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Enhancing CRISPR deletion via pharmacological delay of DNA-PKcs

CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of...

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Detalles Bibliográficos
Autores principales: Bosch-Guiteras, Núria, Uroda, Tina, Guillen-Ramirez, Hugo A., Riedo, Rahel, Gazdhar, Amiq, Esposito, Roberta, Pulido-Quetglas, Carlos, Zimmer, Yitzhak, Medová, Michaela, Johnson, Rory
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Cold Spring Harbor Laboratory Press 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7919447/
https://www.ncbi.nlm.nih.gov/pubmed/33574136
http://dx.doi.org/10.1101/gr.265736.120
Descripción
Sumario:CRISPR-Cas9 deletion (CRISPR-del) is the leading approach for eliminating DNA from mammalian cells and underpins a variety of genome-editing applications. Target DNA, defined by a pair of double-strand breaks (DSBs), is removed during nonhomologous end-joining (NHEJ). However, the low efficiency of CRISPR-del results in laborious experiments and false-negative results. By using an endogenous reporter system, we show that repression of the DNA-dependent protein kinase catalytic subunit (DNA-PKcs)—an early step in NHEJ—yields substantial increases in DNA deletion. This is observed across diverse cell lines, gene delivery methods, commercial inhibitors, and guide RNAs, including those that otherwise display negligible activity. We further show that DNA-PKcs inhibition can be used to boost the sensitivity of pooled functional screens and detect true-positive hits that would otherwise be overlooked. Thus, delaying the kinetics of NHEJ relative to DSB formation is a simple and effective means of enhancing CRISPR-deletion.