Researchers from the University of Illinois Chicago have discovered a new gene-editing technique that allows for the programming of sequential edits over time.
RNA-guided nucleases have opened up opportunities to manipulate the mammalian genome for research and therapies at diverse scales. Compared to other nucleases, RNA-guided nucleases benefit from the simplicity in which they can be programmed to target specific DNA sequences.
The spCas9 nuclease is used for most genome editing in mammals. It was derived from the CRISPR-Cas9 system in Streptococcus pyogenes. In addition to genome editing, spCas9 has been used for synthetic biology purposes, such as storing information in genomic DNA. However, the ability of spCas9 to simultaneously work with each different single-guide RNA (sgRNA) in a cell poses a barrier. This is particularly a problem when constructing circuits that need to be sequentially or temporarily triggered.
In this study, published in Molecular Cell, researchers described a new system that enables multiple genetic manipulations to be executed as a pre-programmed cascade of events. The system consists of an inactive sgRNA-like component that is converted into an active state through the effect of another sgRNA. The authors referred to this component as proGuide. These conditional sgRNAs can be combined to form ‘daisy chains’ or cascades to target spCas9 activities throughout the genome. This specifically allows for sequential genetic manipulations without the need for engineering cell-type-specific promoters or gene regulatory sequences.
ProGuide cascades provide new opportunities for engineering mammalian cells and tissues. It enables the delivery of gene regulatory activities to sites via pre-programmed sequential orders. While proGuide is still in the prototype phase, the team hope to further develop their concept so researchers can use the technique soon.
Bradley Merril, lead author, stated:
“The ability to pre-program the sequential activation of Cas9 at multiple sites introduces a new tool for biological research and genetic engineering.
The time factor is a critical component of human development and also disease progression, but current methods to genetically investigate these processes don’t work effectively with the time element. Our system allows for gene editing in a pre-programmed fashion, enabling researchers to better investigate time-sensitive processes like how cancer develops from a few gene mutations and how the order in which those mutations occur may affect the disease.”
Image by Rafael Zajczewski from Pixabay