ArticleBMC biology2018
Comparative analysis of single-stranded DNA donors to generate conditional null mouse alleles.
Article in BMC biology, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 51 papers.
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Who cites it
51 citing papers in PubMed, 78 citations in OpenAlex.
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- A CRISPR/Cas9-engineered mouse carrying a conditional knockout allele for the early growth response-1 transcription factor.Genesis (New York, N.Y. : 2000) · 2023Article
- Evidence-Based Guide to Using Artificial Introns for Tissue-Specific Knockout in Mice.International journal of molecular sciences · 2023Review
- The roles of Cyp1a2 and Cyp2d in pharmacokinetic profiles of serotonin and norepinephrine reuptake inhibitor duloxetine and its metabolites in mice.European journal of pharmaceutical sciences : official journal of the European Federation for Pharmaceutical Sciences · 2023Article
- Gene Editing in Mouse Zygotes Using the CRISPR/Cas9 System.Methods in molecular biology (Clifton, N.J.) · 2023Article
- Genetic and Molecular Quality Control of Genetically Engineered Mice.Methods in molecular biology (Clifton, N.J.) · 2023Article
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Authors and funding
15 authors at 2 institutions in 1 country.
Funding
Abstract
backgroundThe International Mouse Phenotyping Consortium is generating null allele mice for every protein-coding gene in the genome and characterizing these mice to identify gene-phenotype associations. While CRISPR/Cas9-mediated null allele production in mice is highly efficient, generation of conditional alleles has proven to be more difficult. To test the feasibility of using CRISPR/Cas9 gene editing to generate conditional knockout mice for this large-scale resource, we employed Cas9-initiated homology-driven repair (HDR) with short and long single stranded oligodeoxynucleotides (ssODNs and lssDNAs).
resultsUsing pairs of single guide RNAs and short ssODNs to introduce loxP sites around a critical exon or exons, we obtained putative conditional allele founder mice, harboring both loxP sites, for 23 out of 30 targeted genes. LoxP sites integrated in cis in at least one mouse for 18 of 23 genes. However, loxP sites were mutagenized in 4 of the 18 in cis lines. HDR efficiency correlated with Cas9 cutting efficiency but was minimally influenced by ssODN homology arm symmetry. By contrast, using pairs of guides and single lssDNAs to introduce loxP-flanked exons, conditional allele founders were generated for all four genes targeted, although one founder was found to harbor undesired mutations within the lssDNA sequence interval. Importantly, when employing either ssODNs or lssDNAs, random integration events were detected.
conclusionsOur studies demonstrate that Cas9-mediated HDR with pairs of ssODNs can generate conditional null alleles at many loci, but reveal inefficiencies when applied at scale. In contrast, lssDNAs are amenable to high-throughput production of conditional alleles when they can be employed. Regardless of the single-stranded donor utilized, it is essential to screen for sequence errors at sites of HDR and random insertion of donor sequences into the genome.
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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.