ArticleStem cell reports2021
Efficient ssODN-Mediated Targeting by Avoiding Cellular Inhibitory RNAs through Precomplexed CRISPR-Cas9/sgRNA Ribonucleoprotein.
Article in Stem cell reports, 2021. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 29 papers.
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Who cites it
29 citing papers in PubMed, 51 citations in OpenAlex.
- Generation of a Patient-Derived Inducible Pluripotent Stem Cell Model for Studying theInternational journal of stem cells · 2026Article
- CRISPR/Cas9-based repair of a heterozygous HNF1A mutation in patient-derived hiPSCs.Human genetics · 2026Article
- Strategies and considerations for the generation of ssDNA-Based HDR templates for CRISPR-based genome editing.BMC genomics · 2026Review
- Systemic cyst(e)inase administration induces ferroptosis and synergizes with temozolomide in glioblastoma.iScience · 2026Article
- An iPSC-based in vitro model recapitulates human thymic epithelial development and multi-lineage specification.Nature communications · 2025Article
- Improving the Efficiency of CRISPR/Cas9-Mediated Non-Homologous End Joining Gene Knockout Using Small Molecules in Porcine Cells.Biomolecules · 2025Article
- Focused ultrasound and microbubble-mediated delivery of CRISPR-Cas9 ribonucleoprotein to human induced pluripotent stem cells.Molecular therapy : the journal of the American Society of Gene Therapy · 2025Article
- Optimized simple culture protocol for inducing mature myotubes from MYOD1-overexpressed human iPS cells.Scientific reports · 2024Article
- Article
- Electroporation Delivery of Cas9 sgRNA Ribonucleoprotein-Mediated Genome Editing in Sheep IVF Zygotes.International journal of molecular sciences · 2024Article
- MAGIK: A rapid and efficient method to create lineage-specific reporters in human pluripotent stem cells.Stem cell reports · 2024Article
- Possible involvement of zinc transporter ZIP13 in myogenic differentiation.Scientific reports · 2024Article
- Escaping from CRISPR-Cas-mediated knockout: the facts, mechanisms, and applications.Cellular & molecular biology letters · 2024Review
- Improving the Efficiency of CRISPR Ribonucleoprotein-Mediated Precise Gene Editing by Small Molecules in Porcine Fibroblasts.Animals : an open access journal from MDPI · 2024Article
- CRISPR Ribonucleoprotein-Mediated Precise Editing of Multiple Genes in Porcine Fibroblasts.Animals : an open access journal from MDPI · 2024Article
- Increasing Gene Editing Efficiency via CRISPR/Cas9- or Cas12a-Mediated Knock-In in Primary Human T Cells.Biomedicines · 2024Review
- Using Ribonucleoprotein-based CRISPR/Cas9 to Edit Single Nucleotide on Human Induced Pluripotent Stem Cells to Model Type 3 Long QT Syndrome (SCN5AStem cell reviews and reports · 2023Article
- Recent Advances in CRISPR/Cas9 Delivery Approaches for Therapeutic Gene Editing of Stem Cells.Stem cell reviews and reports · 2023Review
- Trichostatin A for Efficient CRISPR-Cas9 Gene Editing of Human Pluripotent Stem Cells.The CRISPR journal · 2023Article
- iPSC-derived type IV collagen α5-expressing kidney organoids model Alport syndrome.Communications biology · 2023Article
Corrections and comments
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Authors and funding
10 authors at 1 institution in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Combined with CRISPR-Cas9 technology and single-stranded oligodeoxynucleotides (ssODNs), specific single-nucleotide alterations can be introduced into a targeted genomic locus in induced pluripotent stem cells (iPSCs); however, ssODN knockin frequency is low compared with deletion induction. Although several Cas9 transduction methods have been reported, the biochemical behavior of CRISPR-Cas9 nuclease in mammalian cells is yet to be explored. Here, we investigated intrinsic cellular factors that affect Cas9 cleavage activity in vitro. We found that intracellular RNA, but not DNA or protein fractions, inhibits Cas9 from binding to single guide RNA (sgRNA) and reduces the enzymatic activity. To prevent this, precomplexing Cas9 and sgRNA before delivery into cells can lead to higher genome editing activity compared with Cas9 overexpression approaches. By optimizing electroporation parameters of precomplexed ribonucleoprotein and ssODN, we achieved efficiencies of single-nucleotide correction as high as 70% and loxP insertion up to 40%. Finally, we could replace the HLA-C1 allele with the C2 allele to generate histocompatibility leukocyte antigen custom-edited iPSCs.
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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.