CRISPR/Cas9-Directed Genome Editing of Cultured Cells

Curr Protoc Mol Biol. 2014 Jul 1;107:31.1.1-17. doi: 10.1002/0471142727.mb3101s107.

Abstract

Human genome engineering has been transformed by the introduction of the CRISPR (clustered regularly interspaced short palindromic repeats)/Cas (CRISPR-associated) system found in most bacteria and archaea. Type II CRISPR/Cas systems have been engineered to induce RNA-guided genome editing in human cells, where small RNAs function together with Cas9 nucleases for sequence-specific cleavage of target sequences. Here we describe the protocol for Cas9-mediated human genome engineering, including construct building and transfection methods necessary for delivering Cas9 and guide RNA (gRNA) into human-induced pluripotent stem cells (hiPSCs) and HEK293 cells. Following genome editing, we also describe methods to assess genome editing efficiency using next-generation sequencing and isolate monoclonal hiPSCs with the desired modifications for downstream applications.

Keywords: CRISPR; genome engineering; human stem cells.

Publication types

  • Review

MeSH terms

  • Animals
  • CRISPR-Cas Systems*
  • Cell Culture Techniques
  • Cells, Cultured
  • Genome, Human*
  • HEK293 Cells
  • Humans
  • Induced Pluripotent Stem Cells*
  • RNA, Guide / biosynthesis
  • RNA, Guide / genetics
  • Transfection / methods

Substances

  • RNA, Guide