Nanoblades are retroviral particles engineered to encapsidate the active Cas9/sgRNA ribonucleoprotein complex and deliver it to a wide range of immortalised and primary cells as well as embryos and tissues.
This is achieved through the fusion of Murine Leukemia Virus (MLV) Gag protein to Cas9. Upon expression of the Gag:Cas9 fusion protein together with the wild-type Gag and Gag-Pol, VSV-G and BRL envelope proteins and the sgRNA in HEK293T cells, pseudo-viral particles (Nanoblades) are released to the cell culture medium and can be collected similarly to traditional retroviral vectors. Upon purification, Nanoblades can be used to transduce cells and deliver their protein cargo (the Cas9/sgRNA complex) in a rapid and efficient manner (Cas9 activity can be detected as soon as 4 hours upon transduction). Because Nanoblades do not rely on transgene delivery but rather on protein delivery, their effect is transitory and does not leave any genetic material within targeted cells.
The Gag:Cas9 plasmids to produce Nanoblades can be obtained from :
In addition to the Gag:Cas9 plasmid, the following plasmids are essential:
pMD2.G (VSV-G expressing plasmid): Addgene plasmid #12259
pBS-CMV-gagpol (MLV Gag/Pol expressing plasmid): Addgene plasmid #35614
sgRNA expression plasmid (anyone as long as it does not express the Cas9 protein): Addgene plasmid #112915 for example
BRL expression plasmid (essential to produce efficient Nanoblades): Please contact Dr. Els Verhoeyen
Production protocol:
This is achieved through the fusion of Murine Leukemia Virus (MLV) Gag protein to Cas9. Upon expression of the Gag:Cas9 fusion protein together with the wild-type Gag and Gag-Pol, VSV-G and BRL envelope proteins and the sgRNA in HEK293T cells, pseudo-viral particles (Nanoblades) are released to the cell culture medium and can be collected similarly to traditional retroviral vectors. Upon purification, Nanoblades can be used to transduce cells and deliver their protein cargo (the Cas9/sgRNA complex) in a rapid and efficient manner (Cas9 activity can be detected as soon as 4 hours upon transduction). Because Nanoblades do not rely on transgene delivery but rather on protein delivery, their effect is transitory and does not leave any genetic material within targeted cells.
The Gag:Cas9 plasmids to produce Nanoblades can be obtained from :
In addition to the Gag:Cas9 plasmid, the following plasmids are essential:
pMD2.G (VSV-G expressing plasmid): Addgene plasmid #12259
pBS-CMV-gagpol (MLV Gag/Pol expressing plasmid): Addgene plasmid #35614
sgRNA expression plasmid (anyone as long as it does not express the Cas9 protein): Addgene plasmid #112915 for example
BRL expression plasmid (essential to produce efficient Nanoblades): Please contact Dr. Els Verhoeyen
Production protocol: