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You are here: Home / Teams / RNA metabolism in immunity and infection (RMI2) - E. Ricci / Publications / Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades").

Delivery of the Cas9/sgRNA Ribonucleoprotein Complex in Immortalized and Primary Cells via Virus-like Particles ("Nanoblades").

Philippe E Mangeot, Laura Guiguettaz, Thibault JM Sohier, and Emiliano P Ricci (2021)

J Vis Exp(169).

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas systemhas democratized genome-editing in eukaryotic cells and led to the development ofnumerous innovative applications. However, delivery of the Cas9 protein andsingle-guide RNA (sgRNA) into target cells can be technically challenge. Classicalviral vectors, such as those derived from lentiviruses (LVs) or adeno-associatedviruses (AAVs), allow for efficient delivery of transgenes coding for the Cas9protein and its associated sgRNA in many primary cells and in vivo. Nevertheless,these vectors can suffer from drawbacks such as integration of the transgene in thetarget cell genome, a limited cargo capacity, and long-term expression of the Cas9protein and guide RNA in target cells. To overcome some of these problems, adelivery vector based on the murine Leukemia virus (MLV) was developed to packagethe Cas9 protein and its associated guide RNA in the absence of any codingtransgene. By fusing the Cas9 protein to the C-terminus of the structural proteinGag from MLV, virus-like particles (VLPs) loaded with the Cas9 protein and sgRNA(named "Nanoblades") were formed. Nanoblades can be collected from the culturemedium of producer cells, purified, quantified, and used to transduce target cellsand deliver the active Cas9/sgRNA complex. Nanoblades deliver theirribonucleoprotein (RNP) cargo transiently and rapidly in a wide range of primary andimmortalized cells and can be programmed for other applications, such as transienttranscriptional activation of targeted genes, using modified Cas9 proteins.Nanoblades are capable of in vivo genome-editing in the liver of injected adult miceand in oocytes to generate transgenic animals. Finally, they can be complexed withdonor DNA for "transfection-free" homology-directed repair. Nanoblade preparation issimple, relatively low-cost, and can be easily carried out in any cell biologylaboratory.

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