Resumen
CRISPR is a genome-editing platform that makes use of the bacterially-derived endonuclease Cas9 to introduce DNA double-strand breaks at precise locations in the genome using complementary guide RNAs. We developed a nuclear domain knock-in screen, whereby the insertion of a gene encoding the green fluorescent protein variant Clover is inserted by Cas9-mediated homology directed repair (HDR) within the first exon of genes that are required for the structural integrity of subnuclear domains such as the nuclear lamina and promyelocytic leukemia nuclear bodies (PML NBs). Using this approach, we compared strategies for enhancing CRISPR-mediated HDR, focusing on known genes and small molecules that impact non-homologous end joining (NHEJ) and homologous recombination (HR). Ultimately, we identified the small molecule RS-1 as a potent enhancer of CRISPR-based genome editing, enhancing HDR 3- to 6-fold depending on the locus and transfection method. We also characterized U2OS human osteosarcoma cells expressing Clover-tagged PML and demonstrate that this strategy generates cell lines with PML NBs that are structurally and functionally similar to bodies in the parental cell line. Thus, the nuclear domain knock-in screen that we describe provides a simple means of rapidly evaluating methods and small molecules that have the potential to enhance Cas9-mediated HDR.
Idioma original | English |
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Páginas (desde-hasta) | 9379-9392 |
Número de páginas | 14 |
Publicación | Nucleic Acids Research |
Volumen | 43 |
N.º | 19 |
DOI | |
Estado | Published - oct. 30 2015 |
Nota bibliográfica
Funding Information:Canadian Institutes of Health Research (CIHR) [operating grant MOP-84260 to G.D.]; Harvey Graham Cancer Research Fund as part of the Terry Fox Foundation Strategic Health Research Training Program in Cancer Research at CIHR [trainee awards from the BHCRI to J.S. and J.P.]. Funding for open access charge: CIHR [MOP-84260]. Conflict of interest statement. None declared.
Publisher Copyright:
© The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research.
ASJC Scopus Subject Areas
- Genetics
PubMed: MeSH publication types
- Journal Article
- Research Support, Non-U.S. Gov't