VECTOR CONSTRUCTION SERVICE
Independent of our mouse line generation service we can also perform the cloning work for your research project. Depending on the project and complexity of the donor construct we take advantage of several cloning techniques to generate your donor plasmid:
1) Recombineering is a revolutionary method for DNA engineering using homologous recombination in E.coli.
Advantages over conventional methods:
Independent of restriction sites
No size limits
No unwanted mutations
2) Gibson assembly
3) Restriction digestion and ligation
4) Synthesis (gBlock, ssOligo, long single-strand Oligo)
PRODUCTION OF gRNA
For plasmid delivery
We use a one-step recombineering protocol (Baker et al., 2016) or clone into BbsI compatible vectors
pBR322-U6-ccdB-cm-tracrRNA is digested with restriction enzymes to release the ccdB-cm cassette and then co-electroporated with a single stranded oligonucleotide (ssOligo) into GB05 after arabinose induction of pSC101-BAD-ETγA. The ssOligo contains the 20nt gRNA sequence flanked by ~ 25nt of sequence identity (= homology arms) to the U6 promoter and tracrRNA regions.
Anneal oligos & Digest plasmid
Ligation + Re-transformation
For mRNA delivery
The gRNA can be in-vitro transcripted from a T7 or T3 promoter using standard Kits. We will either clone your gRNA(s) into a plasmid containing a T7 promoter or provide the T7 promoter in the forward primer to generate a PCR product.
Following linearization of the plasmid with an unique restriction enzyme located after the polyA tail, gRNA is transcribed with T7 or T3 polymerase following the manufactory's protocol. Purified gRNA can be co-delivered with Cas9 mRNA or protein.
The gRNA can also be synthesized as 2 separate RNA molecules (crRNA + tracrRNA). The crRNA includes your gRNA target site and nucleotides that are complementary to the tracrRNA (generic).
crRNA: N20 + 16nt complementary to tracrRNA = 36nt
Note, no addition of a G nucleotide at the 5' end of the crRNA is required for synthetic gRNAs
Anneal crRNA + tracrRNA (1:1 molar ratio) at 95C for 5 min and then ramp down to 25C at 5C/min. Co-transfect with Cas9 mRNA or pre-complex with Cas9 protein (= Ribonuclearprotein (RNP) complex)
PRODUCTION OF CAS9 mRNA
Following linearization of the plasmid with an unique restriction enzyme located after the polyA tail, Cas9 is transcribed with T7 or T3 polymerase following the manufactory's protocol. Purified Cas9 mRNA can be co-delivered with gRNA or synthetic gRNA.