ArticleBMC biotechnology2014
A single blastocyst assay optimized for detecting CRISPR/Cas9 system-induced indel mutations in mice.
Article in BMC biotechnology, 2014. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 64 papers.
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Who cites it
64 citing papers in PubMed, 94 citations in OpenAlex.
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- Neuromodulation of voltage-gated sodium channels by Gβ1γ2 subunits: Implications for GNB1-linked encephalopathy.Neurobiology of disease · 2025Article
- Lack of glutamate neurotransmission in melanin-concentrating hormone neurons alters mouse reproduction and metabolism in a sex-specific manner.Biology of sex differences · 2025Article
- Enhancing gene expression studies in bovine embryos fertilized in vitro: Identifying stable reference genes across blastocysts with different developmental speeds.The Journal of reproduction and development · 2025Article
- MARTRE family proteins negatively regulate CCR4-NOT activity to protect poly(A) tail length and promote translation of maternal mRNA.Nature communications · 2025Article
- Overcoming the Delivery Challenges in CRISPR/Cas9 Gene Editing for Effective Cancer Treatment: A Review of Delivery Systems.International journal of medical sciences · 2025Review
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- State of the art CRISPR-based strategies for cancer diagnostics and treatment.Biomarker research · 2024Review
- Efficient genome editing of two-cell mouse embryos via modified CRISPR/Cas electroporation.Scientific reports · 2024Article
- Artificial Insemination as a Possible Convenient Tool to Acquire Genome-Edited Mice via In Vivo Fertilization with Engineered Sperm.Biotech (Basel (Switzerland)) · 2024Review
- Regulation of Sex-biased Gene Expression by the Ancestral X-Y Chromosomal Gene PairbioRxiv : the preprint server for biology · 2024Article
- Generation of LEPR Knockout Rabbits with CRISPR/CAS9 System.Doklady biological sciences : proceedings of the Academy of Sciences of the USSR, Biological sciences sections · 2024Article
- Navigating the CRISPR/Cas Landscape for Enhanced Diagnosis and Treatment of Wilson's Disease.Cells · 2024Review
- Taf1 knockout is lethal in embryonic male mice and heterozygous females show weight and movement disorders.Disease models & mechanisms · 2024Article
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- Human SIRT5 variants with reduced stability and activity do not cause neuropathology in mice.iScience · 2024Article
- An oocyte-specific Cas9-expressing mouse for germline CRISPR/Cas9-mediated genome editing.Genesis (New York, N.Y. : 2000) · 2024Article
- Asynchronous microexon splicing ofbioRxiv : the preprint server for biology · 2024Article
- TIGER: Single-step in vivo genome editing in a non-traditional rodent.Cell reports · 2023Article
4 more citing papers are in PubMed but not listed here.
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Authors and funding
5 authors at 3 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
backgroundMicroinjection of clustered regulatory interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 (Cas9)-related RNA and DNA into fertilized eggs is a novel approach for creating gene-modified mice. Blastocysts obtained just before implantation may be appropriate for testing the fidelity of CRIPSR/Cas9-mediated genome editing because they can be individually handled in vitro and obtained 3days after microinjection, thus allowing researchers to check mutations rapidly. However, it is not known whether indel mutations caused by the CRISPR/Cas9 system can be reproducibly detected in embryos. In this study, we assessed the detection of CRISPR/Cas9-induced mutations in embryos.
resultsT7 endonuclease I was more effective than Surveyor nuclease for detecting mutations in annealed fragments derived from 2 plasmids, which contained nearly identical sequences. Mouse fertilized eggs were microinjected with CRISPR/Cas9-related RNA/DNA to examine whether non-homologous end joining-mediated knockout and homologous recombination-mediated knockin occurred in the endogenous receptor (G protein-coupled) activity modifying protein 2 (Ramp2) gene. Individual blastocysts were lysed to obtain crude DNA solutions, which were used for polymerase chain reaction (PCR) assays. T7 endonuclease I-based PCR and sequencing analysis demonstrated that 25-100% of the embryos were knockout embryos and 7-57% of the embryos were knockin embryos. Our results also established that crude DNA from a single blastocyst was an appropriate template for Whole genome amplification and subsequent assessment by PCR and the T7 endonuclease I-based assay.
conclusionsThe single blastocyst-based assay was useful for determining whether CRISPR/Cas9-mediated genome editing worked in murine embryos.
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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.