Nanoblades Are Another Delivery Option for Gene Editing into Live Organisms

Targeted genome editing tools, such as meganucleases (MGN), zinc-finger nucleases (ZFN), transcription activator-like effector nucleases (TALENs) and more recently the clustered regularly interspaced short palindromic repeats (CRISPR) have revolutionized most biomedical research fields. Such tools allow to precisely edit the genome of eukaryotic cells by inducing double-stranded DNA (dsDNA) breaks at specific loci. Relying on the cell endogenous repair pathways, dsDNA breaks can then be repaired by non-homologous end-joining (NHEJ) or homology-directed repair (HDR) allowing the removal or insertion of new genetic information at a desired locus.

Among the above-mentioned tools, CRISPR-Cas9 is currently the most simple and versatile method for genome engineering. Indeed, in the two-component system, the bacterial-derived nuclease Cas9 (for CRISPR-associated protein 9) associates with a single-guide RNA (sgRNA) to target a complementary DNA sequence and induce a dsDNA break. Therefore, by the simple modification of the sgRNA sequence, users can specify the genomic locus to be targeted. Consistent with the great promises of CRISPR-Cas9 for genome engineering and gene therapy, considerable efforts have been made in developing efficient tools to deliver the Cas9 and the sgRNA into target cells ex vivo either by transfection of plasmids coding for the nucleases, transduction with viral-derived vectors coding for the nucleases or by direct injection or electroporation of Cas9-sgRNA complexes into cells.

Researchers have designed Nanoblades, a protein-delivery vector based on friend murine leukemia virus (MLV) that allows the transfer of Cas9-sgRNA ribonucleoproteins (RNPs) to cell lines and primary cells in vitro and in vivo. Nanoblades deliver the ribonucleoprotein cargo in a transient and rapid manner without delivering a transgene and can mediate knock-in in cell lines when complexed with a repair template. Nanoblades can also be programmed with modified Cas9 proteins to mediate transient transcriptional activation of targeted genes.

* similarly to other protocols that lead to transient delivery of the Cas9-sgRNA RNP, Nanoblades display low off-target effects.
* Nanoblades can represent a viable alternative to classical microinjection experiments for the generation of transgenic animals, in particular for species with fragile embryos or with poorly visible pronuclei. 16 out of 40 blastocysts were correctly gene edited in tests and 13 out of 40 had partially successful gene editing.
* The work validates the use of Nanoblades in vivo (in live animals) for generating transgenic mice upon embryo injection in the perivitelline space or in the liver of injected animals. Although, other recent methods for in vivo genome editing of zygotes and animals have reached higher editing rates. Nanoblades represent a viable, inexpensive, and accessible alternative that can still benefit from further improvements.
Programmable nucleases have enabled rapid and accessible genome engineering in eukaryotic cells and living organisms. However, their delivery into target cells can be technically challenging when working with primary cells or in vivo. Researchers used engineered murine leukemia virus-like particles loaded with Cas9-sgRNA ribonucleoproteins (Nanoblades) to induce efficient genome-editing in cell lines and primary cells including human induced pluripotent stem cells, human hematopoietic stem cells and mouse bone-marrow cells. Transgene-free Nanoblades are also capable of in vivo genome-editing in mouse embryos and in the liver of injected mice. Nanoblades can be complexed with donor DNA for “all-in-one” homology-directed repair or programmed with modified Cas9 variants to mediate transcriptional up-regulation of target genes. Nanoblades preparation process is simple, relatively inexpensive and can be easily implemented in any laboratory equipped for cellular biology.

Nature Communications – Genome editing in primary cells and in vivo using viral-derived Nanoblades loaded with Cas9-sgRNA ribonucleoproteins

SOURCE- Nature Communications

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