TECHNICAL SUPPORT

CRISPR gRNA Design Tools

Selecting gRNAs is a critical early step of your CRISPR experiment. Here, we provide a brief introduction to some of our favorite CRISPR design tools for AccuBase™, eSpOT-ON, hfCas12Max, and SpCas9.

For recommendations on complex edits and non-editing CRISPR applications, please visit CRISPR gRNA design for diverse applications.

Design Considerations

CRISPR-Cas Systems. Most CRISPR-Cas systems are comprised of a guide RNA (gRNA) and CRISPR enzyme, which together form a ribonucleoprotein (RNP) complex. eSpOT-ON, SpCas9, hfCas12Max, and AccuBase RNP complexes are shown here. CRISPR enzymes are depicted in a light green/blue gradient color. Each gRNA includes the target sequence (green) and scaffold sequence (dark blue). The genomic DNA (light blue) and PAM sequence (pink) are shown for each enzyme. For eSpOT-ON, SpCas9, and hfCas12Max, the predicted cut site locations on the genomic DNA are indicated by gray scissors. For AccuBase, the deaminase component and editing window are emphasized using a peach color.

gRNA components. Most gRNA molecules have two components: the target sequence that hybridizes with the genomic target and a constant sequence, or scaffold sequence, that enables the gRNA to complex with the nuclease. When we discuss gRNA design here, we will focus on the target sequence.
PAM availability. Most nucleases have a unique protospacer adjacent motif (PAM) sequence. For example, SpCas9 has an NGG PAM. The availability of gRNA target sequences in your region of interest will depend on the presence and location of PAM sequences in your target region. This chapter of our How to Use CRISPR guide explains the importance of the PAM sequence.
Specificity. A good gRNA will guide your nuclease to create high-efficiency edits at the intended target site (on-target) while minimizing editing at other sites (off-target). Most design tools provide information about the predicted off-target sites for each suggested gRNA design. This blog discusses the significance of off-target editing in CRISPR experiments and methods for predicting or detecting these undesirable edits.
Target site location. The ideal target site will depend on your experimental application. Before starting the gRNA design process, consider your experimental needs.
1. CRISPR nucleases (eSpOT-ON, hfCas12Max, and SpCas9) that form double-strand breaks (DSBs) are frequently used for knockout and knock-in edits.
  1. Knockout edit: We recommend creating a cut site in an early exon shared by as many transcripts as possible. This will maximize your chances of creating an early frameshift to disrupt gene expression.
  2. Knock-in edit: We recommend selecting gRNAs with target sites as close to the site of your intended insertion as possible (creating a cut site <10 bp away from the intended insertion is ideal).
2. Base editors (AccuBase) create base conversions within a specific window relative to the gRNA target site.
  1. AccuBase creates cytosine (C) to thymine (T) base edits within a 3-12 base editing window, where position 1 is furthest from the PAM. Within the window, the rate of C to T conversions is highest in positions 7-11.
3. For most applications, we recommend targeting exons shared by as many transcripts as possible. This will ensure a functional edit across isoforms. Databases like Ensembl can help you identify suitable exons for this purpose.
Other considerations. When selecting your gRNAs, you may wish to account for parameters that are not discussed here. These include the predicted activity of your gRNA and other experiment-specific considerations. For CRISPR experiments beyond knockout, knock-in, and base edits, we recommend reviewing our CRISPR gRNA design for diverse applications resource or contacting our technical support team for guidance.

CRISPR Design Tools

There are many excellent CRISPR gRNA design tools available. We’ve named some of our favorite free online tools here.

CRISPOR
CRISPOR supports gRNA design for a wide range of nucleases. We recommend CRISPOR for CRISPR experiments using Synthego’s CRISPR enzymes, eSpOT-ON, hfCas12Max, and SpCas9, which can be selected in CRISPOR’s nuclease dropdown menu.

To design gRNAs, you upload the genomic sequence you intend to target (e.g., the exon of interest). This is an advantage of CRISPOR because you are not restricted to the reference sequences pre-loaded into the tool. CRISPOR’s output provides a ranked list of gRNAs, including information about guide specificity, predicted activity, and predicted editing outcomes.

