Gene Editing Technology Improves Human Anti-Aging Ability and Extends Lifespan## 1. Background The aging process involves multiple complex physiological mechanisms, including cell metabolism disorders, DNA damage accumulation, telomere shortening, increased oxidative stress, cell aging and apoptosis. Gene editing technology provides us with a potential means to intervene in these aging-related processes, and is expected to slow down the aging process by precisely regulating specific genes, thereby significantly extending human lifespan. ## 2. Objectives Through gene editing technology, key genes related to aging are modified to improve cell metabolism, repair DNA damage, stabilize telomere length, reduce oxidative stress, delay cell aging and apoptosis, and ultimately achieve a significant improvement in human anti-aging ability and a significant extension of lifespan. ## 3. Technical route### 1. Gene screening 1. **Literature research** Comprehensively sort out existing scientific literature and collect gene information closely related to the aging process, including but not limited to genes involved in cell cycle regulation, DNA repair, telomere maintenance, redox balance, cell apoptosis, etc. 2. **High-throughput sequencing and data analysis** Perform high-throughput sequencing on cell or tissue samples of people of different age groups, analyze changes in gene expression profiles, and screen out genes whose expression levels change significantly during the aging process and whose functions are clearly related to aging. ### (II) Determination of gene editing targets Based on the key aging-related genes screened out, combined with gene functions and mechanisms of action, determine the target regions suitable for gene editing. These targets should be located in the key regulatory regions or functional domains of the genes, and editing of the targets can effectively regulate the expression level or activity of the genes. ### (III) Selection of gene editing technology Currently commonly used gene editing technologies include zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR/Cas9) systems. Considering the advantages of CRISPR/Cas9 system such as easy operation, high efficiency, and low cost, this plan gives priority to this technology for gene editing. ### (IV) Construction of gene vector 1. **Design of sgRNA** For the selected gene target, use online tools to design specific single-stranded guide RNA (sgRNA) to ensure that it has high affinity and specificity with the target sequence while avoiding off-target effects. 2. **Construction of expression vector** Connect the designed sgRNA and Cas9 protein coding sequence to a suitable expression vector to construct a CRISPR/Cas9 gene editing expression vector. The vector should have the ability to efficiently express sgRNA and Cas9 protein in human cells and drive gene expression through a suitable promoter. ### (V) Establishment and verification of cell model 1. **Cell line selection** Human cell lines, such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs) or fibroblasts, are selected as experimental models for gene editing. These cell lines have the advantages of easy culture, strong proliferation ability, and relatively clear genetic background, and can better simulate the physiological characteristics of human cells. 2. **Cell transfection** The constructed CRISPR/Cas9 expression vector is introduced into the selected cell line through liposome transfection and other methods, so that the cells express sgRNA and Cas9 protein and edit the target gene. 3. **Editing effect verification** After the transfected cells are cultured for a period of time, the gene editing effect is verified by the following methods: - **Genomic DNA extraction and PCR amplification**: Extract cell genomic DNA, amplify the region containing the gene editing target with specific primers, and determine whether the gene editing is successful and the type of editing (such as insertion, deletion or base substitution) through sequencing analysis. - **Protein expression analysis**: Use Western blot and other methods to detect changes in the expression level of the target gene and its related proteins, and evaluate the effect of gene editing on protein expression. ### (VI) Animal model construction and experiment 1. **Animal model selection** Select model animals that are similar to human physiological characteristics, such as mice. Mice have a short breeding cycle, low breeding costs, and a clear genetic background, which is convenient for the overall animal level evaluation of gene editing effects. 2. **Preparation of gene-edited mice** Introduce the CRISPR/Cas9 expression vector into mouse fertilized eggs through methods such as fertilized egg injection to obtain gene-edited mice. Perform genotyping and identification on the mice after birth, and screen out positive mice carrying the target gene editing. 3. **Detection of aging-related indicators** Gene-edited mice and wild-type control mice are tracked and observed for a long time, and a series of aging-related indicators are regularly tested, including but not limited to: - **Physiological function indicators**: such as motor ability (rotarod test, open field test, etc.), cognitive ability (Morris water maze test, etc.), muscle strength (grip strength test, etc.), to evaluate the changes in the overall physiological function of mice with age. - **Cell and tissue level indicators**: Take different tissues (such as liver, muscle, brain, etc.) for histopathological analysis to observe changes in cell morphology, structure and function; detect oxidation