Generation of a conditional analogsensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering

Tyler C. Moyer, Andrew J. Holland

Research output: Chapter in Book/Report/Conference proceedingConference contribution

Abstract

The ability to rapidly and specifically modify the genome of mammalian cells has been a long-term goal of biomedical researchers. Recently, the clustered, regularly interspaced, short palindromic repeats (CRISPR)/Cas9 system from bacteria has been exploited for genome engineering in human cells. The CRISPR system directs the RNA-guided Cas9 nuclease to a specific genomic locus to induce a DNA double-strand break that may be subsequently repaired by homology-directed repair using an exogenous DNA repair template. Here we describe a protocol using CRISPR/Cas9 to achieve bi-allelic insertion of a point mutation in human cells. Using this method, homozygous clonal cell lines can be constructed in 5e6 weeks. This method can also be adapted to insert larger DNA elements, such as fluorescent proteins and degrons, at defined genomic locations. CRISPR/Cas9 genome engineering offers exciting applications in both basic science and translational research.

Original languageEnglish (US)
Title of host publicationMethods in Cell Biology
Subtitle of host publicationCentrosome
EditorsKaren Oegema, Renata Basto
PublisherAcademic Press Inc.
Pages19-36
Number of pages18
ISBN (Print)9780128024492
DOIs
StatePublished - 2015

Publication series

NameMethods in Cell Biology
Volume129
ISSN (Print)0091-679X

ASJC Scopus subject areas

  • Cell Biology

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