Services » Direct genome modifications using targeted endonucleases

The Core carries out targeted genome modifications using Zn finger, TALEN and CRISPR/Cas9 technologies. By far the most heavily utilized is CRISPR-based approaches.

CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) consists of two components: a “guide” RNA (gRNA) and a non-specific CRISPR-associated endonuclease (Cas9). The gRNA is a short synthetic RNA composed of a scaffold sequence “spacer” or “targeting” sequence which defines the genomic target to be modified.

A major use of CRISPR-mediated modifications is the generation of gene knockout animals. Double-stranded breaks generated by targeted Cas9 cleavage are repaired by the error-prone Non-Homologous End-Joining pathway, resulting in indel (insertion/deletion) generation. When targeting within the coding region of a gene, indels frequently result in a frameshift and loss of function. This is the most direct and efficient form of Cas9-mediated genome editing, requiring only the injection of Cas9 and single sgRNA. Defined deletions can be generated by introducing two sgRNA’s flanking the region to be removed.  Alternatively, Cas9 can be used to introduce defined deletiond or short artificial sequences (Knock-in) by injecting Cas(, sgRNA (one or two) and a ssDNA oligo containing the desired nucleotide modification and arms flanking the break. The double-stranded break generated by Cas9 cleavage in this setting can be repaired by the host DNA repair machinery using the oligo as a template.

What the Facility will Provide

Upon receipt of the injection mix, proven to be of high quality and suitable for microinjection (see below), we will proceed to inject a minimum of 150 fertilized eggs. After overnight incubation, normal 2-cell embryos will be transferred into foster females and their pregnancy will be monitored (the gestation period of the mouse is 19-20 days). 10-12 days after birth of the pups, the project scientist will be notified of the number of pups. The facility will carry out tail clipping, animal tagging and genotyping by PCR according to the conditions set by the project scientist. At weaning, the mice positive for the genome-editing will be transferred to the care of the individual investigators as per ULAR procedures. If these studies (with adequate controls) fail to demonstrate the existence of at least one transgenic animal, we will re-inject at least 80 eggs and ask the investigator to cover the cost of the additional animals used. If, however, no positive animal is detected after the second trial, a meeting will be held between the scientist, the Facility Director, and the Technical Director to discuss the project, the tail biopsy analysis, and further plans to be agreed upon.

It is well understood that many factors can affect the production efficiency of genome-edited mice. Such factors include:

  • Number of eggs surviving the injection and developing to 2-cell stage; this will determine the number of transfers into foster females. It is important to have a RNA mix preparation pure of any contaminant and at the appropriate concentration so that toxic effects to the eggs can be avoided.
  • Successful pregnancies: although every transfer promises a certain number of pups, that number may vary greatly due to embryo death in utero. This lethality may be closely associated with the type of RNA mix used.

Investigator’s Responsibilities

In general terms, the investigator must prepare the RNA mix to be injected and assume responsibility for screening and care of the weaned animals. Specifically, in order to provide you with this service, it is requested that you:

  • Contact Jean Richa (215-573-3023) to discuss the RNA construct and the schedule of injections. If you wish to use the services of the CRISPR Core, please follow this link: https://pathbio.med.upenn.edu/crispr/site/ for more information.
  • Provide the facility with a high-quality RNA mix preparation (See the recommended protocol: RNA preparation for CRISPR injection in the “Protocols” web page.)
  • Submit Online a service request form at: http://www.med.upenn.edu/apps/my/transcore/user including evidence of approval of the IACUC and IBC committees for the project.
  • You can have the core screen the tail biopsies at no added cost by providing PCR primers as well as negative and positive control samples.
  • Undertake the care of the potential transgenic pups submitted to the project scientist. These mice can no longer reenter the barrier colony. It is the responsibility of the individual investigator to arrange for animal housing.

Please also consult additional guidelines related to this service under the "Protocols" section of this site.