Designing effective CRISPR experiments involves various steps, one of which is effective design of single guide RNAs (sgRNAs). sgRNAs are programmable sequences that guide nucleases, such as CRISPR-Cas9, to a specific DNA target. Efficient sgRNA design is critical to executing a precise genome editing experiment.
Why is sgRNA design important?
To optimize sgRNA design, the following factors should be considered:
- PAM compatibility: The Protospacer Adjacent Motif (PAM) is required for the Cas protein to recognize, bind to the target DNA and initiate the cleavage process. Different Cas variants recognize different PAM sequences (e.g., Cas9 uses NGG). It is important to confirm that your target site includes a compatible PAM. If your target site lacks the required PAM sequence, consider using TALENs to achieve your edit. TrueDesign Genome Editor also designs TALEN pairs for the region of interest.
- On-target activity: The efficacy of a sgRNA is determined by its sequence, surrounding DNA context, and other features such as sgRNA:DNA melting temperature, minimum free energy of the folded spacer sequence and chromatin accessibility. Various machine-learning algorithms have been developed using these factors to predict a sgRNA’s on-target activity. When designing sgRNAs to target a region of interest, the predicted on-target activity scores can help the user pick optimal sgRNAs. In TrueDesign Genome Editor, we use Rule Set 3 [1] to determine on-target scores.
- Off-target activity: The off-target profile of a sgRNA should also be assessed during the design process. This involves scanning the genome of interest for regions with sequence similarity. Off-target searches should account for mismatches as nucleases like Cas9 enable sgRNAs to bind even with mismatches leading to unintended edits. Additionally, Cas9 recognizes alternative PAM sequences such as NAG and NGA, which should be included in the off-target analysis to comprehensively evaluate potential off-target effects. TrueDesign Genome Editor evaluates off-target sites by allowing up to 3 mismatches in the sgRNA sequence, and includes sites with NGG, NGA, and NAG PAM sequences. TrueDesign Genome Editor scores off-target sites using the Cutting Frequency Determination (CFD) score [2] and annotates the location of the off-target site indicating if it is in the exon or intron of a gene and if the gene is an oncogene or tumor suppressor gene. These details can help users choose sgRNAs with low number of off-target sites, sgRNAs with off-target sites with low CFD scores, sgRNAs with off-target sites in introns or intergenic regions, or a combination of these factors.
- Distance from edit site: Flexibility in distance of the target sequence from the desired edit site depends on the experiment type. For a knockout experiment, sgRNAs should ideally be designed to target sequences in the early exons of a gene. For knock-in experiments, sgRNAs should be designed to target as close to the desired insertion site as possible. Apart from sgRNA activity, knock-in efficiency also depends on the length of the knock-in and the distance of the insertion site from the sgRNA cut site. For insertion sequences with shorter homology arms, knock-in efficiencies are lower if the sgRNA cut site is more than 10 bp away from the insertion site. However, for sequences with longer homology arms, higher efficiencies can be achieved even when the cut site is up to 40 bp away from the desired insertion site.
- Variant overlap: sgRNAs designed using reference genomes may exhibit reduced efficacy and specificity due to the presence of genetic variants. Genetic variants can lead to disruption of PAM sites, creation of novel PAM sites or introduction of mismatches in the target sequence. Therefore, it is important to consider genetic variation in a specific target population (if known) or the overall population when designing sgRNAs. TrueDesign Genome Editor makes it easy to visualize genetic variation during the guide design process and allows users to perform off-target searches including genetic variants.
Validating sgRNA design
While a multitude of algorithms can be used to design and choose the best guide RNAs, functionally testing them is ultimately the definitive method for validation. Algorithms are designed to be predictive, but factors such as cell context and delivery methods can significantly influence sgRNA efficiency. We recommend functionally testing your sgRNAs.
Validation is key to ensuring your sgRNA’s functionality. When measuring the efficiency of a sgRNA, it is critical to have appropriate positive, negative and delivery controls. Efficiency of a sgRNA can be measured in various ways:
- T7 endonuclease-based assay: The Invitrogen GeneArt Genomic Cleavage Detection (GCD) Kit can be used to quickly and confidently confirm the editing efficiency of your sgRNA. TrueDesign Genome Editor designs GCD primers for your selected sgRNAs.
- Sequencing: Sanger or next-generation sequencing can be used to determine sgRNA efficiency and the nature of edits at the region of interest. Insertions or deletions causing frameshift mutations or disruption of splice sites can lead to knockout of the gene. If a knock-in experiment is performed, sequencing can confirm whether the intended template was successfully inserted at the desired location. TrueDesign Genome Editor also designs primers for sequencing of the edit location.
- Flow cytometry: Fluorescently tagged antibodies or reporter genes can be used to validate sgRNA editing efficiency in cells where specific phenotypic changes are expected due to genome editing.
Conclusion
Designing and validating effective sgRNAs is critical to executing accurate and efficient CRISPR experiments. Following the suggested sgRNA design considerations and validation steps can help ensure creation of effective sgRNAs. To simplify the design of sgRNAs, Thermo Fisher Scientific offers a free platform for building sgRNAs and gene editing experiments – TrueDesign Genome Editor. This genome editing design tool incorporates the recommended considerations for sgRNA design optimization and provides off-target evaluation and validation support to enable researchers to build sgRNAs with precision and ease. Visit thermofisher.com/truedesign to learn more. To watch videos on how to use TrueDesign Genome Editor, explore the playlist here.
For Research Use Only. Not for use in diagnostic procedures.
References
[1] DeWeirdt, P. C., et al. (2022). Accounting for small variations in the tracrRNA sequence improves sgRNA activity predictions for CRISPR screening. *Nature Biotechnology, 40*(4), 570-578. https://doi.org/10.1038/s41587-021-01107-5
[2] Doench, J. G., et al. (2016). Optimized sgRNA design to maximize activity and minimize off-target effects of CRISPR-Cas9. *Nature Biotechnology, 34*(2), 184-191. https://doi.org/10.1038/nbt.3437