The CRISPR-Cas9 system enables researchers to edit DNA sequences from many organisms and cell types. However, scientists are increasingly recognizing that it can be used to activate gene expression. To this end, they have constructed a number of synthetic genes that activate the Cas9 protein to study gene function or to compensate for inadequate gene expression in potential therapeutic approaches.
"The use of various engineering variants of the CRISPR-Cas9 system to selectively activate genes," said Dr. George Church, a core faculty member at Harvard University's Wyss Institute for Biological Inspiration Engineering and head of systems biology at Harvard Medical School. Sexuality has led many researchers to ask which of the available synthetic Cas9 activators can be used to achieve their goals. Uniquely designing all synthetic Cas9 activators and testing them in different environments is an important challenge; No one has compared their relative potential side by side. We hope to provide such a comparison table for the biomedical research community."
In a study published in the May 23 issue of Nature Methods, the Wyss team reported a rigorous comparison and listed the various organisms from humans, mice, and fruit flies. The most commonly used artificial Cas9 activator of the cell type. The findings provide researchers with a valuable guide to simplifying research efforts.
The Cas9 protein was engineered to disrupt its DNA editing ability, and the Cas9 proteins of these activatable genes were subsequently fused together with the variable domains of some proteins known to have gene activation potential. In some cases, it is also possible to engineer a second element of the CRISPR-Cas9 system - targeting this complex to a guide RNA (gRNA) of a specific DNA sequence to bind some gene activators.
Marcelle Tuttle, a co-first author of the study and a researcher at the Wyss Institute, said: "We first investigated seven advanced Cas9 activators, compared them to the original Cas9 activator, and compared the original Cas9 activator. To provide proof of concept for the activation potential of the CRISPR-Cas9 gene. Three activators provide higher gene activation than other candidate activators and maintain high specificity for the target gene."
The team further demonstrated that the top three candidate activators similarly drive the highest levels of gene expression in human, mouse, and Drosophila cells, regardless of their tissue and developmental origin. The researchers also found ways to use these three major candidate activators to further extend gene activation to the maximum extent.
Alejandro Chavez, co-first author and postdoctoral researcher of the study, said: "In some cases, maximizing the activation of a target gene is necessary to obtain a cellular or therapeutic effect. When we use three different gRNAs, When three copies of the best performing activators were used to target specific genes, we managed to synergistically increase their expression."
“The ease of use of CRISPR-Cas9 offers tremendous possibilities for the development of some genomic therapies. This research provides valuable new design criteria that will help synthetic biologists and bioengineers design more effectively in the future. Targeted genome engineering," said Dr. Donald Ingber, founding director of the Wyss Institute.
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