A systematic CRISPR screen defines mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage

Zou, Xueqing; Koh, Gene; Nanda, Arjun Scott; Degasperi, Andrea; Urgo, Katie; Roumeliotis, Theodoros I.; Agu, Chukwuma A.; Badja, Cherif; Momen, Sophie; Young, Jamie; Amarante, Tauanne Dias; Side, Lucy; Brice, Glen; Pérez‐Alonso, Vanesa; Rueda, Daniel; Gomez, Céline; Bushell, Wendy; Harris, Robert G.; Choudhary, Jyoti; Jiricny, Josef; Skarne, William C

doi:10.1038/s43018-021-00200-0

Cited by 111 publications

(185 citation statements)

References 85 publications

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“…Future work in post-mitotic MMR-deficient cells could illuminate the requirement of cellular factors or DNA replication for resolving the heteroduplex intermediate. In addition, we showed that transient MLH1dn expression minimally perturbs microsatellites sensitive to MMR activity, though the potential impact of MMR deficiency on genomic mutation rates is well documented ( Lujan et al., 2014 ; Zou et al., 2021 ). An analysis of genome-wide mutations induced by transient PE4 and PE5 expression would more sensitively quantify their off-target editing consequences.…”

Section: Limitations Of the Studymentioning

confidence: 97%

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes

Chen

Hussmann

Yan

et al. 2021

Cell

425

576

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While prime editing enables precise sequence changes in DNA, cellular determinants of prime editing remain poorly understood. Using pooled CRISPRi screens, we discovered that DNA mismatch repair (MMR) impedes prime editing and promotes undesired indel byproducts. We developed PE4 and PE5 prime editing systems in which transient expression of an engineered MMR-inhibiting protein enhances the efficiency of substitution, small insertion, and small deletion prime edits by an average 7.7-fold and 2.0-fold compared to PE2 and PE3 systems, respectively, while improving edit/indel ratios by 3.4-fold in MMR-proficient cell types. Strategic installation of silent mutations near the intended edit can enhance prime editing outcomes by evading MMR. Prime editor protein optimization resulted in a PEmax architecture that enhances editing efficacy by 2.8-fold on average in HeLa cells. These findings enrich our understanding of prime editing and establish prime editing systems that show substantial improvement across 191 edits in seven mammalian cell types. ll

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Section: Limitations Of the Studymentioning

confidence: 97%

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes

Chen

Hussmann

Yan

et al. 2021

Cell

425

576

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“…1). A signature reported in human cells with in vitro engineered biallelic OGG1 deletion is also primarily characterised by C>A mutations at GCA and ACA trinucleotides 51 . It is, therefore, possible that mutagenesis due to the germline OGG1 variant(s) in PD44890 (see above) is superimposed on the mutational signature produced by the MUTYH germline mutations to generate N2 (see Supplementary Information for further analysis and discussion).…”

Section: Mutational Signaturesmentioning

confidence: 99%

Inherited MUTYH mutations cause elevated somatic mutation rates and distinctive mutational signatures in normal human cells

Robinson

Thomas

Abascal

et al. 2021

Preprint

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Cellular DNA damage caused by reactive oxygen species is repaired by the base excision repair (BER) pathway which includes the DNA glycosylase MUTYH. Inherited biallelic MUTYH mutations cause predisposition to colorectal adenomas and carcinoma. However, the mechanistic progression from germline MUTYH mutations to MUTYH-Associated Polyposis (MAP) is incompletely understood. Here, we sequenced normal tissue DNAs from 10 individuals with MAP. Somatic base substitution mutation rates in intestinal epithelial cells were elevated 2 to 5-fold in all individuals, except for one showing a 33-fold increase, and were also increased in other tissues. The increased mutation burdens were of multiple mutational signatures characterised by C>A changes. Different mutation rates and signatures between individuals were likely due to different MUTYH mutations or additional inherited mutations in other BER pathway genes. The elevated base substitution rate in normal cells likely accounts for the predisposition to neoplasia in MAP. Despite ubiquitously elevated mutation rates, individuals with MAP do not display overt evidence of premature ageing. Thus, accumulation of somatic mutations may not be sufficient to cause the global organismal functional decline of ageing.

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“…Although these repair mechanisms are highly effective, some DNA lesions escape repair, are incorrectly repaired or are fixed as DNA mutations following mispairings generated during replication, resulting in an annual accumulation of 15–40 mutations in healthy human stem cells ( Blokzijl et al, 2016 ). Indeed, loss of DNA repair activity results in a tremendously increased rate of mutation accumulation, depending on the affected pathway and presence of DNA damage ( Drost et al, 2017 ; Zou et al, 2018 ; Zou et al, 2021 ; Sanders et al, 2021 ). Therefore, the mutational landscape of a cell is shaped by a balance between DNA damage induction and the efficiency of the repair thereof ( Volkova et al, 2020 ).…”

Section: Introductionmentioning

confidence: 99%

The Mutagenic Impact of Environmental Exposures in Human Cells and Cancer: Imprints Through Time

2021

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During life, the DNA of our cells is continuously exposed to external damaging processes. Despite the activity of various repair mechanisms, DNA damage eventually results in the accumulation of mutations in the genomes of our cells. Oncogenic mutations are at the root of carcinogenesis, and carcinogenic agents are often highly mutagenic. Over the past decade, whole genome sequencing data of healthy and tumor tissues have revealed how cells in our body gradually accumulate mutations because of exposure to various mutagenic processes. Dissection of mutation profiles based on the type and context specificities of the altered bases has revealed a variety of signatures that reflect past exposure to environmental mutagens, ranging from chemotherapeutic drugs to genotoxic gut bacteria. In this review, we discuss the latest knowledge on somatic mutation accumulation in human cells, and how environmental mutagenic factors further shape the mutation landscapes of tissues. In addition, not all carcinogenic agents induce mutations, which may point to alternative tumor-promoting mechanisms, such as altered clonal selection dynamics. In short, we provide an overview of how environmental factors induce mutations in the DNA of our healthy cells and how this contributes to carcinogenesis. A better understanding of how environmental mutagens shape the genomes of our cells can help to identify potential preventable causes of cancer.

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A systematic CRISPR screen defines mutational mechanisms underpinning signatures caused by replication errors and endogenous DNA damage

Cited by 111 publications

References 85 publications

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes

Enhanced prime editing systems by manipulating cellular determinants of editing outcomes

Inherited MUTYH mutations cause elevated somatic mutation rates and distinctive mutational signatures in normal human cells

The Mutagenic Impact of Environmental Exposures in Human Cells and Cancer: Imprints Through Time

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