Amazing stuff!
"... Using an approach based on CRISPR proteins, MIT researchers have developed a new way to precisely control the amount of a particular protein that is produced in mammalian cells.
This technique could be used to finely tune the production of useful proteins, such as the monoclonal antibodies used to treat cancer and other diseases, or other aspects of cellular behavior. ...
The promoter sites used for this synthetic system were designed to be distinct from naturally occurring promoter sites, so that the system won’t affect genes in the cells’ own genomes. Each operator includes between two and 16 copies of the guide RNA binding site, and the researchers found that their system could initiate gene transcription at rates that linearly correspond to the number of binding sites, allowing them to precisely control the amount of the protein produced. ..."
The system that the researchers designed includes several components. One is the gene to be transcribed, along with an “operator” sequence, which consists of a series of artificial transcription factor binding sites. Another component is a guide RNA that binds to those operator sequences. Lastly, the system also includes a transcription activation domain attached to a deactivated Cas9 protein. When this deactivated Cas9 protein binds to the guide RNA at the synthetic promoter site, the CRISPR-based transcription factor can turn on gene expression.
From the abstract:
"Precise, scalable, and sustainable control of genetic and cellular activities in mammalian cells is key to developing precision therapeutics and smart biomanufacturing. Here we create a highly tunable, modular, versatile CRISPR-based synthetic transcription system for the programmable control of gene expression and cellular phenotypes in mammalian cells. Genetic circuits consisting of well-characterized libraries of guide RNAs, binding motifs of synthetic operators, transcriptional activators, and additional genetic regulatory elements express mammalian genes in a highly predictable and tunable manner. We demonstrate the programmable control of reporter genes episomally and chromosomally, with up to 25-fold more activity than seen with the EF1α promoter, in multiple cell types. We use these circuits to program the secretion of human monoclonal antibodies and to control T-cell effector function marked by interferon-γ production. Antibody titers and interferon-γ concentrations significantly correlate with synthetic promoter strengths, providing a platform for programming gene expression and cellular function in diverse applications."
A synthetic transcription platform for programmable gene expression in mammalian cells (open access)
Fig. 1: Design and development of the crisprTF promoter system
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