Compact Cas9-based transcriptional regulators for in vivo gene regulation

Cas9-derived constructs can be used to modulate gene expression, but physical limitations restrict the application of this technique. Researchers at The Ohio State University have overcome this obstacle by engineering new Cas9-derived transcription factors.

The Need

CRISPR/Cas9 systems have revolutionized the field of programmable DNA technology. Recently, a version of the Cas9 protein with a deactivated nuclease domain called “dCas9” was developed. Without this domain, dCas9’s range of activity is limited to recognizing and binding sequences specified by guide RNAs. By designing guide RNAs that direct dCas9 complexes to loci that regulate gene expression, one may use dCas9 as a transcription factor to regulate a gene of interest. However, a dCas9 vector that contains the coding sequences for the protein and guide RNA cannot be directly introduced to complex, postnatal organisms. Instead, dCas9 requires a delivery system. An ideal choice for this system is the vector of adeno-associated virus (AAV). However, the dCas9 construct is too large to fit on the AAV vector, which prevents the use of dCas9 in the development of new gene therapies. Therefore, a solution to this physical limitation would extend gene therapy research by facilitating development of dCas9-dependent therapies.

The Technology

Researchers at The Ohio State University have engineered a compact dCas9 system that overcomes this limitation and can be placed on an AAV vector. Like a standard dCas9 system, it uses the nuclease-inactive dCas9 protein and a guide RNA. However, a 144 amino acid section of the dCas9 protein called the HNH domain has been deleted from the protein-coding DNA sequence. This new, compact dCas9 maintains the transcription-regulating activity of a normal dCas9 system, but can be inserted on an AAV vector. Therefore, in vivo dCas9 delivery via AAV becomes possible with this method, which will allow for the development of new technologies in gene therapy.

Commercial Applications

  • Allows for the development of new gene therapies that modulate transcriptional regulation


  • Circumvents a physical limitation that has restricted the development of dCas9-dependent gene therapy.

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