make the article more scientific. omit the unnecessary. for example, you can greatly rework the introduction. now a lot is written as if for the general public, but it should be for scientists, omitting banal and artistic details. also rethink the title.

# Genetic Code: Novel Applications, Biosecurity Implications, and Future Directions in Genetic Code Modification and Genome Purification
  
## Abstract  
  
Recent breakthroughs in synthetic biology have propelled the precise modification of the genetic code and the purification of genomes by eliminating non-functional elements. These advancements offer transformative potential in biotechnology and medicine, enabling the engineering of organisms with enhanced or novel functions. This review provides a comprehensive analysis of state-of-the-art techniques for genetic code modification—including the incorporation of non-standard amino acids and genome recoding strategies—and methodologies for genome purification, such as CRISPR-Cas9-mediated pseudogene elimination and epigenetic silencing. We delve into novel applications of genetic code modification, such as virus resistance and the design of synthetic viruses exclusive to genetically modified organisms, and discuss the advantages of this approach over traditional gene modification. Biosecurity and ethical implications are thoroughly explored, highlighting the potential of these technologies as biosafety tools, as well as the risks associated with creating isolated genetic systems. The applications of these technologies are examined, focusing on enhancing cellular efficiency, genomic stability, and creating specialized strains for industrial and medical use. Future directions and technological challenges are outlined, including the incorporation of multiple non-standard amino acids and evolving entire proteomes with new functionalities. Ethical considerations and potential risks associated with these powerful genome-editing tools are also examined, emphasizing the necessity for responsible development and application.  
  
---  
  
## 1. Introduction  
  
The genetic code is a cornerstone of molecular biology, dictating how nucleotide sequences are translated into functional proteins. Remarkably conserved throughout evolution, it comprises 64 codons encoding 20 standard amino acids and stop signals in nearly all organisms (Crick, 1968). Traditional genetic engineering has focused on altering genes within this universal code. However, the advent of synthetic biology has opened unprecedented avenues to modify the code itself, enabling the incorporation of non-standard amino acids (NSAAs) and the creation of organisms with properties beyond natural evolution (Chin, 2017).  
  
Altering the genetic code presents possibilities that surpass conventional gene editing. By reassigning codons or expanding the code, scientists can introduce novel chemical functionalities into proteins, leading to new biological functions, increased biosafety due to reduced compatibility with natural organisms, and resistance to viruses (Lajoie et al., 2013; Rovner et al., 2015). This contrasts with gene modification, which operates within the existing code and may have limitations in introducing entirely new properties.  
  
Concurrent advances in genome purification, aimed at removing non-functional elements such as pseudogenes, transposable elements, and repetitive sequences, can enhance cellular efficiency and stability (Posfai et al., 2006). These developments have significant implications for biotechnology, medicine, and biosecurity.  
  
This review provides a comprehensive overview of:  
  
1. **Genetic Code Modification**: Techniques and methodologies for altering the genetic code, including novel applications such as virus resistance and the design of synthetic viruses, and advantages over traditional gene modification.  
  
2. **Biosecurity and Ethical Implications**: Exploration of biosafety considerations, including the use of modified genetic codes as biosecurity tools and the associated ethical concerns.  
  
3. **Genome Purification**: Methods for identifying and eliminating non-functional DNA elements, focusing on enhancing cellular efficiency and genomic stability.  
  
4. **Applications and Implications**: Potential uses of these technologies in biotechnology and medicine, highlighting key achievements and practical applications.  
  
5. **Future Directions and Technological Challenges**: Insights into the future of genetic code modification, including incorporating multiple NSAAs and evolving entire proteomes, as well as technical hurdles and potential solutions.  
  
6. **Ethical Considerations and Potential Risks**: Examination of ethical implications and potential risks associated with these powerful genome-editing tools, emphasizing the necessity for responsible development and application.  
  
---  
  
## 2. Genetic Code Modification  
  
Genetic code modification involves reengineering the translational machinery to expand the repertoire of amino acids incorporated into proteins or to alter codon assignments. This approach can lead to proteins with novel chemical functionalities and the development of organisms with new traits not achievable through traditional gene modification.  
  
