Cas9 Nucleases

Cas9 nuclease formats for CRISPR Cas9 gene editing

In CRISPR-Cas9 gene editing experiments, Cas9 nuclease binds to gRNA and induces a double-stranded break at a specific genomic target sequence. For the Cas9 enzyme to function, this target sequence must have a proto-spacer adjacent motif (PAM) site in the vicinity of the desired break. Cas9 nuclease is available in several formats to fit your experiment design and preferred workflow, allowing for precise DNA cleavage and gene knockout.

Cas9 nuclease selection guide

	Cas9 protein	Cas9 mRNA	Cas9 plasmid	Cas9 lentivirus
Designed for	Most CRISPR research and pre-clinical applications			Stable cell line creation
Key advantages	Bypasses transcription and translation Minimizes off-target effects Available in three formats: Wild-type TrueCut Cas9 Protein v2 for most common research applications TrueCut HiFi Cas9 Protein for applications that are sensitive to off-target effects CTS TrueCut Cas9 Protein for clinical research applications	Fits into existing mRNA protocols Ideal for microinjection Bypasses transcription Can be co-transfected with multiple gRNAs	Fits into existing plasmid protocols Can be incorporated as an all-in-one Cas9 and gRNA vector using the GeneArt CRISPR Nuclease Vector Kits	Ideal for arrayed or pooled gRNA libraries Drives stable Cas9 expression in a wide variety of cell types Includes a human codon-optimized version of Cas9 with two nuclear localization signals
Key limitations	None	Requires translation	Takes time and effort to prepare May exacerbate off-target effects May incorporate into genomic DNA Requires transcription and translation	Potential for cellular response to viral transduction
Recommended delivery options	Lipofectamine CRISPRMAX Transfection Reagent	Lipofectamine MessengerMAX Transfection Reagent	Lipofectamine 3000 Transfection Reagent	Lentiviral transduction
Recommended delivery options	Neon Transfection System CTS Xenon Electroporation System (for large-scale electroporation)			Lentiviral transduction
Learn more	Cas9 protein options	Cas9 mRNA options	Cas9 plasmid options	Cas9 lentivirus options

INFOGRAPHIC: Cas9 plasmids vs proteins

Discover how your CRISPR gene editing experiments can benefit from Cas9 proteins and how the popular nuclease compares to Cas9 plasmids.

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Cellular processing of various CRISPR-Cas9 gene editing modalities

In contrast to the plasmid and mRNA formats, transfection of our TrueCut Cas9 protein complexed with our TrueGuide synthetic gRNA bypasses transcription and translation, helping to sharply increase editing efficiency by the Cas9 enzyme in your experiments. Furthermore, while CRISPR plasmids remain in the cell for more than 72 hours and may exacerbate off-target cleavages, TrueCut Cas9 proteins are cleared from the cell within 24 hours, minimizing off-target effects.

Methods of nuclear entry and genome targeting for three different Cas9 nuclease formats

Figure 1. Comparing CRISPR-Cas9 gene editing formats. Cas9 and guide RNA can be delivered to cells as DNA, RNA or pre-formed ribonucleoprotein complexes (RNPs) formats. When using a plasmid DNA vector (upper left), the Cas9 gene must first be transcribed in the nucleus, exported to the cytoplasm, and translated, while an mRNA/gRNA mix (bottom) must be translated. A reagent-protein complex (upper right), like a TrueCut Cas9 protein coupled with our a TrueGuide synthetic gRNA, bypasses these preliminary steps (i.e., transcription and translation) for simpler and more efficient gene editing.