathies: NCT04438083 for beta-thalassemia,
and NCT03164135 for sickle cell anemia via *BCL11A* dis-
ruption.²⁴ China achieved only moderate success in *CCR5*
deletion in patients with HIV-1 and acute lymphoblastic
leukemia (NCT03164135).²⁵ Finally, two recent *in vivo*
protocols have been developed for Leber’s congenital
amaurosis to correct an abnormal splice in the *CEP290*
gene (NCT03872479) and for ATTR amyloidosis
(NTLA-2001).²⁶,²⁷

Despite successes and rapid development, several ob-
stacles must be overcome before these strategies can be
fully exploited. Indeed, the efficacy of HDR-mediated
gene correction remains low in most primary cells of in-
terest, such as HSCs. It is poorly understood how cells
choose between NHEJ and HDR. Although CRISPR-
Cas9 may function efficiently within eukaryotic cells,
there remain concerns about the accuracy of Cas endonu-
clease effectors and their use for precise gene editing.
Because CRISPR-induced modifications in the genome
are definitive, it is important to evaluate the undesired
genotoxicity risk associated with therapeutic CRISPR-
Cas9 genome editing.

The OFF-T Genotoxicity Risk

The first serious concern about CRISPR-Cas9, the OFF-T
activity, is due to unwanted DSBs in a non-targeted geno-
mic locus.⁹,¹⁰,¹²⁻¹⁴ The specificity of DNA cleavage is de-
termined by Cas9 structure, gRNA complementarity to
DNA, and the presence of a PAM sequence adjacent to
the target sequence.

However, hybridization tolerates mismatches between
the gRNA and DNA or in the recognition of PAM.<sup>28,29</sup>
The ‘5’ non-seed sequence,” which contains the other
10 nucleotides, is not very specific.<sup>13,30–32</sup> Conversely,

the distal 3' part of the gRNA located next to the PAM
('10 nt seed sequence') tolerates very few mismatches.
Another factor is the number of mismatches, which influ-
ences OFF-T activity. Indeed, with two or more mis-
matches, the endonuclease activity is considerably
reduced.<sup>13</sup> The final issue is the recognition of alternative
PAM by SpCas9, which can accept PAM sequences other
than 5'-NGG-3', but with lower cleavage efficiencies like
5'-NAG-3'.<sup>29</sup>

Chromatin inaccessibility is not a critical parameter,
and it does not absolutely prevent OFF-T editing. There
is only a slight preference of OFF-T editing to open chro-
matin.<sup>33</sup> Cas9 is still able to access heterochromatin but
less efficiently.<sup>34</sup>

These OFF-T cuts can be potentially deleterious. The
most well-known consequence of this OFF-T genotoxicity
is gene inactivation. CRISPR-Cas9 may trigger random
mutations by error-prone NHEJ repair pathways at these
OFF-T cut sites.⁹ These unwanted OFF-T DSBs can
sometimes lead to complex chromosomal rearrangements,
even in primary cells.

For example, Park *et al*. reported frequent chromosomal rearrangements in HSC when the OFF-T cut was in the same chromosome as the targeted one. They observed 2% of cells with an extra-large (54 Mb) pericentric deletion due to the loss of genetic material between the desired DSB (*HBB* locus) on the short arm of chromosome 11 and an OFF-T cut on the long arm of chromosome 11. This deletion resulted in a shortened chromosome 11 without a centromere by joining the non-homologous ends. In addition, in 3% of cases, there was an extra-large inversion between the two cuts. Deletions and inversions were prevented using a high-fidelity Cas9 variant, demonstrating that these chromosomal rearrangements were due to OFF-T cleavage.³⁵ To minimize the risk of chromosomal instability in clinical trials for hemoglobinopathies, high-fidelity Cas9 should be used.

