Cas9-NLS Nuclease (10 mg / ml)

This product is a combination of a wild-type Streptococcus pyogenes-derived (S. pyogenesIt is a protein obtained by adding a nuclear localization signal (NLS) modification at the N-terminus to the sequence of Cas9 endonuclease and purified by recombinant expression in E. coli. The product has good in vitro shearing and in vivo gene editing functions. It can recognize the PAM sequence of NGG under the guidance of guide RNA (sgRNA), and specifically cleave it by binding to double-stranded DNA to produce a double-stranded break about 3 bases upstream from the PAM sequence in the target DNA. In addition, this product has crRNA and tracrRNA-dependent, target DNA-activated trans ssDNA cutting activity.

1. High cutting efficiency: NLS localization ensures efficient entry of Cas9 into the nucleus.
2. Low off-target effects: The transient entry of Cas9 nuclease improves cleavage specificity.
3. High trans-activity: can be used for high sensitivity and high specificity nucleic acid detection.
4. High concentration: can be highly diluted to reduce the effect of glycerol on in vivo experiments.
5. Stable activity: Repeated freezing and thawing, low-grade oscillation have no significant effect on the shear activity.
6. Short time: no need for transcription and translation.

Storage temperature: -20℃ storage
Transportation temperature: dry ice transportation or -20℃ ice bag transportation

This product has the property of specific double-stranded DNA shearing, the target DNA is about 700 bp in length, under the designed sgRNA guidance, this product can double-stranded shear the target DNA, which will form two bands at about 200 bp and 500 bp on the gel. (Note: Cas9, sgRNA and target DNA are required for double-stranded shearing to occur.)

0.5 μg of the product and sgRNA targeting the EGFP locus were mixed with Lipofectamine.^TM CRISPRMAX^TM Cas9 Transfection Reagent (Invitrogen)^TM) was transfected to 1.6 × 10⁵ In the 12-well plate system, the green fluorescence of the experimental group was significantly reduced compared with the blank control group (as shown in Figure A). In the fluorescence microscopy results, the green fluorescence of the experimental group was significantly reduced compared with that of the blank control group (as shown in Figure A), and the amount of fluorescent cells in the experimental group decreased by 74.37% compared with that of the blank control group under the counting of 15,000 cell passages by flow cytometry (as shown in Figure B). All the experimental results indicated that Cas9-NLS Nuclease produced effective gene editing at the EGFP site under the guidance of sgRNA.