Introduction and considerations
Gene editing is a powerful technique often used to elucidate gene functions. NTS has established protocols for direct gene editing in embryos based on CRISPR-Cas9 methodology (see “Pronucleus injection CRISPR”). This system relies on carefully designed small RNA (sgRNA) specifically targeting a particular locus in the genome.
Customers with experience in CRISPR-Cas9 techniques may design and synthesize their own sgRNAs and deliver these to NTS for injection into embryos. Laboratories with less experience in the technique may order this service from NTS. NTS may assist in the design, vector cloning, in vitro transcription, and RNA purification steps required to produce working sgRNAs.
Provided by the customer
The customer must decide on strategy and the exact gene locus that should be targeted (250 bp region). NTS may assist with selection of gene regions to target. NTS will design up to three sgRNAs estimated to bind to the genome segment of interest with high specificity. NTS may also design replacement fragments for introduction of mutations.
Benefits
- Design is performed by persons with long experience in the techniques and other transgenic techniques. This increases the chance for a successful design.
Requirements and time frame
- The project involves many molecular steps and sequence validation.
- The whole process is typically completed in 2-3 weeks.
Technical service performed by NTS
NTS performs all steps from design to purification of the working sgRNAs.
- Step 1: Design
- Design of specific sgRNAs (up to three sgRNAs).
- Design oligos for in vitro transcription.
- Design replacement fragment.
- Order replacement fragment.
- Order oligos.
- Step 2: Cloning of sgRNA expression vector
- Anneal sgRNA oligos.
- Ligate sgRNA oligos.
- Transform sgRNA oligos.
- Set up mini-prep sgRNAs.
- Perform mini-prep plasmids.
- Measure plasmid concentration.
- Send for sequencing.
- Validate sequencing results.
- Validate vector that may be used for transfection of cells.
- Step 3: In vitro transcription of RNA
- PCR amplify sgRNA template.
- Separate on 2 % agarose gel.
- Purify from agarose (beads).
- Measure template concentration.
- In vitro transcription of sgRNA.
- Purification of in vitro transcribed sgRNA suitable for injection intoembryos (or transfection into cells).
Guarantee
- Proper design and the quality of the produced sgRNA is an important component of the CRISPR-Cas9 technique. NTS will therefore only guaranty delivery of genetically modified mice from an injection when NTS is responsible for sgRNA design and synthesis.
Conditions
- This service is considered as scientific input to the research project. Such projects will be performed under agreement that one or two persons from NTS will be co-authored on the first publication.
Service Fees
Estimated costs for the above service can be found on our Service Fees page.