Production of Purified CasRNPs for Efficacious Genome Editing

Curr Protoc Mol Biol. 2017 Oct 2;120:31.10.1-31.10.19. doi: 10.1002/cpmb.43.

Abstract

CRISPR-Cas systems have been harnessed as modular genome editing reagents for functional genomics and show promise to cure genetic diseases. Directed by a guide RNA, a Cas effector introduces a double stranded break in DNA and host cell DNA repair leads to the introduction of errors (e.g., to knockout a gene) or a programmed change. Introduction of a Cas effector and guide RNA as a purified Cas ribonucleoprotein complex (CasRNP) has recently emerged as a powerful approach to alter cell types and organisms. Not only does CasRNP editing exhibit increased efficacy and specificity, it avoids optimization and iteration of species-specific factors such as codon usage, promoters, and terminators. CasRNP editing has been rapidly adopted for research use in many contexts and is quickly becoming a popular method to edit primary cells for therapeutic application. This article describes how to make a Cas9 RNP and outlines its use for gene editing in human cells. © 2017 by John Wiley & Sons, Inc.

Keywords: CRISPR; Cas9; RNP; genome editing; ribonucleoprotein.

MeSH terms

  • Bacterial Proteins / genetics
  • Bacterial Proteins / isolation & purification
  • Bacterial Proteins / metabolism
  • CRISPR-Associated Protein 9
  • CRISPR-Cas Systems*
  • Cell-Free System
  • Endonucleases / genetics
  • Endonucleases / isolation & purification
  • Endonucleases / metabolism
  • Escherichia coli / genetics
  • Escherichia coli / metabolism
  • Gene Editing* / methods
  • Gene Expression
  • Gene Targeting / methods
  • Humans
  • RNA, Guide / genetics
  • RNA, Guide / isolation & purification
  • Recombinant Proteins
  • Transcription, Genetic

Substances

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