Genome-wide microhomologies enable precise template-free editing of biologically relevant deletion mutations

Grajcarek, Janin; Monlong, Jean; Nishinaka‐Arai, Yoko; Nakamura, Michiko; Nagai, Miki; Matsuo, Shiori; Lougheed, David; Sakurai, Hiroyuki; Saito, Megumu K.; Bourque, Guillaume; Woltjen, Knut

doi:10.1038/s41467-019-12829-8

Cited by 26 publications

(16 citation statements)

References 57 publications

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“…MMEJ has been exploited to precisely correct frameshift mutations resulting from microduplications in patient-derived cells for diverse rare diseases without the need for additional donor templates [ 125 ]. Recently, a bioinformatics tool has been described that allows the identification of targets which are amenable for MMEJ repair, further promoting the possibility of achieving accurate repair in an HDR-independent way [ 126 ]. This will certainly provide in the future new exciting opportunities for preclinical exploitation of DMD therapeutics based on genome editing.…”

Section: Exploiting Dsb Repair To Develop Innovative Therapeutic Smentioning

confidence: 99%

DNA Damage: From Threat to Treatment

Carusillo

Mussolino

2020

Cells

136

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DNA is the source of genetic information, and preserving its integrity is essential in order to sustain life. The genome is continuously threatened by different types of DNA lesions, such as abasic sites, mismatches, interstrand crosslinks, or single-stranded and double-stranded breaks. As a consequence, cells have evolved specialized DNA damage response (DDR) mechanisms to sustain genome integrity. By orchestrating multilayer signaling cascades specific for the type of lesion that occurred, the DDR ensures that genetic information is preserved overtime. In the last decades, DNA repair mechanisms have been thoroughly investigated to untangle these complex networks of pathways and processes. As a result, key factors have been identified that control and coordinate DDR circuits in time and space. In the first part of this review, we describe the critical processes encompassing DNA damage sensing and resolution. In the second part, we illustrate the consequences of partial or complete failure of the DNA repair machinery. Lastly, we will report examples in which this knowledge has been instrumental to develop novel therapies based on genome editing technologies, such as CRISPR-Cas.

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Section: Exploiting Dsb Repair To Develop Innovative Therapeutic Smentioning

confidence: 99%

DNA Damage: From Threat to Treatment

Carusillo

Mussolino

2020

Cells

136

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“…The ability to predict the mutational outcomes of CRISPR-Cas9 edits and, in certain cases, to achieve a single dominant mutant product introduces the possibility of precisely creating or fixing pathogenic mutations. In fact, such precise generation and correction of pathogenic alleles through template-free Cas9-nuclease editing has been demonstrated 16 – 18 .…”

Section: Introductionmentioning

confidence: 99%

Small molecule inhibition of ATM kinase increases CRISPR-Cas9 1-bp insertion frequency

et al. 2021

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Mutational outcomes following CRISPR-Cas9-nuclease cutting in mammalian cells have recently been shown to be predictable and, in certain cases, skewed toward single genotypes. However, the ability to control these outcomes remains limited, especially for 1-bp insertions, a common and therapeutically relevant class of repair outcomes. Here, through a small molecule screen, we identify the ATM kinase inhibitor KU-60019 as a compound capable of reproducibly increasing the fraction of 1-bp insertions relative to other Cas9 repair outcomes. Small molecule or genetic ATM inhibition increases 1-bp insertion outcome fraction across three human and mouse cell lines, two Cas9 species, and dozens of target sites, although concomitantly reducing the fraction of edited alleles. Notably, KU-60019 increases the relative frequency of 1-bp insertions to over 80% of edited alleles at several native human genomic loci and improves the efficiency of correction for pathogenic 1-bp deletion variants. The ability to increase 1-bp insertion frequency adds another dimension to precise template-free Cas9-nuclease genome editing.

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“…This is based on the fact that MMEJ typically results in relatively large deletions flanked by microhomology. For example, recent studies demonstrate that microhomology tracts (≥3 bp) are efficiently used to repair CRISPR-Cas9-induced DSBs in human cells by MMEJ, resulting in predictable microhomology-mediated deletions ( Grajcarek et al, 2019 ).…”

Section: Resultsmentioning

confidence: 99%

Polθ promotes the repair of 5′-DNA-protein crosslinks by microhomology-mediated end-joining

et al. 2021

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DNA polymerase q (Polq) confers resistance to chemotherapy agents that cause DNA-protein crosslinks (DPCs) at double-strand breaks (DSBs), such as topoisomerase inhibitors. This suggests Polq might facilitate DPC repair by microhomology-mediated end-joining (MMEJ). Here, we investigate Polq repair of DSBs carrying DPCs by monitoring MMEJ in Xenopus egg extracts. MMEJ in extracts is dependent on Polq, exhibits the MMEJ repair signature, and efficiently repairs 5 0 terminal DPCs independently of non-homologous endjoining and the replisome. We demonstrate that Polq promotes the repair of 5 0 terminal DPCs in mammalian cells by using an MMEJ reporter and find that Polq confers resistance to formaldehyde in addition to topoisomerase inhibitors. Dual deficiency in Polq and tyrosyl-DNA phosphodiesterase 2 (TDP2) causes severe cellular sensitivity to etoposide, which demonstrates MMEJ as an independent DPC repair pathway. These studies recapitulate MMEJ in vitro and elucidate how Polq confers resistance to etoposide.

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Genome-wide microhomologies enable precise template-free editing of biologically relevant deletion mutations

Cited by 26 publications

References 57 publications

DNA Damage: From Threat to Treatment

DNA Damage: From Threat to Treatment

Small molecule inhibition of ATM kinase increases CRISPR-Cas9 1-bp insertion frequency

Polθ promotes the repair of 5′-DNA-protein crosslinks by microhomology-mediated end-joining

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