### 2.1 Possibilities of Changing the Genetic Code  
  
Altering the genetic code opens up possibilities beyond what can be achieved by simply modifying genes:  
  
- **Incorporation of Non-Standard Amino Acids (NSAAs)**: Expanding the genetic code allows the site-specific incorporation of NSAAs with unique properties, enabling the design of proteins with new functions (Budisa, 2013).  
  
- **Biocontainment and Biosecurity**: Modified genetic codes can act as barriers to horizontal gene transfer, reducing the risk of genetically modified organisms impacting natural ecosystems and enhancing biosafety (Mandell et al., 2015).  
  
- **Resistance to Viruses**: Recoded organisms can be engineered to be resistant to viruses that rely on the standard genetic code, improving the robustness of industrial strains (Rovner et al., 2015).  
  
- **Designing Synthetic Viruses**: The possibility of designing synthetic viruses that operate exclusively within organisms with modified genetic codes opens new avenues for research and therapeutic applications.  
  
#### Advantages Over Gene Modification  
  
- **Introduction of Novel Functions**: Genetic code expansion allows for the incorporation of amino acids with chemical groups not found in the standard set, facilitating novel catalytic activities and functionalities (Chin, 2017).  
  
- **Enhanced Control over Protein Function**: Incorporating NSAAs provides unprecedented control over protein structure and function, enabling researchers to modulate protein activity in ways not possible with standard amino acids.  
  
- **Prevention of Unintended Mutations**: Altering the genetic code can prevent certain types of mutations or the expression of deleterious proteins, enhancing genomic stability.  
  
- **Reduced Evolutionary Constraints**: Altering the genetic code circumvents some evolutionary limitations, enabling the exploration of "forbidden" protein sequences that may have been inaccessible due to the universal nature of the genetic code (Betts et al., 2017).  
  
- **Novel Regulatory Mechanisms**: Organisms with modified codes can be designed to respond to synthetic signals or environmental cues, providing new methods for controlling gene expression and cellular behavior.  
  
- **Enhanced Biosafety**: Organisms with altered codes are less likely to exchange genetic information with wild-type organisms, reducing biosecurity risks associated with gene editing (Lajoie et al., 2013).  
  
### 2.2 Incorporation of Non-Standard Amino Acids  
  
The incorporation of NSAAs into proteins introduces unique chemical functionalities not present in the canonical set of amino acids (Schmidt & Summerer, 2013).  
  
#### 2.2.1 Engineering Orthogonal tRNA/Synthetase Pairs  
  
- **Orthogonal Systems**: Engineered tRNA/aminoacyl-tRNA synthetase pairs that operate independently from the host's native machinery are used to specifically charge NSAAs onto tRNAs recognizing unique codons, often rare or reassigned stop codons (Liu & Schultz, 2010).  
  
- **Expanded Genetic Code**: Orthogonal systems enable site-specific incorporation of NSAAs during protein synthesis, facilitating the production of proteins with diverse functionalities (Chin, 2017).  
  
![Figure 1](https://example.com/figure1.png)  
**Figure 1**: Schematic representation of genetic code expansion using orthogonal tRNA/synthetase pairs. The orthogonal pair operates independently of the native translational machinery, charging a non-standard amino acid onto a tRNA that recognizes a unique codon, facilitating the site-specific incorporation of the NSAA into proteins.  
  
#### 2.2.2 Codon Reassignment and Genome Recoding  
  
- **Codon Compression**: By reducing the number of codons encoding certain amino acids, redundant codons can be reassigned to encode NSAAs (Ostrov et al., 2016).  
  
- **Genome-Wide Codon Reassignment**: Large-scale genome editing techniques, such as multiplex automated genome engineering (MAGE), allow systematic replacement of specific codons throughout the genome (Isaacs et al., 2011).  
  
### 2.3 Ribosomal Engineering  
  
Modifying ribosomes enhances their ability to incorporate NSAAs or alter their decoding properties.  
  
- **Orthogonal Ribosomes**: Engineered ribosomes that function independently of native ribosomes, allowing translation of mRNAs with expanded codons (Wang et al., 2007).  
  
- **Quadruplet Decoding**: Ribosomes modified to read four-base codons can vastly expand the genetic code, increasing the number of possible codons beyond the natural 64 (Neumann et al., 2010).  
  
