GenCRISPR gRNA & HDR Template Design Tools

How do GenScript’s CRISPR design tools compare to other available design tools?

FAQs

• Why Use Our CRISPR Design Tools?

  Our free and user-friendly CRISPR design tools generate experimentally validated gRNA and HDR template designs, enabling gene editing with Cas9, Cas12, and prime editing systems across a variety of species. Interactive design maps, updated on- and off-target scores, and a host of additional features help ensure precise and efficient editing.

• Which Cas nucleases/editing approaches do your CRISPR design tools support?

  Currently, our design tools support knockout or knock-in with Cas9 or Cas12, as well as knockout, knock-in, or sequence replacement via prime editing.

• How do I design a good sgRNA for my target?

  Designing a good sgRNA for your target depends on several factors, such as the specificity, efficiency, and compatibility of the sgRNA with your CRISPR system. Here are some general guidelines for sgRNA design:

  • Choose a sgRNA sequence that is complementary to your target DNA sequence and contains a protospacer adjacent motif (PAM) that matches the Cas protein you are using.
  • Avoid sgRNA sequences that have high similarity with non-target DNA sequences, as this may cause off-target effects. A good sgRNA has a high on-target score and low off-target score.
  • Optimize the GC content and length of the sgRNA sequence to improve the stability and activity of the sgRNA. A general rule of thumb is to have between 40% and 80% GC content and 17 to 24 nucleotides of spacer RNA.
• How are the On Target and Off Target scores calculated? Are high or low numbers more favorable?

  A Doench Rule Set score is used to measure on-target efficiency; the higher the score, the higher the efficiency. Off-target scores are calculated based on the number of potential off-target binding sites within three (3) base mismatches. A lower score denotes lower off-target potential.

• Can I design gRNA for base editing/prime editing or other Cas proteins?

  Yes! We can design gRNA for ANY gene editing system.

  Currently, our online design tools support gRNA designs for Cas9, Cas12a, and prime editing. If you need to design gRNA for base editing or another gene editing system, please reach out a CRISPR expert at crispr@genscript.com.

• Which species do your design tools support?

  Currently, our design tools support editing in the following species:

  • For Cas9 and Cas12a editing: 14 species including human, mouse, rat, fruit fly, and Chinese hamster.
  • For prime editing: 8 species including human, mouse, and fruit fly.

  If you need to design gRNA for another species, please reach out to a CRISPR expert at crispr@genscript.com.

• Can I design sgRNA for a knock-in experiment?

  Yes, our HDR knock-in design tool can be used to design HDR templates and compatible sgRNAs for KI experiments.

• What should I do if I can't find my target gene?

  Our gRNA and HDR knock-in design tools accept gene symbols and gene IDs approved by HGNC. If you cannot find your target gene, please search the gene database for the correct gene name and ID. If you still can’t find your gene, please contact us for technical support at crispr@genscript.com.

• I already have a sgRNA design that I want to use for my knockout experiments. Can I order this design from you?

  Yes, you can easily order sgRNA using your own sequence. Simply add your sequence to our sgRNA Ordering form here to get started.

• Is the PAM sequence included in my sgRNA design?

  No. You do not need to include the PAM site in your synthesized or expressed gRNA. However, you must ensure the presence of an intact PAM, corresponding to the Cas nuclease variant used for your editing project, in your target sequence adjacent to the gRNA target.

• What is the best method for measuring cleavage efficiency?

  A genomic cleavage detection assay, such as our GenCrispr Mutation Detection Kit, is a fast, T7 endonuclease I-based method to quantify how well your genome editing protocol causes insertions and deletions (indels) in the genome of your cell line. It is the quickest way to validate and select the best CRISPR-Cas9 gRNA and nuclease for your genome editing experiments.

• If there are additional PAM sites near my gRNA target, will they trigger off-target cleavage?

  The presence of additional PAM sites does not necessarily trigger off-target cleavage. PAM sites, defined as NGG, are quite common within any genome. Cleavage specificity is determined by the juxtaposition of your specific target to an NGG site. If there is some redundancy in the region you are trying to edit, you may be at a higher risk of off-site cleavage. If this is a particular problem, you may wish to explore an alternate Cas protein that uses a larger, less common PAM site.

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