dCas9 binding inhibits the initiation of base excision repair in vitro

Antony, Jacob S.; Roberts, Steven A.; Wyrick, John J.; Hinz, John M.

doi:10.1016/j.dnarep.2021.103257

Cited by 5 publications

(8 citation statements)

References 63 publications

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“…We have compared the resistance of Cas9-generated cleavage product to degradation by HEK293 and HEK293 PARP1 −/− cell extracts using 32 proteins in an asymmetric manner, maintaining stronger contacts with the target DNA strand hybridized with sgRNA. This result is consistent with previously published data shown that the 5′terminal part of the protospacer region of the non-target strand in the Cas9/sgRNA complex is accessible for cleavage by P1 nuclease and DNA glycosylases UDG and SMUG1 [38,47]. The observed DNA protection by Cas9 is most likely independent of PARP1.…”

Section: Cas9-generated Dsbs Are Shielded From Parp1 Bindingsupporting

confidence: 93%

“…The protospacer sequences of H4, H6 and H9 (S1 Table) human genomic targets [47] were cloned at the Bbs I site in pSpCas9(BB)-2A-GFP (pX458) plasmid [89] (a gift from Dr. Feng Zhang; Addgene plasmid #48138; http://n2t.net/addgene:48138; RRID: Addgene_48138). To assess the editing efficiency, 5×10 5 HEK293 or HEK293 PARP1 −/− cells were seeded per well of a six-well plate and transfected with 2.5 µg of the plasmid 24 h later using Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA).…”

Section: Methodsmentioning

confidence: 99%

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Cas9 is mostly orthogonal to human systems of DNA break sensing and repair

Maltseva,

Vasil’eva,

Moor

et al. 2023

Preprint

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CRISPR/Cas9 system is а powerful gene editing tool based on the RNA-guided cleavage of target DNA. The Cas9 activity can be modulated by proteins involved in DNA damage signalling and repair due to their interaction with double- and single-strand breaks (DSB and SSB, respectively) generated by wild-type Cas9 or Cas9 nickases. Here we address the interplay between Streptococcus pyogenes Cas9 and key DNA repair factors, including poly(ADP-ribose) polymerase 1 (SSB/DSB sensor), its closest homolog poly(ADP-ribose) polymerase 2, Ku antigen (DSB sensor), DNA ligase I (SSB sensor), replication protein A (DNA duplex destabilizer), and Y-box binding protein 1 (RNA/DNA binding protein). None of those significantly affected Cas9 activity, while Cas9 efficiently shielded DSBs and SSBs from their sensors. Poly(ADP-ribosyl)ation of Cas9 detected for poly(ADP-ribose) polymerase 2 had no apparent effect on the activity. In cellulo, Cas9-dependent gene editing was independent of poly(ADP-ribose) polymerase 1. Thus, Cas9 can be regarded as an enzyme mostly orthogonal to the natural regulation of human systems of DNA break sensing and repair.

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Section: Cas9-generated Dsbs Are Shielded From Parp1 Bindingsupporting

confidence: 93%

Section: Methodsmentioning

confidence: 99%

Cas9 is mostly orthogonal to human systems of DNA break sensing and repair

Maltseva,

Vasil’eva,

Moor

et al. 2023

Preprint

View full text Add to dashboard Cite

show abstract

“…Moreover, they agree with other studies showing that Cas9 (or dCas9) can inhibit other endogenous cellular processes such as double strand break repair ( Clarke et al, 2018 ; Xu et al, 2020 ), replication ( Wiktor et al, 2016 ; Whinn et al, 2019 ; Doi et al, 2021 ), and transcription ( Gilbert et al, 2013 ; Qi et al, 2013 ; Clarke et al, 2018 ; Vigouroux et al, 2018 ). Along with our recent study showing that dCas9 targeting inhibits the initiation of BER in vitro ( Antony et al, 2022 ), we speculate that the mechanism by which background mutations arise during genome editing may be more complex than originally anticipated. Understanding how these mutations arise in yeast or other eukaryotes can be helpful towards designing genome editing experiments that reduce or even eliminate unwanted mutations, therefore elevating the precision and accuracy of the intended edits.…”

