SpCas9 D10A Nickase

SpCas9 is derived from the S. pyogenes strain and is an RNA-guided DNA endonuclease that depends on crRNA and tracrRNA (or a fusion sgRNA) for guidance. In the presence of a PAM (NGG) sequence in the target dsDNA, it specifically cleaves the target DNA, creating a double-strand break and generating blunt ends. PAM is essential for both the recognition and cleavage by Cas9, with the cleavage site located within the target sequence, three bases away from the PAM region. The HNH and RuvC domains of SpCas9 enzyme are responsible for cleaving the target dsDNA that pairs with the sgRNA (target sequence or TS chain) and the non-paired strand (non-target sequence or NTS chain), respectively. In this product, the D10 of SpCas9 has been mutated to A, rendering its RuvC domain inactive. As a result, SpCas9 D10A Nickase only has cleaving activity in the HNH domain and cleaves only the TS chain of the target dsDNA, creating a single-strand nick.

• The Difference Between nCas9 and Cas9:
Cas9 can recognize and cleave dsDNA targets, and its mechanism involves two structural domains, HNH and RuvC. HNH cleaves the target strand (TS), while RuvC cleaves the non-target strand (NTS), together generating double-strand DNA breaks (DSBs) and producing blunt ends. However, nCas9, due to mutations like H840A or D10A in the amino acid sequence, inactivates either the HNH or RuvC domain, allowing it to cleave only one strand of the target dsDNA, creating a single-strand cut. Specifically, SpCas9 H840A Nickase can only cleave the non-target strand (NTS) of the target dsDNA that doesn't pair with the sgRNA, while SpCas9 D10A Nickase can only cleave the target strand (TS) that pairs with the sgRNA. Additionally, dCas9, with mutations in both H840A and D10A, can bind to the target dsDNA but cannot cleave either strand.

• The sgRNA for nCas9 can be designed based on the following sequence:
5′–NNNNNNNNNNNNNNNNNNNNUUUCGUGGCUGAGCCACGGUGAAAAAGUUCAACUAUUGCCUGAUCGGAAUAAAAUUGAACGAUAAAGAUCGAGAUUUUG–3′, (The underlined portion represents the spacer region that complements the specific target sequence).

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