ArticleNature protocols2018
Generation and validation of homozygous fluorescent knock-in cells using CRISPR-Cas9 genome editing.
Article in Nature protocols, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 81 papers.
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Who cites it
81 citing papers in PubMed, 145 citations in OpenAlex.
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- Rapid and Robust Generation of Homozygous Fluorescent Reporter Knock-In Cell Pools by CRISPR-Cas9.Cells · 2025Article
- Dual Ribosome Profiling reveals metabolic limitations of cancer and stromal cells in the tumor microenvironment.Nature communications · 2025Article
- MARK2 regulates Golgi apparatus reorientation by phosphorylation of CAMSAP2 in directional cell migratio.eLife · 2025Article
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- Quantitative imaging of loop extruders rebuilding interphase genome architecture after mitosis.The Journal of cell biology · 2025Article
- Rapid generation of homozygous fluorescent knock-in human cells using CRISPR-Cas9 genome editing and validation by automated imaging and digital PCR screening.Nature protocols · 2025Article
21 more citing papers are in PubMed but not listed here.
Corrections and comments
PubMed lists nothing against this paper. Absence here is not a guarantee, only a check that was made.
Authors and funding
6 authors at 1 institution in 1 country.
Funding
Abstract
Gene tagging with fluorescent proteins is essential for investigations of the dynamic properties of cellular proteins. CRISPR-Cas9 technology is a powerful tool for inserting fluorescent markers into all alleles of the gene of interest (GOI) and allows functionality and physiological expression of the fusion protein. It is essential to evaluate such genome-edited cell lines carefully in order to preclude off-target effects caused by (i) incorrect insertion of the fluorescent protein, (ii) perturbation of the fusion protein by the fluorescent proteins or (iii) nonspecific genomic DNA damage by CRISPR-Cas9. In this protocol, we provide a step-by-step description of our systematic pipeline to generate and validate homozygous fluorescent knock-in cell lines.We have used the paired Cas9D10A nickase approach to efficiently insert tags into specific genomic loci via homology-directed repair (HDR) with minimal off-target effects. It is time-consuming and costly to perform whole-genome sequencing of each cell clone to check for spontaneous genetic variations occurring in mammalian cell lines. Therefore, we have developed an efficient validation pipeline of the generated cell lines consisting of junction PCR, Southern blotting analysis, Sanger sequencing, microscopy, western blotting analysis and live-cell imaging for cell-cycle dynamics. This protocol takes between 6 and 9 weeks. With this protocol, up to 70% of the targeted genes can be tagged homozygously with fluorescent proteins, thus resulting in physiological levels and phenotypically functional expression of the fusion proteins.
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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.