ArticleMolecular therapy : the journal of the American Society of Gene Therapy2023
An optimized SpCas9 high-fidelity variant for direct protein delivery.
Article in Molecular therapy : the journal of the American Society of Gene Therapy, 2023. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 12 papers.
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Who cites it
12 citing papers in PubMed, 20 citations in OpenAlex.
- CRISPR in clinical oncology: translational advances from molecular diagnostics to therapeutics.Nature reviews. Clinical oncology · 2026Review
- Highly Efficient Site-Specific and Cassette Mutagenesis of Plasmids Harboring GC-Rich Sequences.Cells · 2025Article
- Quantifying Protein-Nucleic Acid Interactions for Engineering Useful CRISPR-Cas9 Genome-Editing Variants.Methods in molecular biology (Clifton, N.J.) · 2025Article
- Development of artificial transcription factors and their applications in cell reprograming, genetic screen, and disease treatment.Molecular therapy : the journal of the American Society of Gene Therapy · 2024Review
- Safety and efficacy studies of CRISPR-Cas9 treatment of sickle cell disease highlights disease-specific responses.Molecular therapy : the journal of the American Society of Gene Therapy · 2024Article
- Development and IND-enabling studies of a novel Cas9 genome-edited autologous CD34Molecular therapy : the journal of the American Society of Gene Therapy · 2024Article
- Prime editing functionally corrects cystic fibrosis-causing CFTR mutations in human organoids and airway epithelial cells.Cell reports. Medicine · 2024Article
- Engineering Cas9: next generation of genomic editors.Applied microbiology and biotechnology · 2024Review
- Challenges of CRISPR/Cas-Based Cell Therapy for Type 1 Diabetes: How Not to Engineer a "Trojan Horse".International journal of molecular sciences · 2023Review
- Unlocking the secrets of ABEs: the molecular mechanism behind their specificity.Biochemical Society transactions · 2023Review
- Enrichment strategies to enhance genome editing.Journal of biomedical science · 2023Review
- Genome editing for sickle cell disease: still time to correct?Frontiers in pediatrics · 2023Review
Corrections and comments
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Authors and funding
12 authors at 4 institutions in 4 countries.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Electroporation of the Cas9 ribonucleoprotein (RNP) complex offers the advantage of preventing off-target cleavages and potential immune responses produced by long-term expression of the nuclease. Nevertheless, the majority of engineered high-fidelity Streptococcus pyogenes Cas9 (SpCas9) variants are less active than the wild-type enzyme and are not compatible with RNP delivery. Building on our previous studies on evoCas9, we developed a high-fidelity SpCas9 variant suitable for RNP delivery. The editing efficacy and precision of the recombinant high-fidelity Cas9 (rCas9HF), characterized by the K526D substitution, was compared with the R691A mutant (HiFi Cas9), which is currently the only available high-fidelity Cas9 that can be used as an RNP. The comparative analysis was extended to gene substitution experiments where the two high fidelities were used in combination with a DNA donor template, generating different ratios of non-homologous end joining (NHEJ) versus homology-directed repair (HDR) for precise editing. The analyses revealed a heterogeneous efficacy and precision indicating different targeting capabilities between the two variants throughout the genome. The development of rCas9HF, characterized by an editing profile diverse from the currently used HiFi Cas9 in RNP electroporation, increases the genome editing solutions for the highest precision and efficient applications.
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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 Socratic graph.