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Engineered DNase-inactive Cpf1 variants to improve targeting scope for base editing in E. coli.

ABSTRACT: The development of base editing (BE) technology has opened a new avenue for research studies in bacteriology, particularly for bacterial species in which the DNA double-strand breaks (DSBs) introduced by CRISPR/Cas system would lead to cell death. However, a major limitation of BE-mediated gene editing is the restricted editable sites in the target bacterial genome due to highly diverse genomic compositions, such as GC content. Herein, we developed a broad-spectrum DNase-inactive Cpf1 (dCpf1) variant from Francisella novicida (bsdFnCpf1) through directed evolution. The resulting optimized mutant showed a substantially expanded targeting range, including previously non-canonical protospacer-adjacent motifs (PAMs), especially the GC-rich PAMs. Cytidine deaminase APOBEC1 and uracil DNA glycosylase inhibitor (UGI) were fused with bsdFnCpf1 to achieve specific C to T mutations at multiple target sites with canonical or non-canonical PAMs in the E. coli genome without compromising cell growth. We anticipate that bsdFnCpf1 could be applied for multiplex gene regulation and BE in species that have been reported to be suitable for Cpf1.

SUBMITTER: Chen Z

PROVIDER: S-EPMC8484740 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Expanding the base editing scope in rice by using Cas9 variants.

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Increasing Cytosine Base Editing Scope and Efficiency With Engineered Cas9-PmCDA1 Fusions and the Modified sgRNA in Rice.

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Design and assessment of engineered CRISPR-Cpf1 and its use for genome editing.

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Project description:Recent advances in CRISPR-Cas9 techniques, especially the discovery of base and prime editing, have significantly improved our ability to make precise changes in the genome. We hypothesized that modulating certain endogenous pathway cells could improve the action of those editing tools in mammalian cells. We established a reporter system in which a small fragment was integrated into the genome by prime editing (PE). With this system, we screened an in-house small-molecule library and identified a group of histone deacetylase inhibitors (HDACi) increasing prime editing. We also found that HDACi increased the efficiency of both cytosine base editing (CBE) and adenine base editing (ABE). Moreover, HDACi increased the purity of cytosine base editor products, which was accompanied by an upregulation of the acetylation of uracil DNA glycosylase (UNG) and UNG inhibitor (UGI) and an enhancement of their interaction. In summary, our work demonstrated that HDACi improves Cas9-mediated prime editing and base editing.

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