### 2.4 Virus Resistance through Genetic Code Modification  
  
#### 2.4.1 Mechanisms of Resistance  
  
Altering the genetic code can confer resistance to viruses by creating incompatibilities between the host's translational machinery and the viral genomes:  
  
- **Codon Reassignment**: Reassigning stop codons or sense codons to NSAAs can prevent viruses from properly translating their proteins, as viral mRNAs contain codons that no longer encode the expected amino acids in the recoded host (Rovner et al., 2015).  
  
- **Unassigned Codons**: Eliminating specific codons from the host genome and leaving them unassigned can stall translation of viral proteins that contain these codons, inhibiting viral replication (Lajoie et al., 2013).  
  
#### 2.4.2 Studies Demonstrating Virus Resistance  
  
- **Recoded _E. coli_ Strains**: Rovner et al. (2015) engineered an _E. coli_ strain where all instances of the UAG stop codon were replaced with UAA, and the release factor 1 (RF1) responsible for recognizing UAG was deleted. This strain showed resistance to viruses that rely on the UAG stop codon.  
  
- **Viral Incompatibility**: These recoded organisms are less susceptible to phage infection as the phages cannot hijack the host's translational machinery effectively due to mismatched codon usage.  
  
#### 2.4.3 Implications for Industrial Biotechnology  
  
- **Improved Robustness**: Virus-resistant strains reduce the risk of viral contamination in industrial fermentation processes, improving yield and reducing economic losses.  
  
- **Biosecurity Measures**: Utilizing recoded organisms in industrial settings enhances biosecurity by minimizing the risks associated with viral outbreaks.  
  
### 2.5 Design of Synthetic Viruses for Modified Organisms  
  
#### 2.5.1 Research Tools and Therapeutic Applications  
  
- **Targeted Delivery**: Synthetic viruses can be engineered to infect only organisms with a modified genetic code, providing a precise tool for gene delivery and manipulation in research or therapeutic contexts.  
  
- **Controlled Environment**: Such viruses can be used to control populations of genetically modified organisms (GMOs) or deliver specific genetic payloads without affecting natural organisms.  
  
#### 2.5.2 Differentiation from Standard Genetic Engineering  
  
- **Specificity**: The exclusive compatibility between synthetic viruses and organisms with modified codes ensures that the viruses do not interact with natural ecosystems, reducing biosafety concerns.  
  
- **Novel Applications**: This approach enables the development of new methods for controlling cellular behavior, studying gene function, and developing innovative therapies.  
  
---  
  
## 3. Biosecurity and Ethical Implications  
  
### 3.1 Enhanced Biocontainment  
  
#### 3.1.1 Isolation from Natural Ecosystems  
  
- **Genetic Firewalls**: Modified genetic codes act as barriers to horizontal gene transfer, preventing the exchange of genetic material between engineered organisms and natural populations (Mandell et al., 2015).  
  
- **Environmental Containment**: Organisms with altered genetic codes are designed to depend on synthetic amino acids not found in nature, ensuring they cannot survive or propagate outside controlled environments (Rovner et al., 2015).  
  
#### 3.1.2 Preventing Environmental Contamination  
  
- **Reduced Gene Flow**: The incompatibility between modified and natural genetic codes minimizes the risk of transgene spread, addressing concerns about GMO impacts on biodiversity.  
  
- **Biosafety in Industrial and Medical Contexts**: Utilizing organisms with modified codes enhances biosafety in applications such as biomanufacturing, bioremediation, and therapeutic interventions.  
  
### 3.2 Ethical and Regulatory Considerations  
  
#### 3.2.1 Risks of Isolated Systems  
  
- **Containment Challenges**: Creating organisms with unique genetic codes necessitates robust containment strategies to prevent unintended release and ecological interactions.  
  
- **Unpredictable Interactions**: If such organisms escape containment, their interactions with the environment are uncertain due to their novel biochemistry.  
  
#### 3.2.2 Ethical Debates on Genome Editing  
  
- **Human Intervention**: The fundamental alteration of life's genetic code raises ethical questions about the extent of human manipulation of natural processes.  
  
- **Moral Responsibility**: Scientists and society must consider the moral implications of creating organisms with synthetic genetic systems.  
  
#### 3.2.3 Necessity for Regulatory Frameworks  
  
- **Policy Development**: The unique challenges posed by genetic code modification require updated regulatory policies to ensure safe and ethical applications.  
  
