LentiSH-Cas9-sgRNA-Puro enables the expression of Cas9, single guide RNA (sgRNA), and Puromycin resistance gene through a lentiviral expression system. This construct contains the basic structure as the following:
5’LTR-Psi element-RRE-cPPT/CTS-Cas9 & sgRNA expression cassette-PuroR-WPRE-3’LTR
Cas9 fused with a nuclear localization signal is expressed under the EF1α promoter, and target-specific sgRNA is expressed under a U6 promoter. Each sgRNA contains a 20-nucleotide spacer (N)20 followed by a gRNA scaffold. In the constructs provided by our panels, this spacer is replaced by guide sequences targeting specific genomic location.
LentiSH-Cas9-sgRNA-Puro also carries Ampicillin resistance gene and should be propagated in NEB stable cells at 32 °C for plasmid stability. Annotated sequence can be downloaded here . We recommend using the free DNA editing software ApE to open the sequence file.
LentiSH-Cas9-sgRNA-Puro is compatible with the 2nd and 3rd generation lentiviral packaging plasmids. Compatible packaging plasmids, pSH-VSV-G (Env expression) and pSH-PAX2 (Gag & Pol expression) , can be purchased from our website; their sequences can be downloaded here .