title: Generation of a conditional analog-sensitive kinase in human cells using CRISPR/Cas9-mediated genome engineering.
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 5-6 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.
authors.affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Tyler C
authors.initials: TC
authors.lastname: Moyer
authors.affiliation: Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, Baltimore, MD, USA.
authors.forename: Andrew J
authors.initials: AJ
authors.lastname: Holland
mesh_terms: D019943:Amino Acid Substitution; D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D000076987:CRISPR-Associated Protein 9; D002478:Cells, Cultured; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics*; D004926:Escherichia coli; D015894:Genome, Human; D006801:Humans; D008969:Molecular Sequence Data; D017354:Point Mutation; D017346:Protein Serine-Threonine Kinases / Q000235:genetics*
chemical_list: D001426:Bacterial Proteins; C490219:PLK4 protein, human; D017346:Protein Serine-Threonine Kinases; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases
keywords: Analog-sensitive kinase; CRISPR; Centrosome; Chemical genetics; Genome editing; Plk4