Recently there have been several studies that show, using a truncated sgRNA with a wild type CAS9 can deter the nuclease activity and can potentially be used for epigenetic regulation.
Kiani et al.(2015) claimed that 'that the lack of DNA cleavage with 16-nt gRNA was due not to a lack of DNA binding but to an inability of Cas9 to cleave the target substrate after binding.'
I wish to know how and why does the size of sgRNA affects the nuclease activity.
If I would have to suggest an answer, although the guide area itself is shortened, the trcRNA is still full length and will incorporate into the Cas9 protein and the Cas9/RNA complex can scan the DNA and stop when the guide sequence matches its target. However, as the PAM motif is a critical component for nucleases activity, the shortened guide sequence in complex with Cas9 most likely does not have a favorable thermodynamic potential to fully unwind the DNA near the PAM motif to allow for the actually cutting. Therefore, this complex effectively acts like a dCas9 variant.
As to which two species is better for epigenetic regulation (dCas9/full guide versus wt Cas9/shortguide), I don't think there has been a good study.
Based on my understanding of the gRNA designing and the related stuff, truncated gRNA are the ones which are devoid of nearly 3 nucleotides (17nt gRNA) in the 5` end. Many studies have shown that excluding the initial 3 nucleotides from the gRNA will increase its specificity without compromising its indel efficiency. On the other side, the last three to five nucleotides at 3' end of gRNA are called as seed regions. Particularly the last 3 nucleotides of the gRNA should not be paired/complementary to the 51–53 nucleotides of the tracrRNA. If it is paired, the sgRNA will lose its ability to create ds breaks or might show less efficiency.
Please read this one to understand more Article Rational design of highly active sgRNAs for CRISPR-Cas9-medi...
https://www.nature.com/articles/nbt.2969
https://www.addgene.org/guides/crispr/
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