A universal method for the purification of C2H2 zinc finger arrays

PLoS ONE, Journal Year: 2025, Volume and Issue: 20(2), P. e0318295 - e0318295

Published: Feb. 4, 2025

Zinc fingers (ZFs) are compact, modular, sequence-specific polynucleotide-binding domains uniquely suited for use as DNA probes and the targeted delivery of effector purposes such gene regulation editing. Despite recent advances in both design application ZF-containing proteins, there is still a lack general method their expression purification. Here we describe simple method, involving two chromatographic steps, production homogeneous, functional ZF proteins high yield (one milligram per liter bacterial culture), demonstrate generality this by applying it to diverse set eight C2H2-type proteins. By incorporating surface-exposed terminal cysteine residue that enables site-specific conjugation with maleimide-activated fluorophores, confirm suitability these situ labeling specific sequences human cells.

Language: Английский

---

Language models for protein design

Current Opinion in Structural Biology, Journal Year: 2025, Volume and Issue: 92, P. 103027 - 103027

Published: March 6, 2025

Language: Английский

---

Molecular Functions and Potential Utilization of Zinc Finger Proteins in Protozoan Parasites

Published: April 10, 2025

Zinc finger proteins (ZFP) are metalloproteins whose zinc atom interacts with side chains of specific histidines and cysteines the motif, generating functional three dimensional structures. A good number these from different species similar while others divergent in structure function. Existing literature on structural features, functions their potential to be targeted for drug development or used as molecular tools parasitic protozoa was explored. Evidence reviewed articles revealed that, just like other eukaryotes, function various parasite cellular processes such as: transcription, RNA editing, mRNA processing & export and, turn-over among others. Some them had same features homologues eukaryotes including hosts. However, there were which studies aiming at replacing reported positive results some parasites Giardia lamblia, Trichomonas vaginalis Leishmania donovani though this is promising, off- target challenge that can cause effects, must solved before wide application achieved. Successful engineering use genome editing also Plasmodium falciparum. Since nucleases a powerful research medical tool, repurposing ZFPs useful therapy discovery. For succeed, more knowledge how regulated needed.

Language: Английский

---

A Decade of Genome Editing: Comparative Review of Zfn, Talen, and Crispr/Cas9

Published: May 15, 2025

Recent advances in genome editing technologies, including ZFNs, TALENs, and CRISPR/Cas9 systems, have redefined our ability to probe precisely modify the epigenome vivo vitro. ZFNs TALENs pioneered targeted through engineered nucleases, offering high specificity accuracy, while RNA-guided system has revolutionized field with its simplicity, efficiency, adaptability across diverse biological systems. Emerging innovations enhance precision. Broader applicability enable gene even traditionally intractable models. This collection highlights progress, comparative strengths, expanding applications of these tools research, therapeutic, agricultural fields

Language: Английский

---

Systematic Development of Reprogrammed Modular Integrases Enables Precise Genomic Integration of Large DNA Sequences

bioRxiv (Cold Spring Harbor Laboratory), Journal Year: 2024, Volume and Issue: unknown

Published: May 10, 2024

Summary Despite recent progress in the ability to manipulate genomes of eukaryotic cells 1–3 , there is still no effective and practical method precisely integrate large synthetic DNA constructs into desired chromosomal sites using a programmable integrase. Serine integrases can perform necessary molecular steps 4 but only if their natural target site first installed recipient genome by other methods. A more elegant approach would be directly reprogram serine integrase itself endogenous that different from recognition 5 . Here, we describe development platform Modular Integrases (the MINT platform), versatile protein-guided editing facilitate site-directed targeted integration sites. Through combination structural modeling, directed evolution, screening human have reprogrammed specificity Bxb1. We then utilized these Bxb1 variants enable precise kilobase-sized multiple locations within with up 35% efficiency promising genome-wide specificity. demonstrate therapeutic potential retargeting TRAC AAVS1 loci where wild-type has detectable activity.

Language: Английский

---