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

doi:10.3791/62583

Cited by 3 publications

(6 citation statements)

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“…Radiation mimic reagent zeocin was used to induce DSBs to study how HPV8 E6 deregulates NHEJ [ 99 ]. sgRNA/CAS9 was used to induce DSB at a single genomic locus in cells expressing HPV8 E6 [ 86 , 114 ]. Their ability to cause DSBs in different manners can be used to examine alterations in DSB repair caused by beta HPVs.…”

Section: Approaches To Study Dsb Repairmentioning

confidence: 99%

“…A limitation of this assay is that it can only be used in cells not already expressing CD4. However, because it does not use a reporter cassette, it has the advantage of measuring NHEJ frequency at DSBs occurring in the unaltered host genome [ 114 ]. Further, all of these systems rely on non-physiological relevant mechanisms of inducing DSBs, (e.g., I-SceI or sgRNA/CAS9), producing lesions that are often “cleaner” than naturally occurring DSBs and thus do not represent the complex nature of naturally occurring DSBs [ 114 ].…”

Section: Approaches To Study Dsb Repairmentioning

confidence: 99%

“…However, because it does not use a reporter cassette, it has the advantage of measuring NHEJ frequency at DSBs occurring in the unaltered host genome [ 114 ]. Further, all of these systems rely on non-physiological relevant mechanisms of inducing DSBs, (e.g., I-SceI or sgRNA/CAS9), producing lesions that are often “cleaner” than naturally occurring DSBs and thus do not represent the complex nature of naturally occurring DSBs [ 114 ]. Finally, reporter assays can only detect the type of repair that they are designed for, thus variations that produce unexpected DNA products will not be seen.…”

Section: Approaches To Study Dsb Repairmentioning

confidence: 99%

“…Targeted NGS can provide a robust analysis of a small region of interest allowing deep sequencing at an affordable price [ 140 ]. Targeted NGS can be paired with sgRNA/CAS9 technology to induce a single DSB at a defined locus and then obtain an in-depth analysis of the mutations associated with repair at that locus under experimental conditions of interest [ 86 , 114 ]. There are limitations to NGS approaches.…”

Section: Approaches To Study Dsb Repairmentioning

confidence: 99%

“…NGS at a known region (CD4) around a single DSB induced by sgRNA/CAS9 shows that HPV8 E6 increases multiple types of mutations [ 86 , 114 ]. NGS could also be used to examine how common mutagenic events are in cells with beta HPVs, whether expressing the whole viral genome leads to similar mutations induced by HPV8 E6, and how the pattern of mutations changes with preexisting mutations in the host genome.…”

Section: Approaches To Study Dsb Repairmentioning

confidence: 99%

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Beta HPV Deregulates Double-Strand Break Repair

Wallace

2022

Viruses

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Beta human papillomavirus (beta HPV) infections are common in adults. Certain types of beta HPVs are associated with nonmelanoma skin cancer (NMSC) in immunocompromised individuals. However, whether beta HPV infections promote NMSC in the immunocompetent population is unclear. They have been hypothesized to increase genomic instability stemming from ultraviolet light exposure by disrupting DNA damage responses. Implicit in this hypothesis is that the virus encodes one or more proteins that impair DNA repair signaling. Fluorescence-based reporters, next-generation sequencing, and animal models have been used to test this primarily in cells expressing beta HPV E6/E7. Of the two, beta HPV E6 appears to have the greatest ability to increase UV mutagenesis, by attenuating two major double-strand break (DSB) repair pathways, homologous recombination, and non-homologous end-joining. Here, we review this dysregulation of DSB repair and emerging approaches that can be used to further these efforts.

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Section: Approaches To Study Dsb Repairmentioning

confidence: 99%

Section: Approaches To Study Dsb Repairmentioning

confidence: 99%

Section: Approaches To Study Dsb Repairmentioning

confidence: 99%

Section: Approaches To Study Dsb Repairmentioning

confidence: 99%

Section: Approaches To Study Dsb Repairmentioning

confidence: 99%

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Beta HPV Deregulates Double-Strand Break Repair

Wallace

2022

Viruses

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Beta human papillomavirus 8E6 promotes alternative end-joining

Bugbee

Palinski

et al. 2022

Preprint

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Double strand breaks (DSBs) are one of the most lethal DNA lesions in cells. Previous studies show that the E6 protein of beta-human papillomavirus (HPV8 E6) impairs two major DSB repair pathways homologous recombination (HR) and non-homologous end-joining (NHEJ). However, HPV8 E6 delays but does not eliminate DSB repair capability of cells. How DSBs are repaired in cells with HPV8 E6 remains to be studied. We hypothesis that HPV8 E6 promotes a backup DSB repair pathway, alternative end-joining (Alt-EJ). Using CAS9 based Alt-EJ reporters, we show that HPV8 E6 promotes Alt-EJ. Further, using small molecule inhibitors, CRISPR/CAS9 gene knockout, and HPV8 E6 mutant, we find that HPV8 E6 promotes Alt-EJ by binding p300, an acetyltransferase that facilitates DSB repair by HR and NHEJ. Finally, we analyzed whole genome sequencing data from genomes of human foreskin keratinocytes expressing HPV8 E6 and found they displayed an increased frequency of deletions bearing the microhomology signatures of Alt-EJ. This study fills the knowledge gap how DSB is repaired in cells with HPV8 E6 and the mutagenic consequences of HPV8 E6 mediated p300 destabilization. Broadly, this study supports the hypothesis that beta-HPV promotes cancer formation by increasing genomic instability.

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Beta human papillomavirus 8E6 promotes alternative end joining

Bugbee

Palinski

et al. 2023

eLife

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Double strand breaks (DSBs) are one of the most lethal DNA lesions in cells. The E6 protein of beta-human papillomavirus (HPV8 E6) impairs two critical DSB repair pathways; homologous recombination (HR) and non-homologous end-joining (NHEJ). However, HPV8 E6 only delays DSB repair. How DSBs are repaired in cells with HPV8 E6 remains to be studied. We hypothesize that HPV8 E6 promotes a less commonly used DSB repair pathway, alternative end-joining (Alt-EJ). Using CAS9 based Alt-EJ reporters, we show that HPV8 E6 promotes Alt-EJ. Further, using small molecule inhibitors, CRISPR/CAS9 gene knockout, and HPV8 E6 mutant, we find that HPV8 E6 promotes Alt-EJ by binding p300, an acetyltransferase that facilitates DSB repair by HR and NHEJ. At least some of this repair occurs through a subset of Alt-EJ known as polymerase theta dependent end joining. Finally, whole genome sequencing analysis showed HPV8 E6 caused an increased frequency of deletions bearing the microhomology signatures of Alt-EJ. This study fills the knowledge gap of how DSB is repaired in cells with HPV8 E6 and the mutagenic consequences of HPV8 E6 mediated p300 destabilization. Broadly, this study supports the hypothesis that beta-HPV promotes cancer formation by increasing genomic instability.

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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

Cited by 3 publications

References 0 publications

Beta HPV Deregulates Double-Strand Break Repair

Beta HPV Deregulates Double-Strand Break Repair

Beta human papillomavirus 8E6 promotes alternative end-joining

Beta human papillomavirus 8E6 promotes alternative end joining

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