Enhanced homology-directed human genome engineering by controlled timing of CRISPR/Cas9 delivery

Elife. 2014 Dec 15;3:e04766. doi: 10.7554/eLife.04766.

Abstract

The CRISPR/Cas9 system is a robust genome editing technology that works in human cells, animals and plants based on the RNA-programmed DNA cleaving activity of the Cas9 enzyme. Building on previous work (Jinek et al., 2013), we show here that new genetic information can be introduced site-specifically and with high efficiency by homology-directed repair (HDR) of Cas9-induced site-specific double-strand DNA breaks using timed delivery of Cas9-guide RNA ribonucleoprotein (RNP) complexes. Cas9 RNP-mediated HDR in HEK293T, human primary neonatal fibroblast and human embryonic stem cells was increased dramatically relative to experiments in unsynchronized cells, with rates of HDR up to 38% observed in HEK293T cells. Sequencing of on- and potential off-target sites showed that editing occurred with high fidelity, while cell mortality was minimized. This approach provides a simple and highly effective strategy for enhancing site-specific genome engineering in both transformed and primary human cells.

Keywords: CRISPR/Cas9; cell biology; cell cycle synchronization; chromosomes; genes; genome engineering; homologous recombination; human; nocodazole; non-homologous end joining.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Base Sequence
  • Cell Cycle / genetics
  • Cell Survival
  • Clustered Regularly Interspaced Short Palindromic Repeats*
  • DNA / chemistry
  • DNA / genetics*
  • DNA / metabolism
  • DNA Breaks, Double-Stranded
  • Embryo, Mammalian
  • Embryonic Stem Cells / cytology
  • Embryonic Stem Cells / metabolism
  • Endonucleases / genetics*
  • Endonucleases / metabolism
  • Fibroblasts / cytology
  • Fibroblasts / metabolism
  • Gene Expression
  • Gene Transfer Techniques
  • Genetic Engineering / methods
  • Genome, Human*
  • HEK293 Cells
  • Humans
  • Infant, Newborn
  • Molecular Sequence Data
  • RNA, Guide / chemistry
  • RNA, Guide / genetics*
  • RNA, Guide / metabolism
  • Recombinational DNA Repair*
  • Sequence Analysis, DNA
  • Signal Transduction
  • Staphylococcus / chemistry
  • Staphylococcus / enzymology
  • Time Factors

Substances

  • RNA, Guide
  • DNA
  • Endonucleases

Grant support

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.