Genome editing via delivery of Cas9 ribonucleoprotein

Methods. 2017 May 15;121-122:9-15. doi: 10.1016/j.ymeth.2017.04.003. Epub 2017 Apr 12.

Abstract

The CRISPR-Cas genome editing system is very powerful. The format of the CRISPR reagents and the means of delivery are often important factors in targeting efficiency. Delivery of recombinant Cas9 protein and guide RNA (gRNA) as a preformed ribonucleoprotein (RNP) complex has recently emerged as a powerful and general approach to genome editing. Here we outline methods to produce and deliver Cas9 RNPs. A donor DNA carrying desired sequence changes can also be included to program precise sequence introduction or replacement. RNP delivery limits exposure to genome editing reagents, reduces off-target events, drives high rates of homology-dependent repair, and can be applied to embryos to rapidly generate animal models. RNP delivery thus minimizes some of the pitfalls of alternative editing modalities and is rapidly being adopted by the genome editing community.

MeSH terms

  • Bacterial Proteins / genetics*
  • Bacterial Proteins / metabolism
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems*
  • Clustered Regularly Interspaced Short Palindromic Repeats
  • DNA / genetics
  • DNA / metabolism
  • Endonucleases / genetics*
  • Endonucleases / metabolism
  • Gene Editing / methods*
  • Gene Targeting / methods
  • Gene Transfer Techniques*
  • Genome, Human
  • HEK293 Cells
  • Hematopoietic Stem Cells / cytology
  • Hematopoietic Stem Cells / metabolism
  • Humans
  • Jurkat Cells
  • K562 Cells
  • Primary Cell Culture
  • RNA, Guide / genetics*
  • RNA, Guide / metabolism
  • Recombinant Proteins / genetics
  • Recombinant Proteins / metabolism
  • Ribonucleoproteins / genetics*
  • Ribonucleoproteins / metabolism

Substances

  • Bacterial Proteins
  • RNA, Guide
  • Recombinant Proteins
  • Ribonucleoproteins
  • DNA
  • CRISPR-Associated Protein 9
  • Cas9 endonuclease Streptococcus pyogenes
  • Endonucleases