CRISPRi is based on the CRISPR-Cas system and contains gene scissors without a cutting function, a so-called ‘dead CAS’ (dCAS), which has been fused with a KRAB repressor domain. By using a molecular guide, a guide RNA, dCas9 can bind specifically to DNA sequences. As a result, the target region in the genome is epigenetically silenced and a gene can no longer be read as well. This blockade is called ‘epigenetic silencing’.
To introduce the CRISPRi system into mouse heart muscle cells, the researchers used adeno-associated viruses (AAVs), 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 are not able to reach heart muscle cells efficiently or integrate into the genome.
Effective blockade in the heart muscle
Dr Patrick Laurette and his colleagues at Heidelberg University Hospital were able to demonstrate 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 per cent. Enhancers are regulatory elements that can fine-tune gene expression from distant genomic distances.
‘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. He and his colleagues are now focussing on these regulatory elements if they want to use their new method to influence diseases such as cardiac insufficiency or cardiac arrhythmia.
Translational perspective
Gilsbach emphasises 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 the knock-out method, in which genes are destroyed, the AAV-CRISPRi system offers a more precise imitation of the natural regulatory mechanisms in cells.
Methodologically optimised 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 adeno-associated viruses and CRISPR in particular 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
The publication was named Paper of the Month January 2024.
Scientific contact: Prof Ralf Gilsbach, Heidelberg University Hospital, Institute of Experimental Cardiology, ralf.gilsbach@uni-heidelberg.de
Contact: Christine Vollgraf, Press and Public Relations, German Centre for Cardiovascular Research (DZHK), Tel.: 030 3465 529 02, presse@dzhk.de