Section: Discussionmentioning

confidence: 58%

“…The PAM motif is indicated by the green box. For more details see Gilbert et al, 2013 ; Clarke et al, 2018 ; Laughery et al, 2019 ; Doi et al, 2021 ; Antony et al, 2022 .…”

Section: Potential Pitfalls To Genome Editing In Saccharomy...mentioning

confidence: 99%

“…Given the importance of DNA repair for mediating both intended and unwanted mutations with Cas9 (and dCas9), understanding how Cas9 interacts with endogenous DNA repair pathways is becoming increasingly important. A recent in vitro study from our group demonstrates that BER enzymes like uracil DNA glycosylase (UDG) are inhibited by dCas9 binding in a position-dependent manner ( Antony et al, 2022 ). This could be important towards understanding both base editing outcomes as well as unanticipated mutations resulting from dCas9 (or Cas9) targeting within cells ( Figure 6 ).…”

Section: Potential Pitfalls To Genome Editing In Saccharomy...mentioning

confidence: 99%

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Tips, Tricks, and Potential Pitfalls of CRISPR Genome Editing in Saccharomyces cerevisiae

Antony

Hinz

Wyrick

2022

Front. Bioeng. Biotechnol.

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The versatility of clustered regularly interspaced short palindromic repeat (CRISPR)-associated (Cas) genome editing makes it a popular tool for many research and biotechnology applications. Recent advancements in genome editing in eukaryotic organisms, like fungi, allow for precise manipulation of genetic information and fine-tuned control of gene expression. Here, we provide an overview of CRISPR genome editing technologies in yeast, with a particular focus on Saccharomyces cerevisiae. We describe the tools and methods that have been previously developed for genome editing in Saccharomyces cerevisiae and discuss tips and experimental tricks for promoting efficient, marker-free genome editing in this model organism. These include sgRNA design and expression, multiplexing genome editing, optimizing Cas9 expression, allele-specific editing in diploid cells, and understanding the impact of chromatin on genome editing. Finally, we summarize recent studies describing the potential pitfalls of using CRISPR genome targeting in yeast, including the induction of background mutations.

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Sensing the DNA-mismatch tolerance of catalytically inactive Cas9 via barcoded DNA nanostructures in solid-state nanopores

Sandler,

Weckman,

Yorke

et al. 2023

Nat. Biomed. Eng

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Single-molecule quantification of the strength and sequence specificity of interactions between proteins and nucleic acids would facilitate the probing of protein–DNA binding. Here we show that binding events between the catalytically inactive Cas9 ribonucleoprotein and any pre-defined short sequence of double-stranded DNA can be identified by sensing changes in ionic current as suitably designed barcoded linear DNA nanostructures with Cas9-binding double-stranded DNA overhangs translocate through solid-state nanopores. We designed barcoded DNA nanostructures to study the relationships between DNA sequence and the DNA-binding specificity, DNA-binding efficiency and DNA-mismatch tolerance of Cas9 at the single-nucleotide level. Nanopore-based sensing of DNA-barcoded nanostructures may help to improve the design of efficient and specific ribonucleoproteins for biomedical applications, and could be developed into sensitive protein-sensing assays.

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dCas9 binding inhibits the initiation of base excision repair in vitro

Cited by 5 publications

References 63 publications

Cas9 is mostly orthogonal to human systems of DNA break sensing and repair

Cas9 is mostly orthogonal to human systems of DNA break sensing and repair

Tips, Tricks, and Potential Pitfalls of CRISPR Genome Editing in Saccharomyces cerevisiae

Sensing the DNA-mismatch tolerance of catalytically inactive Cas9 via barcoded DNA nanostructures in solid-state nanopores

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