Two distinct DSBs located in two different chromos-
omes that are not immediately rejoined can also partici-
pate in chromosomal translocations.³⁶ The frequency of
translocations between two sites in the genome is depen-
dent on the frequency of DSBs at the two sites, how long
they persist before being rejoined, and their physical jux-
taposition (“synapse”).³⁷

Recently, Turchiano *et al.* used single targeted linker-
mediated polymerase chain reaction (PCR) sequencing
(CAST-seq) to quantify chromosomal rearrangements
and found that up to 0.5% of cells harbor translocations,
increasing to 1.6% when a gene with a sequence homol-
ogy is present.³⁸ For example, they reported a 0.5% risk
of chromosomal translocations in their study on HSC be-
tween the DSB site of Cas9 (CCR5) and an OFF-T site
located on the same chromosome 3 that can result in acentric or dicentric chromosomes. These events can persist long term *in vivo* (in macaques, for as long as 12 months post transplantation).³³

Accurate predictive methods for CRISPR-Cas9 OFF-T site identification are crucial prior to applying such systems therapeutically. Detecting OFF-T sites in a highly sensitive and comprehensive manner remains a key challenge in the field of gene editing. Several tools have been developed to predict and detect them.³⁹⁻⁴⁵

Fortunately, these OFF-T outcomes can be limited by optimizing the design of CRISPR-Cas9 single gRNA (sgRNA),⁴⁶ double nickase,⁴⁷ enhanced-specific Cas9,²⁸,⁴⁸⁻⁵¹ and more recently by using CRISPR GUARD,⁵² which protects OFF-T sites from gRNA activity.

## The ON-T Genotoxicity Risk

While much attention has been paid to unintended OFF-T genotoxicity, an increasingly abundant literature is emerging on the ON-T risks. ON-T genotoxicity is defined by presence of unwanted genomic modifications at the targeted locus. It is very heterogeneous, with many different outcomes and on a variable scale, from a few bases to the chromosome scale (Fig. 1 and

Table 1). Unlike the OFF-T risk, the ON-T risk cannot be reduced by more specific DSB approaches. To understand the ON-T risk, it is important to examine many types of abnormalities: small (few bases), kilobase-scale, and megabase-scale rearrangements.

### Small rearrangements

After a DSB, the most efficient repair pathway is NHEJ. NHEJ can cause various lengths of indel mutations, which can lead the targeted gene to lose its function by shifting the open reading frame. Most of these indels correspond to nonrandom small rearrangements (indel of <50 bp).⁸,⁹,⁵³ NHEJ is used to invalidate genes in order to study their functional consequences.

However, NHEJ is undesirable in the event of gene correction by HDR with a repair template. In addition to correction by HDR, the concomitant NHEJ-dependent indels frequently inactivate the other targeted allele and decrease or even eliminate the residual activity of the protein, which is a serious concern for the edited cell. For example, in an ornithine transcarbamylase (OTC) mouse model, Yang *et al.* found that the correction by the adeno-associated virus (AAV) of the *OTC* mutation was accompanied by deleterious deletions.⁵⁴ These rearrangements ablated residual expression from the

