CRISPR off-targets: how can I evaluate my gRNA sequence if it contains a mutation sequence that is not recognized by common prediction tools?

TL/DR: online gRNA design tools aren't the best, use BLAST to search for your gRNA sequence with PAM in the WT genome.

Working in yeast, I've never had any luck with online tools for gRNA design. I've put in gRNAs that I've designed myself and it's unable to distinguish between those that work and those that don't. So far, at least in yeast, I've found those tools to be a waste of time and give you false confidence.

I generally follow the Church lab protocol for gRNA design (link below). Typically, I look at my target sequence and find all the PAMs (NGG in my case), on both the coding and non-coding strand (strand doesn't really influence efficiency). I then take the 20bp directly upstream of the PAM as my candidate gRNA sequence (XXXXXXXXXXXXXXXXXXXX-NGG). DON'T include the PAM in the sgRNA that you order. Design several gRNAs for sites near your target sequence; in my experience, about 1 of 4 gRNA designs yields a high-efficiency cutting.

To check for off-target effects, I BLAST the 20bp sequence against the entire yeast genome. Anything that binds all 20bp AND has a downstream NGG, will be off-target. Cas9 can occasionally tolerate ~1-2 mismatches in the 5' region of the gRNA (see the Anderson paper), but none in the 3' region. There will be no off-target effects if there is a perfect match but no PAM. You mention that you're using a mutant- are you just trying to cut a single, known mutation site in an otherwise WT genome, or does your genome contain other mutations? If the former, simply BLAST your potential gRNA against the WT genome. If your genome has other (known) mutations, you may just have to search those mutated regions manually.

Good luck!

https://media.addgene.org/cms/filer_public/13/af/13af84f5-50ee-472e-96f1-35c5b0533645/hcrispr-grna-synthesis.pdf

Article Systematic Analysis of CRISPR-Cas9 Mismatch Tolerance Reveal...

Yang Yu

Evaluating guide RNA (gRNA) sequences for potential off-target effects, especially when the sequence contains mutations not recognized by common prediction tools, requires a thorough and methodical approach. Here’s how to proceed:

Manual Inspection of gRNA Sequence: Begin by manually inspecting the gRNA sequence against the target genome. Pay special attention to regions with high sequence similarity, particularly those with mismatches near the PAM (Protospacer Adjacent Motif) site, as they are more likely to tolerate mismatches.

Utilize Multiple Prediction Tools: Even though common tools might not recognize specific mutations, using a range of prediction tools can provide a broader analysis of potential off-target sites. Tools like CRISPOR, Cas-OFFinder, and Benchling offer different algorithms and may provide complementary insights.

In Silico Analysis of Mutated Sequences: If available, use in silico tools that allow customization of the gRNA sequence, including the mutation. This can help predict off-target effects more accurately in the context of the mutation.

Consideration of PAM Proximity: Focus on potential off-target sites that are close to a PAM sequence, as CRISPR-Cas9 efficiency is highly dependent on the presence of a PAM site near the target sequence.

Secondary Structure Analysis: Evaluate the secondary structure of the gRNA. Mutations can alter the folding and stability of gRNA, potentially affecting its specificity and efficiency.

Empirical Validation: In the absence of reliable prediction, empirical validation becomes crucial. Design and perform experiments to test the specificity of the gRNA. This can include T7 Endonuclease I assay, Surveyor assay, or targeted deep sequencing of potential off-target sites.

Use of High-Fidelity Cas Variants: Consider using high-fidelity Cas9 variants, which have been engineered to have reduced off-target effects, as they might be more specific even in the presence of mutations.

Modification of gRNA Design: If the risk of off-target effects is high, redesign the gRNA. Try to choose a target site with fewer potential off-target sites, or modify the gRNA to reduce similarity with non-target sequences.

Functional Assays for Off-Target Effects: Post-editing, conduct functional assays to detect any unintended effects. This could include phenotype screening, transcriptome analysis, or other relevant assays depending on your experimental system.

Literature Review and Expert Consultation: Review current literature for any similar studies or unique strategies for handling mutated gRNA sequences. Consulting with experts in the field or colleagues who have tackled similar challenges can also provide valuable insights.

By combining these strategies, you can thoroughly evaluate the specificity of a gRNA sequence with mutations and minimize the risk of off-target effects in your CRISPR experiments. It’s important to remember that gRNA design and validation is an iterative process that may require multiple rounds of optimization and testing.

With this protocol list, we might find more ways to solve this problem.

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