Detection of Deleterious On-Target Effects after HDR-Mediated CRISPR Editing

Weisheit, Isabel; Kroeger, Joseph A.; Malik, Rainer; Klimmt, Julien; Crusius, Dennis; Dannert, Angelika; Dichgans, Martin; Paquet, Dominik

doi:10.1016/j.celrep.2020.107689

Cited by 101 publications

(125 citation statements)

References 19 publications

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“…Copy-neutral LOH is common in cancer and can result in tumor suppressor inactivation (48). Our findings therefore provide a clear mechanistic explanation for similar patterns that had been noted, but not explained, after CRISPR-Cas9 genome editing (28, 49, 50). In summary, on-target Cas9 genome editing can generate micronuclei, which in turn can induce large-scale DNA copy number alterations as well as copy-number neutral LOH.…”

Section: Resultssupporting

confidence: 80%

“…On-target DNA breakage can induce the TP53 tumor suppressor and potentially create selective pressure for TP53 loss followed by tumorigenesis (18–21). Additionally, there have been recent reports of local DNA rearrangements and deletions up to several kilobases in length (22–25), as well as regional megabase-scale deletions telomeric to the on-target DSB (26–28). However, the mechanisms leading to these DNA alterations remain poorly defined, in part because of the lack of high-depth whole genome sequencing.…”

Section: Introductionmentioning

confidence: 99%

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Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing

Leibowitz

Papathanasiou

Doerfler

et al. 2020

Preprint

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Genome editing has promising therapeutic potential for genetic diseases and cancer (1, 2). However, the most practicable current approaches rely on the generation of DNA double-strand breaks (DSBs), which can give rise to a poorly characterized spectrum of structural chromosomal abnormalities. Here, we show that a catastrophic mutational process called chromothripsis is a previously unappreciated consequence of CRISPR-Cas9-mediated DSBs. Chromothripsis is extensive chromosome rearrangement restricted to one or a few chromosomes that can cause human congenital disease and cancer (3–6). Using model cell systems and a genome editing protocol similar to ones in clinical trials (7) (NCT03655678, NCT03745287) we show that CRISPR-Cas9-mediated DNA breaks generate abnormal nuclear structures—micronuclei and chromosome bridges—that trigger chromothripsis. Chromothripsis is an on-target toxicity that may be minimized by cell manipulation protocols or screening but cannot be completely avoided in many genome editing applications.

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Section: Resultssupporting

confidence: 80%

Section: Introductionmentioning

confidence: 99%

Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing

Leibowitz

Papathanasiou

Doerfler

et al. 2020

Preprint

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“…For instance, the CRISPR/Cas9 targeting of the HTT translation starting site followed by NHEJ may generate truncated proteins with polyserine or polyalanine expansions, which have been shown to play a role in the disease (Berger et al, 2006). Furthermore, when attempting HDR-based strategies, NHEJ and HDR are competing pathways, and DSBs may be repaired by both mechanisms in the presence of a repair template (Weisheit et al, 2020). Likewise, bystander editing during base editing may give rise to unintended edited products which might even intensify the pathological processes.…”

Section: Future Perspectives In Genome Editing For Cns Disordersmentioning

confidence: 99%

Genome Editing for CNS Disorders

Duarte

Déglon

2020

Front. Neurosci.

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Central nervous system (CNS) disorders have a social and economic burden on modern societies, and the development of effective therapies is urgently required. Gene editing may prevent or cure a disease by inducing genetic changes at endogenous loci. Genome editing includes not only the insertion, deletion or replacement of nucleotides, but also the modulation of gene expression and epigenetic editing. Emerging technologies based on ZFs, TALEs, and CRISPR/Cas systems have extended the boundaries of genome manipulation and promoted genome editing approaches to the level of promising strategies for counteracting genetic diseases. The parallel development of efficient delivery systems has also increased our access to the CNS. In this review, we describe the various tools available for genome editing and summarize in vivo preclinical studies of CNS genome editing, whilst considering current limitations and alternative approaches to overcome some bottlenecks.

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“…These unintended events may occur as a consequence of both HDR and NHEJ repair pathways induced after CAS9 generated DSBs. The extent of these unintended events may be more substantial when CAS9 is active for extended periods of time as in plasmid based CAS9 delivery to cells but also occur with RNP-and mRNA-based delivery methods [7,[27][28][29]. Failure to detect unintended editing can have significant consequences, especially affecting health and pharmaceutical related fields, such as pre-clinical model generation and gene therapy but also in basic research where undetected rearrangements may lead to erroneous conclusions drawn from experiments performed with edited cells.…”

Section: Discussionmentioning

confidence: 99%

Verification of CRISPR editing and finding transgenic inserts by Xdrop™ Indirect sequence capture followed by short- and long- read sequencing

Blöndal¹,

Gamba²,

Møller³

et al. 2020

Preprint

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Validation of CRISPR-Cas9 editing typically explore the immediate vicinity of the target site and distal off-target sequences, which have led to the conclusion that CRISPR-Cas9 editing is very specific. However, an increasing number of studies suggest that on-target unintended editing events like deletions and insertions are relatively frequent but unfortunately often missed in the validation of CRISPR-Cas9 editing. The deletions may be several kb and only affect one allele. The golden standard in molecular validation of gene editing is direct sequencing of relatively short PCR amplicons. This approach will allow detection of smaller editing events but will fail to detect larger rearrangements in particular when these only affect one of the two alleles. Detection of larger rearrangements requires that an extended region is analyzed and the characterization of events may benefit from long-read sequencing. Here we present a new microfluidic technology Xdrop™ that allows targeted enrichment of longer regions (~ 100 kb) using just a standard PCR primer set. We sequenced the enriched region on long-and short-read sequencing platforms to unravel unknown and unintended genome edits resulting from CRISPR-Cas9 gene editing. We show how loss of heterozygosity, due to an accidental insertion in one of the alleles, remained undetected using standard procedures but was discovered implementing the Xdrop™ system. This demonstrates the potential of such technology in CRISPR gene editing assessments, providing an extremely accurate tool.

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Detection of Deleterious On-Target Effects after HDR-Mediated CRISPR Editing

Cited by 101 publications

References 19 publications

Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing

Chromothripsis as an on-target consequence of CRISPR-Cas9 genome editing

Genome Editing for CNS Disorders

Verification of CRISPR editing and finding transgenic inserts by Xdrop™ Indirect sequence capture followed by short- and long- read sequencing

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