<figure><img src="image_3.png" /><figcaption>**FIG. 1.** Schematic representation of on-target (ON-T) genotoxic events induced by one CRISPR-Cas9 double-strand break (DSB) reported in the literature. CRISPR can induce kilobase-scale rearrangements (interstitial deletion in white and interstitial copy-neutral loss of heterozygosity [CN-LOH] by gene conversion in red). It can also induce megabase-scale ON-T events (terminal deletion, CN-LOH in red with risk of loss of imprinting, or chromothripsis). Finally, chromosome loss is described when the DNA cut is near the centromere.</figcaption></figure>
<table><caption>Table 1. ON-Target Large Rearrangement Types of Genotoxicity</caption><thead><tr><th>Genotoxicity type</th><th>Size</th><th>Cell type</th><th>References</th></tr></thead><tbody><tr><td rowspan="2">Deletions</td><td>Kilobase scale</td><td>Human HSPCs, human cell lines (HUDEP-2, RPE1, HEK293T), mouse zygotes, mESCs, mouse bone marrow cells</td><td>38,60–71</td></tr><tr><td>Megabase scale</td><td>Human cell lines (COLO320, HCT116, HEK293T, K562), human fibroblasts, human embryos, hESCs, human HSPCs, hiPSCs</td><td>57,75–78,89</td></tr><tr><td rowspan="2">Copy-neutral LOH</td><td>Chromosome loss</td><td>hiPSCs, human zygotes, hESCs, mouse zygotes, mESCs</td><td>74,75,102</td></tr><tr><td>Kilobase scale</td><td>Human zygotes, human HSPCs, primary human T cells, human cell lines (HEK293T, U2OS), human lung-derived stromal cells, mouse embryos</td><td>72,81–86</td></tr><tr><td rowspan="3">Complex rearrangements</td><td>Megabase scale</td><td>hESCs, hiPSCs, human cell line (HT1080 fibrosarcoma cell), human HSPCs</td><td>76,78,90,91</td></tr><tr><td>Gain and deletion</td><td>Human cell lines (HEK293T, K562, RPE1), human fibroblasts, human embryos, mESCs, mouse bone marrow cells</td><td>57,65,71</td></tr><tr><td>Chromothripsis</td><td>Human cell line (RPE1), human fibroblasts, human HSPCs</td><td>96,97</td></tr></tbody></table>

ESC, embryonic stem cells; HSPCs, hematopoietic stem/progenitor cells; iPSCs, induced pluripotent stem cells; LOH, loss of heterozygosity.

endogenous *OTC* gene, leading to diminished protein tolerance and lethal hyperammonemia. This imprecise genome editing results from simultaneous NHEJ and HDR repair pathways.

Numerous studies have demonstrated that HDR is less efficient than the NHEJ pathway. Without selection, it is difficult to obtain homozygously corrected cells by HDR. The study of the HDR/NHEJ ratio is a way to assess the efficiency of gene correction based on the amount of HDR obtained while considering the NHEJ, which can be harmful.⁵⁵⁻⁵⁷ To improve the HDR/NHEJ ratio, methods may be used to inhibit NHEJ or enhance HDR, such as chemical modulation, synchronized expression, overlapping homology arm, or modified Cas9 nucleases.⁵⁸,⁵⁹

## Kilobase-scale rearrangements

Alternatively, CRISPR-Cas9 DSB can induce larger events, in particular larger deletions (from 0.5 kb to several kilobases; Fig. 1).

## Interstitial deletions

With paired sgRNAs and two DSBs. To delete a specific genomic region using Cas9 nuclease, two (paired) sgRNAs are typically used to generate DSBs flanking a target region that is to be removed. Using paired Cas9 nuclease, Owens et al. observed unintended larger deletions that were expected at high frequency targeting different loci in different chromosomes. These deletions induce large interstitial loss of heterozygosity (LOH). They seemed to be dependent on the presence of micro-homology domains at the breakpoints, suggesting the implication of the MMEJ pathway.⁶⁰

Parikh et al. were the first to observe a high rate of large deletions from 600 bp to 42 kb in mice zygotes using a sgRNA targeting the *Tyr* gene.⁶¹ Such unanticipated large (kilobase-scale) deletions may be difficult to detect using classical PCR screening methods, as

primer-binding sites may be lost, resulting in the failure to amplify these alleles. They also observed a 2 kb inversion and insertion up to 5.5 kb, demonstrating the possibility of more complex rearrangements. Similar genomic events were observed during long-term evaluation of AAV-CRISPR *in vivo* genome editing for Duchenne muscular dystrophy. They can induce unintended transcript alterations.⁶²

With one sgRNA but two DSBs by homology. Studies have shown that these large deletions may be the result of a second cut site close to the target in the same chromosome due to sequence homology. Unlike classical OFF-T, enhanced specific Cas9 does not prevent this second cut without mismatch.

