The Precision gRNA Synthesis Kit is a complete system for rapid synthesis of guide RNA (gRNA) ready to complex with TrueCut™ Cas9 Protein v2 for transfection-ready Cas9 protein/gRNA ribonucleoprotein (Cas9 RNP). This Cas9 RNP format, with our TrueCut Cas9 Protein v2, has been tested in a variety of suspension and adherent cell lines with >70% cleavage efficiencies and no indications of toxicity. Starting with two short single-stranded oligos that code for the target sequence, the gRNA template is assembled with a T7 promoter in a short ‘one-pot’ PCR reaction. The assembled product is then used as template in an in vitro transcription (IVT) reaction followed by a rapid purification step, yielding transfection-ready gRNA in as little as four hours. Resulting gRNA can also be co-transfected with our ready-to-transfect Invitrogen™ CRISPR Nuclease mRNA. Both protein and mRNA Cas9 formats require no plasmid manipulation and so are amenable to high throughput and multiplex genome-wide cell engineering approaches.

Features of the Precision gRNA Synthesis Kit include:
• Fast assembly and synthesis of any gRNA target in as little as four hours including template assembly
• High yield (>10 ug) and concentration (>200 ng/uL) of gRNA

How to obtain a gRNA sequence
Genome editing with CRISPR technology requires a noncoding guide RNA (gRNA) in order to cleave genomic DNA at a target sequence of interest. The gRNA has two molecular components: a target-specific CRISPR RNA (crRNA) and a trans-activating crRNA (tracrRNA) that have been combined into one transcript. The target sequence (20 bases) must be immediately upstream of a PAM motif (NGG) which allows the Cas9 to initiate binding. The PAM is only on the target DNA and not part of the target specific CRISPR sequence. The gRNA and the PAM motif guide the Cas9 nuclease to the target genomic sequence to form a complex and create a double-stranded blunt DNA break (DSB) three nucleotides upstream from the PAM site.

Use our CRISPR Search and Design Tool to search our database of >600,000 gRNA sequences specific to every gene in the human and mouse genomes. Invitrogen predesigned gRNAs are optimized for gene knockout and typically target the first three transcribed exons per gene. Search results include recommendations based on minimizing potential off-target effects, potential binding sites, and exon maps with gRNA locations. This tool can also be used to analyze any sequence of interest to design unique CRISPR sequences.

How to make gRNA
Once gRNA sequences have been selected, choose from three options for making gRNA:
1. TrueGuide synthetic guide RNA—choose from our catalog of predesigned gRNAs or upload your sequence to our TrueGuide gRNA Ordering Tool
2. Precision gRNA Synthesis Kit (this page)—for transfection-ready gRNA in as little as four hours including template assembly
3. Genome Engineering Services—save time and effort and have our custom services team design, synthesize, and purify in vitro transcribed (IVT) gRNA sequences for you. To obtain a services quotation, or to order, please contact our Custom Services department at 1-800-955-6288 x45682 or gemservices@thermofisher.com.