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Original link:Metabiology - Chemical Genetics Technology
Chemical genetics technology (also known as pharmacological genetics technology) is an important new technology that has emerged in recent years along with photogenetics. This technology modifies some biomolecules to interact with previously unrecognizable small molecules, achieving controllable and reversible control of their activity (compounds can be added or removed at any time to initiate or interrupt specific reactions). This technology has been widely applied in signal transduction, drug development, functional genomics, and other research areas.
They are widely used to enhance or inhibit neuronal activity in a cell specific and non-invasive manner. Although DREADDs lack precise time control capabilities like photogenetics, they are most likely to require long-term regulation of neuronal circuits during disease treatment, and DREADDs are highly suitable for such applications. In addition, many FDA approved drugs target GPCRs, and DREADDs are modified GPCRs, so DREADDs may provide rich possibilities in drug development.
Currently, the modified biomolecules include nucleic acid hybridization, protein kinases, various metabolic enzymes, and G protein coupled receptors (GPCRs). There are many chemical genetics platforms based on GPCRs modification, such as Allele specific activation of genetically encoded receptors constructed in 1991, Receptors activated solely by synthetic ligands (RASSLs) constructed in 1998, and Engineering modified receptors (RASSLs). And the Designer receptors exclusively activated by designer drugs (DREADDs) constructed in 2007, DREADDs have become the most widely used chemical genetics technology, and this article will mainly discuss DREADDs technology.
图1.化学遗传学技术
1) DREADDs technology
DREADDs technology was invented by Bryan L. Roth et al., who altered the structure of the G protein coupled receptor acetylcholine receptor, which can only be activated or inhibited by a specific compound Clozapine N-oxide (CNO). The receptor of such changes selectively acts on different GPC R-cascade reactions, including Gq, Gi, Gs, Golf, and β - arrestin, with Gq DREADD and Gi DREADD being the most widely used. By expressing the above receptors in cells, the results produced by CNO vary.
Figure 2: Principles of action of several commonly used DREADDs receptors（Scott M. Sternson & Bryan L. Roth, Annu. Rev. Neurosci., 2014）
2) Common DREADDs receptors
1、Gq-DREADD和hM3Dq：
The initial Gq-DREADD, also known as hM3Dq, was modified from the human muscarinic acetylcholine receptor (mAchRs) subtype M3 (also known as hM3). Under normal physiological conditions, hM3 binds to acetylcholine and then couples with Gq class G protein coupled receptors, acting on the signaling pathway of phospholipase C, inositol triphosphate, and intracellular calcium ions (Figure 3).
Surprisingly, as long as the Y3.33C and A5.46 sites are mutated, the hM3 receptor cannot couple with acetylcholine, but will bind to CNO at a nanomolar concentration level. This mutated hM3 receptor is named hM3Dq (human M3 mucosal DREADD receptor coupled to Gq). Due to the conservatism of Y3.33 and A5.46 in different subtypes of human muscarinic acetylcholine receptors, M1 and M5 have also been successfully transformed into Gq-DREADD (hM1Dq and hM5Dq). However, so far, hM3Dq remains the most widely used Gq-DREADD.
The results of CNO induced hM3Dq vary among different cell types, for example: 1) In mature neurons, CNO induced hM3Dq results in depolarization of neurons, enhancing neuronal excitability, which is also the most commonly used function of hM3Dq, which is to promote the discharge activity of divine meridians; 2) In astrocytes, it has been reported that CNO induced hM3Dq results in increased release of Ca+from astrocytes, thereby altering the physiological conditions of the autonomic nervous system; 3) Outside the nervous system, there are also some studies, such as expressing hM3Dq in pancreatic beta cells. Acute CNO treatment promotes insulin release, while chronic CNO treatment leads to an increase in beta cell numbers; In liver cells, activation of hM3Dq increases blood glucose levels, possibly due to increased glycolysis and gluconeogenesis.
Figure 3. hM3 binds to acetylcholine as a signaling molecule to activate the Gq coupled GPCRs signaling pathway
2、Gi-DREADD和hM4Di:
Y3.33 and A5.46 are conserved in different subtypes of human muscarinic acetylcholine receptors, so scientists can also mutate the Y3.33 and A5.46 sites on M2 and M4 mAchRs. As downstream of M2 and M4 activate the Gi channel, Gi DREADDs are generated, named hM2Di and hM4Di, which can activate the signaling pathway regulated by Gi (Figure 4).