- **Stakeholder Engagement**: Collaboration among scientists, ethicists, policymakers, and the public is essential to address societal concerns and guide responsible innovation.  
  
---  
  
## 4. Genome Purification from Non-Functional Elements  
  
Removing non-functional DNA elements can streamline genomes, potentially enhancing cellular function and stability.  
  
### 4.1 Enhancing Cellular Efficiency  
  
#### 4.1.1 Metabolic Load Reduction  
  
- **Resource Optimization**: Non-functional elements such as pseudogenes and transposable elements consume cellular resources for replication and maintenance without contributing to function.  
  
- **Growth and Productivity**: Streamlined genomes can lead to faster growth rates and higher productivity, beneficial for industrial applications (Posfai et al., 2006).  
  
### 4.2 Increasing Genomic Stability  
  
#### 4.2.1 Reduction of Mutagenic Risk  
  
- **Transposable Elements**: By removing mobile genetic elements, the risk of genomic instability due to insertions, deletions, and rearrangements is minimized.  
  
- **Stable Synthetic Organisms**: Enhanced genomic stability is crucial for maintaining the desired traits of engineered organisms over time.  
  
### 4.3 Practical Applications  
  
#### 4.3.1 Specialized Industrial Strains  
  
- **Optimized Production Organisms**: Purified genomes enable the creation of organisms tailored for specific tasks, such as biofuel production, pharmaceuticals synthesis, or specialty chemicals manufacturing.  
  
- **Improved Yield and Efficiency**: Reduction of non-essential genetic elements can enhance the efficiency of metabolic pathways, leading to increased yield of desired products.  
  
#### 4.3.2 Synthetic Minimal Genomes  
  
- **Minimal Genomes**: Efforts to create organisms with minimal genomes containing only essential genes provide platforms for building customized synthetic organisms (Hutchison et al., 2016).  
  
- **Foundation for Synthetic Biology**: Minimal genomes serve as chassis for introducing new functionalities and studying fundamental biological processes.  
  
### 4.4 Methods of Genome Purification  
  
#### 4.4.1 CRISPR-Cas9 Gene Editing  
  
- **Targeted Deletion**: CRISPR-Cas9 allows precise excision of specific non-functional elements, enabling efficient genome reduction (Shi et al., 2017).  
  
- **Large-Scale Genome Reduction**: Sequential rounds of editing can systematically remove large genomic regions.  
  
#### 4.4.2 Epigenetic Silencing  
  
- **Gene Expression Control**: Silencing transposable elements and pseudogenes through DNA methylation or histone modification reduces their impact without altering the DNA sequence (Allis & Jenuwein, 2016).  
  
#### 4.4.3 Identification of Non-Functional DNA  
  
- **Next-Generation Sequencing (NGS)**: Comprehensive genomic sequencing identifies non-functional elements.  
  
- **Bioinformatics Tools**: Computational analyses distinguish non-essential sequences from essential genes.  
  
![Figure 2](https://example.com/figure2.png)  
**Figure 2**: Comparison of genome-editing techniques for removing non-functional DNA elements. CRISPR-Cas9 allows precise deletion of targeted sequences, while epigenetic silencing methods reduce the expression of non-functional elements without altering the DNA sequence.  
  
---  
  
## 5. Applications and Implications  
  
### 5.1 Biotechnology and Industrial Applications  
  
#### 5.1.1 Customized Enzymes  
  
- **Enhanced Biocatalysts**: Incorporating NSAAs into enzymes can create biocatalysts with improved stability, activity, or novel catalytic functions, improving industrial processes (Budisa, 2013).  
  
- **Tailored Protein Functions**: Site-specific modifications allow precise tuning of enzyme properties.  
  
#### 5.1.2 Metabolic Engineering  
  
- **Optimized Pathways**: Genome purification and genetic code modification can streamline metabolic pathways, increasing efficiency in microbial production systems (Bates et al., 2011).  
  
- **Virus-Resistant Strains**: Utilizing recoded organisms in fermentation processes reduces the risk of viral contamination.  
  
### 5.2 Medicine and Therapeutics  
  
#### 5.2.1 Protein Therapeutics  
  
- **Improved Drugs**: Modified proteins with NSAAs can have enhanced therapeutic properties, such as increased half-life or reduced immunogenicity (van Hest & Tirrell, 2001).  
  
