Nature Biotechnology: new progress in CRISPR-Cas9
Cas9 is an RNA-directed DNA binding and cutting protein that can be used for gene and non-coding genetic elements in a variety of organisms. At present, Cas9-mediated genomic editing technologies have been applied to the treatment of eye, ear, liver and muscle related diseases, but the fact that tissue-specific transmission is not ideal is still a problem. One of the solutions is the use of non-genetic coding, preassembling and short-lived Cas9 ribosomal protein (RNP) complexes. In this latest article, the researchers first performed on Ai9 tdTomato mice in order to detect the activity of Cas9 RNP in vivo. The improved mouse model provides a high-throughput and quantitative sequence reading of site-specific genome editing that enables the function of the red fluorescent protein in the gene-modified cells to be obtained.
Researchers have developed a single-directed sgRNA through recombinant horse proteins, which they called sgRNAtdTom, and sgRNAtdTom can remove the terminal box and activate the tdTomato expression in cells. After they were subjected to nuclear transfection of neural progenitor cells (NPCs) using this sgRNAtdTom RNP complex, the researchers observed a dose-dependent activation of RNP by microscopic and flow cytometry, while the analysis of NGS sequence of the locus also showed that Cas9 RNP induced insertion or deletion mutations.
These series of studies have shown that tdTomato mice can be used for visual inspection of the Cas9 RNP genome editing and the editable cells can be observed in vivo, although the number of tdTomato + cells is lower than the reported total number of RNP genome editing.
Through this study, Jennifer Doudna demonstrated that injecting the Cas9 ribosomal protein (RNP) complex into the adult hippocampus, striatum and cortex can complete the gene editing of the mitotic neurons in the mouse brain, and SV4 core positioning sequence of Cas9 editing can improve the efficiency of neuronal editing in vivo by ten times.
“These findings demonstrate that the genomic editing of the neural progenitor cells mediated by Cas9 RNP can be used in vitro, as well as in many different brain regions in the body. Although the 4x NLS-Cas9–2 x NLS RNP Neurotropism is still unclear, this can be used for neuronal-specific targeting, suggesting that this technology can be used in the future for the treatment of neurological diseases of different neurons, correcting or inactivating the potential genetic factors of neurological diseases of the brain,” said the author. In addition, the study also shows that functional Cas9 RNP can be delivered in non-genetic coding, effective, accurate and safe delivery to the adult animal brain neurons to solve the problem raised earlier, contributing to future use of Cas9-RNP complexes for the treatment of neurological disorders and general tissue-specific gene editing. By the way, Flarebio provides you with superior recombinant proteins like recombinant ITGB4 at good price.