Cas9 Nuclease

Reference: CAS9-100

Cas9 Nuclease, Streptococcus pyogenes, is an RNA-guided endonuclease that catalyzes site-specific cleavage of double stranded DNA.

More info

Introduction
Cas9 (CRISPR associated protein9) is a RNA-guided DNA endonuclease enzyme associated with the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system in Streptococcus pyogenes.The CRISPR/Cas9 system is the latest RNA-guided, endonuclease tool in genome editing which allows for very specific genomic disruption and replacement. Guided by a target-specific, single guide RNA (sgRNA), the Cas9 nuclease protein serves to unwind the genomic DNA duplex and cleave both strands upon recognition of the target sequence by the sgRNA, resulting in double-stranded breaks. The resulting double-stranded break gets repaired by the non-homologous end joining (NHEJ) pathway, leading to a disruption in the open reading frame of the targeted gene.

Description
Recombinant Cas9-NLS comprises the entire Cas9 protein sequence (1368 amino acids) fused to a proprietary nuclear localization sequence (NLS) and a 6 X His tag at the C-terminus. The protein has an apparent molecular weight of 162 kDa on SDS-PAGE.

Cas9 variants for various genome manipulations are available in 8 forms

Cas9 (WT)

Cas9 with nuclear localization signal (NLS)

Cas9 with 3 NLS (on C-terminus) for better nuclear localization

NickaseCas9 (D10A) for nicking

dCas9 (D10A/H840A) for simple binding

Hi-Fi1 Cas9 3NLS (K848A, K1003A, R1060A) and Hi-Fi2 Cas9 3NLS (N497A, R661A, Q695A, Q926A) for reducing genome

wide off-target effects

dCas9-biotinylated for immunoprecipitation

Features
Low endotoxin Level
High Biological activity
Consistent from lot to lot

Application

Genome editing

Source
Cas9 gene from Streptococcus pyogenes expressed in E. coli

Purity
Greater than 97% as determined by SDS-PAGE.

Activity
96% of PCR product digestion after 30 minutes at 37°C. The reaction is performed as follow; 20µl reaction, including 100nM Cas9 protein, 200nM tracrRNA, 200nM EMX1-specific crRNA, 200ng EMX1-specific PCR product and 1x digestion buffer (20mM HEPES, 100mM NaCl and 5mM MgCl2, pH 6.5). Incubate 30 minutes at 37°C, and stop reaction with 1µl RNAse A (1mg/ml).

Storage condition: -20°C

10X Cas9 Reaction Buffer:
200 mM HEPES, 50 mM MgCl2, 1 M NaCl, 1 mM EDTA, pH 6.5

Storage Buffer:
50 mM Tris-HCl, 50 M KCl, 1 mM DTT, 1 mM EDTA, 50% Glycerol, pH 7.5

Name	Cat#	Size	c
Cas9 Nuclease (wt)	CAS9-100	250 pmol, 50 µl	5 µM
Cas9 Nuclease (wt)	CAS9-200	1 nmol, 50 µl	20 µM
Cas9 Nuclease NLS	CAS9N-100	250 pmol, 50 µl	5 µM
Cas9 Nuclease NLS	CAS9N-200	1 nmol, 50 µl	20 µM
Cas9 Nuclease 3NLS	CAS9N3-100	250 pmol, 50 µl	5 µM
Cas9 Nuclease 3NLS	CAS9N3-200	1 nmol, 50 µl	20 µM
Nickase Cas9 3NLS	NICKASE-100	250 pmol, 50 µl	5 µM
Nickase Cas9 3NLS	NICKASE-200	1 nmol, 50 µl	20 µM
dCas9 3NLS	dCAS9-100	250 pmol, 50 µl	5 µM
dCas9 3NLS	dCAS9-200	1 nmol, 50 µl	20 µM
Hi-Fi1 Cas9 3NLS (K848A, K1003A, R1060A)	HF1CAS9-100	250 pmol, 50 µl	5 µM
Hi-Fi1 Cas9 3NLS (K848A, K1003A, R1060A)	HF1CAS9-200	1 nmol, 50 µl	20 µM
Hi-Fi2 Cas9 3NLS (N497A, R661A, Q695A, Q926A)	HF2CAS9-100	250 pmol, 50 µl	5 µM
Hi-Fi2 Cas9 3NLS (N497A, R661A, Q695A, Q926A)	HF2CAS9-200	1 nmol, 50 µl	20 µM
dCas9-biotinylated 3NLS	dCAS9B-100	250 pmol, 50 µl	5 µM
dCas9-biotinylated 3NLS	dCAS9B-200	1 nmol, 50 µl	20 µM