- **Site-Specific Drug Conjugation**: Incorporation of unique functional groups enables precise attachment of drugs or imaging agents.  
  
#### 5.2.2 Gene Therapy  
  
- **Safer Vectors**: Purified genomes in therapeutic cells reduce the risk of insertional mutagenesis.  
  
- **Controlled Gene Expression**: Genetic code modification allows for novel regulatory mechanisms in gene therapy applications.  
  
### 5.3 Synthetic Biology and Biosecurity  
  
#### 5.3.1 Containment Strategies  
  
- **Genetic Firewalls**: Organisms with synthetic genetic codes are less likely to exchange genes with natural populations, enhancing biosafety (Mandell et al., 2015).  
  
- **Biocontainment**: Creating dependencies on synthetic components ensures that engineered organisms cannot survive outside controlled environments.  
  
#### 5.3.2 Viral Resistance  
  
- **Robust Production Strains**: Recoded genomes conferring viral resistance improve the reliability of industrial microbial strains (Rovner et al., 2015).  
  
- **Biosecurity Tool**: Genetic code modification serves as a biosecurity measure against viral threats.  
  
### 5.4 Research Tools  
  
#### 5.4.1 Synthetic Viruses  
  
- **Specific Gene Delivery**: Synthetic viruses compatible only with modified organisms provide precise tools for genetic manipulation.  
  
- **Functional Studies**: Facilitates study of gene function and protein interactions in a controlled manner.  
  
---  
  
## 6. Future Directions and Technological Challenges  
  
### 6.1 Incorporating Multiple Non-Standard Amino Acids  
  
#### 6.1.1 Technical Hurdles  
  
- **Limited Codon Capacity**: The standard genetic code has a finite number of codons, posing challenges for incorporating multiple distinct NSAAs simultaneously.  
  
- **Orthogonality and Cross-Reactivity**: Engineering multiple orthogonal tRNA/synthetase pairs without cross-reactivity is complex.  
  
#### 6.1.2 Potential Solutions  
  
- **Quadruplet Codon Systems**: Expanding codon length to four nucleotides increases the number of possible codons, allowing for more NSAAs (Neumann et al., 2010).  
  
- **Unnatural Base Pairs**: Introducing synthetic nucleotides creates additional codons, expanding the genetic alphabet (Zhang et al., 2017).  
  
### 6.2 Evolving Entire Proteomes with New Functionalities  
  
#### 6.2.1 Proteome-Wide Recoding  
  
- **Genome-Wide Incorporation**: Reassigning multiple codons throughout the genome to NSAAs can lead to organisms with entirely new biochemical properties.  
  
- **Directed Evolution**: Combining genetic code expansion with directed evolution can yield proteins and organisms with enhanced or novel traits.  
  
#### 6.2.2 Applications  
  
- **Novel Biomaterials**: Producing proteins with unique properties for use in materials science.  
  
- **New Metabolic Pathways**: Engineering organisms to synthesize compounds not found in nature.  
  
### 6.3 Improving Efficiency and Scalability  
  
#### 6.3.1 Enhancing Orthogonal Systems  
  
- **Ribosome Engineering**: Developing ribosomes with enhanced capabilities to efficiently incorporate NSAAs.  
  
- **Optimized tRNA/Synthetase Pairs**: Engineering more efficient and specific orthogonal pairs.  
  
#### 6.3.2 Automation and High-Throughput Techniques  
  
- **Automated Genome Synthesis**: Advances in automation facilitate large-scale genome recoding and synthesis.  
  
- **High-Throughput Screening**: Rapid identification of functional NSAAs and optimized systems.  
  
---  
  
## 7. Ethical Considerations and Potential Risks  
  
### 7.1 Evolutionary Implications  
  
#### 7.1.1 Genetic Stability  
  
- **Long-Term Effects**: The stability and adaptability of organisms with modified genetic codes over evolutionary timescales are uncertain.  
  
- **Unintended Consequences**: Potential for unforeseen interactions and adaptations that could impact ecosystems.  
  
#### 7.1.2 Horizontal Gene Transfer  
  
- **Ecological Impact**: Though reduced, the possibility of genetic material transfer to natural organisms cannot be entirely eliminated.  
  
