SpCas9 H840A Nickase
$640.00 - $1,280.00
$1,600.00
All products have special prices for bulk purchase, please contact for more details if required.
Cat. No.: Cas9HAN-100 (for 100pmol)
Cat. No.: Cas9HAN-1k (for 1000pmol)
Description
SpCas9 originates from the S. pyogenes strain and is a DNA endonuclease guided by crRNA and tracrRNA (or the fused sgRNA). In the presence of a PAM (NGG) in the target double-stranded DNA, SpCas9 selectively cleaves the target DNA, causing a break in the DNA double-strand and generating blunt ends. PAM is essential for both the recognition and cleavage of SpCas9, with the cleavage site located within the target sequence, three bases away from the PAM region. The HNH and RuvC domains of the SpCas9 enzyme are responsible for cleaving the strands of the target double-stranded DNA that are paired and unpaired with sgRNA, respectively. In this product, the H840 of SpCas9 is mutated to A, rendering its HNH domain inactive. As a result, SpCas9 H840A Nickase only retains cleavage activity in the RuvC domain, cutting the non-target strand (NTS) of the dsDNA, creating a single-strand nick.
Storage
Recommended to be stored at -20°C.
Q1: What are the differences between nCas9 and Cas9?
A1: Cas9 can recognize and cleave the double-stranded DNA (dsDNA) target. Its mechanism involves two domains, HNH and RuvC, where HNH cleaves the target strand (TS), and RuvC cleaves the non-target strand (NTS). Together, they generate double-strand breaks (DSBs) in dsDNA, producing blunt ends. However, nCas9, due to amino acid mutations like H840A or D10A, results in the inactivation of either the HNH or RuvC domain. As a consequence, nCas9 can only cut one strand of the target dsDNA, creating a single-strand break. Specifically, SpCas9 H840A Nickase can only cleave the non-target strand (NTS) of the target dsDNA that is not complementary to sgRNA, while SpCas9 D10A Nickase can only cleave the target strand (TS) that is complementary to sgRNA. Additionally, there is dCas9, where mutations in both H840A and D10A render dCas9 incapable of cleaving either strand of the target dsDNA, only allowing binding.
Q2: How should sgRNA for nCas9 be designed?
A2: The sgRNA for nCas9 can be designed using the following sequence: 5′–NNNNNNNNNNNNNNNNNNNNUUUCGUGGCUGAGCCACGGUGAAAAAGUUCAACUAUUGCCUGAUCGGAAUAAAAUUGAACGAUAAAGAUCGAGAUUUUG–3′ (Underscores indicate the spacer region complementary to the specific target sequence).
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Related: Synthetic sgRNAs
