CRISPR/Cas9 Knockout Adenovirus Vectors
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Introduction to CRISPR/Cas9 Knockout Adenovirus Vectors
Recombinant adenovirus (Adv), a replication-defective adenoviral vector system, is widely used for gene delivery in most cell types. The adenoviral vectors provided by Genemedi are based on human adenovirus type 5 (Ad5), which is replication-incompetent (-E1/-E3) and can’t be integrated into host genome, guaranteeing the security for subsequent operations. Adv packaging by Genemedi shows almost 100% gene delivery in most cell types in the recombinant protein expression system both in vivo and in vitro.
Derived from the type II CRISPR-Cas bacterial adaptive immune system, Cas9, an RNA-guided endonuclease, has emerged as a versatile genome-editing platform. Several microbial species possess this system, among which, Streptococcus pyogenes Cas9 (SpCas9), is the most robust and widely used Cas9 to date, primarily recognizes NGG PAMs and is consequently restricted to sites that contain this motif.
By combining Adv packaging system with Cas9 genome-editing platform, Genemedi has further developed Adv-Cas9 system to achieve gene knockout with high efficiency, which is one of the top advanced gene knockout technology in the world.
|Sipping and Storage Guidelines||Shipped by dry ice, stored at -80 ° C, effective for 1 year. Avoid repeatedly freezing and thawing|
|Titer||> 1*10^10 PFU/ml|
1. Genome editing system of somatic tissues in postnatal animals.
2. Broad range of host. Adv has the ability to infect dividing, quiescent cells, stem cells, and primary cells, allowing genetic materials to be delivered to a highly diverse range of cell types and tissues.
3. High infection efficiency. As high as 100% gene delivery in most cell types, completely surpassing other viral vector tools and liposome transfection.
4. Without integration. No alteration to host genome.
5. High titer. Up to 1011 pfu/ml.
Applications and Figures
Quality control description
Our optimized custom adenoviral vector production and strict quality control systems provide customers a high titer of functional recombinant adenoviral vectors. Viral titers are determined by TCID50 method, which is the most accurate way to measure the titer of adenovirus.
1. For detailed protocol about how to package and purify Adv, please see the Adenovirus User Manuall on the Genemedi website.
2. For further information about how to design Adv-Cas9 system, please refer to the CRISPR/Cas9 Knockout Adenovirus User Manual on the Genemedi website.
Frequently Asked Questions(FAQs)
- 1. Why does adenovirus have a relatively higher immunogenecity compared with rAAV?
The adenovirus without E1/E3 can express all of the other genes in the viral backbone and hence induces immunogenic responses, while rAAV does not have any of the AAV genes, thus no immunogenicity from viral protein.
- 2. What is the role of the E1 Gene in adenoviruses?
Simply, the E1 gene products are early proteins that are transcribed in the early transcribed regions and required for proceeding subsequent steps in viral replication. The E1 gene contains E1A and E1B, involved in the replication of adenovirus. E1A is critical to start viral replication by promoting transcription from rep gene promoters, P5 and P19, and facilitate viral replication by activating the early adenovirus promoters.
- 3. How to store adenovirus?
It would be better to store adenovirus in PBS at -80oC. Sucrose or DMSO may help to stabilize the vector.
- 4. How can you tell if your vector is lentiviral, retroviral, or adenoviral?
You should blast your vector sequence and see if there’re sequences of reverse transcriptase and integrase (gene names: gag and pol), which are for lentiviral/retroviral vectors, but not for adenoviral. For another, if your plasmid is around 30-35kb in size, it's certainly adenoviral.
- 5. Is adenovirus a useful tool to study primary macrophage functions?
In RAW264.7 and PM cells, adenovirus works very well, and it seems that IL1β expression is increased slightly after adenovirus transfection compared with negative control. While for the BMDM, adenovirus does not work, it may be better to use lentivirus instead, which gives a pretty good transduction efficacy and less inflammatory response.
- 6. Is it possible to infect a tissue preparation with lentivirus and afterwards with adenovirus and getting high efficiency in transduction?
It is definitively possible to perform sequential transductions/infections. Polybreen, protamine sulfate or other transduction enhancing reagents are recommended to enhance viral particle infectivity.
- 7. Is it still possible to get the same mRNA accumulation in RT PCR after CRISPR/Cas9 induced gene deletion?
Yes. The transcript will continue to be transcribed as nothing has changed with respect to the promoter of the gene. Besides the indel you introduced, the transcript will be unchanged and your primers (presumably not overlapping indel) will detect the transcript. You'll want to use Western blot to observe the effect of the indel.
- 8. I am trying to design gRNA for TLR4 gene CRISPR cas9 genome editing. TLR4 has multiple transcript variants. Which one should I choose?
You should use a gRNA which target as many transcript variants as possible. To make it clear, target those exon(s) which can be found in the most variants of TLR4. As I see exon 1 and exon 4 are the best choices to target TLR4.
- 9. Which sequencing depth should I use for my CRISPR-Cas9 screen?
PCR amplification and Sanger sequencing are the main CRISPR KO screening methods.
- 10. Does the CRISPR/Cas9 system work for non-sequenced organisms?
In theory it should be able to work with any organism. However, there may be some modifications that you will need to work out. You may need to codon optimize the Cas9 gene for your organism and confirm if there are many rare codons used in your organism. Next, you'll need to find a good promoter in your organism that you can use to make your crRNA. Maybe you'll need to add a nucleus localization signal to the Cas9 to make sure it stays in the nucleus, for Cas9 won't work if it is in the cytoplasm. Another issue you need to determine is what protospacer to use. If your organism is similar to one that is sequenced, then this may work, otherwise if you don't know the DNA sequence of your gene, it will be impossible to design a good protospacer against it. Lastly, you'll need a way to transfer Cas9 and crRNA into your organism, which should not hurt your organism.
- 11. Why is spCas9 the dominant CRISPR system?
One of the main reasons is the relatively abundant PAM (protospacer adjacent motif) sequence of spCas9: NGG. This sequence is highly abundant throughout the genome, so this will give you a large amount of options for the sequence to target your sgRNA against. For instance, ST1 and NM require PAMs NNAGAAW and NNNNGATT respectively. These are far less abundant, and thus give far less targets in the genome to work with. SaCas9 is indeed a lot smaller than spCas9, and this can be Advantageous. However, its optimal PAM is NNGRRT.
The second is that SpCas9 outperforms Sa/Nm in generating DSBs in the genome.
The third part of the reason is also for historical reasons. There may be Cas9 species with other 3nt PAM sites. However, since the first one that was discovered was SpCas9, most systems have made adaptations of this Cas9 protein, so sgRNAs (and sgRNA design tools) can be used for multiple systems (including dCas9 activation / inhibition).
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