CAS9 S. Pyogenes

CRISPR-Associated Protein-9 Nuclease S. Pyogenes Recombinant

Cat. No.

BT13840

Source

Escherichia Coli.

Synonyms

CRISPR-associated endonuclease Cas9/Csn1, SpyCas9, cas9, csn1.

Appearance

Sterile Filtered colorless solution.

Purity

Greater than 90% as determined by SDS-PAGE.

Usage

THE BioTek's products are furnished for LABORATORY RESEARCH USE ONLY. The product may not be used as drugs, agricultural or pesticidal products, food additives or household chemicals.

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Description

Recombinant Cas9-NLS produced in E.coli, comprises the entire Cas9 protein sequence (1368 amino acids) joined to a proprietary nuclear localization sequence (NLS) and a 6xHis tag at the C-terminus, having a total of 1414 amino acids. The protein has an apparent molecular weight of 163kDa.

Product Specs

Introduction

Cas9, short for CRISPR associated protein 9, is an RNA-guided DNA endonuclease enzyme. It is part of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) adaptive immunity system found in bacteria like Streptococcus pyogenes. This system helps bacteria like S. Pyogenes to remember, identify, and eliminate foreign DNA, such as that from invading bacteriophages or plasmids. When the DNA substrate matches the guide RNA, Cas9 cuts the foreign DNA. Beyond its natural role in bacterial defense, Cas9 has become a crucial tool in genome engineering. It is used to create targeted double-strand breaks in DNA, enabling researchers to inactivate genes or introduce new genes through non-homologous end joining and homologous recombination, respectively. This technology has revolutionized genetic research in various laboratory model organisms.

Description

This product is a recombinant Cas9-NLS protein expressed in E. coli. It consists of the complete Cas9 protein sequence (1368 amino acids) linked to a proprietary nuclear localization sequence (NLS) and a 6xHis tag at the C-terminus, totaling 1414 amino acids. The protein has an apparent molecular weight of 163 kDa.

Physical Appearance

A clear, colorless solution that has been sterilized by filtration.

Formulation

This CAS9 solution is buffered with 10mM Tris-HCl (pH 7.5) and contains 300mM NaCl, 0.1mM EDTA, 50% glycerol, and 1mM DTT.

Stability

For optimal storage, keep the CAS9 solution at 4°C if you plan to use the entire vial within 2-4 weeks. For longer storage, freeze the solution at -20°C. Adding a carrier protein like HSA or BSA (0.1%) is recommended for extended storage. Avoid repeated freezing and thawing to maintain protein stability.

Purity

The purity of this CAS9 protein is greater than 90%, as assessed by SDS-PAGE analysis.

Endonuclease Activity

To assess endonuclease activity, 20 pmol of CAS9 protein was incubated with 1 µg of supercoiled pUC19 plasmid DNA at 37°C for 4 hours. Under these conditions, less than 10% of the pUC19 plasmid was converted to the open circular form, as determined by gel electrophoresis.

Non-specific Dnase Activity

To test for non-specific DNase activity, 20 pmol of CAS9 protein was incubated with 1 µg of lambda DNA at 37°C for 4 hours. No detectable DNA degradation was observed under these conditions, as visualized by agarose gel electrophoresis.

Synonyms

CRISPR-associated endonuclease Cas9/Csn1, SpyCas9, cas9, csn1.

Source

Escherichia Coli.

Enzymatic Activity

97% of PCR product digestion after 30 minutes at 37°C.

Product Science Overview

Discovery and Function

Cas9 was initially discovered as part of the CRISPR system, which bacteria use to defend against invading viruses and plasmids. The system works by capturing snippets of DNA from invaders and incorporating them into the bacterial genome as “spacers” between the CRISPR sequences. When the bacterium encounters the same invader again, it transcribes these spacers into RNA, which guides the Cas9 protein to the matching DNA sequence in the invader. Cas9 then cleaves the DNA, neutralizing the threat .

Mechanism of Action

The Cas9 protein operates by unwinding the target DNA and checking for a sequence complementary to the guide RNA (gRNA). If a match is found, Cas9 induces a double-strand break in the DNA. This mechanism is highly specific due to the RNA-DNA complementarity, making Cas9 a powerful tool for precise genome editing .

Applications in Genetic Engineering

The ability of Cas9 to induce site-specific double-strand breaks has been harnessed for various applications in genetic engineering. By designing specific gRNAs, scientists can target almost any gene in a genome, allowing for gene knockout, insertion, or correction. This has profound implications for research, medicine, and biotechnology .

Recombinant Cas9

Recombinant Cas9 refers to the Cas9 protein that has been produced through recombinant DNA technology. This involves inserting the Cas9 gene into an expression system, such as Escherichia coli, to produce large quantities of the protein. Recombinant Cas9 is essential for laboratory applications, where it is used in conjunction with synthetic gRNAs to edit genes in various organisms .

Advances and Variants

Research on Cas9 has led to the development of various Cas9 variants to overcome limitations and enhance its functionality. For example, Cas9 nickase (Cas9n) induces single-strand breaks instead of double-strand breaks, reducing off-target effects. Other variants have been engineered to recognize different PAM (Protospacer Adjacent Motif) sequences, broadening the range of targetable DNA sequences .

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CAS9 S. Pyogenes

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