• banner:Other Nucleases for Gene Editing

    S. aureus Cas9 nuclease for Gene Editing

    The Edit-R™ CRISPR Design Tool allows you to quickly and easily generate guide RNA sequences for S. aureus Cas9, MAD7 and other CRISPR-Cas systems such as Cas12a, Cas12c, Cas13, or other novel CRISPR enzymes.

    Explore our products for successful CRISPR-Cas genome engineering

    CRISPR genome engineering

    Genome engineering has advanced tremendously with the characterization of bacterial and archaeal CRISPR (clustered regularly interspaced short palindromic repeats) systems and their adapted usage in mammalian cells. The ability to precisely and permanently alter endogenous genomic sequence through targeted genome editing is a highly effective genetics tool.

    In addition to the canonical Cas9 nuclease, other editing nucleases have been discovered including Cas12a (includes MAD7), Cas12b, Cas12c, Cas13, and many more.

    Gene editing with S. aureus Cas9 nuclease

    Staphylococcus aureus Cas9 is a programable DNA endonuclease guided by a single guide RNA (sgRNA). DNA targeting requires a 20-nucleotide guide RNA that is complementary to the DNA target site and a 5’ NNGRRT protospacer adjacent motif (PAM). S. aureus Cas9 has a cleavage site at position 17 from the 5’ end of the target sequence and produces a blunt double-stranded break.

    We suggest using EnGen® Sau Cas9 protein from New England Biolabs (NEB) for gene editing experiments in which S. aureus Cas9 is required.

    Highlights of EnGen® Sau Cas9 protein:

    • Longer 5′ NNGRRT 3′ PAM sequence allows for higher specificity
    • Two nuclear localization signals for improved transport into the nucleus
    • High concentration liquid format option can be used for microinjection, electroporation, or lipofection
    • Compatible with the EnGen® Mutation Detection Kit

    We recommend testing 6-8 guide RNAs to find one that is most efficient.

    The following synthetic RNA products may be ordered through the CRISPR Design Tool. Contact technical support if larger quantities are needed.

    Synthetic crRNA is also available at a discounted price in 96-well plates, when ordering a minimum of 20 wells.

    1-2 x 105 HEK293T cells were added to electroporation mixes containing 10 µl Nucleofector™ Solution (Lonza), 2.5 µl NEB EnGen SauCas9 protein (20 uM), and 2 µl of each individual synthetic sgRNAs (50 µM) targeting DNMT1, EMX1, FANCF, HPRT1, RUNX1, or WTAP.

    The RNP and cell mixtures were electroporated using a 4D-Nucleofector™ System (Lonza) and plated into 96-well plates. Genomic DNA was harvested after 48-72 hours of cell growth. Targeted gene loci were amplified using Q5® Hot Start High-Fidelity 2X Master Mix (NEB; M0494) and indel frequency was determined using T7 Endonuclease I assays (NEB).

    All sgRNA targets except EMX1 showed indel frequencies >25%. FANCF was the only target to show high variability between the sgRNAs that targeted it, sgRNA 1 (65% editing) vs. sgRNA 2 (~10% editing). Note: WTAP only had one synthetic sgRNA design run unlike the other targets which all had 2 guides.