Cloning single gRNA plasmids
To introduce target sites into the single gRNA vectors pCFD1-3 we are using a cloning strategy that ligates two annealed oligos into the backbone that has been digested with a type II-S restriction enzyme. It allows seamless cloning and is fast, very efficient and cheap.
The cloning site of plasmids pCFD1-3 can be digested with BbsI to create seamless sticky-ends, which will accept two annealed oligos containing the gRNA target site. Note that the exact oligo design is slightly different for each of the three vectors (consult the individual cloning protocols).
Cloning protocols
Cloning two gRNAs into plasmid pCFD4
To clone two different gRNAs into the pCFD4 plasmid we PCR amplify a fragment of that vector and insert the two target sites into the forward and reverse primers. The PCR product is then inserted into a pCFD4 backbone that has been digested with BbsI.
Cloning of two gRNAs is done by homology directed cloning. The primers contain homologies to either end of the digested backbone at their 5′ end, followed by the gRNA target sites and homologous 3′ sequences for primer extension during PCR.
Plasmid maps
Cloning gRNAs into tRNA::gRNA plasmid pCFD5
pCFD5 contains tRNAs flanking the gRNAs and allows expression of several (we have tested up to 6) gRNAs from the strong ubiquitous U6:3 promoter.
Cloning protocols
Cloning gRNAs into tRNA::gRNA plasmid pCFD6
pCFD6 contains tRNAs flanking the gRNAs and allows expression of several gRNAs under control of the Gal4/UAS system. We currently use pCFD6 to create a large-scale library of transgenic gRNA lines.
Cloning protocols