CRISPR on a gold basis
Gold ensured the entry of CRISPR systems into cells with mutations
Anna Kaznadzei, N+1
Biologists from UC Berkeley and Lawrence National Laboratory together with colleagues from The University of Tokyo has developed a new way to deliver CRISPR systems that edit the genome to cells. The Cas9 protein, together with the guide RNA (gRNA) and donor DNA (dDNA), was attached to gold nanoparticles and surrounded with a special charged polymer, obtaining a complex that effectively penetrates cells and simultaneously delivers all the necessary macromolecules to a given target.
As a result, such a tool turned out to be able to edit mutations, for example, in a gene mutations in which cause Duchenne muscular dystrophy, without turning it off, but performing repair due to homologous recombination and achieving the restoration of the correct gene sequence. The study is published in Nature Biomedical Engineering (Lee et al., Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair.
Genomic editing can be a solution in the fight against many diseases, including chronic ones. The CRISPR-Cas9 genome editing system, first developed for use in the laboratory by one of the co-authors of this work, Jennifer Dudnoy and her colleagues, currently offers two main techniques: cutting out a piece of a gene, leading to its shutdown; or homologous recombination, as a result of which the sequence of a gene with a mutation is replaced by the correct one, and the gene begins work the way a healthy person works.
The second technique, despite its obvious advantages, is still rarely used, since it is rarely possible to achieve the desired level of its effectiveness and safety in laboratory conditions. This is due, in particular, to the fact that it requires the delivery of several macromolecules to the mutated DNA simultaneously: the Cas9 protein, which will recognize the sequence with the mutation and cut it, gRNA (the guide RNA in which the mutation is recorded for recognition) and dDNA (donor DNA).
At the moment, in experiments with Duchenne muscular dystrophy in mice, where the CRISPR-Cas9 system was injected directly into the animal body, it was possible to achieve only 0.3 percent of the efficiency of editing the dystrophin gene, mutations in which cause this incurable fatal disease.
One of the alternative methods of delivering the CRISPR-Cas9 system to the body is to package it in adenovirus particles, but new difficulties arise here: for most people, for example, a strong immune reaction begins in response to the ingestion of such viral particles into the body; in addition, the particles used are often not large enough and the material has to be distributed unevenly in them, which reduces the efficiency of its delivery.
In this work, scientists used gold nanoparticles to which thiol-DNA was bound, and then hybridized it with dDNA. Then Cas9 complexes with gRNA (RNP) were attached to this system due to affinity to DNA; finally, a positively charged polymer (poly(N-(N-(2-aminoethyl)-2-aminoethyl) aspartamide) PAsp(DET) was applied externally, providing electrostatic interactions inside the complex, increasing its stability.
The structure of the CRISPR-Cas9 complex with a gold nanoparticle.
Drawings from the article by Lee et al.
The complex, as was shown separately, penetrated into cells due to caveolar endocytosis, after which PAsp(DET) destroyed endosomes, and all components of the CRISPR-Cas9 system were inside the cell simultaneously. A series of in vitro experiments with human and mouse cells showed that the system successfully penetrates cells and edits the specified genes.
Delivery of the CRISPS-Cas9 complex with a gold nanoparticle into the cell due to endocytosis,
exit from the endosome and subsequent genomic editing.
After that, the scientists conducted a study with live mice suffering from Duchenne muscular dystrophy and analyzing the effect of CRISPR-Cas9 on the dystrophin gene after one injection. It turned out that the efficiency of gene editing with gold nanoparticles is 18 times higher than with the direct introduction of the system into the body (5.4 percent of successfully edited genes versus 0.3 percent). Moreover, the standard test for mice, which determines their ability to hang on four limbs for a certain time, demonstrated twice the best performance compared to this control. The level of fibrosis in the muscle tissues of mice also decreased markedly. Scientists note that this indicates significant progress in the treatment of the disease.
Results of genomic editing using CRISPR-Cas9
and gold nanoparticles in mice with Duchenne myodystrophy.
Additional studies have shown that the introduction of gold nanoparticles does not lead to significant outbreaks of the immune response, as happens with the introduction of viral carriers, although the level of CD45+ and CD11+ leukocytes in the body increases slightly. In addition, scientists have found that even repeated administration of gold nanoparticles does not cause increased synthesis of anti-inflammatory cytokines and weight loss.
Delivery of CRISPR-Cas9 systems in viral particles can also be associated with the subsequent excessively high level of Cas9 synthesis in cells, which can lead to an incorrect course of its work and a change in the host DNA in arbitrary places. Delivery of the system in gold nanoparticles, however, does not involve additional Cas9 synthesis, and an assessment of the number of random mutations after such injections showed that the mutation level does not exceed the usual norm (0.005 — 0.2 percent).
In the future, scientists are going to conduct clinical studies, the ultimate goal of which will be the development of drugs for the treatment of human diseases (in the USA, editing of the human genome was allowed back in 2016, and the first experiments with embryos have already been published, although so far such embryos cannot yet be carried). For this purpose, in particular, the co-authors of the article opened the startup GenEdit. And the Berkeley lab is currently developing new nanoparticles that will deliver genome editing systems directly to stem cells.
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