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

Methods Cell Biol. 2015;129:19-36. doi: 10.1016/bs.mcb.2015.03.017. Epub 2015 May 27.

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.

Keywords: Analog-sensitive kinase; CRISPR; Centrosome; Chemical genetics; Genome editing; Plk4.

MeSH terms

  • Amino Acid Substitution
  • Bacterial Proteins / genetics*
  • Base Sequence
  • CRISPR-Associated Protein 9
  • Cells, Cultured
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Endonucleases / genetics*
  • Escherichia coli
  • Genome, Human
  • Humans
  • Molecular Sequence Data
  • Point Mutation
  • Protein-Serine-Threonine Kinases / genetics*

Substances

  • Bacterial Proteins
  • PLK4 protein, human
  • Protein-Serine-Threonine Kinases
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases