Recurrent deletions in clonal hematopoiesis are driven by microhomology-mediated end joining

Feldman, Tzah; Bercovich, Akhiad; Moskovitz, Yoni; Chapal-Ilani, Noa; Mitchell, Amanda; Medeiros, Jessie J. F.; Biezuner, Tamir; Kaushansky, Nathali; Minden, Mark D.; Gupta, Vikas; Milyavsky, Michael; Livneh, Zvi; Tanay, Amos; Shlush, Liran I.

doi:10.1038/s41467-021-22803-y

Cited by 28 publications

(15 citation statements)

References 54 publications

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“…Noteworthy, identical deletions of 7 bp flanked by homologous TG sequences were highly overrepresented. This is in agreement with similar natural mutations observed in vivo 41 , 42 and induced in gene editing experiments 32 . This reinforces the observation that INDEL profiles can be non-random depending on target 43 .…”

Section: Discussionsupporting

confidence: 92%

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: 92%

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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“…1A). The region exhibited mostly high levels of methylation, consistent with homozygote gene body methylation (S1 Fig) . To induce DSB in a controlled manner, we used the CRISPR/CAS9 system that recapitulates the naturally occurring deletions in ASXL1 [29]. After introducing DSBs, single-cell clones were established.…”

Section: Epigenetic Stability After Dsb In Highly Methylated Gene Bod...mentioning

confidence: 99%

“…Recently, we demonstrated that the most common deletions in myeloid malignancies and CH are the result of MMEJ repair. A CRISPR based system induced the formation of the naturally occurring recurrent deletions in ASXL1, SRSF2 and CALR together with small indels, which are the result of NHEJ repair [29]. To study methylation patterns accurately after different DSB repair pathways we created single cell clones following DSB, so that all the cells in the clone underwent the same repair.…”

Section: Mmej and Methylation In Myeloid Malignanciesmentioning

confidence: 99%

“…To address this question, we chose to focus in the unmethylated SRSF2 gene in K562 cells. SRSF2 plays an important role in pathogenesis of AML and was part of the MMEJ induction system established previously [29,38]. The high density of CpG sites around the canonical MMEJ deletion in SRSF2 limited our ability to explore the region directly with targeted BS-seq.…”

Section: Methylation Restoration After Dsb Is Stable On Dnmt3a R882c ...mentioning

confidence: 99%

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DNA Methylation is stable after MMEJ and NHEJ double strand break repair

Bnaya

Shilo

Feldman

et al. 2022

Preprint

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DNA double strand breaks (DSBs) are a major source of mutations. Both non-homologous-end-joining (NHEJ) and microhomology-mediated-end-joining (MMEJ) DSB repair pathways are error prone and produce deletions, which can lead to cancer. DSBs also lead to epigenetic changes, including demethylation, which is involved in carcinogenesis. Of specific interest is the MMEJ repair pathway, as it requires methylation restoration around the break, as a result of the resection and formation of single stranded (ssDNA) intermediates. While, methylation patterns after homologous recombination (HR) have been partially studied, the methylation status after MMEJ and NHEJ remains poorly reported, and can be relevant for cancer. To study methylation patterns around DSB after NHEJ and MMEJ repair, we used targeted bisulfite-sequencing (BS-seq) to quantify methylation of dozens of single cell clones after induction of DSB by CRISPR. Each single cell clone was classified according to the sequence signature to a specific repair mechanism: NHEJ or MMEJ. Comparison of single cell clones after DSB to control cells, without DSB, demonstrated correct restoration of the methylation levels. No difference in methylation patterns was noticed when comparing NHEJ to MMEJ. Methylation levels in gene body, highly methylated CpGs (n=61, 4000 base pairs around DSB) and in low methylation CpGs (n=19), remained stable after both MMEJ and NHEJ. Gene body methylation persisted even on the background of DNMT3A R882C mutation, the most prevalent preleukemic mutation, in which the de novo methylation machinery is compromised. An exception observed in a single CpG site (ASXL1 995) which demonstrated elevated methylation rate after DSB repair only in the presence of WT DNMT3A. In summary, DNA methylation restoration demonstrated high fidelity after DSB both in methylated and unmethylated gene body, even in cases where DNA resections and deletions occurred.

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“… 12 Interestingly, some CHIP‐associated mutations in genes such as ASXL1 and SRSF2 are caused by PARP1 dependent microhomology‐mediated end joining (MMEJ) double‐stranded break repair, indicating that DNA damage repair can also contribute to mutations associated with CHIP. 25 …”

Section: Recurrent Mutations In Chipmentioning

confidence: 99%