ArticleScientific reports2015
CRISPR/Cas9-based generation of knockdown mice by intronic insertion of artificial microRNA using longer single-stranded DNA.
Article in Scientific reports, 2015. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 72 papers.
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Who cites it
72 citing papers in PubMed.
- Strategies and considerations for the generation of ssDNA-Based HDR templates for CRISPR-based genome editing.BMC genomics · 2026Review
- Generation of mice expressing liver-specific fluorescent genes and the optimal conditions for signal detection via in vivo imaging.Experimental animals · 2026Article
- Generation of high-quality single-stranded DNA for full-length and truncated genome standards of recombinant adeno-associated viruses.Molecular therapy. Methods & clinical development · 2025Article
- Efficient Delivery of CRISPR-Cas9 RNP Complexes with Cyclodextrin-Based Nanosponges for Enhanced Genome Editing: TILD-CRISPR Integration.International journal of molecular sciences · 2025Article
- Bridging biological samples to functional nucleic acid biosensor applications: current enzymatic-based strategies for single-stranded DNA generation.Analytical sciences : the international journal of the Japan Society for Analytical Chemistry · 2024Review
- Targeted insertion of conditional expression cassettes into the mouse genome using the modified i-PITT.BMC genomics · 2024Article
- SMARCA4 is a haploinsufficient B cell lymphoma tumor suppressor that fine-tunes centrocyte cell fate decisions.Cancer cell · 2024Article
- Increasing Knockin Efficiency in Mouse Zygotes by Transient Hypothermia.The CRISPR journal · 2024Article
- Long-read sequencing for fast and robust identification of correct genome-edited alleles: PCR-based and Cas9 capture methods.PLoS genetics · 2024Article
- Donor template delivery by recombinant adeno-associated virus for the production of knock-in mice.BMC biology · 2024Article
- A method for generating user-defined circular single-stranded DNA from plasmid DNA using Golden Gate intramolecular ligation.Biotechnology and bioengineering · 2023Article
- ssDNA is not superior to dsDNA as long HDR donors for CRISPR-mediated endogenous gene tagging in human diploid RPE1 and HCT116 cells.BMC genomics · 2023Article
- Engineering CpG island DNA methylation in pluripotent cells through synthetic CpG-free ssDNA insertion.Cell reports methods · 2023Article
- Comparison of CRISPR-Cas9-mediated megabase-scale genome deletion methods in mouse embryonic stem cells.DNA research : an international journal for rapid publication of reports on genes and genomes · 2023Article
- Screening DNA aptamers that control the DNA cleavage, homology-directed repair, and transcriptional regulation of the CRISPR-(d)Cas9 system.Molecular therapy : the journal of the American Society of Gene Therapy · 2023Article
- Efficient DNA knock-in using AAV-mediated delivery with 2-cell embryo CRISPR-Cas9 electroporation.Frontiers in genome editing · 2023Article
- Floxing by Electroporating Single-Cell Embryos with Two CRISPR RNPs and Two ssODNs.Methods in molecular biology (Clifton, N.J.) · 2023Article
- Easily-controllable, helper phage-free single-stranded phagemid production system.Genes and environment : the official journal of the Japanese Environmental Mutagen Society · 2022Article
- CRISPR-KRISPR: a method to identify on-target and random insertion of donor DNAs and their characterization in knock-in mice.Genome biology · 2022Article
- Efficient Homology-Directed Repair with Circular Single-Stranded DNA Donors.The CRISPR journal · 2022Article
12 more citing papers are in PubMed but not listed here.
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Authors and funding
5 authors.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Knockdown mouse models, where gene dosages can be modulated, provide valuable insights into gene function. Typically, such models are generated by embryonic stem (ES) cell-based targeted insertion, or pronuclear injection, of the knockdown expression cassette. However, these methods are associated with laborious and time-consuming steps, such as the generation of large constructs with elements needed for expression of a functional RNAi-cassette, ES-cell handling, or screening for mice with the desired knockdown effect. Here, we demonstrate that reliable knockdown models can be generated by targeted insertion of artificial microRNA (amiRNA) sequences into a specific locus in the genome [such as intronic regions of endogenous eukaryotic translation elongation factor 2 (eEF-2) gene] using the Clustered Regularly Interspaced Short Palindromic Repeats/Crispr associated 9 (CRISPR/Cas9) system. We used in vitro synthesized single-stranded DNAs (about 0.5-kb long) that code for amiRNA sequences as repair templates in CRISPR/Cas9 mutagenesis. Using this approach we demonstrate that amiRNA cassettes against exogenous (eGFP) or endogenous [orthodenticle homeobox 2 (Otx2)] genes can be efficiently targeted to a predetermined locus in the genome and result in knockdown of gene expression. We also provide a strategy to establish conditional knockdown models with this method.
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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.