“Nanoparticle delivery of Cas9 ribonucleoprotein and donor DNA in vivo induces homology-directed DNA repair”
CRISPR-Cas9 is a powerful therapeutic tool that can be used to edit and repair DNA and cure genetic diseases. One difficulty in this tool is delivering a complete package of the Cas9 protein, guide RNA and donor RNA to the gene site that needs to be fixed. In this paper, Kunwoo Lee, et al. show that this package can be delivered using gold nanoparticles as a vehicle to hold the pieces together (2017). They have shown this to work for many different cell types with success in correcting DNA mutations that cause Duchenne muscular dystrophy, a genetic disease, in mice.
In this study, researchers aimed to “induce homology directed DNA repair”. As opposed to therapies that would mute disease causing DNA sequences, homology directed repair has the amazing ability to correct DNA sequences by replacing problem causing or broken sections of a gene with a desired “donor RNA” sequence. Currently, the best method of applying homology directed repair is to bring the guide RNA, donor RNA, and Cas9 editing tool to the desired DNA site via special viruses. This is a problematic method because many humans are immune to the viruses, rendering the process ineffective.
By incorporating materials science and engineering to this biological therapy, homology directed repair becomes a much more feasible and efficient method. Rather than using one or more viruses as a delivery tool, Lee shows gold nanoparticles bonded to the guide RNA, Cas9, donor RNA complex as an efficient and accurate vehicle for inducing homology directed repair. This vehicle, named “CRISPR-Gold” by Lee and colleagues, is a simple 15 nm particle. Gold was chosen for its ability to be accepted by cells and its ability to bond with necessary proteins. Gold-DNA conjugation is the base of the CRISPR-Gold delivery mechanism. A single strand of donor (desired sequence) DNA coats the cold particle and binds to both Cas9 and guide RNA. To ensure acceptance into the cell, this package is coated with silicate and a positively charged polymer PAsp(DET).
The resulting “CRISPR-Gold” structure was analyzed during processing via spectroscopy, where red shifts were recorded after each added layer, resulting in an overall peak shift from 518 nm for pure gold nanoparticles to 546 nm for the final “CRISPR-Gold” structure. The resulting vehicle was then tested both in and out of living organisms (in vivo and in vitro) and in a variety of cell types (primary, stem, etc). The system was proven to be effective, most notably in treating muscular dystrophy in mice by editing DNA affected tissue via local injection. This shows both great promise and broad power in the future of health care. Lee’s work is a great model for the potential of an interdisciplinary materials-biology approach to curing genetic diseases.
Reference
Lee et al., “Nanoparticle Delivery of Cas9 Ribonucleoprotein and Donor DNA in Vivo Induces Homology-Directed DNA Repair.”
CRISPR-Cas9 is a powerful therapeutic tool that can be used to edit and repair DNA and cure genetic diseases. One difficulty in this tool is delivering a complete package of the Cas9 protein, guide RNA and donor RNA to the gene site that needs to be fixed. In this paper, Kunwoo Lee, et al. show that this package can be delivered using gold nanoparticles as a vehicle to hold the pieces together (2017). They have shown this to work for many different cell types with success in correcting DNA mutations that cause Duchenne muscular dystrophy, a genetic disease, in mice.
In this study, researchers aimed to “induce homology directed DNA repair”. As opposed to therapies that would mute disease causing DNA sequences, homology directed repair has the amazing ability to correct DNA sequences by replacing problem causing or broken sections of a gene with a desired “donor RNA” sequence. Currently, the best method of applying homology directed repair is to bring the guide RNA, donor RNA, and Cas9 editing tool to the desired DNA site via special viruses. This is a problematic method because many humans are immune to the viruses, rendering the process ineffective.
By incorporating materials science and engineering to this biological therapy, homology directed repair becomes a much more feasible and efficient method. Rather than using one or more viruses as a delivery tool, Lee shows gold nanoparticles bonded to the guide RNA, Cas9, donor RNA complex as an efficient and accurate vehicle for inducing homology directed repair. This vehicle, named “CRISPR-Gold” by Lee and colleagues, is a simple 15 nm particle. Gold was chosen for its ability to be accepted by cells and its ability to bond with necessary proteins. Gold-DNA conjugation is the base of the CRISPR-Gold delivery mechanism. A single strand of donor (desired sequence) DNA coats the cold particle and binds to both Cas9 and guide RNA. To ensure acceptance into the cell, this package is coated with silicate and a positively charged polymer PAsp(DET).
The resulting “CRISPR-Gold” structure was analyzed during processing via spectroscopy, where red shifts were recorded after each added layer, resulting in an overall peak shift from 518 nm for pure gold nanoparticles to 546 nm for the final “CRISPR-Gold” structure. The resulting vehicle was then tested both in and out of living organisms (in vivo and in vitro) and in a variety of cell types (primary, stem, etc). The system was proven to be effective, most notably in treating muscular dystrophy in mice by editing DNA affected tissue via local injection. This shows both great promise and broad power in the future of health care. Lee’s work is a great model for the potential of an interdisciplinary materials-biology approach to curing genetic diseases.
Reference
Lee et al., “Nanoparticle Delivery of Cas9 Ribonucleoprotein and Donor DNA in Vivo Induces Homology-Directed DNA Repair.”