For example, in a preclinical study of CRISPR-mediated beta-hemoglobinopathy gene therapy, kilobase-scale deletions were observed using a sgRNA targeting *HBG* genes encoding fetal hemoglobin. Due to structural homology between the *HBG1* and *HBG2* promoters, gRNA against *HBG* induced two DSBs and created a 5.2 kb deletion by loss of genetic material contained between both cuts. This undesired rearrangement has been detected at a frequency of 20% of the erythroid human cell line (HUDEP-2).⁶³ These deletions were detected by qPCR and fluorescence *in situ* hybridization (FISH).

Moreover, kilobase-scale deletions at the *CCR5* locus were observed in human embryonic kidney (HEK) cells and in around 10% of edited human hematopoietic cells by CAST-Seq.³⁸,⁶⁴ They are due to a second cut in *CCR2*, with sequence homology with *CCR5*. Regions that share substantial homology with the ON-T region (pseudogene) are subject to RAD51-dependent chromosomal rearrangements.³⁸

Altogether, these data show that a CRISPR-Cas9 system with only one gRNA may cause kilobase-scale deletions if it induces cleavage at two defined locations. To prevent such unwanted chromosomal aberrations, it is
prudent whenever possible to avoid target loci that share
stretches of sequence homology with chromosomal re-
gions somewhere else in the genome.

*With one sgRNA and one DSB.* In 2018, Kosicki *et al.* demonstrated that CRISPR-Cas9 can induce large deletions after a single DSB in human cells.⁶⁵ After gene editing of the X-linked *PIGA* gene and the *CD9* gene on chromosome 12, they detected kilobase deletions up to 9.5 kb by long-range PCRs in murine embryonic stem (ES) cells, primary mouse cells from the bone marrow, and human retinal pigment epithelial (RPE) cells. Consistent with this, other studies then confirmed the existence of these large deletions at the cleavage site from 100 bases to a few kilobases targeting many loci, in different chromosomes and in different cell types.

For example, Adikusuma *et al.* delivered CRISPR-Cas9 into mouse embryos by microinjection to target six different loci. They confirmed frequent large deletions from 100 bb to 2.3 kb in 35/127 (45%) zygotes.⁶⁶ In the mouse genome, CRISPR-mediated large deletions induced by a sgRNA are mostly asymmetric, starting at the cutting site, with extension either toward the 5' end or the 3' end of the gRNA. They can be detected by UDitas, HTGTS,⁶⁷ and nanopore sequencing.⁶⁸ Symmetric deletions with equal loss upstream and downstream of the cut site are less frequent.⁶⁹,⁷⁰

Recently, ON-T damage was observed in human em-
bryos targeting the *POUF5 (OCT4)* gene on chromosome
6. Eight out of 25 embryos had abnormalities on chromo-
some 6. Four (16%) presented a segmental loss or gain
(4–20 kb) adjacent to *POUF5*.⁷¹ Interestingly, similar to
deletions after paired DSB, MMEJ is also implicated in
these large deletions after a single DSB.⁶⁰ Indeed, they
preferentially occur at repeat sequences. Significant
microhomologies are detected at almost all large dele-
tion breakpoint junctions.⁷⁰ Bridging of broken ends by
template could decrease large deletions induced by
CRISPR-Cas9.⁶⁸

In addition to kilobase-scale deletions, some studies
reported more complex rearrangements, including gain.⁶⁵,⁷¹

Copy neutral LOH

In parallel, other interstitial LOH without loss of genetic
material, called copy-neutral LOH (CN-LOH), have been
reported after CRISPR-mediated DSB (Fig. 1). They are
due to gene conversion or interhomolog repair (IHR),
which involves the unidirectional transfer of genetic ma-
terial from intact homologous sequences to regions con-
taining DSBs. In mammalian cells, gene-conversion
tracts are usually short and rarely exceed 1 kb. They are
mainly described in meiotic cells but can also occur

more rarely in mitotic cells. The importance of gene
conversion and, in general, the use of an endogenous re-
pair template for gene editing experiments came to light
recently.

