TrueGuide Synthetic gRNA

gRNA, or guide RNA, is part of the CRISPR-Cas9 system. The gRNA molecule guides the Cas9 protein to a specific genomic locus via base pairing between the crRNA sequence and the target sequence. The CRISPR-Cas9 system is composed of a short noncoding gRNA that has two components: a target-specific CRISPR RNA (crRNA) and an auxiliary trans-activating crRNA (tracrRNA).

sgRNA stands for single guide RNA. As opposed to the native S. pyogenes CRISPR-Cas9 system that uses a two-part guide (CRISPR RNA [crRNA] hybridized with a tracrRNA), an sgRNA combines both RNAs into a single, long RNA. sgRNA are typically 100 nt in length and can be expressed from a vector or chemically synthesized.

crRNA is one strand of the conventional two-part guide RNA system. The TrueGuide crRNA:tracrRNA two-part guide RNA product format has been discontinued to simplify our synthetic gRNA offering and focus on the modified sgRNA, which demonstrates superior efficiency across more cell types than the two-part system.

tracrRNA is one strand of the conventional two-part guide RNA system. The TrueGuide crRNA:tracrRNA two-part guide RNA product format has been discontinued to simplify our synthetic gRNA offering and focus on the modified sgRNA, which demonstrates superior efficiency across more cell types than the two-part system.

gRNAs are available as chemically synthesized RNA oligos or generated as in vitro transcribed (IVT) gRNAs using the GeneArt Precision gRNA synthesis kit. The IVT gRNA format can also be purchased as a custom ready-to-use option from GEMServices@thermofisher.com. Chemically synthesized gRNA are available from Thermo Fisher Scientific in three different formats:

• Modified sgRNA format: Invitrogen customers can purchase the single guide RNA (sgRNA), which is comprised of both the crRNA and tracrRNA as a single long RNA with 2′ O-Methyl and phosphorothioate modifications on both 5′ and 3′ ends of the molecule. These modifications enhance editing efficiency by increasing binding to the target site and inhibiting nuclease degradation, respectively.
• Two-part format: The TrueGuide crRNA:tracrRNA two-part guide RNA product format has been discontinued to simplify our synthetic gRNA offering and focus on the modified sgRNA, which demonstrates superior efficiency across more cell types than the two-part system.
• Unmodified sgRNA format: Customers can purchase a single guide RNA (sgRNA) without any chemical modifications from the Custom gRNA Order tool. The predesigned option for unmodified sgRNA has been discontinued. To simplify our synthetic gRNA offering and focus on the modified sgRNA, which demonstrates superior efficiency across more cell types than the two-part system.

Chemical modifications include 2’ O-Methyl analogs and phosphorothioate internucleotide linkages at the 5′ and 3′ ends of the molecule. These modifications enhance editing efficiency by increasing binding to the target site and inhibiting nuclease degradation, respectively.

TrueGuide synthetic gRNA along with TrueCut Cas9 Protein V2 offers maximum editing efficiency across broad cell types while minimizing cell toxicity and innate immune responses. Unlike a plasmid-based system, the RNP system is transient with faster turnover. The result is that it minimizes random integrations and off-target events.

TrueGuide Synthetic gRNA sequences are analyzed by mass spectrometry to verify sequence integrity.

To estimate CRISPR/Cas9 mediated editing efficiency in a pooled cell population, one could use GeneArt Cleavage Detection kit (Part No. A24372), perform next generation sequencing (NGS), or use Sanger sequencing-based analysis. Using NGS of the amplicons from edited populations or Sanger sequencing of amplicons cloned into plasmids gives a more accurate estimate of % editing efficiency and indel types.