The Core carries out targeted genome editing using several CRISPR-Cas9 based approaches (see below).
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 application of CRISPR-Cas9 genome editing is the generation of gene knockout animals. Targeted double-stranded breaks induced by Cas9 are repaired via the error-prone Non-Homologous End-Joining (NHEJ) pathway, leading to the formation of insertions or deletions (indels). When these indels occur within a gene's coding region, they often cause a frameshift mutation, resulting in loss of gene function. This approach is the most straightforward and efficient method of Cas9-based editing, requiring only the introduction of Cas9 and a single guide RNA (sgRNA) via injection or electroporation.
Targeted deletions can be achieved by using two sgRNAs flanking the region to be excised. In addition, Cas9 can facilitate the knock-in of short exogenous sequences—such as reporter genes, protein tags, or loxP sites—by co-delivering Cas9, one or two sgRNAs, and a single-stranded DNA oligonucleotide (ssODN) containing the desired modification flanked by homology arms. In this context, the Cas9-induced break is repaired using the ssODN as a template, a method known as “Efficient additions with ssDNA inserts–CRISPR” (Easi-CRISPR), which is effective for knock-ins up to 2 kb in size (Miura et al., 2018). For larger knock-ins (up to 4.5 kb), the CRISPR RNP electroporation combined with AAV donor delivery, known as the CRISPR-READi protocol, is employed (Chen et al., 2019).
GEMC offers the following genome editing services:
- CRISPR-Cas9 genome editing via RNP electroporation or injection
- SHORT:KI (Knock-In, up to 200bp)
- SHORT:KO (Knock-Out, up to 1Kb)
- LARGE:KO (Knock-Out, >1Kb up to 100Kb)
- SHORT:Flox (up to 200bp per loxP site)
- Efficient additions with ssDNA inserts–CRISPR (Easi-CRISPR)
- LONG:Flox-EASI-CRISPR (>200bp up to 2Kb)
- LONG:KI-EASI-CRISPR (>200bp up to 2Kb)
- CRISPR RNP electroporation and AAV donor infection (CRISPR-READi)
- LONG:Flox-CRISPR-READI (>200bp up to 4.5Kb)
- LONG:KI-CRISPR-READI (>200bp up to 4.5Kb)
What the Facility will Provide
Upon receipt of the CRISPR-Cas9 RNP complex—validated for high quality and microinjection suitability (see below)—we will proceed to inject a minimum of 150 fertilized mouse eggs. For projects utilizing electroporation to introduce RNPs, fewer embryos are typically required. Following overnight incubation, viable 2-cell stage embryos will be transferred into pseudopregnant foster females, and pregnancies will be monitored (mouse gestation lasts approximately 19–20 days).
Approximately 10–12 days after birth, the project scientist will be informed of the number of pups born. The facility will perform animal identification (tagging) and provide tail biopsies to the investigator for genotyping purposes. At weaning, genome-edited mice will be transferred to the investigator’s animal room in accordance with ULAR protocols.
If the initial cohort fails to yield at least one genome edited animal (based on appropriate controls), we will perform a second round of injections with at least 80 additional embryos. In this case, the investigator will be responsible for covering the cost of the extra animals. Should the second attempt also fail to produce positive results, a consultation will be scheduled with the project scientist, Facility Director, and Technical Director to review the project, genotyping results, and determine appropriate next steps.
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 an RNP 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 gene editing event or type of RNP mix used.
Investigator’s Responsibilities
In general, the investigator is responsible for preparing the CRISPR-Cas9 RNP mix for injection, as well as for the genetic screening and ongoing care of the weaned animals. To facilitate the provision of this service, we kindly request that you:
- Submit an online service request including IACUC and IBC approvals.
- Contact Kathleen Moosbrugger (kthompso@pennmedicine.upenn.edu – 215-573-3023) to discuss the project details and the schedule of injections. Visit the CRISPR-Cas9 Mouse Targeting Core for assistance with project design selection of reagents, and screening founders.
- Provide the facility with a high-quality CRISPR-Cas9 RNP mix
- Undertake the care of founder pups at the time of weaning. It is the responsibility of the individual investigator to arrange for animal housing.
