Mutation-specific reporter for optimization and enrichment of prime editing

Abstract Prime editing is a versatile genome-editing technique. However, some genomic sites remain difficult to edit and optimal design of prime-editing tools is elusive. We present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranked the efficiency of prime edit guide RNAs (pegRNAs) and any prime editor protein, including novel variants with flexible protospacer adjacent motif (PAM) recognition. We applied fluoPEER to instruct correction of pathogenic variants in patient cells and found that plasmid-editing enriched for editing at the genomic target site. Transcriptomic analysis of reporter-edited cells revealed that successful prime editing was associated with expression of DNA repair genes. Together, our results show that fluoPEER can be employed for rapid and efficient correction of patient cells, selection of gene-edited cells, and elucidation of cellular mechanisms needed for successful prime editing.

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Germline Pathogenic Variants in Homologous Recombination and DNA Repair Genes in an Asian Cohort of Young-Onset Colorectal Cancer

JNCI Cancer Spectrum ◽

10.1093/jncics/pky054 ◽

2018 ◽

Vol 2 (4) ◽

Cited By ~ 5

Author(s):

Ming Ren Toh ◽

Jian Bang Chiang ◽

Siao Ting Chong ◽

Sock Hoai Chan ◽

Nur Diana Binte Ishak ◽

...

Keyword(s):

Colorectal Cancer ◽

Dna Repair ◽

Homologous Recombination ◽

Cancer Predisposition ◽

Colorectal Cancers ◽

Dna Repair Genes ◽

Young Onset ◽

Pathogenic Variants ◽

Predisposition Genes ◽

Repair Genes

Abstract Background Growing evidence suggests a role for cancer susceptibility genes such as BRCA2 and PALB2 in young-onset colorectal cancers. Using a cohort of young colorectal cancer patients, we sought to identify and provide functional evidence for germline pathogenic variants of DNA repair genes not typically associated with colorectal cancer. Methods We recruited 88 patients with young-onset colorectal cancers seen at a general oncology center. Whole-exome sequencing was performed to identify variants in DNA repair and colorectal cancer predisposition genes. Pathogenic BRCA2 and PALB2 variants were analyzed using immunoblot and immunofluorescence on patient-derived lymphoblastoid cells. Results In general, our cohort displayed characteristic features of young-onset colorectal cancers. Most patients had left-sided tumors and were diagnosed at late stages. Four patients had familial adenomatous polyposis, as well as pathogenic APC variants. We identified 12 pathogenic variants evenly distributed between DNA repair and colorectal cancer predisposition genes. Six patients had pathogenic variants in colorectal cancer genes: APC (n = 4) and MUTYH monoallelic (n = 2). Another six had pathogenic variants in DNA repair genes: ATM (n = 1), BRCA2 (n = 1), PALB2 (n = 1), NTHL1 (n = 1), and WRN (n = 2). Pathogenic variants BRCA2 c.9154C>T and PALB2 c.1059delA showed deficient homologous recombination repair, evident from the impaired RAD51 nuclear localization and foci formation. Conclusion A substantial portion of pathogenic variants in young-onset colorectal cancer was found in DNA repair genes not previously associated with colorectal cancer. This may have implications for the management of patients. Further studies are needed to ascertain the enrichment of pathogenic DNA repair gene variants in colorectal cancers.

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Germline DNA repair mutations in men with prostate cancer are less frequent in African Americans as compared with Caucasians.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.7_suppl.258 ◽

2019 ◽

Vol 37 (7_suppl) ◽

pp. 258-258

Author(s):

Piper L.W. Nicolosi ◽

Elisa Ledet ◽

Shan Yang ◽

Scott T. Michalski ◽

James Vu ◽

...

Keyword(s):

Prostate Cancer ◽

Dna Repair ◽

Dna Repair Genes ◽

Chi Square ◽

Pathogenic Variants ◽

Gene Panels ◽

Sparse Methods ◽

Repair Genes ◽

Germline Testing ◽

Identical Gene

258 Background: Pathogenic DNA repair defects are well described in Caucasian (C) men with prostate cancer (PCa) but comparative data on African-American (AA) men are sparse. Methods: Germline testing for DNA repair defects were assayed by Invitae (Invitae.com). Pathogenic variants in 14 genes were compared between AA and C men with PCa; these particular genes were also assessed by Pritchard et al NEJM 375:443, 2016. Identical gene panels were not used in all men, thus variations in assay numbers from gene to gene are noted. Chi square was used to compare proportions. Results: In the 14 genes, there were 16/214 (7.5%) AA men and 347/2488 (13.9%) C men with pathogenic findings (p=0.008). As shown in the Table, the most common pathogenic variants in AA men were BRCA2 (6/214, 2.8%), BRCA1 (3/213, 1.4%), PALB2 (2/182, 1.1%), ATM (2/206, 1.0%), RAD51C (1/148, 0.68%), CHEK2 (1/207, 0.48%), and PMS2 (1/212, 0.47%). In contrast to C men in this series, no AA men had pathogenic mutations in BARD1, BRIP1, MLH1, MSH2, MSH6, NBN, or RAD51D. Pathogenic CHEK2 was less commonly detected in AA as compared to C (p=0.03) in this dataset. Comparing our C men revealed no differences in the proportion of men with pathogenic findings compared to Pritchard et al (13.9% here vs 11.8%, P=0.15). Conclusions: AA men with PCa are less likely to have pathogenic DNA repair mutations among these 14 assayed DNA repair genes compared to C men. Limitations regarding lack of stage, Gleason scores, and FH are notable. Details on gene assays and comparison to Pritchard et al NEJM 375:443, 2016 [Table: see text]

