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Cat. No.: Cas9HN-500 (for 500pmol)

Cat. No.: Cas9HN-2500 (for 2500pmol)

Description

Cas9 HF-NLS (SpCas9 HF-NLS) is a high-fidelity mutant of CRISPR-associated endonuclease Cas9 (also known as Csn1) containing a nuclear localization signal (NLS). Derived from Streptococcus pyogenes, this nuclease can specifically cut double-stranded DNA guided by gRNA. It significantly reduces nonspecific DNA cleavage and can be used for CRISPR/Cas9-mediated gene editing within cells, screening efficient guide RNA (gRNA) sequences in vitro, cleaving specific DNA sequences guided by gRNA, and linearizing circular double-stranded DNA containing specific sequences.

Cas9 HF-NLS, containing four mutation sites (N497A/R661A/Q695A/Q926A), has SV40 T-antigen nuclear localization signals (NLS) on both its N- and C-termini. This allows the Cas9-gRNA complex to quickly move from the cytoplasm to the nucleus post-transfection, greatly enhancing gene editing efficiency. Cas9 HF-NLS can enter cells through microinjection, electroporation, or liposome-mediated methods, eliminating the risk of exogenous DNA integration into the cell genome.

CRISPR/Cas9 is a groundbreaking genome editing technology known for its simplicity and wide applicability. CRISPR (clustered regularly interspaced short palindromic repeats) is an adaptive immune system in prokaryotes that uses RNA-guided DNA nucleases, such as Cas9, to silence genes in foreign bacteriophage or viral DNA. This system has evolved into a mature gene editing technology applicable to both prokaryotic and eukaryotic organisms. The technique enables site-specific cutting of genomic DNA guided by gRNA through Cas9, followed by error-prone repair or homologous recombination to alter or insert sequences at the cut site, resulting in gene knockout. The gRNA ensures the specificity of the target site. With CRISPR's advancement, it can now achieve point mutations, insertions, and various mutation types, particularly for clinical applications like repairing deleterious mutations. Moreover, constructing Cas9 mutants without endonuclease activity (dCas9) allows for transcriptional activation or repression of targeted genes by directly fusing or indirectly recruiting transcription activators or repressors.

The CRISPR/Cas9 system consists of the Cas9 Nuclease and gRNA complex. gRNA, also known as single guide RNA (sgRNA), comprises an 18-20bp CRISPR RNA (crRNA) sequence complementary to the target gene and a trans-activating crRNA (tracrRNA) sequence that binds specifically to Cas9. The gRNA pairs with the target sequence to guide Cas9 Nuclease to the target DNA. The PAM-interacting domain at the C-terminal of Cas9 recognizes the G-rich PAM sequence (5'-NGG-3'), and the HNH and RuvC domains collaborate to create a DNA double-strand break (DSB) approximately three bases upstream of the PAM sequence. If this DSB occurs within a cell, gene insertions, deletions, or replacements at the target site may occur during DNA repair, potentially leading to frameshift mutations and gene knockout.

Source

Obtained through E. coli recombination, expression, and purification, with the expression gene originating from Streptococcus pyogenes.

Applications

Suitable for cell gene editing, in vitro screening of efficient gRNA sequences, guided cleavage of specific double-stranded DNA by gRNA, and selective linearization of double-stranded DNA containing specific sequences.

Purity

Free from DNA exonuclease, non-gRNA-dependent DNA endonuclease, and RNase.

Storage