First, Ma *et al.* reported the correction of a heterozygous paternally inherited *MYBPC3* dominant mutation responsible for a severe congenital hypertrophic cardiopathy in human zygotes by CRISPR-Cas9 through an endogenous HDR mechanism using the maternal wild-type allele as a repair template.⁷² The authors hypothesized that the maternal allele was exchanged through IHR. However, some scientists commented that PCR used to analyze correction cannot discriminate editing by the healthy allele and a large deletion.⁷³⁻⁷⁵ Recent data confirm that both events can occur.⁷⁶⁻⁷⁸ Array comparative genomic hybridization (CGH) could resolve this interesting issue.

Meiotic gene conversions between *HBB* and *HBD* have already been described. The gene conversion transfers the *HBB* sequence into *HBD*.⁷⁹,⁸⁰ Several groups observed *HBD* footprints in the *HBB* gene when using CRISPR-Cas9 to target the human *HBB* gene in different human cell types.⁸¹⁻⁸⁴

Recently, Wilde *et al.* observed gene conversion in early murine embryos that targeted the albinism-associated *Tyr* mutation.⁸⁵ Interestingly, the gene conversion rate seemed to be correlated with the cut efficacy and was unidirectional. Indeed, introducing a DSB in *HBB* leads to *HBD* footprints in *HBB*. When the DSB was in the *HBD* gene, genetic information flowed in the opposite direction (*HBB* to *HBD*). The frequency of events was lower in human primary cells (0.15%) than in p53-deficient HEK293T cells (2.3%), suggesting the role of p53. Another major actor seems to be Rad51.⁸³,⁸⁴

CRISPR-Cas9 can also mediate mitotic gene conversion between highly homologous sequences, an effect that has been overlooked so far.⁸⁶ These events are probably underestimated because they are not detectable by regular methods such as array CGH. If gene conversion were to be more prevalent than originally thought, it would pave the way for a new type of gene correction based on homologous regions in the genome as repair templates.

Megabase-scale rearrangements

*Terminal deletions.* Cullot *et al.* were the first to demonstrate the existence of extra-large deletions up to 7.5 Mb after a single DSB targeting the *UROS* gene on chromosome 10 (Fig. 1). These terminal deletions start at the *UROS* breakpoint to the Chr10q distal telomere and induce LOH of 43 genes. Around 10% of cells (human cell lines and human P53-deficient fibroblasts) exhibited
truncations that were identified by FISH and confirmed by array CGH.⁵⁷ In contrast, these events were under the FISH detection threshold in p53-proficient human fibroblasts.

It is possible that DSB and truncations induced a p53-mediated DNA damage response and apoptosis, whereas survival is possible in p53-deficient cells. Indeed, low P53 activity promoted cell editing by CRISPR-Cas9 and their survival.⁸⁷,⁸⁸ These results were then confirmed by targeting other loci in p53-deficient cells.⁷⁶,⁸⁹ More recently, the same genomic outcomes were observed in p53-competent cells. Przewrocka *et al.* reported 6% of edited human colon carcinoma 116 cancer cells with chromosome arm truncations from the targeted *ZFN516* locus to the chromosome 18q telomere,⁷⁷ Weisheit *et al.* observed chromosome arm truncations in 16–40% of primary human stem cells (induced pluripotent stem cells [iPSC]) targeting *APP* and *HDAC9*,⁷⁸ and Zuccaro *et al.* showed that CRISPR leads to frequent loss of the targeted chromosome 6 arm in human embryos when targeting the *EYS* pericentromeric locus.⁷⁵

These recent data suggest that *TP53* alone may not predict the predisposition of cells to large ON-T events, and that *TP53* is not the only promoter of this genotoxicity.

