ArticleMolecular therapy. Nucleic acids2022
A dual conditional CRISPR-Cas9 system to activate gene editing and reduce off-target effects in human stem cells.
Article in Molecular therapy. Nucleic acids, 2022. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 11 papers.
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Who cites it
11 citing papers in PubMed, 19 citations in OpenAlex.
- CRISPR/Cas9 in perspective: evaluating efficacy, delivery methods, and ethical challenges in genome editing.Molecular biology reports · 2026Review
- Chemically-inducible CRISPR/Cas9 circuits for ultra-high dynamic range gene perturbation.Nature communications · 2025Article
- Optimization of gene knockout approaches and sgRNA selection in hPSCs with inducible Cas9 expression.Scientific reports · 2025Article
- Controlling CRISPR-Cas9 genome editing in human cells using a molecular glue degrader.Molecular therapy. Nucleic acids · 2025Article
- Gene Editing: An Effective Tool for the Future Treatment of Kidney Disease.Journal of inflammation research · 2025Review
- Current Strategies for Increasing Knock-In Efficiency in CRISPR/Cas9-Based Approaches.International journal of molecular sciences · 2024Review
- CRISPR/Cas9: a powerful tool in colorectal cancer research.Journal of experimental & clinical cancer research : CR · 2023Review
- Small Molecules for Enhancing the Precision and Safety of Genome Editing.Molecules (Basel, Switzerland) · 2022Review
- CRISPR/Cas9-mediated editing of double loci ofFrontiers in plant science · 2022Article
- The evolution and polymorphism of mono-amino acid repeats in androgen receptor and their regulatory role in health and disease.Frontiers in medicine · 2022Review
- Gene editing of putative cAMP and CaThe Journal of eukaryotic microbiologyArticle
Corrections and comments
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Authors and funding
9 authors at 1 institution in 1 country.
Funding
Abstract
The CRISPR-Cas9 system has emerged as a powerful and efficient tool for genome editing. An important drawback of the CRISPR-Cas9 system is the constitutive endonuclease activity when Cas9 endonuclease and its sgRNA are co-expressed. This constitutive activity results in undesirable off-target effects that hinder studies using the system, such as probing gene functions or its therapeutic use in humans. Here, we describe a convenient method that allows temporal and tight control of CRISPR-Cas9 activity by combining transcriptional regulation of Cas9 expression and protein stability control of Cas9 in human stem cells. To achieve this dual control, we combined the doxycycline-inducible system for transcriptional regulation and FKBP12-derived destabilizing domain fused to Cas9 for protein stability regulation. We showed that approximately 5%-10% of Cas9 expression was observed when only one of the two controls was applied. By combining two systems, we markedly lowered the baseline Cas9 expression and limited the exposure time of Cas9 endonuclease in the cell, resulting in little or no undesirable on- or off-target effects. We anticipate that this dual conditional CRISPR-Cas9 system can serve as a valuable tool for systematic characterization and identification of genes for various pathological processes.
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Registered trials
Read under generation 80e0d062 · epoch 390. Bibliography from PubMed, PubMed Central and OpenAlex; grants from NIH RePORTER; trial links from ClinicalTrials.gov; estimates, votes and beliefs from the OpenQuestion graph.