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

  1. Jin-Soo Kim1,2,3
  1. 1Department of Chemistry, Seoul National University, Seoul 151-747, South Korea;
  2. 2Center for Genome Engineering, Institute for Basic Science, Seoul 151-747, South Korea

    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.

    Footnotes

    • 3 Corresponding author

      E-mail jskim01{at}snu.ac.kr

    • [Supplemental material is available for this article.]

    • Article published online before print. Article, supplemental material, and publication date are at http://www.genome.org/cgi/doi/10.1101/gr.171322.113.

      Freely available online through the Genome Research Open Access option.

    • Received December 17, 2013.
    • Accepted March 25, 2014.

    This article, published in Genome Research, is available under a Creative Commons License (Attribution-NonCommercial 4.0 International), as described at http://creativecommons.org/licenses/by-nc/4.0/.

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