ArticleThe Journal of cell biology2020
CRISPR-Cas12a-assisted PCR tagging of mammalian genes.
Article in The Journal of cell biology, 2020. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 38 papers.
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Who cites it
38 citing papers in PubMed, 73 citations in OpenAlex.
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- There and back again: a cell biologist's journey from organelles to molecules.Biological chemistry · 2025Review
- RNA-mediated condensation of TFE3 oncofusions facilitates transcriptional hub formation to promote translocation renal cell carcinoma.Nature communications · 2025Article
- PKD3 localizes to late endosomes to maintain Rab7-dependent endolysosomal homeostasis.iScience · 2025Article
- Induced proximity to PML protects TDP-43 from aggregation via SUMO-ubiquitin networks.Nature chemical biology · 2025Article
- Rapid depletion and super-resolution microscopy reveal dual roles of SRSF5 in coordinating nuclear speckle-paraspeckle crosstalk during cellular stress.Nucleic acids research · 2025Article
- Nondisruptive inducible labeling of ER-membrane contact sites using the Lamin B receptor.PLoS biology · 2025Article
- Dual Mode Analysis of Methicillin-ResistantACS omega · 2025Article
- Engineering Exosomes for CNS Disorders: Advances, Challenges, and Therapeutic Potential.International journal of molecular sciences · 2025Review
- Triple-Branch Catalytic Assembly DNAzyme Motivated DNA Tweezer for Sensitive and ReliableJournal of microbiology and biotechnology · 2024Article
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- C16ORF70/MYTHO promotes healthy aging in C.elegans and prevents cellular senescence in mammals.The Journal of clinical investigation · 2024Article
- Characterization of nucleolar SUMO isopeptidases unveils a general p53-independent checkpoint of impaired ribosome biogenesis.Nature communications · 2023Article
- CRISPR-Cas-mediated transcriptional modulation: The therapeutic promises of CRISPRa and CRISPRi.Molecular therapy : the journal of the American Society of Gene Therapy · 2023Review
- Fast, precise and cloning-free knock-in of reporter sequences in vivo with high efficiency.Development (Cambridge, England) · 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
- SAYSD1 senses UFMylated ribosome to safeguard co-translational protein translocation at the endoplasmic reticulum.Cell reports · 2023Article
- Abnormal triaging of misfolded proteins by adult neuronal ceroid lipofuscinosis-associated DNAJC5/CSPα mutants causes lipofuscin accumulation.Autophagy · 2023Article
Corrections and comments
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Authors and funding
11 authors at 2 institutions in 1 country.
Funding
No grant is acknowledged in the PubMed record.
Abstract
Here we describe a time-efficient strategy for endogenous C-terminal gene tagging in mammalian tissue culture cells. An online platform is used to design two long gene-specific oligonucleotides for PCR with generic template cassettes to create linear dsDNA donors, termed PCR cassettes. PCR cassettes encode the tag (e.g., GFP), a Cas12a CRISPR RNA for cleavage of the target locus, and short homology arms for directed integration via homologous recombination. The integrated tag is coupled to a generic terminator shielding the tagged gene from the co-inserted auxiliary sequences. Co-transfection of PCR cassettes with a Cas12a-encoding plasmid leads to robust endogenous expression of tagged genes, with tagging efficiency of up to 20% without selection, and up to 60% when selection markers are used. We used target-enrichment sequencing to investigate all potential sources of artifacts. Our work outlines a quick strategy particularly suitable for exploratory studies using endogenous expression of fluorescent protein-tagged genes.
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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.