## Terminal CN-LOHs

Like kilobase-scale genotoxicity, megabase-scale CN-LOH without loss of genomic material can be induced by a single CRISPR cut (Fig. 1). The first observations were made in ES cells. Ikeda *et al.* first reported rare CN-LOHs induced by CRISPR-Cas9 in human ES cells.⁹⁰ This side effect was then detected in iPSC (8–40%) targeting *APP* and *HDAC9*.⁷⁸ More recently, these extra-large rearrangements were observed in non-embryonic cells, in cancer cell lines, and in hematopoietic stem/progenitor cells (HSPC).⁷⁶,⁹¹ Targeting the beta-globin locus, 1% of edited cells presented CN-LOH.⁷⁶

These observations suggest that CN-LOH may not be limited to one cell type or locus. However, frequency, rearrangement type, and functional consequences will probably be cell and locus dependent. This induced CN-LOH always starts from the cut site and extends to the telomere, without deletion, leading to partial uniparental disomy. These genomic outcomes are difficult to detect because FISH, karyotype, and array CGH findings are normal. Only SNP analysis can reveal this type of genomic abnormality.

CN-LOHs result from interhomolog recombination to prevent loss of a chromosomal end after DSB. The mechanism is probably consistent with the break-induced replication recently described in eukaryotic cells.⁹² CN-LOH might be a survival repair response to the loss of an allele in p53-proficient cells. It involves extensive DNA synthesis from the DSB to the telomere. The CN-

LOH cells then retain two copies derived from one parental source and no copy from the other parent. The duplication of the remaining allele leads to acquired homozygosity that can contribute to tumorigenesis by activating potential oncogenes or by unmasking mutated tumor suppressor genes. A growing list of cancer types present frequent and recurring CN-LOH.⁹³,⁹⁴

Moreover, these large CN-LOHs can erase parent-specific epigenetic DNA modifications and lead to loss of imprinting.⁷⁶ For example, the beta-globin locus, which is the target of numerous gene therapies of beta-hemoglobinopathies, had imprinting centers telomeric to the breakpoint. CN-LOHs (5.2 Mb induced) lead to chromosome 11p15.5 partial uniparental disomy, comprising two chromosome 11p15.5 imprinting centers (*H19/IGF2:IG-DMR/IC1* and *KCNQ1OT1:TSS-DMR/IC2*), thereby impacting their methylation and resulting in *H19* and *IGF2* mis-expression.⁷⁶

## Chromothripsis

Chromothripsis is an unexpected novel form of complexity in chromosomal rearrangements. Up to thousands of clustered chromosomal rearrangements occur in a single event in localized and confined genomic regions in one chromosome. This mutational process dismantles a part of the chromosome and then reassembles it in random order resulting in “a chromosomal patchwork” (Fig. 1). This massive genomic reorganization is frequently accompanied by micronuclei.⁹⁵

Recently, two publications reported that Cas9 cuts can induce chromosome bridges and micronuclei formation in dividing cells (up to 8% of edited immortalized human RPE, in edited foreskin fibroblasts, in edited primary HSC, and in mouse embryos).⁹⁶,⁹⁷ FISH analysis showed that in >80% of cases, micronuclei contained the acentric chromosomal fragment relative to the CRISPR-Cas9 cut site.

The authors hypothesized that Cas9 cleaves the targeted chromosome into two segments: one with the centromeric region and one with the telomeric region. If the DSB is not repaired before cell division, the telomeric fragment lacking a functional centromere can mis-segregate, forming chromosome bridges and micronucleus. Of note and major concern, in several cases, the evicted chromosome fragment was reincorporated into the genome but in a seriously rearranged form, in some cases leading to LOH without a loss of copy number, in accordance with the observation of CN-LOH previously described. Due to mis-segregation, abnormal copy numbers (loss or gain) or CN-LOH (with uniparental disomy) may occur.

In this study, p53 activity did not influence the formation of micronuclei.⁹⁶ Only half of the micronucleated
p53-proficient cells did not undergo cell division, suggesting that p53 was not sufficient to induce apoptosis of these rearranged cells. These micronuclei appeared to target four loci on four chromosomes and even to target the clinically relevant *BCL11A* gene.

