Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins

Genome Res. 2014 Jun;24(6):1012-9. doi: 10.1101/gr.171322.113. Epub 2014 Apr 2.

Abstract

RNA-guided engineered nucleases (RGENs) derived from the prokaryotic adaptive immune system known as CRISPR (clustered, regularly interspaced, short palindromic repeat)/Cas (CRISPR-associated) enable genome editing in human cell lines, animals, and plants, but are limited by off-target effects and unwanted integration of DNA segments derived from plasmids encoding Cas9 and guide RNA at both on-target and off-target sites in the genome. Here, we deliver purified recombinant Cas9 protein and guide RNA into cultured human cells including hard-to-transfect fibroblasts and pluripotent stem cells. RGEN ribonucleoproteins (RNPs) induce site-specific mutations at frequencies of up to 79%, while reducing off-target mutations associated with plasmid transfection at off-target sites that differ by one or two nucleotides from on-target sites. RGEN RNPs cleave chromosomal DNA almost immediately after delivery and are degraded rapidly in cells, reducing off-target effects. Furthermore, RNP delivery is less stressful to human embryonic stem cells, producing at least twofold more colonies than does plasmid transfection.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • CRISPR-Cas Systems*
  • Cell Line, Tumor
  • Cells, Cultured
  • Fibroblasts / metabolism
  • Genome, Human*
  • Humans
  • Mutagenesis, Site-Directed / methods
  • Pluripotent Stem Cells
  • Point Mutation
  • RNA Editing*
  • RNA, Guide, Kinetoplastida / genetics*
  • Ribonucleoproteins / genetics*
  • Ribonucleoproteins / metabolism

Substances

  • RNA, Guide
  • Ribonucleoproteins

Associated data

  • SRA/SRX473144