New CRISPR-Cas9 System Combines Epigenetic Editing Domains

The ongoing effort to explore further possibilities of the CRISPR-Cas9 system is ushering in newer developments to this tool. In one of the latest refinements of the technique, as illustrated in a study published in BioDesign Research, scientists from Stanford University, have developed a CRISPR-Cas9 system that induces highly effective silencing of target genes.

For practical purposes, such as suppressing the effect of a disease-causing mutation,  longer-lasting effects of silencing are desirable. The corresponding author of the study, Lei S. Qi explains this concept further, "The Cas9 endonuclease, that stops the expression of a target gene, is analogous to a brake which stops the car by breaking its engine. In a modified dCas9-repressor fusion system, that contains a transcriptional repressor, the car remains stopped as long as one holds down the brake pedal actively. However, we aimed to develop a silencing system that, like a parking gear, prevents wheel movement until you switch the car out of it".

To achieve this goal, the researchers adopted an approach based on the epigenetic regulation of the eukaryotic genome where gene expression is altered stably and reversibly by the direct modification of genomic regions. The team fused dCas9 protein with a transcription repressor domain KRAB (Krüppel-associated box) and DNA methylating domains of DNMT3L and DNMT3A—two potent epigenetic modifiers. They named the construct dCas9-KAL and tested its silencing capacity in a cell-based reporter system. When stably integrated into human cells expressing fluorescent protein EGFP, the dCas9-KAL construct, designed to localize at the promoter of EGFP, repressed fluorescence for weeks.

The success in developing a robust and long-term epigenetic repressor has multifold implications.  Muneaki Nakamura from Stanford University, the lead author of the study, explains, "Since its adoption, CRISPR-Cas9 has revolutionized the face of genetic modification. Our system, as a powerful addition to the CRISPR toolbox, will facilitate further research in the field. It can be used to better engineer cells with desired behaviors, which could find use in the development of custom cell types with wide-ranging research and therapeutic applications.”