RNA-guided human genome engineering via Cas9

Science. 2013 Feb 15;339(6121):823-6. doi: 10.1126/science.1232033. Epub 2013 Jan 3.

Abstract

Bacteria and archaea have evolved adaptive immune defenses, termed clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems, that use short RNA to direct degradation of foreign nucleic acids. Here, we engineer the type II bacterial CRISPR system to function with custom guide RNA (gRNA) in human cells. For the endogenous AAVS1 locus, we obtained targeting rates of 10 to 25% in 293T cells, 13 to 8% in K562 cells, and 2 to 4% in induced pluripotent stem cells. We show that this process relies on CRISPR components; is sequence-specific; and, upon simultaneous introduction of multiple gRNAs, can effect multiplex editing of target loci. We also compute a genome-wide resource of ~190 K unique gRNAs targeting ~40.5% of human exons. Our results establish an RNA-guided editing tool for facile, robust, and multiplexable human genome engineering.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Chromosomes, Human, Pair 19 / genetics
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • Codon / genetics
  • DNA Cleavage
  • Exons
  • Gene Targeting / methods*
  • Genetic Engineering / methods*
  • Genetic Loci
  • Genome, Human*
  • Humans
  • Induced Pluripotent Stem Cells
  • Inverted Repeat Sequences / genetics
  • K562 Cells
  • RNA / chemistry*
  • RNA / genetics

Substances

  • Codon
  • RNA