Useful hairpin
RNA hairpin increased CRISPR accuracy by 50 times
Daria Spasskaya, N+1
American researchers have shown that adding a small element of the secondary structure to the guide RNA can significantly increase the accuracy of DNA editing by CRISPR systems. As scientists explain in an article in Nature Biotechnology (Kocak et al., Increasing the specificity of CRISPR systems with engineered RNA secondary structures), the RNA hairpin interferes with the nuclease activity of the Cas protein on the "wrong" targets, but does not interfere in the case of an exact sequence match between the guide RNA and the target.
A drawing from the Duke University Engineering press release "Hairpins" Increases CRISPR Accuracy - VM.
Despite a significant breakthrough in the field of therapeutic use of CRISPR editing (these systems are already used for ex vivo editing of the genome of individual cells, for example, leukocytes, and even editing human embryos), scientists are still concerned about the accuracy of CRISPR, which often shows inappropriate activity in the human genome. Increasing the accuracy of DNA recognition and cutting is most often attempted by introducing mutations into CRISPR effectors, in particular, the Cas9 protein. Nevertheless, this approach, although it has led to the creation of many Cas variants with increased specificity, most often leads to a significant decrease in the efficiency of editing on the right targets.
Scientists from Duke University approached the problem from the other side and started tuning the RNA component of CRISPR. The "simplified" version of the CRISPR system, which is used as an editing tool in eukaryotic cells, includes an effector (Cas9 or Cas12a) and a guide RNA that contains a 20-nucleotide spacer – a site complementary to the sequence in the genome that needs to be cut. It is known that the first part of the spacer makes the main contribution to the accuracy of recognition. The researchers suggested that if the remaining end is hidden inside the secondary structure, the editing accuracy will increase.
The scheme of hairpin formation at the 5'-end of the RNA spacer (here and below are the drawings from the article in Nature Biotechnology).
To test the hypothesis, the authors predicted in silico and showed experimentally that the addition of a short sequence forming a small and not very strong hairpin with the 5’ end of the RNA spacer does not reduce the effectiveness of binding Cas9 to the target, but at the same time affects the efficiency of editing. The latter parameter was tested on guide RNAs against VEGFA and EMX1 genes, which each have several known "off-targets" (sites of nonspecific binding in the genome).
It turned out that for such sections, the hairpin significantly reduces unwanted activity – both in comparison with the control unmodified spacer, and in comparison with the shortened version of the spacer, where the extra letters were simply cut off, and in comparison with a spacer with a surplus that does not form a hairpin. At the same time, CRISPR activity did not change significantly at the desired site (although calculations predicted its decrease). On average, for a number of off-targets, an increase in the accuracy of the "canonical" SpCas9 from Streptococcus pyogenes occurred 55 times. The authors also saw an increase in accuracy in the presence of a hairpin for other effectors used in practice – SaCas9 from Staphylococcus aureus and various forms of Cas12a.
The effectiveness of different guide RNAs at the target locus in the EMX1 (ON) gene and several non–target loci (OT). WT is an unmodified spacer, Hairpin is an RNA with a hairpin, Truncated is a trimmed spacer.
Discussing the mechanism of the observed phenomenon, the authors of the work suggested that the hairpin at the end of the spacer inhibits the formation of the RNA-DNA complex (the so-called R-loop), which is necessary for the nuclease activity of the Cas protein, in the case of inaccurate DNA-RNA pairing, and thus protects the system from inappropriate activity. The most important thing in this method of increasing specificity is its versatility, since the guide RNA is used by all CRISPR systems, and it is easy to synthesize.
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