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Cat. No.: SRYC9N-500 (for 500pmol)

Cat. No.: SRYC9N-2500 (for 2500pmol)

Description

SpRY Cas9 NLS is a nuclear localization signal (NLS)-tagged mutant Cas9 nuclease derived from Streptococcus pyogenes. It carries 11 amino acid substitutions within its PAM-interacting domain: A61R/L1111R/D1135L/S1136W/G1218K/E1219Q/N1317R/A1322R/R1333P/R1335Q/T1337R.

Unlike wild-type SpCas9, which recognizes the canonical 5′-NGG-3′ PAM sequence, SpRY Cas9 has been demonstrated to exhibit nearly no PAM dependency in vitro, enabling cleavage at target sites bearing the 5′-NNN-3′ PAM motif. While it preferentially targets sites with 5′-NRN-3′ (R = A/G) PAMs over 5′-NYN-3′ (Y = C/T) PAMs in vivo, this broad PAM compatibility vastly expands the pool of editable target loci. When guided by ~100-nucleotide gRNA, SpRY Cas9 nuclease introduces a DNA double-strand break (DSB) three nucleotides upstream of the 5′-NNN-3′ PAM.

The C-terminus of SpRY Cas9 NLS harbors the nuclear localization signal (NLS) from SV40 large T antigen. Following cellular transfection, the ribonucleoprotein complex formed by SpRY Cas9 NLS and gRNA rapidly translocates from the cytoplasm into the nucleus, substantially boosting genome editing efficiency. SpRY Cas9 NLS can be delivered into cells via microinjection, electroporation, or liposome-mediated transfection.

CRISPR/Cas9 represents a transformative genome-editing tool characterized by straightforward workflows and versatile applicability. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) constitutes an adaptive immune system native to prokaryotes: prokaryotes deploy the RNA-guided DNA nuclease Cas9 to silence foreign genetic material from invading phages and viruses. This natural defense mechanism has been engineered into a sophisticated, widely adopted gene-editing platform functional in both prokaryotic and eukaryotic organisms.

Under gRNA guidance, Cas9 mediates site-specific cleavage of target genomic DNA sequences. Subsequent cellular repair pathways—error-prone non-homologous end joining or homologous recombination—introduce insertions, deletions or sequence substitutions at breakpoints, frequently inducing frameshift mutations and achieving targeted gene knockout; gRNA confers strict target recognition specificity. Advancements in CRISPR technology have expanded its utility beyond gene knockout, enabling precise generation of point mutations and knock-in insertions. Notably, the platform holds promising clinical potential for correcting pathogenic genetic variants.

Furthermore, catalytically dead Cas9 (dCas9)—an endonuclease-deficient Cas9 mutant—can be genetically fused to transcriptional activators or repressors, or used to indirectly recruit such regulatory factors. This system allows sgRNA-directed transcriptional activation or silencing of target genes without inducing DNA strand breaks.

The SpRY Cas9 NLS-based CRISPR/Cas9 editing system consists of a ribonucleoprotein complex assembled from SpRY Cas9 NLS and gRNA. Also termed single guide RNA (sgRNA), gRNA comprises two core segments: an 18–20 bp crRNA (CRISPR RNA) domain complementary to the target gene, and a tracrRNA (trans-activating crRNA) domain that specifically binds SpRY Cas9 NLS. Base-pairing between gRNA and its complementary genomic target recruits SpRY Cas9 NLS to the intended DNA locus.

Distinct from wild-type SpCas9, SpRY Cas9 NLS recognizes the unrestricted 5′-NNN-3′ PAM motif, eliminating rigid PAM constraints and dramatically broadening the scope of accessible editing sites. Its C-terminal PAM-interacting domain mediates recognition of the 5′-NNN-3′ PAM. Coordinated catalytic activity of the HNH and RuvC nuclease domains generates a DNA double-strand break approximately three bases upstream of the PAM sequence. Intracellular DSBs trigger endogenous DNA repair machinery, which introduces random insertions, deletions or base substitutions at the targeted locus. These lesions often generate frameshift mutations, culminating in loss-of-function knockout of the target gene.

Source

Obtained via recombinant expression and purification in E. coli. The encoding gene is derived from a mutant variant of Cas9 originating from Streptococcus pyogenes.

Applications

Cellular genome editing, in vitro screening of high-efficiency gRNA sequences, gRNA-guided cleavage of target double-stranded DNA, and selective linearization of double-stranded DNA bearing specific sequences. Its prominent advantages are as follows: it recognizes the unrestricted 5′-NNN-3′ PAM motif, eliminating sequence constraints for targeted double-stranded DNA cleavage; it has been successfully applied to digest large-fragment plasmids in cloning workflows.

Purity Specifications

Free of DNA exonuclease activity, gRNA-independent DNA endonuclease activity, and RNase contamination.

Storage