Gi coupled GPCRs can activate G protein inward rectifying potassium channels (GIRK), and under the action of CNO, hM2Di and hM4Di receptors are activated to inhibit neuronal firing activity, with hM4Di being the most commonly used Gi DREADD. There are also studies indicating that hM4Di can inhibit the release of neurotransmitters, thereby achieving the effect of inhibiting neuronal activity.
Figure 4: hM4 binds to acetylcholine as a signaling molecule to activate the Gi coupled GPCRs signaling pathway
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3) Application Strategies of DREADDs Technology
The application of DREADDs technology using viral vectors generally includes the following key steps (Figure 5):
1. According to the experimental purpose, determine the appropriate DREADDs receptors. Generally speaking, activate neurons by selecting hM3Dq and inhibit neurons by selecting hM4Di;
2. Expressing DREADDs receptors in animals using viral vectors;
3. Design an experimental plan to administer CNO drugs to animals within an appropriate time window, activate receptors, and administer CNO through brain targeted injection, intraperitoneal injection, and water feeding;
4. Phenotypic detection, which detects changes in neuronal activity through behavioral or electrophysiological methods.
Figure 5. General strategy of DREADDs technology
4) DREADDs technology advantages
The application of DREADDs technology and photogenetics technology is very similar, and their purposes are also the same. So, how do we choose them in our daily research? The selection criteria are an analysis of the advantages and disadvantages of two technologies:
The advantages of DREADDs technology mainly include:
1. The experimental requirements are relatively low and the operation is simple: unlike optogenetics technology that requires fiber optics, laser controllers, etc., DREADDs technology only requires conventional pharmacological techniques such as injection or feeding of CNO;
2. Non invasive: Unlike optical genetic technology, which requires craniotomy surgery to embed optical fibers, it will not affect mouse behavior due to additional load, and can regulate the activity of specific brain regions and neurons in mice with complete free movement;
3. Realizing long-term activation or inhibition of neuronal activity: Due to the different principles of the two technologies, photogenetics technology relies on the opening of photosensitive channels, which require the flow of ions on the cell membrane to generate potential changes and affect neuronal activity. However, long-term ion reverse concentration differences require the consumption of a large amount of ATP (such as ion pumps), which can cause cell damage and death. In addition, the thermal effect of light stimulation can also damage cells. In contrast, DREADDs express a receptor that can continuously activate or inhibit neuronal activity for several hours without affecting normal cellular physiology;
4. High safety: CNO is a metabolite of the FDA approved drug Clozapine, which is relatively safe for in vivo use. In addition, many FDA approved drugs target GPCRs, and DREADDs are modified GPCRs, so DREADDs may provide rich possibilities in drug development.
Table 1 Comparison of advantages and disadvantages between photogenetics and chemical genetics.
Photogenetic technology DREADDs technology
Time accuracy can reach millisecond or even sub millisecond hour levels, enabling sustained activation or inhibition of neuronal activity for several hours without affecting normal cellular physiology
Through brain localization injection, specific promoters, and subcellular organelle localization peptides in space, photosensitive proteins can be anchored to target cells or organelles for operation, reaching the level of a single cell. DREADDs receptors can be anchored to a specific type of cell through localization injection and specific promoters
Surgical technique for implanting fiber optic cables in craniotomy requires fiber optic cables, laser controllers, etc., which is relatively difficult and non immersive. Conventional pharmacological techniques such as injection or feeding of CNO are sufficient
Application Cases of DREADDs Technology
1、hM4Di
① Customers publish articles：Science. (IF=41.058). Mu D,et.al. (2017). A central neural circuit for itch sensation. [Adenovirus, itching, photogenetics, chemical genetics]
Injection site: PBN in mice
carrier：AAV-hSyn-HA-hM4Di-IRES-mCitrine
Serotype：AAV2/9
Virus titer：1.0× 1013 VG/mL
Injection volume：150nl
Observation time: 3 weeks
② Customers publish articles：Neuron . (IF=14.319). Xu HF, et al. (2019) A Disinhibitory Microcircuit Mediates Conditioned Social Fear in the Prefrontal Cortex. [Social phobia, AAV, chemical inheritance]
Injection site: PrL in mice
carrier：AAV2/9-hSyn-DIO-hM4D(Gi)-mCherry
Serotype：AAV2/9
Virus titer：3.44× 1013 VG/mL
Injection volume：100-200nl
Observation time: 4 weeks
