Paper of the month June 2018

Partial DNA-guided Cas9 enables genome editing with reduced off-target activity

CRISPR-Cas9 is a powerful genome editing tool. The system relies on a synthetic guide RNA (sgRNA) to identify and cut the target. To improve the CRISPR system precision, many approaches have been attempted. In this paper, the authors suggest the synthesis of a hybrid sgRNA which is partially made of DNA.

In the first experiment, they test if it is possible to replace some parts of the sgRNA with DNA. The sgRNA can be divided in two different part:

1: The seed region: ten nucleotides at the 3’ end of the guide sequence, it is essential for Cas9-RNA binding and recognition of targeted DNA
2: The tail region: ten nucleotides at the 5´ end which mostly interacts with the Cas9

From previous studies it was known that modification of the seed region can affect Cas9 function. To test the sensibility of this, they use a cell line expressing the GFP protein – therefore the cells will display a green fluorescence. If they supply the Cas9 and the unmodified sgRNA – targeting the GFP protein – they can observe a reduction in the number of GFP positive cells. Then try to substitute the RNA with DNA at different positions of the sgRNA. As expected:

• if the RNA to DNA substitution is made within the tail region, they can still observe a reduction in the GFP fluorescence, similar to the experiment where they use unmodified sgRNA
• if the RNA to DNA substitution is made within the seed region, no drop in the green fluorescence is observed. Strengthening the previous observations that highlight the importance of the seed region.

They also test the approach on the endogenous human genes EMX1 and VEGFA. The results were consistent with the ones coming from the previous experiment, confirming that modifications at the tail region do not impair the cleavage activity of the Cas9.
The test was also repeated by delivering CRISPR as ribonucleoprotein complex (RNP). This consist of the Cas9 protein incubated with the sgRNA before delivering them to the cell. This experiment is important, since delivery of CRISPR as RNP is essential for therapeutic application due to the reduced cell toxicity compared to plasmid DNA. The results of this experiment are consistent with what evidenced so far.
A key point of the paper is also the evidence that partial substitution of RNA with DNA – at the tail region of the sgRNA – leads to a significant reduction in the off-target. In fact, the authors show that compared to an unmodified sgRNA, the use of DNA:RNA hybrid sgRNA leads to a dramatic reduction in the off-targets. The explanation may be that DNA:DNA interaction – between the tail region and the target sequence- are less stable than the RNA:DNA interaction. Thereby, only the desired target is bound stably enough to be than cleaved by the Cas9.

The authors provide an attractive way to synthetize guiding RNA for Cas9 protein. A DNA:RNA chimeras is cheaper than a standard sgRNA. Plus, it was proved to retain cleavage activity as well as to feature reduced off-target activity. This, coupled with the compatibility for RNP delivery, makes the DNA:RNA gRNA a new suitable tool for therapeutic purpose.