The mechanism of shattering and repair in chromothripsis is not well understood. During anaphase, acentric fragment chromosome cleaved by CRISPR-Cas9 is not segregated. This fragment can be encapsulated by the nuclear envelope outside the nucleus to form a micronucleus.

In response to mitotic signals, the DNA can be further fragmented. Micronuclear DNA can be either destroyed inducing extra-large LOH by deletion or CN-LOH or randomly reincorporated into the primary nucleus after cell division. The chromosome is imperfectly restored, the orientation and the order of the fragments can change, and some fragments can be removed (Fig. 2).

Many chromothripsis events may compromise cell fitness, leading to senescence or cell death. Chromothripsis

<figure><img src="image_4.png" /><figcaption>**FIG. 2.** Chromothripsis induced by CRISPR-Cas9 cleavage. (A) During anaphase, acentric fragment chromosome cleaved by CRISPR-Cas9 is mis-segregated. This fragment is encapsulated by the nuclear envelope outside the nucleus and formed a micronucleus. In response to mitotic signals, the DNA can be prematurely compact and fragmented. The further destiny of micronuclear DNA can be different. Its DNA can be randomly reincorporated into the primary nucleus of one of the daughter cells, or destroyed inducing extra-large LOH by deletion or CN-LOH. (B) Scheme of chromosomal rearrangements in chromothripsis. During chromothripsis, the chromosome arm targeted by CRISPR-Cas9 was destroyed, followed by incomplete and random restoration of segments. The order, and/or the orientation of the segments, can change and segment can be lost. This results in an abnormal rearranged chromosome.</figcaption></figure>
is also a frequent event in cancer cells.⁹⁸,⁹⁹ Recently, authors confirmed that chromothripsis events are pervasive across cancers, with a frequency of >50% in several cancer types, in both p53-deficient and -proficient cells.¹⁰⁰ In addition to chromothripsis, more complex rearrangements including both losses and gains (duplications/triplications) region on the rearranged chromosome can occur and could be due to chromoanasynthesis.

Chromothripsis contributes to oncogene amplification and to inactivation of genes such as mismatch repair-related genes.

Although rare, these events induced by CRISPR could facilitate clonal expansion and could be impactful in clinical-scale genome editing. In addition to chromothripsis, even more complex rearrangements, including both losses and gains (duplications/triplications), on the rearranged chromosome can occur in HEK cells.⁵⁷ Mechanism involved in these gains are unknown. They could be due to another mechanism named chromoanasynthesis.¹⁰¹

Research is needed to probe the potential negative consequences of this phenomenon and ways to mitigate them.

## Chromosomal translocations

Chromosomal translocations³⁶ and inversions can be modeled by CRISPR-Cas9 with two cuts.¹⁰² When researchers wish to induce numerous desirable DSBs in the same cells, translocations can occur. During a Phase I clinical trial of NCT03399448, concomitant TRAC, TRBC, and PDCD1 inactivation by CRISPR in T cells for cancer immunotherapy induced translocations in patients' cells.²² Fortunately, these genomic outcomes did not appear to provide a selective advantage because abnormal cells tended to disappear 6 months after grafting.

## Chromosomal losses

CRISPR-Cas9 can also give rise to chromosomal scale abnormalities. Multiple cleavages in the same chromosome by different gRNAs or by gRNAs targeting repeat sequences can induce sex chromosome or autosome elimination in mouse zygotes. Zuo *et al.* achieved complete elimination of the Y chromosome by several CRISPR-Cas9-mediated DNA cleavages on the targeted chromosome in ES cells and in zygotes with high efficiency.¹⁰³ They also promoted extra human chromosome 14 or chromosome 21 loss in aneuploid ES cells, as well as human chromosome 7 loss in human cancer cells.¹⁰³ Adikusuma *et al.* demonstrated that two pericentromeric cuts by the same gRNA due to homology are sufficient to induce loss of the entire Y chromosome in 40% of murine ES cells.⁷⁴

