Description
Genome editing by CRISPR-Cas
Genome editing based on programmable nucleases (CRISPR-Cas systems) overcoming the imprecision of current gene therapy is likely to become the next-generation gene therapy technology. At present, there are four major classes of engineered nucleases: meganucleases, zinc finger nucleases (ZFNs), transcription activator–like effector nucleases (TALENs) and the CRISPR-Cas system[1].
CRISPR-Cas9 has been widely used in the research field as well as in disease treatment. In recent years, CRISPR-Cas9 has quickly progressed to the clinical stage for the treatment.
CRISPR-Cas systems can be used for all kinds of manipulations with the genome:
- Knockdown
- Knockout
- Mutagenesis
- Point mutagenesis
- Epigenetic editing
Аpproaches to genetic editing of living cells
There are several approaches to genetic editing of organisms. It is possible to edit germ cells and somatic cells . Editing a single germ cell appears to be a much simpler task. In the case of successful editing, all cells derived from that edited organism will carry the given mutation. Undoubtedly, editing germ cells can help address a vast number of inherited diseases. However, what should be done if the disease is detected after the organism has grown or if it is associated with changes in the epigenetic status of cells or specific tissues in the organism? In such cases, the problem of editing all cells or specific tissues in an adult organism, which amounts to trillions of cells, needs to be solved. One way to address this problem may involve extracting donor cells from the organism, editing them, and subsequently transplanting them into the patient.
Ex vivo gene editing:
The strategy of altering genes in autologous cells ex vivo is the most straightforward application of gene editing. In this process, somatic cells are isolated first, modified by gene editing tools, and finally delivered back to the patients’ organs.
In vivo gene editing:
Strategies that are used to change genes inside the organisms. To achieve gene editing in vivo, the effective delivery of gene-editing nucleases and donor vectors to target tissues, low off-target frequencies, and low genotoxic effects are all required. It is also possible to classify gene editing as viral delivery systems and non-viral delivery systems.
There are many factors that can affect the successful operation of the genetic editing system:
- The efficiency of homologous recombination
- Knockout
- The duration and magnitude of nuclease expression
- DNA-related cytotoxicity
- and so on
This picture above shows a number of examples of clinical trials in which CRISPR-Cas is used.
For ex vivo research, the modification of T cells and hematopoietic stem cells (HSCs) to treat hematologic disorders, viral infections and some refractory cancers are mainly discussed. Since the first autologous CAR therapies targeting CD19 were approved for the treatment of B-cell lymphoma and leukemia in 2019 , an increasing number of ex vivo studies have come to clinical trials; you can see some of them below.
For in vivo studies, the genome editing technology has also been applied to treat a wide variety of diseases and disorders, mainly liver metabolic disorders , ocular disorders, and neuromuscular disorders. There are a huge number of diseases that can potentially be cured using in vivo genome editing technology viral hepatitis and hepatocellular carcinoma hemophilia A and hemophilia B, hereditary tyrosinemia, lysosomal storage disorders, Ocular disorders, Neuromuscular disorders and so on[2].
Genetic therapy can be directed at various tissues. In particular , now as blood system and immune deficiencies T cells, nerve tissue, muscle, heart, liver cells.
Gene expression control by CRISPR-Cas protein
CRISPR technology in gene circuits is the ability to regulate gene expression. The strategies for transcriptional activation and repression through CRISPR tools are known as CRISPRa (activation) and CRISPRi (interference). In bacteria, the mechanism for CRISPRi is rather simple. In mammalian cells, dCas9 alone is not an efficient repressor, and needs to be fused to a repressive domain such as KRAB (Krüppel-associated box). CRISPRa requires activator domains both in eukaryotes and prokaryotes. A key feature of CRISPR gene control for building complex circuits is multiplexing, i.e. the ability to regulate multiple targets at the same time[3].
The picture below shows examples of many different options for using Cas proteins for gene expression control.
CRISPR-Cas-based genetic circuits
The use of genetic circuits makes it possible to regulate the levels of gene expression inside cells depending on the external or internal signal. The concept of genetic circuits is actively used in the creation of biosensors. The simplest genetic circuits include logical systems : AND, OR, NOT, etc.). Crispr Сas systems are actively used in the creation of genetic circuits[4].
Using knowledge about the work of genetic circuits and using CRISPR-Cas systems for Gene expression control, effective biosensors can be created.
Aim
We have decided to attempt to integrate various approaches to study the activity of Cas protein detection systems, as well as determining and measuring the activity of these proteins within living cells. Our team plans to divide the solution into several stages and, within the framework of this competition, explore the theoretical foundations of creating such systems and implement a basic model demonstrating the possibility of using such systems.
So, for this year our project aims to develop a biosensor that enables non-invasive, real-time monitoring of CRISPR-Cas system activity in living cells. That approach has the potential to revolutionize genetic editing research and applications by providing valuable insights into the dynamics and efficiency of the editing process.
We plan to create a simple system for assembling and confirming the activity of various CRISPR-Cas systems. Our system will help you quickly scan potential targets and select the best options for CRISPR-Cas systems to interact with them. In our opinion, such a system will speed up the process of choosing the most effective system for future therapy, as well as reduce the possible harm from the use of such technologies.
Tasks
Let's dive into the details of the project's tasks!
The first task we undertake is the analysis of the performance of the most popular genome editing systems based on literature data in order to understand the strengths and limitations of these systems. This information serves as a foundation for our subsequent work.
Next, we move on to the selection of the main components of the genetic editing system. These carefully chosen components are instrumental in developing a biosensor that enables us to detect and monitor the activity of the genetic editing system.
