Journal
PLOS ONE
Volume 9, Issue 10, Pages -Publisher
PUBLIC LIBRARY SCIENCE
DOI: 10.1371/journal.pone.0109752
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Categories
Funding
- Marie Curie Intra-European fellowship (FanDamAge)
- Medical Research Council (MRC)
- Medical Research Council [MC_U127070192, MC_UU_12016/1] Funding Source: researchfish
- MRC [MC_U127070192, MC_UU_12016/1] Funding Source: UKRI
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The Cas9/CRISPR system become a popular choice for genome editing. In this system, binding of a single guide (sg) RNA to a cognate genomic sequence enables the Cas9 nuclease to induce a double-strand break at that locus. This break is next repaired by an error-prone mechanism, leading to mutation and gene disruption. In this study we describe a range of refinements of the method, including stable cell lines expressing Cas9, and a PCR based protocol for the generation of the sgRNA. We also describe a simple methodology that allows both elimination of Cas9 from cells after gene disruption and re-intoduction of the disrupted gene. This advance enables easy assessment of the off target effects associated with gene disruption, as well as phenotype-based structure-function analysis. In our study, we used the Fan1 DNA repair gene as control in these experiments. Cas9/CRISPR-mediated Fanl disruption occurred at frequencies of around 29%, and resulted in the anticipated spectrLlm of genotoxin hypersensitivity, which was rescued by re-introduction of Fan1.
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