Recombinant Cas9, genetically engineered production, expressed by E. coli.
一、 Product introduction
Cas9 is an adaptive immune defense formed by bacteria and archaea during the long-term evolution process, which can fight against invading viruses and foreign DNA. It can integrate the fragments of invading phage and plasmid DNA into CRISPR sequence, and guide the degradation of homologous sequence by Cas9 protein through corresponding CRISPR RNAs (crRNAs). Which provides immunity. Based on the properties of the CRISPR/Cas system, scientists have transformed it into the most efficient genome editing tool available.
The Cas9 system consists of three components: tracrRNA+crRNA Mosaic sgRNA (single guide RNA) + protospacer adjacent motif (PAM) +Cas9 scissors on host DNA sequence (most commonly SpCas9 protein from Streptococcus pyogenes). sgRNA guides the Cas9 scissors to the target sequence that is complementary to the 5 ‘terminal base of sgRNA (about 20nt long). Immediately downstream of this complementary sequence, if there is a PAM sequence that can be recognized by Cas9 protein (PAM specifically recognized by SpCas9 is an NGG sequence composed of 3bp, N represents any base), The Cas9 scissors use the two nuclease domains HNH and RuvC (at the sites of 3 to 4nt upstream of PAM) to cut the two strands of DNA, resulting in a double-strandedbreak (DSB).
二、Product characteristics
- Transparent liquid
- Purity ≥90% (SDS-PAGE/SEC-HPLC)
- The residual amount of host protein is less than 100ng/mg
- Bacterial endotoxin less than 10EU/mg
- Host DNA residue is less than 10ng/mg
- DNA enzyme residues are negative
- RNA enzyme residues were negative
- Exonuclease activity is greater than or equal to 80%
- Concentration greater than or equal to 5mg (ABS)
三、Product application
Knock-out is referred to as KO
The Cas9 protein can cut the target genome to form a double-strand break in DNA. Normally, cells repair broken DNA using efficient non-homologous end connections. However, base insertion or deletion mismatch usually occurs in the repair process, resulting in frameshift mutations that cause the target gene to lose function and thus achieve gene knockout. In order to improve the specificity of the CRISPR system, one domain of Cas9 can be mutated to form a Cas9 nuclease that can only cut a single strand of DNA and cause DNA notch. To achieve the effect of double strand break, two sgRNA sequences can be designed to target and bind two complementary strands of DNA. DNA breaks can be formed and the gene is knocked out by frameshift mutations during repair.
Knock-in is called KI for short
When the DNA double strand breaks, if a DNA repair template enters the cell, the broken part of the genome will undergo homologous recombination repair (HDR) according to the repair template, so as to achieve gene knockin. HDR repair patterns occur at a low rate in cells, usually less than 10%. In order to increase the success rate of gene knockin, many scientists are currently committed to improving the efficiency of HDR, synchronizing the edited cells to the most active cell division period of HDR, and promoting the repair method to be carried out in HDR; Or use chemical methods to inhibit genes for NHEJ to improve the efficiency of HDR.
Multiplex Editing
When multiple sgRNA plasmids are transferred into cells, multiple genes can be edited at the same time, which has the function of genome screening. Applications of multiple editing include the use of double Cas9 nickases to improve gene knockout accuracy, large genome deletions, and simultaneous editing of different genes. Typically, two to seven different Sgrnas can be constructed on a single plasmid for multiple CRISPR gene editing.
四、Storage and transport stability
Storage stability: effective for two years at -25 to -15 ° C.
Transport stability: refrigerated transport, stable activity.
Note: This product is not recommended to store at -80℃, repeated freezing and thawing will reduce the enzyme activity of this product.
五、 Product advantages
No animal origin: Reconstituted production, no exogenous virus pollution, the production process does not use any animal source raw materials.
Stable quality: mass production (stable production supply of 100g), can ensure stable and continuous batch production; There is no difference between batches and the quality is stable.
High purity: higher than life; Host protein residues are less than the biological product limit requirements.
Compliance with regulatory requirements: the production equipment and production environment meet the relevant regulatory requirements, and the production process fully complies with the GMP guiding principles.
Complete quality documents: According to customer needs, relevant regulatory support documents can be provided.
六、Related products
Recombinant sumo enzyme;
Recombinant TEV enzyme;
Recombinant enterokinase.
Only for scientific research and production use, can not be used for human body.