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Cancer driver genes in prostate cancer from young men.

Journal of Clinical Oncology ◽

10.1200/jco.2019.37.15_suppl.e16586 ◽

2019 ◽

Vol 37 (15_suppl) ◽

pp. e16586-e16586

Author(s):

Simone Maistro ◽

Camila dos Santos Xavier ◽

Pedro Adolpho MP Serio ◽

Maria Lucia Hirata Katayama ◽

Rosimeire Aparecida Roela ◽

...

Keyword(s):

Dna Repair ◽

Young Adults ◽

Prostate Carcinoma ◽

In Silico ◽

Dna Repair Genes ◽

Cancer Gene ◽

Driver Genes ◽

Cancer Driver ◽

Pathogenic Variants ◽

Repair Genes

e16586 Background: Although prostate carcinoma is the most frequent cancer in older man, it is a rare disease in young adults. Search for global knowledge of the cancer genome is of great importance for diagnosis and/or treatment of these patients. Our goal was to identify the mutation profile of prostate carcinoma from young adults aged ≤49 years, using in silico tools. Methods: In silico analysis of somatic variants derived from exome or genome sequencing, deposited in the COSMIC or CBIOPORTAL database or in published manuscripts was performed. Data was searched for cancer driver genes catalogued in Cancer Gene Census (CGC) or Candidate Cancer Gene Database rank A or B (CCGD) and DNA repair genes. Results: 46 prostate adenocarcinoma samples from patients aged between 23-49 years (median 44.5) were included. A median of 15 variants per patient (minimum: 2 and maximum: 343) was detected. The most frequent events were C to T transitions, representing a median percentage of 39% of the substitutions. A total of 160 genes were affected by pathogenic mutations (splice site, nonsense, frame shift ins, frame shift del, nonstop, in frame del). Ten tumors presented pathogenic variants in at least one cancer driver (CGC); 3 tumors presented SNVs in two CGC. The most frequently altered cancer causing genes (CGC) were MUC16 (17%); TP53 (8%), SPOP (8%), XPC, SYNE1, IDH1, CDMD3, ANAPC1, AHNAK (6%). Seven tumors presented pathogenic variants in at least one in driver candidate from CCGD rank A, and 3 tumors in CCGD rank B. Single nucleotide variants affecting genes involved in DNA repair were found in 35% samples and one tumor presented variants in genes involved in BER, NER, FA, DDC and HRR. Variants in TP53 and XPC wer3e detected in 3 tumors. Conclusions: In prostate carcinoma from young men a wide range of cancer causing genes may be altered, including MUC16 and TP53. Variants in DNA repair genes may be found in 35% of the samples.

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Mutation-specific reporter for the optimization and enrichment of prime editing

10.1101/2021.05.08.443062 ◽

2021 ◽

Author(s):

Imre F. Schene ◽

Indi P. Joore ◽

Jan H.L. Baijens ◽

Sawsan Shehata ◽

Eveline F. Ilcken ◽

...

Keyword(s):

Target Site ◽

Cellular Mechanisms ◽

Guide Rnas ◽

Novel Variants ◽

Genomic Editing

We present a fluorescent prime editing and enrichment reporter (fluoPEER), which can be tailored to any genomic target site. This system rapidly and faithfully ranks the efficiency of prime edit guide RNAs (pegRNAs) and any prime editor protein, including novel variants with flexible PAM recognition. Successful reporter editing enriches for genomic editing. FluoPEER can be employed for efficient correction of patient cells and to elucidate cellular mechanisms needed for successful prime editing.

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Truncating variants in DNA-repair genes and their effect on AAO of hereditary breast cancer

10.1055/s-0038-1651801 ◽

2018 ◽

Author(s):

I Sepahi ◽

U Faust ◽

M Sturm ◽

K Bosse ◽

M Kehrer ◽

...