Recently, even a single DSB in the *EYS* locus near the chromosome 6 centromere was sufficient to delete an entire chromosome in a human zygote.⁷⁵ Indeed, half of the DNA breaks remained unrepaired, leading to one undetectable allele, and after mitosis, resulting in frequent loss of one or both chromosomal arms. Recently, Papathanasiou *et al.* elucidated how CRISPR-Cas9 can lead to whole chromosome loss in mouse embryos. They highlighted the chromosome breakage-fusion-bridge mechanism.⁹⁷

Significant challenges remain for correcting human embryos. Researchers should always be aware that CRISPR cutting can destabilize the entire targeted chromosome. However, these observations offer promise for developing animal models with chromosome deletion and a potential therapeutic strategy for human trisomy diseases.

## Conclusions

Clinical trials of gene therapy with CRISPR-Cas9 have already begun in genetic diseases following the promising results of numerous preclinical studies. While OFF-T genotoxicity now seems to be part of the evaluations following a DSB by CRISPR-Cas9, the awareness of ON-T genotoxicity is more recent. DSBs can generate many genomic rearrangements at the targeted site: small insertions, deletions or point mutations, large deletions up to chromosomal truncations, inversions, CN-LOH, translocations, aneuploidy, and even chromothripsis.

Taking into account the diversity and the complexity of these large ON-T genomic events induced by CRISPR, a single technique alone cannot highlight them all. It is important to combine different approaches such as next-generation sequencing (difficulty to detect large translocation and deletions), array CGH, SNP array, and FISH and karyotyping with G-banding for example (do not detect small aberrations and balanced rearrangements).

While only a small percentage of edited cells present ON-T events, the numbers of CRISPR-edited cells needed for therapeutics are very high. For example, in hematological disorders, $5-20 \times 10^6$ edited HSPC per kilogram are grafted. According to around 1% of megabase scale CN-LOH in edited cells, $0.5-2 \times 10^5$ grafted HSPC per kilogram could present ON-T large genomic rearrangements.⁷⁶ As many ON-T genomic rearrangements are linked to tumorigenesis, the long-term effects of even small percentages of aberrant cells being introduced into patients require further investigation.

ON-T genotoxicity is a severe adverse event that compromises genomic integrity and cell fitness and typically results in cell death. Other functional consequences such
as selective advantage and proliferation require further investigation. They are probably locus-dependent or dependent on the gene environment, with the presence of tumor suppressor genes or oncogenes whose expression may be affected by chromosome remodeling induced by CRISPR-Cas9 nuclease. Frangoul *et al.* reported that patients appeared to be functionally cured of all major symptoms of their disease.²⁴ Fortunately, no treatment-related adverse events due to genome editing have been reported until now in any clinical trials. Nevertheless, long-term follow-up data are not yet available to understand the full contribution of this fascinating technology.

Further explorations are required to understand better the scale and scope of CRISPR-induced undesired ON-T genomic abnormalities and to know whether edited cells are affected in the long term. Relevant quality aspects could then become part not only of the preclinical work required to initiate a clinical trial but also to screen out edited cells that contain ON-T rearrangements before the cells are reintroduced into the patient, and during long-term follow-up in clinical trials. The scientific community must now develop efficient and sensitive tools for searching for this ON-T genotoxicity easily and quickly, as soon as patients begin to be treated. The clinical utility of genome editing will depend on accuracy (ON- vs. OFF-T genetic changes) and precision (ON-T desired vs. unwanted genetic outcomes).

Attempts to circumvent DSBs during gene editing have resulted in the recent development of alternative approaches. Encouragingly, engineered systems that do not rely on a DSB, base-editors, nickase, or prime editing are rapidly advancing toward clinical use.¹⁰⁴

**Author Disclosure Statement**

No competing financial interests exist.

**Funding information**

This work was generously supported by ANR grant no. 21-CE18-002-01 and Ligue Régionale Nouvelle Aquitaine Contre le Cancer.

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