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Published on: March 31, 2022
dCas9 binding inhibits the initiation of base excision repair in vitro
Jacob S Antony1, Steven A Roberts2, John J Wyrick2
1School of Molecular Biosciences, Washington State University, Pullman, WA 99164, USA.
Abstract:
Cas9 targets DNA during genome editing by forming an RNA:DNA heteroduplex (R-loop) between the Cas9-bound guide RNA and the targeted DNA strand. We have recently demonstrated that R-loop formation by catalytically inactive Cas9 (dCas9) is inherently mutagenic, in part, by promoting spontaneous cytosine deamination within the non-targeted single-stranded DNA of the dCas9-induced R-loop. However, the extent to which dCas9 binding and R-loop formation affect the subsequent repair of uracil lesions or other damaged DNA bases is unclear. Here, we show that DNA binding by dCas9 inhibits initiation of base excision repair (BER) for uracil lesions in vitro. Our data indicate that cleavage of uracil lesions by Uracil-DNA glycosylase (UDG) is generally inhibited at dCas9-bound DNA, in both the dCas9:sgRNA-bound target strand (TS) or the single-stranded non-target strand (NT). However, cleavage of a uracil lesion within the base editor window of the NT strand was less inhibited than at other locations, indicating that this site is more permissive to UDG activity. Furthermore, our data suggest that dCas9 binding to PAM sites can inhibit UDG activity. However, this non-specific inhibition can be relieved with the addition of an sgRNA lacking sequence complementarity to the DNA substrate. Moreover, we show that dCas9 binding also inhibits human single-strand selective monofunctional uracil-DNA glycosylase (SMUG1). Structural analysis of a Cas9-bound target site subsequently suggests a molecular mechanism for BER inhibition. Taken together, our results imply that dCas9 (or Cas9) binding may promote background mutagenesis by inhibiting the removal of DNA base lesions by BER.
Insights
Catalytically inactive Cas9 (dCas9) binding inhibits base excision repair (BER) of uracil lesions. This inhibition, observed in both DNA strands, suggests dCas9 may increase genome editing mutagenesis by blocking DNA repair pathways.
Area of Science:
- Molecular Biology
- Genetics
- Biochemistry
Background:
- Cas9 is a key enzyme in genome editing, forming RNA:DNA heteroduplexes (R-loops).
- Catalytically inactive dCas9 can induce R-loops and promote DNA mutagenesis via cytosine deamination.
- The impact of dCas9 binding on DNA repair, particularly for uracil lesions, remains largely uncharacterized.
Purpose of the Study:
- To investigate the effect of dCas9 binding on the initiation of base excision repair (BER) for uracil lesions.
- To determine whether dCas9 inhibits uracil-DNA glycosylase (UDG) activity in vitro.
- To elucidate the molecular mechanisms underlying dCas9-mediated inhibition of BER.
Main Methods:
- In vitro assays measuring uracil lesion cleavage by Uracil-DNA glycosylase (UDG) on dCas9-bound DNA substrates.
- Testing UDG activity on both the target strand (TS) and non-target strand (NT) of dCas9-bound DNA.
- Assessing the effect of dCas9 binding to PAM sites and the role of sgRNA complementarity on UDG inhibition.
- Evaluating the inhibition of human single-strand selective monofunctional uracil-DNA glycosylase (SMUG1) by dCas9.
- Structural analysis of Cas9-bound DNA to understand BER inhibition mechanisms.
Main Results:
- dCas9 binding significantly inhibits UDG-mediated cleavage of uracil lesions on both TS and NT DNA strands.
- UDG activity is less inhibited within a specific 'base editor window' on the NT strand.
- dCas9 binding to PAM sites can non-specifically inhibit UDG, an effect relieved by non-complementary sgRNA.
- dCas9 also inhibits the activity of human SMUG1.
- Structural insights suggest a molecular mechanism for dCas9-induced BER inhibition.
Conclusions:
- dCas9 binding inhibits the initiation of base excision repair for uracil lesions.
- This inhibition of BER by dCas9 may contribute to background mutagenesis during genome editing.
- Understanding these interactions is crucial for optimizing CRISPR-based gene editing technologies and minimizing off-target effects.
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