Keyword(s):

Breast Cancer ◽

Dna Repair ◽

Hereditary Breast Cancer ◽

Dna Repair Genes ◽

Repair Genes

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Comprehensive Analyses of Copy Number Variations In DNA Repair Genes Reveal the Role of Poly(ADP-Ribose) Polymerase in Longevity in Trees

SSRN Electronic Journal ◽

10.2139/ssrn.3751787 ◽

2020 ◽

Author(s):

Yuta Aoyagi Blue ◽

Junko Kusumi ◽

Akiko Satake

Keyword(s):

Dna Repair ◽

Copy Number ◽

Copy Number Variations ◽

Dna Repair Genes ◽

Repair Genes

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Copy number analyses of DNA repair genes reveal the role of poly(ADP-ribose) polymerase (PARP) in tree longevity

iScience ◽

10.1016/j.isci.2021.102779 ◽

2021 ◽

pp. 102779

Author(s):

Yuta Aoyagi Blue ◽

Junko Kusumi ◽

Akiko Satake

Keyword(s):

Dna Repair ◽

Copy Number ◽

Dna Repair Genes ◽

Repair Genes

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Coexistence of SOS-Dependent and SOS-Independent Regulation of DNA Repair Genes in Radiation-Resistant Deinococcus Bacteria

Cells ◽

10.3390/cells10040924 ◽

2021 ◽

Vol 10 (4) ◽

pp. 924

Author(s):

Laurence Blanchard ◽

Arjan de Groot

Keyword(s):

Dna Repair ◽

Deinococcus Radiodurans ◽

Dna Repair Genes ◽

Lesion Bypass ◽

Radiation Resistant ◽

Radiation Induced ◽

Induced Mutagenesis ◽

High Doses ◽

Translesion Polymerases ◽

Repair Genes

Deinococcus bacteria are extremely resistant to radiation and able to repair a shattered genome in an essentially error-free manner after exposure to high doses of radiation or prolonged desiccation. An efficient, SOS-independent response mechanism to induce various DNA repair genes such as recA is essential for radiation resistance. This pathway, called radiation/desiccation response, is controlled by metallopeptidase IrrE and repressor DdrO that are highly conserved in Deinococcus. Among various Deinococcus species, Deinococcus radiodurans has been studied most extensively. Its genome encodes classical DNA repair proteins for error-free repair but no error-prone translesion DNA polymerases, which may suggest that absence of mutagenic lesion bypass is crucial for error-free repair of massive DNA damage. However, many other radiation-resistant Deinococcus species do possess translesion polymerases, and radiation-induced mutagenesis has been demonstrated. At least dozens of Deinococcus species contain a mutagenesis cassette, and some even two cassettes, encoding error-prone translesion polymerase DnaE2 and two other proteins, ImuY and ImuB-C, that are probable accessory factors required for DnaE2 activity. Expression of this mutagenesis cassette is under control of the SOS regulators RecA and LexA. In this paper, we review both the RecA/LexA-controlled mutagenesis and the IrrE/DdrO-controlled radiation/desiccation response in Deinococcus.

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Patients with Systemic Sclerosis Present Increased DNA Damage Differentially Associated with DNA Repair Gene Polymorphisms

The Journal of Rheumatology ◽

10.3899/jrheum.130376 ◽

2014 ◽

Vol 41 (3) ◽

pp. 458-465 ◽

Cited By ~ 11

Author(s):

Gustavo Martelli Palomino ◽

Carmen L. Bassi ◽

Isabela J. Wastowski ◽

Danilo J. Xavier ◽

Yara M. Lucisano-Valim ◽

...

Keyword(s):

Dna Damage ◽

Dna Repair ◽

Systemic Sclerosis ◽

Blood Cells ◽

Gene Polymorphisms ◽

Peripheral Blood Cells ◽

Dna Repair Genes ◽

Repair Gene ◽

Dna Repair Gene ◽

Repair Genes

Objective.Patients with systemic sclerosis (SSc) exhibit increased toxicity when exposed to genotoxic agents. In our study, we evaluated DNA damage and polymorphic sites in 2 DNA repair genes (XRCC1Arg399Gln andXRCC4Ile401Thr) in patients with SSc.Methods.A total of 177 patients were studied for DNA repair gene polymorphisms. Fifty-six of them were also evaluated for DNA damage in peripheral blood cells using the comet assay.Results.Compared to controls, the patients as a whole or stratified into major clinical variants (limited or diffuse skin involvement), irrespective of the underlying treatment schedule, exhibited increased DNA damage.XRCC1(rs: 25487) andXRCC4(rs: 28360135) allele and genotype frequencies observed in patients with SSc were not significantly different from those observed in controls; however, theXRCC1Arg399Gln allele was associated with increased DNA damage only in healthy controls and theXRCC4Ile401Thr allele was associated with increased DNA damage in both patients and controls. Further, theXRCC1Arg399Gln allele was associated with the presence of antinuclear antibody and anticentromere antibody. No association was observed between these DNA repair gene polymorphic sites and clinical features of patients with SSc.Conclusion.These results corroborate the presence of genomic instability in SSc peripheral blood cells, as evaluated by increased DNA damage, and show that polymorphic sites of theXRCC1andXRCC4DNA repair genes may differentially influence DNA damage and the development of autoantibodies.

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