Delivering gene-editing proteins in vivo using a new strategy.

Delivering Genetic Technologies by Microinjection.

Delivering Genetic Technologies by Microinjection.

Delivering genetic technologies to appropriate cells at the appropriate times is, for many insect systems, the biggest obstacle to the widespread use of these technologies.

Microinjecting developing embryos with DNA, RNA or protein remains the dominant mode of delivery in insect systems but this is not practical for many insects.

How to we get around this limitation? Generally speaking, with new modes and mechanisms of delivery.

Transfection - General Strategy

Transfection – General Strategy

Zuris et al (2014) in Nature Biotechnology describe their unique use of cationic lipid-mediated transfection agents to deliver gene-editing proteins in vitro and in vivo.

Zuris et al’s work was done within the broader context of protein therapeutics and there are many challenges facing this field of medicine. There is an active research enterprise looking for ways to effectively deliver proteins not only to extracellular targets (cell receptors etc) but also to intracellular targets. Proteins that are not protected in some way can be degraded rapidly or otherwise ‘neutralized’ so intracellular delivery is a challenging problem.

Proteins have diverse electrostatic properties and what Zuris et al. thought was that if they can either make a protein polyanionic or if they could non-covalently complex it to a polyanionic molecule they might be able to deliver these protein intracellularly using conventional cationic lipid reagents. Protein transfection is not new but performance has been variable and high concentrations of protein are often needed.

Cas9 + sgRNA

Cas9 + sgRNA

Zuris et al. show that Cas9 complexed with a single guide RNA (sgRNA) could be linked to a modified green fluorescent protein that was engineered to be superanionic and that this linked complex could be efficiently delivered to cells using various cationic lipid formulations. Significantly, the genomes of the transfected cells were efficiently edited (compared to when Cas9-encoding plasmids were transfected).  The important part of what Zuris et al. did was to manage protein charges so they could coax the proteins to associate with cationic lipids.

What is particularly impressive about the work of Zuris et al. is that they are able to use this protein transfection system to deliver the CRISPR editing system to hair cells in the inner ear of live mice. This required a surgical procedure and the injection of 0.3 ul of the protein/transfection cocktail into each cochlea.  Their in vivo results were quite impressive. They estimate that 20% of the cells transfected in vivo were genetically modified.

Inner Ear of Mouse

Inner Ear of Mouse

How this technology can be applied to insect systems is not absolutely clear at this point but certainly solving the delivery problem associated with some insects will require thinking “outside the box” (embryo injections) using technologies we might not be currently using.

So maybe this will involve delivering genetic technologies to germ-line stem cells directly, or developing oocytes or immature gonads. Sounds like it might be challenging but so does in vivo delivery of gene editing technology to the cochlea of mice.

John A Zuris, David B Thompson, Yilai Shu, John P Guilinger, Jeffrey L Bessen, Johnny H Hu, Morgan L Maeder, J Keith Joung, Zheng-Yi Chen & David R Liu    (2014)  Cationic lipid-mediated delivery of proteins enables efficient protein-based genome editing in vitro and in vivo.Nature Biotechnology  Oct 2014 doi:10.1038/nbt.3081

Recent Related Technology Topics.

Injectoporation for Gene Delivery 
Embryo Microinjections: High Tech, Low Tech ?
Robotic Injection of Embryos – Ready for Insects?

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