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Using Next Generation Sequencing to Identify Mutations Associated with Repair of a CAS9-induced Double Strand Break Near the CD4 Promoter.
Hu, Changkun; Doerksen, Tyler; Bugbee, Taylor; Wallace, Nicholas A; Palinski, Rachel.
Afiliação
  • Hu C; Division of Biology, Kansas State University.
  • Doerksen T; Kansas State Veterinary Diagnostic Laboratory, Kansas State University.
  • Bugbee T; Division of Biology, Kansas State University.
  • Wallace NA; Division of Biology, Kansas State University.
  • Palinski R; Kansas State Veterinary Diagnostic Laboratory, Kansas State University; Department of Diagnostic Medicine/Pathobiology, Kansas State University; rpalinski@vet.k-state.edu.
J Vis Exp ; (181)2022 03 31.
Article em En | MEDLINE | ID: mdl-35435904
Double strand breaks (DSBs) in DNA are the most cytotoxic type of DNA damage. Because a myriad of insults can result in these lesions (e.g., replication stress, ionizing radiation, unrepaired UV damage), DSBs occur in most cells each day. In addition to cell death, unrepaired DSBs reduce genome integrity and the resulting mutations can drive tumorigenesis. These risks and the prevalence of DSBs motivate investigations into the mechanisms by which cells repair these lesions. Next generation sequencing can be paired with the induction of DSBs by ionizing radiation to provide a powerful tool to precisely define the mutations associated with DSB repair defects. However, this approach requires computationally challenging and cost prohibitive whole genome sequencing to detect the repair of the randomly occurring DSBs associated with ionizing radiation. Rare cutting endonucleases, such as I-Sce1, provide the ability to generate a single DSB, but their recognition sites must be inserted into the genome of interest. As a result, the site of repair is inherently artificial. Recent advances allow guide RNA (sgRNA) to direct a Cas9 endonuclease to any genome locus of interest. This could be applied to the study of DSB repair making next generation sequencing more cost effective by allowing it to be focused on the DNA flanking the Cas9-induced DSB. The goal of the manuscript is to demonstrate the feasibility of this approach by presenting a protocol that can define mutations that stem from the repair of a DSB upstream of the CD4 gene. The protocol can be adapted to determine changes in the mutagenic potential of DSB associated with exogenous factors, such as repair inhibitors, viral protein expression, mutations, and environmental exposures with relatively limited computation requirements. Once an organism's genome has been sequenced, this method can be theoretically employed at any genomic locus and in any cell culture model of that organism that can be transfected. Similar adaptations of the approach could allow comparisons of repair fidelity between different loci in the same genetic background.
Assuntos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Reparo do DNA / Quebras de DNA de Cadeia Dupla Tipo de estudo: Prognostic_studies / Risk_factors_studies Idioma: En Revista: J Vis Exp Ano de publicação: 2022 Tipo de documento: Article País de publicação: Estados Unidos

Texto completo: 1 Coleções: 01-internacional Base de dados: MEDLINE Assunto principal: Reparo do DNA / Quebras de DNA de Cadeia Dupla Tipo de estudo: Prognostic_studies / Risk_factors_studies Idioma: En Revista: J Vis Exp Ano de publicação: 2022 Tipo de documento: Article País de publicação: Estados Unidos