Zinc-finger nucleases (ZFNs) drive efficient genome editing by introducing a double-strand break into the targeted gene. Cleavage is induced when two custom-designed ZFNs heterodimerize upon binding DNA to form a catalytically active nuclease complex. The importance of this dimerization event for subsequent cleavage activity has stimulated efforts to engineer the nuclease interface to prevent undesired homodimerization. Here we report the development and application of a yeast-based selection system designed to functionally interrogate the ZFN dimer interface. We identified critical residues involved in dimerization through the isolation of cold-sensitive nuclease domains. We used these residues to engineer ZFNs that have superior cleavage activity while suppressing homodimerization. The improvements were portable to orthogonal domains, allowing the concomitant and independent cleavage of two loci using two different ZFN pairs. These ZFN architectures provide a general means for obtaining highly efficient and specific genome modification.

Download full-text PDF

Source
http://dx.doi.org/10.1038/nmeth.1539DOI Listing

Publication Analysis

Top Keywords

cleavage activity
8
enhancing zinc-finger-nuclease
4
zinc-finger-nuclease activity
4
activity improved
4
improved obligate
4
obligate heterodimeric
4
heterodimeric architectures
4
architectures zinc-finger
4
zinc-finger nucleases
4
nucleases zfns
4

Similar Publications

A trigger-inducible split-Csy4 architecture for programmable RNA modulation.

Nucleic Acids Res

January 2025

Research Center for Life Sciences Computing, Zhejiang Lab, Kechuang Avenue, Yuhang District, Hangzhou, Zhejiang, 311121, China.

The CRISPR-derived endoribonuclease Csy4 is a popular tool for controlling transgene expression in various therapeutically relevant settings, but adverse effects potentially arising from non-specific RNA cleavage remains largely unexplored. Here, we report a split-Csy4 architecture that was carefully optimized for in vivo usage. First, we separated Csy4 into two independent protein moieties whose full catalytic activity can be restored via various constitutive or conditional protein dimerization systems.

View Article and Find Full Text PDF

The SARS-CoV-2 papain-like protease PLpro has multiple roles in the viral replication cycle, related to both its polypeptide cleavage function and its ability to antagonize the host immune response. Targeting the PLpro function is recognized as a promising mechanism to modulate viral replication, while supporting host immune responses. However, the development of PLpro-specific inhibitors remains challenging.

View Article and Find Full Text PDF

Dual sgRNA-directed knockout gene expression using CRISPR/Cas9 technology for editing gene in triple-negative breast cancer.

Narra J

December 2024

Animal Research Facilities, Indonesia Medical Education and Research Institute (IMERI), Faculty of Medicine, Universitas Indonesia, Jakarta, Indonesia.

Clustered regularly interspaced short palindromic repeats (CRISPR)-associated nuclease 9 (CRISPR/Cas9) offers a robust approach for genome manipulation, particularly in cancer therapy. Given its high expression in triple-negative breast cancer (TNBC), targeting with CRISPR/Cas9 holds promise as a therapeutic strategy. The aim of this study was to design specific single guide ribonucleic acid (sgRNA) for CRISPR/Cas9 to permanently knock out the gene, exploring its potential as a therapeutic approach in breast cancer while addressing potential off-target effects.

View Article and Find Full Text PDF

Carbon-carbon bond formation and cleavage at redox active bis(pyridylimino)isoindole (BPI) germylene compounds.

Dalton Trans

January 2025

Instituto de Investigaciones Químicas (IIQ), Departamento de Química Inorgánica, Facultad de Química, and Centro de Innovación en Química Avanzada (ORFEO-CINQA), Consejo Superior de Investigaciones Científicas (CSIC) and Universidad de Sevilla, 41092 Sevilla, Spain.

Redox-active ligands provide alternative reaction pathways by facilitating redox events. Among these, tridentate bis(piridylimino)isoindole (BPI) fragments offer great potential, though their redox-active behaviour remains largely underdeveloped. We describe herein a family of BPI germanium(II) complexes and the study of their redox properties.

View Article and Find Full Text PDF

Synthetic circuits that regulate protein secretion in human cells could support cell-based therapies by enabling control over local environments. Although protein-level circuits enable such potential clinical applications, featuring orthogonality and compactness, their non-human origin poses a potential immunogenic risk. In this study, we developed Humanized Drug Induced Regulation of Engineered CyTokines (hDIRECT) as a platform to control cytokine activity exclusively using human-derived proteins.

View Article and Find Full Text PDF

Want AI Summaries of new PubMed Abstracts delivered to your In-box?

Enter search terms and have AI summaries delivered each week - change queries or unsubscribe any time!