Multiplexed CRISPR-Cas9 system in a single adeno-associated virus to simultaneously knock out redundant clock genes

Basics of the internal clock in mammals
All higher living beings have a circadian rhythm, i.e. an internal biological clock that has adapted to the day-night cycle on Earth and has a period length of approximately 24 hours. The circadian rhythm controls many physiological processes and determines the behavior of an organism. In mammals, for example, the wake-sleep cycle, body temperature, blood pressure and the immune system are regulated by the internal clock. The circadian rhythm is regulated by the periodic expression of many internal clock genes that are in all the tissues of an organism. The interaction between gene expression of the many different genes is regulated in the brain, more precisely in the hypothalamus of mammals. The most important genes of the internal clock are Cryptochrome (Cry), Period (Per), Bmal1 and Clock. If the gene expression of one of these genes is disturbed, sleep disorders, mood swings or obesity can be the result.

The genes of the internal clock were examined, among other things, in mouse models, in which so far only individual genes could be switched off. The effects of the gene knockout experiments in mice were diverse: the mice showed increased signs of anxiety in their behavior as well as depression, disturbed sleep or wakefulness.

Aims and results of the study by Kim et al.
The authors of the study wanted to knock out several different internal clock genes in mice with the help of CRISPR/Cas9 gene scissors. Until now, the gene scissors have only been used to knock out single internal clock genes or several genes that have a similar DNA sequence. The aim here was to carry out a complex CRISPR/Cas9 application called multiplexing in different mouse models to study the circadian rhythm.

The scientists used mouse cells in cell culture to knock out single genes of the internal clock with CRISPR/Cas9 and to test various gene scissor guide RNAs. The aim was to select the guide RNAs that most efficiently knocked out the target genes. They combined three selected guide RNAs in three different genetic constructs ( CSAC-Crys, CSAC-Pers and CSAC-Bmal1) that recognize different areas of the target genes Cry1/2, Pers1/2 and Bmal1 respectively, and then injected them into the hypothalamus of transgenic mice. They used mice that already had the genetic information needed to form the gene scissors in their genomes. The gene scissors are formed continuously in these mice. Only when guide RNAs are introduced, will the gene scissors recognize certain target sequences and cut there.

The genetic constructs are introduced through the incorporation of the guide RNA into adeno-associated viruses (AAV). Such viruses are no longer infectious and act as ‘transport vehicles’ to introduce DNA or RNA into mammalian cells. Such ‘transport vehicles’ are also known as viral vectors; they are often used in basic research. The AAV vectors are degraded within a certain period of time.

To check whether the genes of the internal clock in the injected mice had actually been knocked out, the brains of some mice were removed, microscopic sections were taken and antibody staining used to see whether the genes had been knocked out. Next, the behavior of the mice was examined more closely to see whether knocking out the genes really had an impact on the internal clock. The mice in the control group displayed completely normal locomotion activity in accordance with their periodic day-night rhythm, even when they were kept completely in the dark for a longer period of time. In contrast, the mice in which various genes of the internal clock were knocked out had impaired locomotor activity as soon as they were kept in the dark for a long time. Similar results were obtained for body temperature: the mice in the control group had a typical periodic change in their body temperature, both in normal day-night conditions and in permanently dark conditions, whereas the CRISPR/Cas-modified mice showed an aperiodic change in body temperature in the dark.

Open questions
The study can be categorized as basic research to investigate the circadian rhythm in mammals. The authors developed a method to include up to three guide RNAs for CRISPR/Cas9 in adeno-associated viruses and to introduce them into living mice. Up to three different target sequences could therefore be altered simultaneously. In previous studies, CRISPR/Cas9 was used to knock out a single internal clock gene. What was not done in the study and is not possible with the system described here, is a combination of more guide RNAs to simultaneously target the different target genes Cry1, Cry2, Pers1, Pers2 and Bmal1. So far, this is a proof of concept study to show that the multiplexing of up to three different target sequences can be used relatively quickly in mouse cells in cell cultures and in living mice.