Look at these websites for guidelines or tools for designing gRNA:

http://www.addgene.org/CRISPR

http://tools.genome-engineering.org

http://crispr-ga.net

http://crispr.mit.edu

https://chopchop.rc.fas.harvard.edu

Dear Subin

I'd appreciate your immediate attention. I'd looked them previously. However, I couldn't find anything about sgRNA offset.

Hi Hadi,

For maximum cutting efficiency I find that an offset of -3 to +20 is optimal. Minimizing off-target potential is also advisable. Even though the nicking Cas9 is less mutagenic it is not perfect. Aside from screening off-target sites for each guide, you should look for potential paired off-target sites.

Dear all,

Thank you very much for sharing. I used DNA2.0 gRNA Design Tool "https://www.dna20.com/eCommerce/cas9/input"  for designing the gRNAs and according to Ran et al. (2013), the offset  with high efficiency for Cas9n fixed on 7-10bp . Morover, the offset for Cas9-Fok1 was chosen between 14-17bp based on  Guilinger et al. (2014) "Fusion of catalytically inactive Cas9 to FokI nuclease improves the specificity of genome modification". Another big thanks to your kindness.

Cordially,

When using Cas9 nickase (Cas9n) for a gene knock-out approach, selecting the optimal single guide RNA (sgRNA) offset is crucial for maximizing efficiency while minimizing off-target effects. Here are some best considerations to keep in mind when planning your experiments with sgRNAs and Cas9 nickase:

**1. **Understanding Cas9 Nickase

Cas9 nickase is a variant of the standard Cas9 protein that has been mutated to nick only one strand of the DNA double helix, rather than creating a double-strand break (DSB). The common Cas9 nickase versions (such as D10A in SpCas9) nick the non-target strand, leaving the target strand intact.

**2. **sgRNA Pairing

For effective gene knock-out using Cas9 nickase, two sgRNAs are typically used. Each sgRNA guides a Cas9 nickase to a specific location on opposite strands of the DNA, ideally spaced closely together. This approach is intended to introduce nicks on both strands of DNA, mimicking a DSB but with reduced risk of off-target cleavage.

**3. **Optimal Offset and Spacing

Offset Range: The optimal distance (offset) between the two nicks should generally be between 10 to 20 base pairs. This range is effective in facilitating efficient DNA repair mechanisms that lead to indel formation, which disrupts the gene sequence.

Directionality: Ensure that the sgRNAs are designed to target opposite strands of DNA, with their PAM sequences oriented outward from the nick site. This configuration helps in producing staggered nicks that effectively mimic a DSB.

**4. **Selection of sgRNA Target Sites

Sequence Selection: Choose sgRNA target sequences that are specific to the region of interest to minimize the potential for off-target effects. Utilizing tools like CRISPR design tools (e.g., CRISPOR, Benchling) can help in identifying sgRNA sequences with high specificity and efficiency scores.

PAM Availability: Consider the availability of suitable PAM sequences (NGG for SpCas9) near your target site, as this will dictate where Cas9 can bind and nick the DNA.

**5. **Considerations for Off-target Effects

sgRNA Design: Design sgRNAs to minimize potential off-target sites by comparing candidate sgRNA sequences against the genome. High-fidelity Cas9 nickase variants can also be used to further reduce off-target activity.

Validation: Validate sgRNA activity using in vitro assays before proceeding to more complex experiments. Checking for off-target effects through methods like genome-wide off-target analysis by next-generation sequencing (NGS) can be beneficial.

**6. **Experimental Controls

Include Controls: Use appropriate controls, such as cells transfected with only one sgRNA and a non-targeting sgRNA control. These controls help in distinguishing between effects caused by targeted nicks versus potential background mutations or cellular responses to Cas9/sgRNA delivery.

**7. **Assessment of Indels

Detection of Indels: Use techniques such as T7 Endonuclease I assay, Surveyor assay, or deep sequencing to detect and quantify indel formation at the target site. This will help assess the efficiency of the sgRNA pair and the nickase activity.

Conclusion

Choosing the right offset for sgRNAs when using Cas9 nickase is critical for achieving effective gene knock-out while maintaining specificity and minimizing off-target effects. Precise control over sgRNA design and spacing, thorough in silico validation, and careful experimental setup will greatly enhance the success of your CRISPR-Cas9n experiments.

l Perhaps this protocol list can give us more information to help solve the problem.