Using A CRISPR Cas9 Edited Sequence
CRISPR technology involves the use of a specific type of molecular tool, and the delivery of this tool into cells is typically facilitated by vectors. Cas9 is an enzyme that acts like molecular scissors, capable of cutting DNA at specific target sequences. Using CRISPR Cas9 technology, researchers can edit a genome to further study how the edited sequence performs in a host cell.
To deliver the gene of interest to a host cell, you will need a vector. Packaging a CRISPR Cas9 plasmid in a lentiviral vector is a highly efficient way to deliver a genetic payload. If you have questions about CRISPR vectors, our team has put together a list of frequently asked questions and answers.
Editing A DNA Sequence
Our CRISPR team can preform a variety of CRISPR services to make sure you get the sequence you need including custom or standard CRISPR vector productions, sgRNA construct cloning services, cassette and donor vector construction design services, and CRISPR knock out and CRISPR knock in services. Learn more about our quick turnaround CRISPR Cas9 services.
When preparing to request a CRISPR production on your plasmid DNA construct, there are a few things to consider, especially if you are interested in packaging your plasmid in a lentiviral vector. Our team has made the below checklist of items to consider:
- Design and validate the guide RNAs (gRNAs) carefully to ensure they specifically target the desired genomic region. If you need help designing your gRNA, our team can help.
- Choose a suitable plasmid backbone for the CRISPR Cas9 construct. The backbone should be compatible with lentiviral packaging systems and contain appropriate regulatory elements such as a promoter for driving Cas9 and gRNA expression.
- Choose an appropriate Cas9 variant based on factors such as its size, activity, and specificity.
- Optimize the expression elements, such as the promoter driving Cas9 and gRNA expression, to ensure robust and balanced expression levels. Avoid strong promoters that may lead to toxicity.
- Consider incorporating reporter genes such as fluorescent proteins or selectable markers into the lentiviral vector to facilitate the identification and enrichment of transduced cells.
- Sequence verify the entire CRISPR Cas9 construct to confirm the correct assembly and absence of unintended mutations. Ensure that the edited plasmid is free of any undesired mutations or rearrangements. Our team can preform a sequencing assay on your DNA construct to ensure your sequence does not have unwanted mutations.
- Remember the size constraints imposed by the lentiviral packaging system. Some lentiviral vectors have limited cargo capacity, so the size of your CRISPR Cas9 construct should be within acceptable limits.
- Minimize the inclusion of repetitive sequences in the CRISPR Cas9 construct, as they may increase the risk of recombination events during lentiviral packaging.
- Use endotoxin free plasmid purification methods to avoid potential immunogenic responses. All of our plasmid prep productions have a 1.8 260/280 Nucleic Acid ratio.
- Validate the editing efficiency of the CRISPR-Cas9 system in relevant cell lines before packaging into lentiviral vectors. This can be done through functional assays, PCR, or sequencing.
Packaging A CRISPR Cas9 Lentivirus
Should you choose to edit your plasmid DNA sequence to match your research application or procure a predesigned plasmid from a vendor like Addgene (such as lentiCRISPR v2), our team can package this plasmid DNA in a lentiviral vector. Packaging a CRISPR Cas9 lentivirus offers several advantages for gene editing applications. Here are some benefits:
Benefits of using a lentiviral vector for CRISPR Cas9:
- Efficient gene delivery: Lentiviral vectors can efficiently transduce a wide range of dividing and non-dividing cells, making them suitable for various cell types, including neurons and other hard-to-transfect cells.
- Integration into the genome: Lentiviruses have the ability to integrate their genetic material into the host genome. This feature is advantageous for stable and long-term expression of the CRISPR Cas9 components, enabling sustained gene editing activity.
- High payload capacity: Lentiviral vectors have a larger cargo capacity compared to AAVs, allowing the inclusion of multiple genetic elements such as Cas9, gRNA, and reporter genes without significant size constraints.
- Sustained expression: Lentiviral vectors often provide sustained and stable expression of the CRISPR Cas9 components, facilitating prolonged gene editing activity over time.
- Versatility: Lentiviral vectors are versatile and can be used for in vivo and ex vivo applications, making them suitable for a wide range of experimental setups.
Comparison with AAV and adenovirus vectors::
- Integration vs. episomal expression: Lentiviruses integrate their genetic material into the host genome, providing stable, long-term expression. AAV vectors, on the other hand, often remain episomal, resulting in transient expression. The choice between integration and episomal expression depends on your experimental requirements.
- Cell type compatibility: Lentiviral vectors are more effective at transducing non-dividing cells, making them suitable for a broader range of cell types. AAV vectors are generally better suited for transducing dividing cells. Our team offers a variety of cell types using HEK293-T cell line for lentivirus packaging.
- Cargo capacity: Lentiviral vectors have a larger cargo capacity compared to AAV vectors, allowing for the inclusion of larger genetic elements or multiple components in a single vector.
- Immunogenicity: Lentiviruses and AAVs are often considered less immunogenic compared to adenoviruses. Adenovirus vectors may trigger an immune response, limiting their usefulness for certain applications.
- Tissue tropism: AAV vectors have different serotypes with varying tissue tropisms, allowing for targeted gene delivery to specific tissues. Lentiviruses and adenoviruses also exhibit tissue tropism, but AAVs are particularly known for their ability to target specific tissues in vivo.
Of course you should always choose the vector system that best matches your research application. However the above reasons are just some of the reasons why cell and gene therapy researchers use lentiviral vectors to deliver a genetic payload.
Let’s get started! Our CRISPR and viral vector teams are excited to bring your project to life.
If you are interested in starting a CRISPR knock in or CRISPR knock out production, please provide the following when requesting a production:
- 3 µg of wildtype plasmid DNA
- The complete genome sequence you want to target
- The desired sequence edits: insertions, deletions, and or other modifications
If you are interested in packaging a CRISPR Cas9 plasmid procured from another vendor, or if you have performed the CRISPR production in house, just let us know when requesting a production. Our team only requires 1 µg of wildtype plasmid DNA to get started on your lentivirus packaging production.
Want to learn more about the latest in CRISPR genome-editing based research? Our colleagues at ScienceDirect and Genetic Engineering & Biotechnology News continuously collect and publish the latest information on CRISPR based research.
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