For information about CRISPOR’s enzyme options, detailed user instructions, and guidance for interpreting your results, please review the CRISPOR Manual.

Not sure how to access your sequence of interest to upload? Recommendations for accessing this data using Ensembl are at the bottom of this page.

CHOPCHOP
CHOPCHOP has a simple interface that only requires you to input the name of your gene, species, nuclease, and edit type. However, CHOPCHOP offers significant search customization through an adjustable ‘Options’ menu. This menu enables custom PAM entries, making CHOPCHOP a good design tool option for novel and engineered nucleases. CHOPCHOP’s output ranks gRNAs according to off-target data, predicted activity, guide location, and sequence-specific characteristics (e.g., self-complementarity and GC content).

CHOPCHOP’s instructions page provides user recommendations, suggestions for avoiding common errors, and guidance for navigating the tool’s output.

Benchling
Benchling is another excellent option for designing gRNAs for a range of nucleases. Like CHOPCHOP, Benchling has a custom PAM option, so you are not restricted to a list of pre-determined nuclease or PAM selections. Benchling also provides a base editing feature that highlights the predicted base edits for each gRNA design.

Benchling allows you to begin your design process with a sequence imported from a genomic database (Ensembl, NCBI GenBank) or a custom sequence, and you may specify your target region within the reference sequence. Like many other design tools, Benchling’s output provides off-target data and predicted activity scores for suggested gRNA designs.

Benchling also offers a homology-directed repair (HDR) template design tool, which is a significant advantage for knock-in experiments.

To begin using Benchling for CRISPR Design, we recommend these blog posts about gRNA design and analysis, gRNA design for base editing, and HDR template design.

To use Benchling’s CRISPR design tools, you must create a free user account.

Cas-Designer
Cas-Designer offers designs for an expansive list of predetermined nucleases (PAM sequences), including eSpOT-ON, hfCas12Max, and SpCas9. Searches are performed using a FASTA sequence uploaded by the user, which provides target sequence flexibility. An advantage of Cas-Designer is that the tool allows you to specify target coordinates within the uploaded sequence. Cas-Designer’s output includes off-target details, sequence information, and an out-of-frame score designed to predict the likelihood of frameshift creation.

To learn more about Cas-Designer, we recommend reviewing the publication introducing the tool.

BE-Designer
Created by the team behind Cas-Designer, BE-Designer is specifically intended to design gRNAs for base editing applications. Like Cas-Designer, BE-Designer searches are performed using a FASTA sequence uploaded by the user. Users can select the base editing type (for AccuBase, select “BE (C to T)”) and enter the base editing window specific to their genome editor. Be-Designer’s results include the predicted base changes within the editing window and off-target data.

For more information about BE-Designer, we recommend reviewing the publication describing the development of this tool.

This is not an exhaustive list of design tools. Please use a tool that meets the needs of your experiment!

Next Steps

Once you are ready to order your gRNAs, you can access our ordering page here.

New To CRISPR?

For a comprehensive guide to Synthego’s CRISPR resources, please visit our Getting Started with CRISPR page.

Accessing Your Sequences Using Ensembl

Ensembl is an online genome browser for vertebrate genomes. The genomes in Ensembl are annotated, which is beneficial when identifying specific transcripts or regions for CRISPR editing.

Below, we have also provided a few steps for accessing your sequence of interest.

Search for your gene name on Ensembl's home page to navigate to your gene of interest. To make your search more efficient, we recommend selecting your species of interest (e.g., human) instead of the default “All species” option.
In the search results, find and open the page for your gene of interest.
On the left side of the page, Ensembl displays a menu (“Gene Based Displays”). In this menu, select “Sequence,” and an annotated version of the sequence will appear on the page below the Transcript table and Summary.
1. Tutorials for accessing, configuring, and downloading sequence data may be found here.
You may copy and paste this sequence or download the sequence using the “Download sequence" button. If you use the latter option, we recommend downloading the whole genomic DNA sequence, not a coding or transcript sequence.
1. Instructions for exporting data may be found here.

Additional Assistance

Having trouble with your gRNA designs? Connect with our scientific support team!

Contact Support