Whole chromosome loss and genomic instability in mouse embryos after CRISPR-Cas9 genome editing

Papathanasiou, Stamatis; Markoulaki, Styliani; Blaine, Logan J.; Leibowitz, Mitchell L.; Chen, Ken; Jaenisch, Rudolf; Pellman, David

doi:10.1038/s41467-021-26097-y

Cited by 99 publications

(78 citation statements)

References 27 publications

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“…Thus, it is very likely that one entire Chromosome 29 got lost in these embryos. This is in agreement to a recent study reporting whole chromosome loss in mouse embryos 35 as well as in human embryos 33 after genome editing. Likewise, 3 out of 21 embryos displayed Loss-of-chromosomal arms.…”

Section: Discussionsupporting

confidence: 93%

The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events

Miskel

Poirier

Beunink

et al. 2022

Sci Rep

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At the embryonic level, CRISPR technologies have been used to edit genomes reliably and efficiently in various mammalian models, with Ribonucleoprotein (RNP) electroporation potentially representing a superior delivery method into mammalian zygotes. However, detailed insights of the interactions between varying technical settings as well as the time point of electroporation in a bovine zygote’s cell cycle on developmental metrics and the frequency and type of editing events are largely unknown. The present study uncovers that increasing pulse lengths result in higher Full Edit rates, with Mosaicism in Full-Edit embryos being significantly affected by adjusting RNP-electroporation relative to zygote cell cycle. A considerable proportion of Full Edit embryos demonstrated loss-of-heterozygosity after RNP-electroporation prior to S-phase. Some of these loss-of-heterozygosity events are a consequence of chromosomal disruptions along large sections of the target chromosomes making it necessary to check for their presence prior use of this technique in animal breeding. One out of 2 of these loss-of-heterozygosity events, however, was not associated with loss of an entire chromosome or chromosomal sections. Whether analysed loss-of-heterozygosity in these cases, however, was a false negative result due to loss of PCR primer sequences after INDEL formation at the target side or indeed due to interhomolog recombination needs to be clarified in follow up studies since the latter would for sure offer attractive options for future breeding schedules.

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Section: Discussionsupporting

confidence: 93%

The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events

Miskel

Poirier

Beunink

et al. 2022

Sci Rep

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show abstract

“…For these reasons, it would be desirable to perform similar experiments also in other sample types and organisms. A further limitation of our study is that long-range PCR is unable to capture large genome aberrations and complex genomic rearrangements, such as chromothripsis 21 and whole chromosome deletions 17 , 20 . Detecting such events requires an additional analysis of the edited samples, for example through long-read WGS as performed here, or by an alternative method for targeted long-read sequencing 42 – 45 .…”

Section: Discussionmentioning

confidence: 99%

CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations

et al. 2022

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CRISPR-Cas9 genome editing has potential to cure diseases without current treatments, but therapies must be safe. Here we show that CRISPR-Cas9 editing can introduce unintended mutations in vivo, which are passed on to the next generation. By editing fertilized zebrafish eggs using four guide RNAs selected for off-target activity in vitro, followed by long-read sequencing of DNA from >1100 larvae, juvenile and adult fish across two generations, we find that structural variants (SVs), i.e., insertions and deletions ≥50 bp, represent 6% of editing outcomes in founder larvae. These SVs occur both at on-target and off-target sites. Our results also illustrate that adult founder zebrafish are mosaic in their germ cells, and that 26% of their offspring carries an off-target mutation and 9% an SV. Hence, pre-testing for off-target activity and SVs using patient material is advisable in clinical applications, to reduce the risk of unanticipated effects with potentially large implications.

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“…Pericentromeric losses of genomic DNA adjacent to the cut site suggests that sister chromatids are linked, followed by rupture of that link during mitosis. DNA bridge formation has been observed in Cas9-mediated chromosomal aneuploidies in mouse embryos 30 as well as in cultured cells 21 . MRE11 forms dimers binding to DSB ends, thereby tethering them 31 .…”

Section: Discussionmentioning

confidence: 99%

DNA Double Strand Breaks cause chromosome loss through sister chromatid tethering in human embryos

Turocy

Marin

et al. 2022

Preprint

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Genome editing by DNA double-strand breaks (DSB) is currently being investigated as a tool to treat or even prevent heritable diseases. However, DNA repair mechanisms in the human embryo remain poorly understood and DSBs may result in chromosome loss. Here we provide evidence of whole and segmental chromosome loss in over one third of chromosomes 16, 17 and X targeted by CRISPR/Cas9-induced DNA DSB, including pericentromeric and mid-arm sites. Chromosomal changes were asymmetric relative to the Cas9 cut site: segmental losses occurred on both centric as well as acentric chromosome arms, while gains were exclusively found on acentric arms, suggesting that centromeres in broken chromosomes continued to mediate sister chromatid separation. Using this pattern of chromosomal errors, we were able to define new genomic coordinates of the active centromere on chromosome 16. Asymmetry was also found in the attrition of gDNA at the break site: attrition occurred centromeric of the DSB, while telomeric to the break, chromosomal ends were protected. Thus, spindle forces at centromeres and end tethering and protection at DSBs are antagonistic forces that interfere with accurate segregation of sister chromatids. Thereby, a single DSB is sufficient to result in the loss of a chromosome from the embryo. These results highlight the risks of aneuploidy in CRISPR/Cas9 genome editing, while also providing a mechanism for mitotically acquired aneuploidy caused by DNA breaks in human embryos.

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Whole chromosome loss and genomic instability in mouse embryos after CRISPR-Cas9 genome editing

Cited by 99 publications

References 27 publications

The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events

The cell cycle stage of bovine zygotes electroporated with CRISPR/Cas9-RNP affects frequency of Loss-of-heterozygosity editing events

CRISPR-Cas9 induces large structural variants at on-target and off-target sites in vivo that segregate across generations

DNA Double Strand Breaks cause chromosome loss through sister chromatid tethering in human embryos

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