Hi Amanda,

An alternative is to test only the sites that are most likely to be affected. I mean, predicting the possible off-target sites in the genome of your organism and using an in vitro assay to validate if a given sgRNA can induce a DSB in that sequence.

This is what we showed in our recent paper:

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0124633

This is fast a very reliable according to our experience so far.

Best,

Juan

Thanks. I suppose I could just check the top 10 likely off target sites. When I blast my gRNA with PAM (23nt) I only get a match for the last 13 nt and it is missing the final G of the PAM site. I assume I should test ones that match closest to the PAM site. Using your tool I suppose I pick the ones that exonic and fewer mismatch in the core region to test yes? Or are they already ranked by likelihood somehow and I should just test the first 10?

Yes, the output that you get from CCTop is ranked based on the position of the mismatches. So, the sites at the top are more likely to be targeted by the Cas9 protein.

On how many to test, it depends on the specific case. Base on many pieces of evidence, a single mismatch in the next couple of based adjacent to the PAM will make that this site is not suitable for DSB. So, if something like this already appears in position 5 of the list, probably it doesn't make sense to test any farther. In other cases you may have dozens of potential off-target sites only differing in the most distal nucleotides to the PAM.

Best,

Juan

you can use T7 Endonuclease assay or CEL1.

there are several experimental tests and approaches for detecting off-target effects in CRISPR experiments. Off-target effects occur when the CRISPR/Cas9 system edits unintended genomic locations, which can lead to unwanted mutations or alterations. Here are some common methods to identify and analyze off-target effects:

**1. Sequence-Specific Methods

**1.1 Targeted Sequencing

**1.1.1 Description:

Capture of Potential Off-Target Sites: Design and sequence regions of the genome that are predicted to be off-target sites based on the CRISPR guide RNA (gRNA) sequence.

**1.1.2 Applications:

Validation of Off-Target Sites: Confirm the presence of mutations at predicted off-target sites.

**1.1.3 Considerations:

Scope: Limited to known or predicted off-target sites.

**1.2 Deep Sequencing

**1.2.1 Description:

Whole-Genome or Exome Sequencing: Perform high-depth sequencing to detect unintended genetic changes throughout the genome.

**1.2.2 Applications:

Comprehensive Off-Target Analysis: Identify both known and unknown off-target effects.

**1.2.3 Considerations:

Cost and Complexity: Requires high sequencing depth and sophisticated data analysis.

**1.3 Guide RNA and PAM Sequence Analysis

**1.3.1 Description:

Bioinformatics Tools: Use computational tools to predict potential off-target sites based on the sequence of the gRNA and the protospacer adjacent motif (PAM).

**1.3.2 Applications:

Preliminary Screening: Predict potential off-target sites before experimental validation.

**1.3.3 Considerations:

Prediction Accuracy: Predictions may not always match actual experimental findings.

**2. Functional Methods

**2.1 T7 Endonuclease I Assay

**2.1.1 Description:

Detection of Indels: Detect small insertions or deletions (indels) at potential off-target sites by cleaving mismatched DNA.

**2.1.2 Applications:

Detection of Indels: Assess the presence of indels at specific genomic locations.

**2.1.3 Considerations:

Sensitivity: Less sensitive compared to sequencing-based methods.

**2.2 Surveyor Assay (Cleavage-Ligation Detection)

**2.2.1 Description:

Detection of Mutations: Detect mutations at specific loci by using a mismatch-sensitive endonuclease.

**2.2.2 Applications:

Mutation Detection: Identify and quantify off-target mutations.

**2.2.3 Considerations:

Specificity: May not detect all types of mutations or off-target effects.

**3. Cellular Phenotypic Assays

**3.1 Reporter Assays

**3.1.1 Description:

Incorporation of Reporter Constructs: Use reporter genes inserted into potential off-target sites to detect functional changes.

**3.1.2 Applications:

Functional Validation: Assess the impact of CRISPR/Cas9 on specific genomic locations based on reporter activity.

**3.1.3 Considerations:

Construction: Requires designing and incorporating reporter constructs.

**3.2 Cellular Phenotype Analysis

**3.2.1 Description:

Assessment of Cell Function: Evaluate changes in cell function, growth, or behavior that may indicate off-target effects.

**3.2.2 Applications:

Phenotypic Impact: Determine the broader impact of potential off-target mutations on cellular processes.

**3.2.3 Considerations:

Indirect Assessment: May not directly identify specific off-target sites.

**4. Other Approaches

**4.1 In Vivo Models

**4.1.1 Description:

Animal Models: Use animal models to assess the impact of CRISPR/Cas9 on off-target effects in a whole organism.

**4.1.2 Applications:

Systemic Effects: Study the broader implications of off-target effects in a physiological context.

**4.1.3 Considerations:

Complexity: Requires extensive validation and ethical considerations.

**4.2 High-Throughput Screening

**4.2.1 Description:

Library-Based Approaches: Use CRISPR libraries or screens to identify off-target effects across the genome.

**4.2.2 Applications:

Comprehensive Screening: Identify and analyze off-target effects systematically.

**4.2.3 Considerations:

Resource-Intensive: Requires significant resources and computational analysis.

Summary

Detecting off-target effects in CRISPR experiments involves a combination of sequence-specific, functional, and phenotypic methods. Techniques such as targeted sequencing, deep sequencing, and T7 endonuclease I assays provide detailed insights into potential off-target sites and mutations. Cellular phenotypic assays and in vivo models help assess the broader impact of off-target effects. Combining these approaches can help ensure the specificity and safety of CRISPR-based interventions.

l This protocol list might provide further insights to address this issue.