Ordering gRNAs

If you select eSpOT-ON, hfCas12Max, AccuBase, or SpCas9 (unmodified or standard modification only), you will provide the ~20 base targeting sequence only. We will automatically synthesize your target sequence attached to a constant or scaffold sequence optimized for the selected Gene Editor. We will also automatically include standard modifications, unless you select SpCas9 from the Gene Editor menu and Unmodified from the Modifications menu.

Example
This example shows the entry of an eSpOT-ON gRNA. The researcher provides the target sequence only. Synthego appends the optimized scaffold and chemical modifications at the time of production.

Note: The synthesized sequence will include the standard modifications for the Gene Editor (if applicable) and Synthego’s predetermined proprietary scaffold sequence for the Gene Editor. The 89-nucleotide eSpOT-ON scaffold sequence is represented here using placeholder text (“eSpOT-ON scaffold”).

If you select Other Gene Editor (unmodified or standard modifications), you must provide the entire sequence of your gRNA molecule (target plus scaffold sequence). If you select standard modifications, they will be automatically added during synthesis.

Example
This example depicts the entry of a hypothetical novel Cas12 variant gRNA sequence with Synthego’s standard modifications. While we have included non-specified bases (N) in this example, full-length sequences must specify the base used at each position. Additionally, the scaffold sequence precedes the targeting sequence in this example. The relative orientation of the target and scaffold sequence will vary depending on your nuclease. If you have questions about the appropriate assembly of your gRNA, please contact our technical support team for guidance.

Note: The synthesized sequence will include the standard modifications for the Gene Editor (if applicable).

If you select SpCas9 or Other Gene Editor and select User-Defined from the modifications menu, you must enter the full-length sequence of your gRNA molecule with 2’-O-methyl analogs and 3’ phosphorothioate linkages at the desired locations. 

1. 2’-O-methyl analogs: To indicate a base with a 2’-O-methyl analog, place an m in front of the base you wish to modify (e.g., mG). 
2. 3’ phosphorothioate linkages: To indicate a 3’ phosphorothioate linkage, place a * between the bases you wish to have the bond (e.g., G*G or mG*mG or G*mG or mG*G). 

If you are interested in chemical modifications beyond 2’-O-methyl analogs and 3’ phosphorothioate bonds, please contact our technical support team for guidance.

Example
This example depicts the entry of a hypothetical novel Cas12 variant gRNA sequence decorated with a custom modification pattern. While we have used non-specified bases (N) for this example, full-length sequences must specify the base used at each position. Additionally, the scaffold sequence precedes the targeting sequence in this example. The relative orientation of the target and scaffold sequence will vary depending on your nuclease. If you have questions about the appropriate assembly of your gRNA, please contact our technical support team for guidance.

It is critical to ensure that your molecules are entered correctly into our system to ensure that you get the RNA molecules you need for your experiments. Below, we’ve described common errors for each sequence entry type and how to avoid them.

All sequence entry types

• Do not use a non-single-letter convention to enter your sequences (e.g., rArCrGrU…).
  • Please indicate the bases in your sequence using only RNA (ACGU) or DNA (ACGT) codes.
• Do not enter a mix of RNA and DNA bases.
  • Use all RNA bases (ACGU) or DNA bases (ACGT). If using DNA code to enter your gRNA sequences, you must click the ‘Convert to RNA’ button before adding your sequencing to your cart.
• Do not include the PAM sequence in your target sequence.
  • The PAM sequence is never part of the gRNA. If the PAM sequence is included in your target or full-length sequence, we will synthesize the sequence as entered and include the PAM bases in your molecule. gRNAs that include the PAM sequence are nonfunctional.
• Do not enter the reverse complement, reverse, or complement of your gRNA sequence.
  • Please enter the sequence as you would like it synthesized in 5’ to 3’ orientation. We will not synthesize the reverse complement, reverse, or complement of the sequence provided. If you provide your sequence using DNA code, we will convert this sequence to RNA code without making any other changes (e.g., CAT will become CAU).

Target sequence entry

• Do not include the PAM sequence in your target sequence.
  • The PAM sequence is never part of the gRNA sequence. Please only enter the target sequence without the PAM.
• Avoid entering an incomplete target sequence or additional non-target sequence.
  • Most target sequences are approximately 20 bases long, though this may vary depending on the gene editor used. If your sequence is too short or too long, an error will occur, and you’ll need to adjust the length.

Full-length sequence entry

• Do not enter an incomplete sequence.
  • Full-length sequences are synthesized exactly as entered, with no additions or changes. Thus, you must provide the targeting and scaffold sequence for your molecule.

Full-length sequence with modifications entry

• Avoid entering modifications in the incorrect position relative to the modified bases.
  • m (2’-O-methyl analog) must be a prefix to any modified bases.
  • * (3’ phosphorothioate linkages) must be located at linkage positions between bases.
• Do not enter modification characters other than m (2’-O-methyl analog) and * (3’ phosphorothioate).
  • Synthego’s online ordering page does not support other modification types at this time. If you would like to include other modifications, please contact our sales team.

Synthego’s standard purification is suitable for most discovery research applications. To purify all gRNAs, Synthego uses solid phase extraction (SPE) to enrich for your full-length gRNA sequences. For some larger yield orders, Synthego automatically adds size exclusion chromatography (SEC) for additional desalting.

HPLC purification is available for researchers who require additional purification steps. HPLC purification removes production contaminants, including sequence contaminants, that may not be completely removed by our standard purification. Please be aware that HPLC purification is available for a subset of yields only. If you are interested in HPLC-purified molecules, please contact our sales team. 

For researchers interested in additional analytical data, Analytical HPLC Reports are available for HPLC-purified molecules. If you want to add analytical HPLC to your gRNA order, please contact our sales team.