I have a plan to knock-in a gene with about 2kb in size into a cancer cell's chromosome. I suppose to make a DSB (double strand break) using CRISPR-Cas9 system then a gene (2kb) will be inserted by HDR (homology directed repair). I am designing 2 arms (left and right with about 1kb) flanking the inserted gene. So, I am wondering what are the important points when I choose a donor vector to put in all my gene insertion construct?
Thank you very much ^^
Hi Vu. The important points are 1) length of the homology arms-which you already covered; b) the selection marker/reporter you want to use; 3) Make sure the sgRNA targets close to the transgene insertion site (within 100 bp), and 4) Mutate the PAM sites in the donor so that CRISPR can't cut the donor. GeneCopoeia can help you with this. We have CRISPR products and kits, including custom HDR donor clones and cloning vectors. Visit http://www.genecopoeia.com/product/crispr-cas9/ to learn more.
Is it necessary to have an antibiotic selection marker in the donor plasmid? In other words is antibiotic selection specifically for the donor plasmid (stable transfection) required during the HDR (transfection of Cas9 + sgRNA + donor)?
Hi, we took the easier path to include a selection marker (fluorescent protein). Makes selection a breeze.
Ah, ok. In that case we have had to do it the hard way by picking clones... Article Biology and Clinical Implications of the 19q13 Aggressive Pr...
For example, or then using an antibiotic such as puromycin, depending on the plasmid used to express gRNAs
When using CRISPR-Cas9 for a knock-in experiment, selecting an appropriate donor vector is a crucial step. The donor vector carries the gene or sequence you want to insert into the genome. Here are key considerations and steps for selecting a donor vector for gene knock-in:
1. Target Gene/Sequence
- Sequence Length: Consider the length of the gene or sequence you want to insert. Larger inserts might require specific types of vectors with higher capacity.
- Sequence Composition: Check for repetitive elements or other features that might affect cloning or recombination efficiency.
2. Homology Arms
- Homologous Recombination: Include homology arms in the donor vector. These are sequences homologous to the target site in the genome and are critical for facilitating the insertion of the gene via homologous recombination.
- Length of Homology Arms: Typically, 500-1000 base pairs (bp) on each side. Longer arms can increase recombination efficiency but might be harder to clone.
3. Vector Backbone
- Type of Vector: Choose between plasmid, BAC (bacterial artificial chromosome), or viral vectors. Plasmids are commonly used for smaller inserts, while BACs can accommodate larger inserts.
- Selection Marker: Include a selection marker gene (like antibiotic resistance) for identifying cells that have successfully integrated the donor DNA.
- Promoter: Ensure that the vector has an appropriate promoter for the expression of the inserted gene, if expression is desired.
4. Cloning Method
- Restriction Cloning or Gibson Assembly: Depending on the cloning method you're comfortable with, select a vector compatible with that method (e.g., containing the necessary restriction sites for restriction cloning).
5. CRISPR-Cas9 System Compatibility
- PAM Sequence: Ensure that the PAM (protospacer adjacent motif) sequence necessary for Cas9 binding is present at the target site.
- gRNA Design: The guide RNA (gRNA) should be designed to target the specific site where you want the gene to be inserted, and it should be compatible with the chosen vector.
6. Safety and Regulatory Compliance
- Biosafety: Choose vectors that are safe and comply with your institutional biosafety regulations.
- Ethical Considerations: If working with human or animal cells, ensure compliance with ethical guidelines.
7. Quality Control
- Sequence Verification: After cloning your gene into the donor vector, verify the sequence to ensure there are no errors.
8. Additional Elements (Optional)
- Reporter Genes: You might want to include a reporter gene (like GFP) to easily identify cells with successful knock-ins.
- Insulators: To prevent unwanted interactions between vector and host DNA, you might include insulator sequences.
9. Vendor Selection
- Reputable Supplier: Acquire vectors from reputable suppliers or academic sources to ensure quality.
10. Consultation
- Expert Advice: If you’re new to CRISPR or gene knock-ins, consult with experienced colleagues or consider outsourcing to a specialized service.
Conclusion
Selecting a donor vector for CRISPR-Cas9 mediated gene knock-in requires careful consideration of the insert size, homology arms, vector type, cloning method, and compatibility with the CRISPR system. It's important to tailor the vector to your specific experimental needs and ensure compliance with all safety and ethical guidelines.
l This protocol list might provide further insights to address this issue.
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