improved-genome-editing-via-oviductal-nucleic-acids-delivery-based

Improved Genome Editing <em>via</em> Oviductal Nucleic Acids Delivery-based <em>In Vivo</em> Electroporation Technique for Knockout Mice Generation

Methods for creating knockout mice typically involve three main steps: (1) collecting embryos from donor females, (2) microinjecting genetic constructs ...

St. Petersburg State University

The Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) - RNA-guided Cas9 endonuclease system has provided a fast and efficient method for precise genome editing in diverse mammalian species, including humans. The CRISPR/Cas9 technology allows generation of modifications into site-specific locations of the selected genes in one major step by carrying deletions, insertions or DNA donor-directed precise sequence modifications. Cas9 forms a nucleoprotein complex with a sequence-specific guide RNA to create double-stranded breaks in complementary DNA target. Further, double-stranded break repair machinery leads to the intended gene modifications. The CRISPR/Cas9 system is widely used technique for genome modification, editing and other biotechnology applications, such as functional annotation, a system for visualization of specific genomic loci and transcriptional control of genes. CRISPR/Cas9-mediated manipulation of the laboratory animal genomes has contributed to the understanding of gene functions and has become a popular approach for modeling human disorders. Furthermore, the growing application of CRISPR-Cas9 system to human genes emerges as an extremely powerful technology for the molecular characterization and treatment of human disease. In this review we present the essential principles of CRISPR/Cas9 technology and the recent advances in its use in translational biomedicine.

CRISPR/Cas9 Technology in Translational Biomedicine.

Cell repair machinery is responsible for protecting the genome from endogenous and exogenous effects that induce DNA damage. Mutations that occur in somatic cells lead to dysfunction in certain tissues or organs, while a violation of genomic integrity during the embryonic period often leads to death. A mammalian embryo's ability to respond to damaged DNA and repair it, as well as its sensitivity to specific lesions, is still not well understood. In this review, we combine disparate data on repair processes in the early stages of preimplantation development in mammalian embryos.

Features of DNA Repair in the Early Stages of Mammalian Embryonic Development.

The identification and characterization of ligand-receptor binding sites are important for drug development. Trace amine-associated receptors (TAARs, members of the class A GPCR family) can interact with different biogenic amines and their metabolites, but the structural basis for their recognition by the TAARs is not well understood. In this work, we have revealed for the first time a group of conserved motifs (fingerprints) characterizing TAARs and studied the docking of aromatic (β-phenylethylamine, tyramine) and aliphatic (putrescine and cadaverine) ligands, including gamma-aminobutyric acid, with human TAAR1 and TAAR6 receptors. We have identified orthosteric binding sites for TAAR1 (Asp68, Asp102, Asp284) and TAAR6 (Asp78, Asp112, Asp202). By analyzing the binding results of 7500 structures, we determined that putrescine and cadaverine bind to TAAR1 at one site, Asp68 + Asp102, and to TAAR6 at two sites, Asp78 + Asp112 and Asp112 + Asp202. Tyramine binds to TAAR6 at the same two sites as putrescine and cadaverine and does not bind to TAAR1 at the selected Asp residues. β-Phenylethylamine and gamma-aminobutyric acid do not bind to the TAAR1 and TAAR6 receptors at the selected Asp residues. The search for ligands targeting allosteric and orthosteric sites of TAARs has excellent pharmaceutical potential.

Search for Structural Basis of Interactions of Biogenic Amines with Human TAAR1 and TAAR6 Receptors.