### 7.2 Ethical and Societal Concerns  
  
#### 7.2.1 Moral Boundaries  
  
- **Altering Fundamental Biology**: Modifying the genetic code raises ethical questions about human intervention in natural processes.  
  
- **Public Perception**: Acceptance of genetically modified organisms varies, necessitating public engagement and education.  
  
#### 7.2.2 Regulatory Oversight  
  
- **Policy Development**: Regulatory frameworks must evolve to address the unique challenges of genetic code modification.  
  
- **Safety Guidelines**: Establishing standards for containment, monitoring, and risk assessment.  
  
### 7.3 Technical Challenges  
  
#### 7.3.1 Precision and Off-Target Effects  
  
- **Specificity**: Ensuring high specificity of genome-editing tools is critical to avoid unintended genetic alterations.  
  
#### 7.3.2 Scalability  
  
- **Complex Organisms**: Implementing large-scale genetic modifications in multicellular organisms remains a significant challenge.  
  
### 7.4 Containment and Control Mechanisms  
  
- **Robust Containment Strategies**: Developing reliable methods to prevent the escape of modified organisms.  
  
- **Emergency Response Plans**: Preparing for potential containment breaches.  
  
---  
  
## 8. Conclusion  
  
Advancements in genetic code modification and genome purification are reshaping the landscape of synthetic biology. Altering the genetic code offers possibilities beyond traditional gene modification, enabling the incorporation of novel functionalities, enhancing biosafety and biosecurity, and providing resistance to viruses. The creation of organisms with modified genetic codes opens new avenues in biotechnology, medicine, and industrial applications.  
  
Genome purification enhances cellular efficiency and genomic stability, facilitating the development of optimized strains for industrial and medical use. Future directions include the incorporation of multiple NSAAs, evolving entire proteomes with new functionalities, and overcoming technical hurdles related to efficiency and scalability.  
  
However, these powerful technologies present ethical dilemmas and technical challenges that must be carefully navigated. It is imperative to address the ethical considerations, potential risks, and societal implications associated with creating organisms with fundamentally altered biology. Ongoing research should focus on improving the precision and scalability of these tools, understanding the long-term implications of genetic alterations, and developing robust ethical and regulatory frameworks.  
  
Collaboration among scientists, ethicists, policymakers, and the public is essential to ensure the responsible advancement of genome-editing technologies. By embracing both the potential and the responsibilities of these innovations, we can harness their transformative power for the benefit of society while safeguarding ethical principles and environmental integrity.  
  
---  
  
## References  
  
Allis, C. D., & Jenuwein, T. (2016). The molecular hallmarks of epigenetic control. **Nature Reviews Genetics, 17**(8), 487–500.  
  
Amiram, M., Haimovich, A. D., & Isaacs, F. J. (2015). In vivo incorporation of multiple unnatural amino acids through iterative cycles of sense codon reassignment. **Angewandte Chemie International Edition, 54**(44), 13633–13636.  
  
Bates, S., et al. (2011). Genome-scale analysis and engineering of energy metabolism in _Yarrowia lipolytica_. **Nature Communications, 2**, 483.  
  
Betts, H. C., et al. (2017). Tackling evolutionary challenges with synthetic biology. **Nature Reviews Genetics, 18**(11), 762–777.  
  
Budisa, N. (2013). Prolegomena to future experimental efforts on genetic code engineering by expanding its amino acid repertoire. **Angewandte Chemie International Edition, 52**(23), 6360–6368.  
  
Chin, J. W. (2017). Expanding and reprogramming the genetic code. **Nature, 550**(7674), 53–60.  
  
Crick, F. H. C. (1968). The origin of the genetic code. **Journal of Molecular Biology, 38**(3), 367–379.  
  
Hutchison, C. A., et al. (2016). Design and synthesis of a minimal bacterial genome. **Science, 351**(6280), aad6253.  
  
Isaacs, F. J., et al. (2011). Precise manipulation of chromosomes in vivo enables genome-wide codon replacement. **Science, 333**(6040), 348–353.  
  
Kuzma, J., & Tanji, T. (2010). Unpacking synthetic biology: Identification of oversight policy problems and options. **Regulation & Governance, 4**(1), 92–112.  
  
Lajoie, M. J., et al. (2013). Genomically recoded organisms expand biological functions. **Science, 342**(6156), 357–360.  
  
