A new Nature Biotechnology study from the lab of Jennifer A Doudna has described a major step toward the in vivo application of CRISPR-Cas9-mediated gene editing to correct the genetic causes of neurological diseases. One major problem is how to deliver Cas9 constructs to the desired tissue, as the application of viral vectors or plasmids encoding Cas9 can lead to integrational mutagenesis, detrimental immune responses caused by persistent expression, and off-target activity.
To avoid these problems, Staahl et al have created a non-genetically encoded, preassembled, and short-lived Cas9 ribonucleoprotein (RNP) complex targeted for cell penetration thanks to the addition of an optimized pattern of Simian vacuolating virus 40 (SV40) nuclear localization sequences. Initial assessments employed neural stem cells and terminally differentiated neurons carrying a fluorescent reporter gene silenced by the inclusion of a stop cassette. Treatment with the engineered Cas9 RNP complex and a guide RNA targeting the stop cassette led to reporter gene expression in both cell types and provided proof-of-concept to move this gene-editing strategy in vivo into a reporter mouse model.
The authors injected the same Cas9-guide RNA RNP complex into the adult mouse hippocampus, striatum, and cortex and assessed gene editing and reporter gene expression after 12–14 days. Excitingly, they observed high levels of reporter gene expression in diverse neuronal subtypes in all regions tested, with no signs of an innate immune response. However, the Cas9 RNP complex did not target the reporter stop cassette in astrocytes, an abundant support cell present in the brain, suggesting that engineered RNPs exhibit preferential neurotropism, a characteristic that may prove advantageous in future cell-specific applications.
While manias and obsessions rarely bear much fruit, this study has discovered that having CRISPR-Cas9 on the brain can produce exciting new results. Engineered Cas9 RNP complexes promise to contribute greatly to tissue-specific editing and to help construct effective in vivo therapeutic applications, so check out this new study at Nat Biotech, 2017.