Rare genetic diseases reduce quality of life and can significantly shorten the lifespan. There are few effective treatment options for these diseases, and existing therapeutic strategies often represent only supportive or palliative care. Therefore, designing genetic-engineering technologies for the treatment of genetic diseases is urgently needed. Rapid advances in genetic editing technologies based on programmable nucleases and in the engineering of gene delivery systems have made it possible to conduct several dozen successful clinical trials; however, the risk of numerous side effects caused by off-target double-strand breaks limits the use of these technologies in the clinic. Development of adenine-to-inosine (A-to-I) and cytosine-to-uracil (C-to-U) RNA-editing systems based on dCas13 enables editing at the transcriptional level without double-strand breaks in DNA. In this review, we discuss recent progress in the application of these technologies in  and  experiments. The main strategies for improving RNA-editing tools by increasing their efficiency and specificity are described as well. These data allow us to outline the prospects of base-editing systems for clinical application.

Cas-Based Systems for RNA Editing in Gene Therapy of Monogenic Diseases:  and  Application and Translational Potential.

Millions of people worldwide have rare genetic diseases that are caused by various mutations in DNA sequence. Classic treatments of rare genetic diseases are often ineffective, and therefore great hopes are placed on gene-editing methods. A DNA base-editing system based on nCas9 (Cas9 with a nickase activity) or dCas9 (a catalytically inactive DNA-targeting Cas9 enzyme) enables editing without double-strand breaks. These tools are constantly being improved, which increases their potential usefulness for therapies. In this review, we describe the main types of base-editing systems and their application to the treatment of monogenic diseases in experiments  and . Additionally, to understand the therapeutic potential of these systems, the advantages and disadvantages of base-editing systems are examined.

Translational potential of base-editing tools for gene therapy of monogenic diseases.

The RNA-guided Cas12a nuclease forms a complex with a CRISPR RNA (crRNA) to cleave the double-stranded DNA target. Among others, Cas12a protein from  (LbCas12a) is widely used for biomedical research. For target recognition, LbCas12a requires a specific nucleotide sequence, named a protospacer adjacent motif (PAM). Besides the canonical TTTV PAM, LbCas12a can recognize other suboptimal PAMs. We examined a novel TTAA PAM for the LbCas12a nuclease and found that the specificity of cleavage was increased. We found that single nucleotide substitutions at all positions of the guide RNA except the 20th position blocked the cleavage of the target DNA. The type of nucleotide substitutions (U-A, U-C or U-G) did not affect the efficiency of cleavage in the 20th position. When we used the canonical PAM under the same conditions, we observed the cleavage of target DNA by LbCas12a in many positions, showing less specificity in given conditions. The efficiency and specificity of the LbCas12a nuclease were evaluated both by gel-electrophoresis and using FAM-labeled single-stranded probes. We were able to assess the change in fluorescence intensity only for several variants of guide RNAs. High specificity allows us to type single nucleotide substitutions and small deletions/insertions (1-2 nucleotides) and look for target mutations when knocking out.

New PAM Improves the Single-Base Specificity of crRNA-Guided LbCas12a Nuclease.

Neurophotonic technology is a rapidly growing group of techniques that are based on the interactions of light with natural or genetically modified cells of the neural system. New optical technologies make it possible to considerably extend the tools of neurophysiological research, from the visualization of functional activity changes to control of brain tissue excitability. This opens new perspectives for studying the mechanisms underlying the development of human neurological diseases. Epilepsy is one of the most common brain disorders; it is characterized by recurrent seizures and affects >1% of the world's population. However, how seizures occur, spread, and terminate in a healthy brain is still unclear. Therefore, it is extremely important to develop appropriate models to accurately explore the causal relationship of epileptic activity. The use of neurophotonic technologies in epilepsy research falls into two broad categories: the visualization of neural epileptic activity, and the direct optical influence on neurons to induce or suppress epileptic activity. An optogenetic variant of the classical kindling model of epileptic seizures, in which activatable cells are genetically defined, is called optokindling. Research is also underway concerning the application of neurophotonic techniques for suppressing epileptic activity, aiming to bring these methods into clinical practice. This review aims to systematize and describe new approaches that use combinations of different neurophotonic methods to work with in vivo models of epilepsy. These approaches overcome many of the shortcomings associated with classical animal models of epilepsy and thus increase the effectiveness of developing new diagnostic methods and antiepileptic therapy.

