CRISPR/Cas9

CRISPRs (clustered regularly interspaced short palindromic repeats) are repetitive nucleotide sequences followed by a short spacer DNA segments integrated into the genomes of many bacteria and archaea upon exposure to invading pathogens.  CRISPR-associated (Cas) nucleases form the CRISPR-Cas machinery to rid the prokaryotes of foreign genomic elements.  In 2012, CRISPR was utilized in eukaryotes and a readily-programmable two-component CRISPR system was used to disrupt targeted genomic sites.

Introduction

The CRISPR-Cas9 system can make genetic manipulations much faster than previous used methods. Instead of 1.5-2 years to make a genetically engineered mouse, it now can take as little as 4 months!
Some examples of modifications this system can create:

  • permanent gene deletion in cell lines
  • genomic knockouts of genes in mice (and other model organisms) in less than 1/3 the normal time
  • genomic deletion of enhancers that drive gene transcription
  • targeted genomic knock-in of reporter and resistance genes or LoxP sites for tissue selective removal
  • disease related genetic mutation correction through targeted homology directed recombination
The list of what CRISPR can do is growing almost daily and represents one of the most rapidly growing techniques in genetic research today.

Designing your CRISPR

Dustin Rubenstein runs the Translational Genomics Facility and will assist you in designing your CRISPR guides and obtaining embryo tested reagents. Before ordering any materials contact Dustin to talk about your project. 

The TGF will create and purify any reagents that will be injected into embryos. The introduction of foreign material into the nucleus of the embryos is a painstaking and delicate process. It is extremely important that the materials being injected are extremely pure to ensure the survival of the embryo. 

For additional background information please go to GeeWisc's Website. They have some useful information about the production of model animals using CRISPR/Cas9.

Microinjection to Produce Mice and Rats

After a purified RNA guide has been created by the TGF an injection day will be scheduled with Kathy Krentz in the Transgenic Animal Facility. The gRNA, Cas9, ssODN, and/or plasmid DNA (if applicable) will be microinjected into fertilized one-cell embryos. The one-cell embryos are then implanted into the host dams and 19 days later the offspring are born. Following genetic testing and breeding the mice are shipped to the investigators.