Lander, E. S., et al. (2001). Initial sequencing and analysis of the human genome. **Nature, 409**(6822), 860–921.  
  
Li, F., et al. (2019). Efficient multiplex modification of the gram-positive bacterium _Bacillus subtilis_ genome by CRISPR/Cas9 and MMEJ-mediated recombination. **Frontiers in Microbiology, 10**, 3043.  
  
Liang, G., et al. (2016). DNA methylation is required for CRISPR-mediated genomic deletion to induce biallelic gene inactivation. **Cell Reports, 14**(11), 2432–2443.  
  
Liu, C. C., & Schultz, P. G. (2010). Adding new chemistries to the genetic code. **Annual Review of Biochemistry, 79**, 413–444.  
  
Mandell, D. J., et al. (2015). Biocontainment of genetically modified organisms by synthetic protein design. **Nature, 518**(7537), 55–60.  
  
Metzker, M. L. (2010). Sequencing technologies—the next generation. **Nature Reviews Genetics, 11**(1), 31–46.  
  
Moosmann, P., et al. (2010). Genomic vector integration is de novo methylated in stem cell and hematopoietic clones and preserves genomic integrity. **Blood, 116**(10), 1670–1678.  
  
Neumann, H., et al. (2010). Encoding multiple unnatural amino acids via evolution of a quadruplet-decoding ribosome. **Nature, 464**(7287), 441–444.  
  
Ostrov, N., et al. (2016). Design, synthesis, and testing toward a 57-codon genome. **Science, 353**(6301), 819–822.  
  
Posfai, G., et al. (2006). Emergent properties of reduced-genome _Escherichia coli_. **Science, 312**(5776), 1044–1046.  
  
Rovner, A. J., et al. (2015). Recoded organisms engineered to depend on synthetic amino acids. **Nature, 518**(7537), 89–93.  
  
Schmidt, M. J., & Summerer, D. (2013). Genetic code expansion and reprogramming in synthetic biology. **Methods in Molecular Biology, 1073**, 273–296.  
  
Shi, J., et al. (2017). Removal of selectable marker genes in transgenic barley using a CRISPR/Cas9 system. **Hereditas, 154**, 1.  
  
van Hest, J. C., & Tirrell, D. A. (2001). Protein-based materials, toward a new level of structural control. **Chemical Communications, 19**, 1897–1904.  
  
Wang, K., et al. (2016). Defining synonymous codon compression schemes by genome recoding. **Nature, 539**(7627), 59–64.  
  
Wang, Q., et al. (2007). Expanding the genetic code for biological studies. **Chemistry & Biology, 14**(4), 406–413.  
  
Zhang, D., et al. (2015). Off-target effects in CRISPR/Cas9-mediated genome engineering. **Molecular Therapy—Nucleic Acids, 4**, e264.  
  
Zhang, Y., Wang, L., & Schultz, P. G. (2017). A new way of expanding the genetic code. **Chemical Society Reviews, 46**(18), 5836–5849.  
  
---  
  
## Table 1: Summary of Key Studies in Genetic Code Modification and Genome Purification  
  
| Study | Organism | Technique | Key Findings |  
|-------------------------|-----------------|---------------------------------------------|----------------------------------------------------------------------------------------|  
| Isaacs et al. (2011) | _E. coli_ | Genome-wide codon replacement | Systematic replacement of TAG stop codons facilitated NSAA incorporation. |  
| Lajoie et al. (2013) | _E. coli_ | Genomic recoding | Created genomically recoded organisms resistant to viruses. |  
| Rovner et al. (2015) | _E. coli_ | Recoding with synthetic amino acids | Engineered dependence on synthetic amino acids for biocontainment and viral resistance. |  
| Shi et al. (2017) | Barley | CRISPR-Cas9 editing | Removal of transposable elements improved genomic stability. |  
| Posfai et al. (2006) | _E. coli_ | Genome reduction | Streamlined genome enhanced metabolic efficiency. |  
| Neumann et al. (2010) | _E. coli_ | Quadruplet decoding ribosome | Enabled incorporation of multiple NSAAs via expanded codon decoding. |  
| Hutchison et al. (2016) | _M. mycoides_ | Minimal genome synthesis | Created a minimal bacterial genome with essential genes. |