Neurophotonic methods in approach to in vivo animal epileptic models: Advantages and limitations.

Many retinal degenerative diseases result in vision impairment or permanent blindness due to photoreceptor loss or dysfunction. It has been observed that Pde6b mice (rd1), which carry a spontaneous nonsense mutation in the  gene, have a strong phenotypic similarity to patients suffering from autosomal recessive retinitis pigmentosa. In this study, we present a novel mouse model of retinitis pigmentosa generated through  gene knockout using CRISPR/Cas9 technology. We compare this Pde6b-KO mouse model to the rd1 mouse model to gain insights into the progression of retinal degeneration. The functional assessment of the mouse retina and the tracking of degeneration dynamics were performed using electrophysiological methods, while retinal morphology was analyzed through histology techniques. Interestingly, the Pde6b-KO mouse model demonstrated a higher amplitude of photoresponse than the rd1 model of the same age. At postnatal day 12, the thickness of the photoreceptor layer in both mouse models did not significantly differ from that of control animals; however, by day 15, a substantial reduction was observed. Notably, the decline in the number of photoreceptors in the rd1 model occurred at a significantly faster rate. These findings suggest that the C3H background may play a significant role in the early stages of retinal degeneration.

Does Background Matter? A Comparative Characterization of Mouse Models of Autosomal Retinitis Pigmentosa rd1 and Pde6b-KO.

The worldwide number of deaths from complications caused by severe influenza and COVID-19 is about 1 million cases annually. Development of the effective antiviral therapy strategies for the disease treatment is one of the most important tasks. Use of the CRISPR/Cas13 system, which specifically degrades viral RNA and significantly reduces titer of the virus, could be a solution of this problem. Despite the fact that Cas13 nucleases have been discovered only recently, they already have shown high efficiency in suppressing viral transcripts in cell cultures. The recent advances in mRNA technology and improvements in non-viral delivery systems have made it possible to effectively use CRISPR/Cas13 in animal models as well. In this review, we analyzed experimental  and  studies on the use of CRISPR/Cas13 systems as an antiviral agent in cell cultures and animal models and discussed main directions for improving the CRISPR/Cas13 system. These data allow us to understand prospects and limitations of the further use of CRISPR/Cas13 in the treatment of viral diseases.

Article	Year
CRISPR/Cas9 Technology in Translational Biomedicine. Cellular physiology and biochemistry : international journal of experimental cellular physiology, biochemistry, and pharmacology\| PubMed ID: 32298553	2020
Features of DNA Repair in the Early Stages of Mammalian Embryonic Development. Genes\| PubMed ID: 32992616	2020
Search for Structural Basis of Interactions of Biogenic Amines with Human TAAR1 and TAAR6 Receptors. International journal of molecular sciences\| PubMed ID: 35008636	2021
Cas-Based Systems for RNA Editing in Gene Therapy of Monogenic Diseases: and Application and Translational Potential. Frontiers in cell and developmental biology\| PubMed ID: 35784464	2022
Translational potential of base-editing tools for gene therapy of monogenic diseases. Frontiers in bioengineering and biotechnology\| PubMed ID: 36032737	2022
New PAM Improves the Single-Base Specificity of crRNA-Guided LbCas12a Nuclease. Life (Basel, Switzerland)\| PubMed ID: 36431062	2022
Neurophotonic methods in approach to in vivo animal epileptic models: Advantages and limitations. Epilepsia\| PubMed ID: 38115808	2024
Does Background Matter? A Comparative Characterization of Mouse Models of Autosomal Retinitis Pigmentosa rd1 and Pde6b-KO. International journal of molecular sciences\| PubMed ID: 38139011	2023
Progress in CRISPR/CAS13-Mediated Suppression of Influenza A and SARS-CoV-2 Virus Infection in and Models. Biochemistry. Biokhimiia\| PubMed ID: 40609996	2025

Elena I. Leonova

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