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In this protocol, engineered Cas9-ribonucleoprotein (Cas9 protein and sgRNA, together called Cas9-RNP) and gold nanoparticles are used to make nanoassemblies that are employed to deliver Cas9-RNP into cell cytoplasm and nucleus. Cas9 protein is engineered with an N-terminus glutamic acid tag (E-tag or En, where n = the number of glutamic acid in an E-tag and usually n = 15 or 20), C-terminus nuclear localizing signal (NLS), and a C-terminus 6xHis-tag. [Cas9En hereafter] To use this protocol, the first step is to generate the required materials (gold nanoparticles, recombinant Cas9En, and sgRNA). Laboratory-synthesis of gold nanoparticles can take up to a few weeks, but can be synthesized in large batches that can be used for many years without compromising the quality. Cas9En can be cloned from a regular SpCas9 gene (Addgene plasmid id = 47327), and expressed and purified using standard laboratory procedures which are not a part of this protocol. Similarly, sgRNA can be laboratory-synthesized using in vitro transcription from a template gene (Addgene plasmid id = 51765) or can be purchased from various sources.
Once these materials are ready, it takes about ~30 min to make the Cas9En-RNP complex and 10 min to make the Cas9En-RNP/nanoparticles nanoassemblies, which are immediately used for delivery (Figure 1). Complete delivery (90–95% cytoplasmic and nuclear delivery) is achieved in less than 3 h. Follow-up editing experiments require additional time based on users’ need.
Synthesis of arginine functionalized gold nanoparticles (ArgNPs) (Yang et al., 2011), expression of recombinant Cas9En, and in vitro synthesis of sgRNA is reported elsewhere (Mout et al., 2017). We report here only the generation of the delivery vehicle i.e., the fabrication of Cas9En-RNP/ArgNPs nanoassembly.
Delivery of Cas9-ribonucleoprotein provides an alternative strategy for CRISPR gene delivery, offering a transient way of editing genes. Although a few strategies for Cas9-RNP delivery have been reported, these strategies suffer from endosomal entrapment of both Cas9 protein and sgRNA (Liu et al., 2015). Mechanical methods including membrane deformation (Han et al., 2015), electroporation (Schumann et al., 2015), and the use of hypertonic agents (D’Astolfo et al., 2015) provide direct delivery, however, they require specialized instrumentations and are generally not practical for in vivo therapeutic applications. Our protocol provides an approach for direct cytoplasmic and nuclear delivery of Cas9-RNP that can find applications in both gene editing and genome imaging.
Final working nanoassembly concentration is 125 nM of ArgNPs and 62 nM of Cas9En-RNP complex, which is at a 2:1 molar ratio of ArgNPs/Cas9En-RNP. The total volume of the nanoassembly samples required for delivery depends on the kind of cell culture plate used. We generally use 1 ml for round bottom 35 mm confocal dishes, 500 μl for 24-well plates, and 200 μl for 96-well plates, per well. Therefore, the nanoassemblies should be made and scaled up according to users’ need. The following calculation is for one sample in a 24-well plate (i.e., 500 μl total volume). Additionally, the following protocol is for HeLa cells, however, we verified the Cas9En-RNP delivery in other cell lines including mouse macrophage RAW 264.7, human embryonic kidney HEK cells, and human primary mammary epithelial cells.
Cytoplasmic delivery efficiency of Cas9En or Cas9En-RNP was determined by confocal microscopy imaging. Even distribution of fluorophore labelled Cas9En in the cytosol and nucleus is considered as effective delivery, whereas any punctate distribution in the cytoplasm is considered as endosomal delivery (Figures 2A and and2B).2B). Briefly, ~400 cells were counted manually to estimate effective cytosolic and nuclear delivery. Any punctate distribution (see Figure 2B) of labelled-Cas9En should be avoided from counting as direct cytoplasmic and nuclear delivery. Please see Mout et al., 2017 for more details.
This research was supported by the NIH (GM077173), NSF (CHE-1307021) and a UMass OTCV grant.
1As a point of reference, gene editing verification is performed 48 h after nanoassembly treatment.
2The ratio ArgNPs/Cas9En is the most crucial factor for delivery efficiency. Every batch of ArgNPs can be slightly deferent in terms of surface ligand coverage and therefore users are suggested to test different ratios of ArgNPs/Cas9En to find out maximum delivery. Fluorophore labelled (FITC/Alexa Fluor 488) Cas9En should be used to evaluate delivery efficiency (see manufacturer’s protocol for labelling: Alexa Fluor 488 NHS Ester, Thermo Fisher Scientific).
3Freshly prepared (not more than a week old, and preserved at 4 °C) Cas9En should be used for better activity. Flash-frozen Cas9En at −80 °C with glycerol should be avoided as our preliminary investigation shows that glycerol hampers assembly formation.