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Solution for Multiple CRISPR-Cas Genome Editing 
Genome editing is a vital technology that modifies genetic information to obtain desired traits, and it is widely applied in various organisms such as microbes, plants, animals, and humans. The advent of CRISPR-Cas technology has made precise sequence editing possible by altering the target recognition sequence in guide RNA (gRNA). By expressing multiple gRNAs simultaneously, multiple targets can be edited in a single cell, significantly reducing the time and cost required to achieve specific traits.
Significance and Development of Multiplex Genome Editing
Multiplex genome editing allows for the simultaneous editing of multiple gene sites within a single cell, greatly accelerating the process of obtaining desired traits. Precision is crucial in genome editing steps because the mismatch tolerance of the CRISPR-Cas system might cut both edited and non-edited targets, leading to cell death. Thus, using highly truncated single guide RNAs (sgRNAs) can overcome this trait of the CRISPR-Cas system. In multiplex editing, cell survival rates and editing efficiency decline as the number of targets increases.
Figure 1. Procedures of classical genome editing and multiplex CRISPR-Cas genome editing methods. (Lim SR, et al., 2024)
Strategies and Improvements in Multiplex Genome Editing
1. Multiple gRNA Expression Methods
To enhance the efficiency of multiplex genome editing, multiple gRNAs need to be stably expressed on a single plasmid. Common methods include expressing tracrRNA and crRNA arrays separately, using elements such as Csy4 or tRNAs for polycistronic transcription, and employing a set of specific monocistronic gRNA arrays.
2. DNA Repair and Recombination Regulation
Regulating the DNA repair process can enhance multiplex genome editing efficiency. Strategies to inhibit the NHEJ pathway or overexpress recombinases required for homologous recombination (such as λ-red proteins or RecET) can improve editing efficiency.
3. Engineering Editing Tools and Optimizing Recovery Conditions
Using engineered Cas9 nuclease variants or deaminase variants, optimizing promoter strength, and extending post-transformation incubation time can improve editing efficiency. AI-driven predictions and optimization of editing efficiency also show great potential.
4. Diverse CRISPR-Cas Systems
Utilizing different CRISPR-Cas systems, such as Cas12a (Cpf1) and the smaller Cas12f for multiplex editing, is also effective. Understanding different DNA structures and repair mechanisms, as well as the factors influencing multiplex editing target selection, is crucial.
General Steps for Implementing Multiplex Genome Editing
1. Target Selection and Design
Target Identification: Identify genome regions to target using genomic analysis tools.
Designing gRNAs: Design gRNA sequences complementary to the DNA target sequence.
Optimizing gRNA Design: Choose suitable gRNAs to improve targeting efficiency and specificity.
2. Constructing the CRISPR/Cas System
Synthesizing or Constructing gRNA Expression Vectors: Clone-designed gRNA sequences into expression vectors.
Building Cas9 Expression Vectors: Select appropriate Cas9 protein variants and clone them into expression vectors.
Constructing Exogenous Donor Nucleic Acid Vectors: If needed, build vectors containing exogenous donor nucleic acids for homologous recombination.
3. Cell Transfection and Expression
Selecting Cells: Choose suitable cell lines for transfection.
Transfection Operations: Introduce gRNA and Cas9 expression vectors into cells using appropriate methods.
Inducing Cas9 Expression: Add inducers if using inducible Cas9 to activate Cas9 protein expression.
4. Genome Editing and Screening
Editing Verification: Verify gene editing effects using PCR and sequencing techniques.
Isolating Single Cell Clones: Sort single cells post-transfection to cultivate high-efficiency edited cell lines.
Clone Screening: Screen high-efficiency edited clones using genotyping methods.
5. Functional Analysis and Validation
Functional Tests: Test if edited cells exhibit the desired phenotype or functional changes.
Genomic Stability Checks: Analyze edited cell genomic stability to ensure no significant off-target mutations.
Long-term Observation: Assess the long-term stability and function of edited cells to ensure persistent editing effects without unintended side effects.
Creative Biogene Optimization Strategies for Multiplex Genome Editing Efficiency
Creative Biogene offers leading multipurpose CRISPR-Cas genome editing services. With our cutting-edge technology, you can efficiently achieve multi-target gene editing and accelerate the development of your ideal engineered strains. We provide comprehensive support in several key areas:
Enhanced Cas Protein Variants
- High-Fidelity Cas9 Variants: HiFi Cas9: Optimized Cas9 proteins like HiFi Cas9 offer reduced off-target effects and improved editing accuracy.
- Other Cas Protein Variants: Cpf1 (Cas12a): Cpf1 can create sticky ends at target sites, different from the blunt ends produced by traditional Cas9.
- Cas13: The Cas13 system targets RNA sequences, enabling RNA-level gene regulation.
- Improved gRNA Designs
Self-Guided RNA Systems
- Single-guide RNA (sgRNA): Simplifies the multiplex editing process and reduces complexity.
- Optimized gRNA Stability:
- Through sequence optimization, enhance gRNA stability and expression within cells.
CRISPR/Cas System Delivery Technologies
- Lentivirus and Adenovirus: Optimized for higher safety and efficiency in CRISPR/Cas system delivery.
- Lipid Nanoparticles (LNPs): Combine with CRISPR/Cas for optimized cell transfection efficiency.
- Novel Plasmid Systems: Improved designs for stable Cas9 and gRNA expression.
High-Throughput Screening Technologies
- Genome-Wide Screening: Quickly identify genes related to specific phenotypes.
- Functional Screening: Combine phenotype analysis to find genes affecting certain biological processes.
AI Integration
- AI technology enhances precision and efficiency in CRISPR-Cas target editing, predicting gRNA efficiency, and optimizing editing tools via machine learning.
Creative Biogene provides comprehensive support, including improved Cas proteins, advanced delivery systems, high-specificity gRNA design, and long-term stability evaluation. Contact Creative Biogene today to learn how we can support your research and innovation needs!
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