Introduction and considerations
Establishment of a transgenic line is a relatively fast approach to investigate the function of a gene. NTS has established protocols for gene editing with CRISPR. This is a relatively new molecular technique already validated to be highly successful in multiple species in a high number of laboratories world-wide. CRISPR can be used for many purposes, e.g. to delete a gene (even multiple genes simultaneously), insert point mutations, insert LoxP sites, or link a tag to the C- or N-terminus of a gene.
NTS has adapted the design-tools and uses the vector system established by the Feng Zhang lab (Ran, F.A., Nature Protocols, 2013), and has based protocols for RNA synthesis, RNA isolation and pronuclear injection by work from the Rudolf Jaenisch lab (Yang, H., Cell, 2103).
The CRISPR-Cas9 system used by NTS originates from Streptococcus pyogenes, a Type II CRISPR system. The nuclease Cas9 promotes genomic editing by stimulating double strain breaks guided by small sgRNA sequences with homology to any genomic locus. The only requirement for the sgRNA sequence is the requirement for a NGG sequence adjacent to the sgRNA target sequence. Double stranded DNA break will be repaired with error-prone non-homologous end joining (NHEJ) or with high-fidelity homology-directed repair (HDR) in the presence of a repair fragment.
NTS may inject sgRNA and CRISPR delivered by customers, or may give a full service where NTS design, clone and synthesize the sgRNA for injection (see NTS- CRISPR design service for further details). Thus far, NTS has generated a handful new animal models with CRISPR-Cas 9.
Description of the service (mice)
- NHEJ: One or two (or more if required) in vitro transcribed sgRNA(s) and Cas9 RNA are co-injected into the pronucleus of 1-cell stage embryos.
- HDR: One or two in vitro transcribed sgRNA(s), Cas9 RNA or Cas9-D10A RNA (nickase), and a repair fragment are co-injected into the pronucleus of 1-cell stage embryos. To enhance HDR, embryos may be cultured until next day in the presence of drugs to prevent NHEJ or stimulate HDR.
- Following injection, embryos are transferred into oviducts of pseudopregnant foster mothers where they develop to term. Offspring is born approximately 20 days later. A high number of mice (usually 30-80%) will be modified by NHEJ, whereas the efficiency for HDR is considerable lower.
- Genetically modified mice can be identified by various screening methods. PCR amplification followed by cloning of PCR products and sequencing of individual clones is one approach.
Requirements and time-frame
- Construct: To minimize off-target cleavage and introduction of unwanted mutations, the designed sgRNA(s) should recognize unique genome sequences. To be able to estimate cleavage efficiency for sgRNAs designed by customers, NTS will co-inject with internally synthesized Cas9 RNA validated to be enzymatically active.
- NTS routinely injects into mice with C57BL/6N background. Constructs may be injected into embryos collected from mice of other genetic backgrounds.
- NTS will inject the construct into 150 embryos for NHEJ projects and >200 embryos for HDR projects. To increase the success-rate for each project, injections will be distributed on 2-4 different injection days within a 2-4 weeks period. The customer should expect delivery of ~25 offspring.
- Customer will receive biopsies and will be responsible for verification of positive founders. Alternatively, NTS may assist with genotyping.
- The project lasts typically for 12-18 weeks.
Technical service performed by NTS
- NTS is responsible for the animal procedure permit (FOTS application) describing all steps needed for generation of the new transgenic line. The customer must obtain their own permit for housing and experimental procedures for phenotypic characterization of the transgenic line (see Running projects with NTS).
- Planning and coordination of the project.
- Superovulation of donor females, mating overnight, dissection of oviducts and collection of embryos.
- Injection of the construct into 150-200 embryos.
- Implantation of embryos into pseudopregnant foster mothers.
- Separation of offspring and collection of ear biopsies.
- Customer will receive biopsies in house. If personal pick-up is not an option, transport will be charged customer.
- NTS expects confirmation of genotyping within a reasonable time (2-3 weeks).
Costs cover the following
- Purchase and transport of donor females from vendors.
- Hosing of vasectomized males and foster mothers.
- Housing of males (studs) for mating with donor females for 4 weeks.
- Up to four rounds of injection or >150/200 embryos injected (whatever comes first).
- Confirmed birth of ~25 offspring and delivery of 3 or more positive founders.
- Hosing of offspring until 6 weeks of age.
Service Fees
Estimated costs for the above service can be found on our Service Fees page.