CRISPRi is based on the CRISPR-Cas genome editing system but lacks the ability to cut DNA, a so-called "dead CAS" (dCAS) and is fused to a KRAB repressor domain. Using a short RNA as a molecular guide dCas9 can bind to specific DNA sequences. As a result, the target region in the genome is epigenetically silenced, and a gene can no longer be read. This blockade is called "epigenetic silencing".
To introduce the CRISPRi system into the heart muscle cells of mice, the researchers used adeno-associated viruses (AAV), which do not integrate into the genome. One challenge was to package the entire system into the limited genomic capacity of the AAVs. The researchers succeeded in doing this by using a particularly small dCAS. Although other viral vectors have more capacity in this respect, they cannot efficiently reach heart muscle cells or integrate into the genome.
Effective blockade in the heart muscle
Dr Patrick Laurette and his colleagues at Heidelberg University Hospital demonstrated how well epigenetic silencing with the AAV-CRISPRi system works in heart cells for several genes and enhancers. The activity of some genes decreased by up to 95 percent. Enhancers are regulatory elements that can fine-tune gene expression from distant genomic regions.
"The complexity of the mammalian organism is the result of around one million regulatory elements. There are between fifty and one hundred thousand of these enhancers in heart muscle cells alone," says Prof. Ralf Gilsbach. They are now focusing on modulating these regulatory elements using their new method to treat diseases such as heart failure or cardiac arrhythmia.
Translational perspective
Gilsbach emphasizes the translational significance of this approach, which makes it possible to specifically influence gene expression in vivo without changing the DNA sequence. The method is also titratable, meaning it can be regulated and its effect can be reversed. Compared to other methods, such as genetic knock-out, in which genes are destroyed, the AAV-CRISPRi system offers a more precise imitation of natural regulatory mechanisms.
Methodically optimized and further developed, the procedure could also be used for human therapy in the long term. "I am convinced that this approach has translational significance, even if it is difficult to predict how quickly development will continue here," says Gilsbach.
There are already numerous companies that are considering AAVs to deliver CRISPR components for therapies. Among other things, they are working on avoiding unwanted antibody reactions. This is because humans have antibodies against AAV and often also against the CRISPR protein derived from bacteria.
Original publication: Laurette P, Cao C, Ramanujam D, et al. In Vivo Silencing of Regulatory Elements Using a Single AAV-CRISPRi Vector. Circ Res. 2024;134(2):223-225. doi: 10.1161/CIRCRESAHA.123.323854
Scientific contact: Prof. Ralf Gilsbach, Heidelberg University Hospital, Institute of Experimental Cardiology, ralf.gilsbach(at)uni-heidelberg.de
Contact: Christine Vollgraf, Press and Public Relations, German Center for Cardiovascular Research (DZHK), Tel.: 030 3465 529 02, presse(at)dzhk.de
