Normally guide RNAs have been 20 nucleotides long, but I am wondering if I can select to make them longer in order to reduce off-target activity. For example, say there is a gene of interest which I want to delete that is 40 nucelotides long. Can I use a 40-nucleotide gRNA to more precisely target that sequence of interest? And is there a maximum length which gRNAs can be?
You can make it longer, but it will not improve the specificity.
It is known that the distal part of the gRNA does not contribute to CRISPR specificity.
I agree with Laurie.
To reduce the off target effects , its better to decrease the length sgRNA rather than increasing it. To avoid the effect of off target site, try to keep the site of mismatch (if any) just adjacent to PAM site. Moreover, some companies have further optimized the particular positions of nt at which the efficiency of sgRNa are high such as G should be at first place and A or T at 17th place.
Best Wishes
This article has shown that longer guides are trimmed down to 20nt in vivo:Article Double nicking by RNA-Guided CRISPR Cas9 for enhanced genome...
Yes, there is a practical limit to the length of guide RNAs (gRNAs) used in the CRISPR-Cas9 system. The maximum length of a gRNA can vary depending on the specific Cas9 protein variant used and the delivery method, but it is generally in the range of 17 to 30 nucleotides. Here are some key points to consider:
Researchers often use bioinformatics tools and software to design gRNAs that are optimized for their specific CRISPR experiments, taking into account the length and sequence requirements of the chosen Cas9 protein. Additionally, advances in CRISPR technology may lead to the development of Cas proteins with different requirements and constraints, so it's important to stay updated with the latest research in the field.
l This protocol list might provide further insights to address this issue.
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