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

Artistic rendering of Cas9 Protein

Cas9 mRNA

Artisitc rendering of Cas9 mRNA

Cas9 plasmid

Artistic rendering of Cas9 plasmid

Cas9 lentivirus

Artistic rendering of Cas9 lentivirus

Designed forMost CRISPR research and pre-clinical applicationsStable cell line creation
Key advantages
  • Fits into existing mRNA protocols
  • Ideal for microinjection
  • Bypasses transcription
  • Can be co-transfected with multiple gRNAs
  • 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 limitationsNoneRequires 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 ReagentLipofectamine MessengerMAX Transfection ReagentLipofectamine 3000 Transfection ReagentLentiviral transduction
Learn moreCas9 protein optionsCas9 mRNA options

Cas9 plasmid options

Cas9 lentivirus options


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.

For Research Use Only. Not for use in diagnostic procedures.