Tissue specificity of DNA repair: the CRISPR compass

Silva, Joana Ferreira da; Meyenberg, Mathilde; Loizou, Joanna I.

doi:10.1016/j.tig.2021.07.010

Cited by 18 publications

(11 citation statements)

References 33 publications

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“…To understand the origins of these deletions, the authors generated a library of mouse embryonic stem cells de cient in 32 DNA repair genes based on a single clone constitutively expressing Cas9, and found that the frequency of large deletions increased when genes essential for non-homologous end-joining (NHEJ) were impaired, and decreased when genes required for microhomology-mediated end joining (MMEJ) were disrupted [76]. Depending on the speci c cell type or stage of cell cycle, some DNA repair mechanisms, whether HDR, NHEJ or MMEJ, may be favored over others which thus may determine the propensity for, and characteristics of, any such on-target effects [77][78][79]. For example, prolonged in vitro intracellular expression of Cas9 in human pluripotent stem cells caused DSBs in DNA that were toxic via P53 inhibition of HDR, a mechanism which appeared to be cell-speci c and related to the early developmental status of the cell types [ As can be the case for ectopic expression of other heterologous proteins, constitutive intracellular expression of Cas9 can also be toxic due to stresses on protein homeostasis.…”

Section: Literature Assessment Of Hcas9 Toxicitymentioning

confidence: 99%

Bioinformatic and literature assessment of toxicity and allergenicity of a CRISPR-Cas9 engineered gene drive to control the human malaria mosquito vector Anopheles gambiae

Qureshi

Connolly

2022

Preprint

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Background Population suppression gene drive is currently being evaluated, including via environmental risk assessment (ERA), for malaria vector control. One such gene drive involves the dsxFCRISPRh transgene encoding (i) hCas9 endonuclease, (i) T1 guide RNA (gRNA) targeting the doublesex locus, and (iii) DsRed fluorescent marker protein, in genetically modified mosquitoes (GMMs). Problem formulation, the first stage of ERA, for environmental releases of dsxFCRISPRh previously identified nine potential harms to the environment or health that could occur, should expressed products of the transgene cause allergenicity or toxicity. Methods Amino acid sequences of hCas9 and DsRed were interrogated against those of toxins or allergens from NCBI, UniProt, COMPARE and AllergenOnline bioinformatic databases and the gRNA was compared with microRNAs from the miRBase database for potential impacts on gene expression associated with toxicity or allergenicity. PubMed was also searched for any evidence of toxicity or allergenicity of Cas9 or DsRed, or of the donor organisms from which these products were originally derived. Results While Cas9 nuclease activity can be toxic to some cell types in vitro and hCas9 was found to share homology with the prokaryotic toxin VapC, there was no evidence of a risk of toxicity to humans and other animals from hCas9. Although hCas9 did contain an 8-mer epitope found in the latex allergen Hev b 9, the full amino acid sequence of hCas9 was not homologous to any known allergens. Combined with a lack of evidence in the literature of Cas9 allergenicity, this indicated negligible risk to humans of allergenicity from hCas9. No matches were found between the gRNA and microRNAs from either Anopheles or humans. Moreover, potential exposure to dsxFCRISPRh transgenic proteins from environmental releases was assessed as negligible. Conclusions Bioinformatic and literature assessments found no convincing evidence to suggest that transgenic products expressed from dsxFCRISPRh were allergens or toxins, indicating that environmental releases of this population suppression gene drive for malaria vector control should not result in any increased allergenicity or toxicity in humans or animals. These results should also inform evaluations of other GMMs being developed for vector control and in vivo clinical applications of CRISPR-Cas9.

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Section: Literature Assessment Of Hcas9 Toxicitymentioning

confidence: 99%

Bioinformatic and literature assessment of toxicity and allergenicity of a CRISPR-Cas9 engineered gene drive to control the human malaria mosquito vector Anopheles gambiae

Qureshi

Connolly

2022

Preprint

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“…HR predominates during S-phase and declines during G 2 , in favor of cNHEJ ( Figure 2 B). This updated view of repair pathway choice plays important roles in current efforts to employ CRISPR/Cas9 for genome editing [ 86 , 87 ].…”

Section: Ddr Responses During the Mitotic Cell Cycle Across Model Systemsmentioning

confidence: 99%

DNA Damage Responses during the Cell Cycle: Insights from Model Organisms and Beyond

Clay

Fox

2021

Genes

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Genome damage is a threat to all organisms. To respond to such damage, DNA damage responses (DDRs) lead to cell cycle arrest, DNA repair, and cell death. Many DDR components are highly conserved, whereas others have adapted to specific organismal needs. Immense progress in this field has been driven by model genetic organism research. This review has two main purposes. First, we provide a survey of model organism-based efforts to study DDRs. Second, we highlight how model organism study has contributed to understanding how specific DDRs are influenced by cell cycle stage. We also look forward, with a discussion of how future study can be expanded beyond typical model genetic organisms to further illuminate how the genome is protected.

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“…Other approaches disrupt regulatory regions on both the normal and diseased allele 42,45 , which could be deleterious as homozygous knockout causes early lethality in mice [37][38][39] . Finally, editing strategies that utilize homology directed repair 44 are inefficient in post-mitotic cells 47 such as neurons, and are therefore not suitable for therapeutic applications.…”

Section: Introductionmentioning

confidence: 99%

Two therapeutic CRISPR/Cas9 gene editing approaches revert FTD/ALS cellular pathology caused by aC9orf72repeat expansion mutation in patient derived cells

Sckaff

Gill

Sachdev

et al. 2022

Preprint

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CRISPR gene editing holds promise to cure or arrest genetic disease, if we can find and implement curative edits reliably, safely and effectively. Expansion of a hexanucleotide repeat in C9orf72 is the leading known genetic cause of frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). We evaluated three approaches to editing the mutant C9orf72 gene for their ability to correct pathology in neurons derived from patient iPSCs: excision of the repeat region, excision of the mutant allele, and excision of regulatory region exon 1A. All three approaches normalized RNA abnormalities and TDP-43 pathology, but only repeat excision and mutant allele excision completely eliminated pathologic dipeptide repeats. Our work sheds light on the complex regulation of the C9orf72 gene and suggests that because of sense and anti-sense transcription, silencing a single regulatory region may not reverse all pathology. Our work also provides a roadmap for evaluating CRISPR gene correction using patient iPSCs.

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Tissue specificity of DNA repair: the CRISPR compass

Cited by 18 publications

References 33 publications

Bioinformatic and literature assessment of toxicity and allergenicity of a CRISPR-Cas9 engineered gene drive to control the human malaria mosquito vector Anopheles gambiae

Bioinformatic and literature assessment of toxicity and allergenicity of a CRISPR-Cas9 engineered gene drive to control the human malaria mosquito vector Anopheles gambiae

DNA Damage Responses during the Cell Cycle: Insights from Model Organisms and Beyond

Two therapeutic CRISPR/Cas9 gene editing approaches revert FTD/ALS cellular pathology caused by aC9orf72repeat expansion mutation in patient derived cells

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