How to Design CRISPR Guide RNAs with the Synthego Design Tool

The CRISPR Design Tool includes the ability to easily validate, share, and purchase synthetic single guide RNA (sgRNA) directly from within the design interface. The “Validate” function enables the user to validate guides designed manually or using other tools, leveraging its built-in algorithms and heuristics. Furthermore, easy sharing of designs with colleagues is enabled using the simple yet unique feature of direct links to guide design results. Finally, when a researcher is ready to begin their CRISPR experimental workflow, this tool enables the purchase of high-quality synthetic sgRNA with the click of a button.

The current version of the Design tool (1.2) is optimized for recommending sgRNAs that are most likely to result in a gene Knockout using S.pyogenes Cas9. The tool can still be used to search for other sgRNA as outlined below.

Once you are in the Synthego CRISPR Design Tool, you will be prompted to enter the genome or organism of interest followed by the gene that you wish to target for your experiment. Be sure to select the correct reference genome if given the option between several different choices.

Next, you will type in the gene name or gene ID. Note that the gene ID needs to match the selected reference genome. If you are not able to find the gene name you are looking for, try any known aliases or synonyms.

Once you’ve selected your genome and genes, the CRISPR Design Tool goes through a series of algorithms to determine the highest ranked guide RNAs for your particular target. The highest ranked guides are displayed in the “Recommended Guides” tab. You can hover over the green stripes which represent the sgRNA to see more detailed information about the sequence, cut site, exon, on-target score, and off-target score.

If desired, you can also view guides that did not quite meet the criteria for being optimal for gene KO by selecting the "All Guides" tab at the top right of the page. On the "All Guides" page you will see a list of additional guides that target the gene, but may not be as optimal as the “Recommended Guides” for the purpose of gene knockout. On this page, you can also navigate through different transcript variants and navigate through different exons. Using these dropdown menus, you can target regions of the gene that are closer to your target site of interest, which is useful in certain cases like knock-in experiments.

On both the "Recommended Guides" and "All Guides" view, you can click on the sgRNA sequence to enter the "Validate Tool" side of the website.

Here, you can find more detailed information about the sequence, cut site, on-target activity as well as the locations and characteristics of possible off-target sites.

The CRISPR Design Tool from Synthego takes a sequential approach to the identification and design of guide RNA sequences to ensure that they have the highest chance of generating a CRISPR knockout while also minimizing potential off-target effects.  

These steps are highlighted below:

1. The CRISPR Design Tool first identifies the sequence of the full gene within the genome, including all its splice and transcript variants.
2. The software identifies the primary transcript from the various alternative transcripts or splice variants that might exist for that gene across multiple databases.
3. The CRISPR Design Tool identifies the exons and the coding sequences for the primary transcript. The algorithm identifies and prioritizes exons that are common across multiple transcripts or splice variants, enabling design of guide RNAs that target all of them.
4. The tool further narrows down the design to exons in the early coding region of the target gene to identify the guide RNAs that have the highest probability of generating a complete functional knockout through insertions or deletions (indels). These indels in the early part of the gene are likely to lead to frameshift mutations that disrupt the translation of the RNA transcript, creating a full knockout of the gene/protein.
5. Once the target exon has been identified, the CRISPR Design Tool then identifies all the potential CRISPR-Cas9 target sites, based on the available PAM sites on the exon.
6. Using the sequences of these guide RNAs, the CRISPR Design Tool identifies all the potential off-target sites across the same genome and determines the number of mismatches between each target guide RNA and all its potential off-target sites. Based on this information, the algorithm is able to score and rank the guides that have the least number of off-target sites.
7. The algorithm then uses the Azimuth 2.0 model from Doench et al., 2016 to calculate the on-target score for each sgRNA.
8. Finally, the algorithm uses all the above information to pick and recommend guide RNAs that have the highest likelihood of knocking out the target gene within the genome, with the least number of off-target effects. Specifically, the recommended guides need to...

• Target a common exon in the primary transcript
• Target an early region of the gene
• Have an on-target score of greater than 0.5 based on the Azimuth 2.0 model
• Have no off-target sites within the same genome that have 0, 1 or 2 mismatches compared to the guide RNA sequence

To add sgRNA sequences to your cart for purchasing you can simply click the "SELECT" button next to your desired sequences then proceed to the next step by clicking the blue "Continue With Guides" button at the bottom right of the page. The Guides will be added to your cart and you can proceed with checking out from there.

The Synthego CRISPR Design Tool sorts the guides by using several heuristics, such as the cut site on the gene, common exon, and high activity. The CRISPR Design Tool preferentially chooses sequences that occur in exons in the 5’ end of the gene because an indel in these regions will improve the chances of knocking out the gene’s function. It then preferentially selects guide sequences in exons that are common to various splice variants. Finally, the CRISPR Design Tool uses on-target score (Doench et al., 2016) and off-targets as a pass/fail criteria to select guide RNA sequences that we recommend for use to knock out a gene.