SpCas9 (D10A)-NLS

Overview:

Cas9 is a multidomain nuclease from the bacterium Streptococcus pyogenes characteristic of type II CRISPR- Cas systems (1). Using single guide RNA (sgRNA), Cas9 is targeted to specific genomic loci where it generates a blunt ended double strand break (2). SpCas9 (D10A)-NLS is a catalytic mutant of Cas9 containing a nuclear localization sequence for optimal activity (3). Unlike wild type Cas9, SpCas9 (D10A)-NLS generates a single strand DNA break on the targeting strand (4). SpCas9 (D10A) can be used for various genome editing applications.

Accession Number:

Q99ZW2

Formulation:

Recombinant protein stored 50mM sodium phosphate, pH 7.5, 300mM NaCl, 200mM imidazole, 1mM DTT, 10% glycerol.

References:

1. Nishimasu H, et.al. Crystal structure of Cas9 in complex with guide RNA and target DNA. 2014. Cell. 156(5):935-49.

2. Sapranauskas R et al. The Streptococcus thermophilus CRISPR/Cas system provides immunity in Escherichia coli. 2011.Nucleic Acids Res. 39(21):9275-82.

3. Jiang F, Doudna JA. 2017. CRISPR-Cas9 Structures and Mechanisms. Annu Rev Biophys.46:505-529.

4. Chiang, TW., et al. CRISPR-Cas9D10A nickase-based genotypic and phenotypic screening to enhance genome editing. Sci Rep 6, 24356 (2016).

Storage and Stability:

Store product at –70oC. For optimal storage, aliquot target into smaller quantities after centrifugation and store at recommended temperature. For most favorable performance, avoid repeated handling and multiple freeze/thaw cycles. Always include RNase inhibitor at manufacturer recommended concentrations in Cas enzyme reactions.