NEW YORK (GenomeWeb) – While many researchers have focused on developing efficient genome editing systems, other teams have looked at the problem of how those systems will be delivered to the cells they are meant to edit. Currently, the most frequently used delivery systems involve viral vectors, including adeno-associated virus (AAV).
However, as a new study published today in Nature Biotechnology noted, an ideal CRISPR-Cas9 delivery system would limit how long cells are exposed to the genome editing technology in order to minimize potential off-target effects. Further, spCas9, is difficult to fit in typical AAV constructs with strong promoters, and patient immune response to AAV capsids can limit repeat dosing.
To address these challenges, a team led by researchers from the Massachusetts Institute of Technology developed a non-viral delivery system. They identified regions of single guide RNAs (sgRNA) that tolerate chemical modification without inhibiting their interaction with Cas9, while maintaining or enhancing genome editing activity. They then demonstrated that a single dose of these enhanced sgRNAs (e-sgRNA) combined with Cas9 mRNA allowed for nearly complete editing of a target gene in the hepatocytes of live mice.
The researchers began by analyzing sgRNAs to study which modifications they could make that the guide could tolerate. They engineered HEK293 cells to stably express GFP and spCas9, and introduced a functional sgRNA targeting GFP to abrogate the expression of GFP through the generation of frameshifting indel mutations. This experiment helped identify which modifications to the sgRNA were well tolerated and how its editing abilities were affected, the researchers noted.
Once they had determined which modifications worked best, the researchers sought to compare the editing efficiency of unmodified sgRNA-targeting GFP, a previously published chemically modified sgRNA termed 5'&3'-sgRNA, and the "enhanced" chemically modified sgRNA they had developed themselves.
"Cas9 mRNA and one of these three sgRNAs were delivered to HEK293 cells expressing GFP. This e-sgRNA generated a significantly higher number of indels than the 5'&3'-sgRNA and the native sgRNA (43 percent, 22 percent, and 20 percent, respectively)," the authors wrote.
The team then moved on to evaluate the e-sgRNA in vivo. The researchers formulated the GFP-targeting sgRNA into a lipid nanoparticle (LNP), and intravenously injected the nanoparticles into the livers of mice constitutively expressing Cas9 and GFP (Cas9-2A-GFP).
After a single injection, unmodified GFP-sgRNA induced a low indel rate of about 5 percent at the GFP locus in the liver tissue, compared with about 22 percent for 5'&3'-sgRNA, the researchers noted. By contrast, e-sgRNA treatment resulted in a significantly higher rate of indels of about 46 percent. After two doses of LNP encapsulated e-sgRNA, indel frequency increased to about 77 percent.
To evaluate the in vivo potential of non-viral delivery of e-sgRNA with Cas9 for a therapeutically relevant target, the team designed two e-sgRNAs targeting the mouse Pcsk9 gene, which is a target for the treatment of familial hypercholesterolemia. They encapsulated Cas9 mRNA and both e-sgRNAs in LNPs, and found that serum Pcsk9 was undetectable five days after a single intravenous administration, and total cholesterol had decreased 35 percent to 40 percent.
"We identified a total of 83 percent (plus or minus 3 percent) editing events in the liver genomic DNA, including small indels, a major genomic deletion induced by two sgRNAs, and lower levels of inversion," the authors wrote.
They also measured gene editing efficiency in the lung and spleen to determine possible off-target events after treatment with Cas9 and e-sgRNAs targeting Pcsk9, and found undetectable levels of such events, indicating the liver specificity of the LNPs used in the study.
"Non-viral genome editing is particularly attractive in a therapeutic setting, given the potential advantages of non-viral delivery systems, including ease of scale-up, speed of customization, lack of pre-existing immunity, and the possibility for limiting exposure to nuclease, among other items," the researchers concluded. "Besides the potential therapeutic applications of e-sgRNA, we anticipate that the highly modified sgRNA could be integrated into a range of CRISPR-associated technologies such as CRISPR-mediated imaging, activation and inhibition of targeted genes. We believe that the ability to use nanoparticles to permanently modify the genome of living animals opens the door to a range of therapeutic and industrial applications, and further advances the utility of the Cas9 genome editing system."