Once the components are selected, the next task at hand is the design of a molecular biosensor system. This system operates based on genetic chains, allowing us to specifically monitor the activity of individual components within the genetic editing system.
Moving forward, we focus on creating a genetic chain capable of detecting the activity of the most popular editing systems.
Lastly, we plan to evaluate the activity of the obtained system in vivo, employing model cellular systems. By conducting experiments within these controlled environments, we can observe and measure the performance of our genetic editing system, further refining and optimizing its functionality.
Description of the operation of the biosensor
The main elements of our biosensor system are shown in the picture above. The genetic construct contains a cassette of sgRNA from various crisp cas systems. These constructs are located under an inducible promoter to control the synthesis of sgRNA. The reporter system consists of two fluorescent proteins. RFP and GFP. sgRNA for individual types, type 2 and type 5 cas proteins target different reporter proteins.
Obtaining cells from the organism is planned to undergo genetic modification.
The main idea of our system is to analyze the work of CRISPR-Cas systems for a specific organism, and in the future the use of such an approach in personalized medicine.
The cells of an adult organism are suitable for obtaining IPScell. To do this, the first stage of our system's work is to isolate cells from the organism.
Induction of modification in given cells. Conversion of these cells into iPS cells.
The second stage of the work is the return of cells to a pluripotent state.
There are different approaches to reprogramming cells. In particular, the use of viruses to deliver reprogramming factors (for example, lentiviruses) that infect the cell genome. This approach is widely used in laboratory practice due to its high efficiency, methodological simplicity and cheapness. iPS cells obtained in this way can be potentially oncogenic. But our task is not to return these cells inside the body. They will only be used to build the model.
After dedifferentiation, it is necessary to make sure that the cells have become pluripotent and do not contain chromosomal abnormalities, and start experiments.
Oct4, Klf4, Sox2 and c-Myc, (limanaki factors)
Adding a genetic circuit to cells. Obtaining a cellular biosensor for measuring activity of CRISPR-Cas systems.
Viral delivery systems will also be used to deliver our genetic construct containing a plasmid with sgRNA and a reporter system. The operation of the system was described above.
Differentiation of cells into the main tissues of the organism and tissue of interest for genetic modification.
Since genetic modifications can be directed at various cells of the body, as well as in order to determine the potential effect that these modifications can cause to other cells, it is necessary to obtain basic tissues. Nervous, muscular, cardiac, as well as liver and blood cells. Cell differentiation is one of the most expensive and time-consuming experiments. But we believe that such a process is necessary when working with genetic editing, since different tissues have different epigenetic status and a set of transcription factors. As a consequence, it can be critical to the functioning of editing systems inside cells of different types.
Use transfection of the obtained tissues in various editing systems.
After obtaining cells of different tissues carrying biosensor elements, it is possible to study the work of different Crispr Cas systems. To do this, it is planned to transform different tissues, different cas systems, and study their activity separately. Various variants of cas 9 will be used for transfection, as the most widely occurring protein in genome editing Type 2, as well as proteins of the system based on cas12a, cas12e proteins belonging to Type 5.
Obtaining data “online” on the function and activity of genetic editing systems through biosensor response.
The activity of the systems will be determined by the presence of the simplest gene circuit in the cells. Depending on the reporter's signal, it will be possible to draw a conclusion about the activity of editing systems and time dependence within specific tissues of a particular organism. At the initial stages of testing the model, we plan to use GFP and RFP fluorescent proteins due to the simplicity of measuring fluorescence, as shown on the slide.
Analysis of data on efficiency of different CRISPR-Cas systems in different tissues. Choose the most appropriate system for the selected target tissue and organism.
The system proposed by us will allow real-time monitoring of the activity of crispus systems inside cells. The data obtained will allow us to understand the efficiency of individual cash register systems in specific types of fabrics. Determine the optimal active operating time of these systems. To identify tissues that are more susceptible to editing and less susceptible. After that, it will be possible to choose the most suitable system for editing a living organism.
Conclusion
When planning any experiment involving genetic editing technologies, it is difficult to anticipate all the hazards and potential consequences that may affect the environment or humans. The potential drive of genes can disrupt the delicate balance of an ecosystem. In addition, the capability of genetic editing is an incredibly powerful tool that requires strict control and regulation.
Nevertheless, these technologies will inevitably become more and more integrated into human life. Genetic editing technologies allow us to effectively treat numerous inherited genetic diseases, cancerous conditions, fight aging, and develop new approaches to treat diseases caused by various pathogens, viruses or microbes.
References
[1] Phan HTL, Kim K, Lee H, Seong JK. Progress in and Prospects of Genome Editing Tools for Human Disease Model Development and Therapeutic Applications. Genes (Basel). 2023 Feb 14;14(2):483. doi: 10.3390/genes14020483. PMID: 36833410; PMCID: PMC9957140.
[2] Van Haasteren, J., Li, J., Scheideler, O.J. et al. The delivery challenge: fulfilling the promise of therapeutic genome editing. Nat Biotechnol 38, 845–855 (2020). https://doi.org/10.1038/s41587-020-0565-5
[3] Santos-Moreno J, Schaerli Y. CRISPR-based gene expression control for synthetic gene circuits. Biochem Soc Trans. 2020 Oct 30;48(5):1979-1993. doi: 10.1042/BST20200020. PMID: 32964920; PMCID: PMC7609024.
[4] Shaytan AK, Novikov RV, Vinnikov RS, Gribkova AK, Glukhov GS. From DNA-protein interactions to the genetic circuit design using CRISPR-dCas systems. Front Mol Biosci. 2022 Dec 14;9:1070526. doi: 10.3389/fmolb.2022.1070526. PMID: 36589238; PMCID: PMC9795063.