ArticleMolecular biology of the cell2018
CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses.
Article in Molecular biology of the cell, 2018. The graph could read no effect estimate from its abstract, so it casts no vote on the map. Cited by 9 papers.
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Who cites it
9 citing papers in PubMed, 18 citations in OpenAlex.
- The Current Situation and Development Prospect of Whole-Genome Screening.International journal of molecular sciences · 2024Review
- Article
- The IRF2/CENP-N/AKT signaling axis promotes proliferation, cell cycling and apoptosis resistance in nasopharyngeal carcinoma cells by increasing aerobic glycolysis.Journal of experimental & clinical cancer research : CR · 2021Article
- Regulating CRISPR/Cas9 Function through Conditional Guide RNA Control.Chembiochem : a European journal of chemical biology · 2021Review
- Development of CRISPR Interference (CRISPRi) Platform for Metabolic Engineering ofInternational journal of molecular sciences · 2020Article
- Beneficial effects of LRP6-CRISPR on prevention of alcohol-related liver injury surpassed fecal microbiota transplant in a rat model.Gut microbes · 2020Article
- Utilizing CRISPR/Cas9 technology to prepare lymphoblastoid cell lines harboring genetic mutations for generating quality control materials in genetic testing.Journal of clinical laboratory analysis · 2020Article
- Inhibition of PLK4 might enhance the anti-tumour effect of bortezomib on glioblastoma via PTEN/PI3K/AKT/mTOR signalling pathway.Journal of cellular and molecular medicine · 2020Article
- Multiplexed CRISPR/Cas9 gene knockout with simple crRNA:tracrRNA co-transfection.Cell & bioscience · 2019Article
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Authors and funding
4 authors at 1 institution in 1 country.
Funding
Abstract
A key goal for cell biological analyses is to assess the phenotypes that result from eliminating a target gene. Since the early 1990s, the predominant strategy utilized in human tissue culture cells has been RNA interference (RNAi)-mediated protein depletion. However, RNAi suffers well-documented off-target effects as well as incomplete and reversible protein depletion. The implementation of CRISPR/Cas9-based DNA cleavage has revolutionized the capacity to conduct functional studies in human cells. However, this approach is still underutilized for conducting visual phenotypic analyses, particularly for essential genes that require conditional strategies to eliminate their gene products. Optimizing this strategy requires effective and streamlined approaches to introduce the Cas9 guide RNA into target cells. Here we assess the efficacy of synthetic guide RNA transfection to eliminate gene products for cell biological studies. On the basis of three representative gene targets (KIF11, CENPN, and RELA), we demonstrate that transfection of synthetic single guide RNA (sgRNA) and CRISPR RNA (crRNA) guides works comparably for protein depletion as cell lines stably expressing lentiviral-delivered RNA guides. We additionally demonstrate that synthetic sgRNAs can be introduced by reverse transfection on an array. Together, these strategies provide a robust, flexible, and scalable approach for conducting functional studies in human cells.
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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.