Paper of the month January 2019

Covalent linkage of the DNA repair template to the CRISPRCas9 nuclease enhances homology-directed repair

Gene-editing via CRISPR-Cas9 is a two-step process: First Cas9 is directed to a particular DNA region, where it works as a pair of molecular scissors and induces a double-strand break (DSB) into the DNA. During the second step the cells own repair mechanisms are activated. Repair of the DSB can be achieved by two alternative pathways, namely homology-directed repair (HDR) or non-homologous end-joining (NHEJ). NHEJ can only imperfectly repair a DSB and leaves a scar in the DNA. HDR makes use of a repair template to restore the original sequence and is thus often the preferred pathway. However, competition of those two pathways leads to low HDR efficiency in mammalian cells and is a major obstacle for the therapeutic use of CRISPR-Cas9. Many researchers, including some of our IMGENE fellows, therefore try to increase HDR efficiency.

In this paper Savic and colleagues demonstrate that spatial and temporal colocalization of Cas9 and repair template can increase HDR efficiency. The scientists chemically fused a repair template to Cas9 and called the new system RNPD¹. Afterwards they measured HDR efficiencies in comparison to Cas9 with unlinked repair template and showed an increase of up to 24-fold in HEK293T cells. Additional experiments showed repeatedly an improvement in correction efficiency for RNPD, although the exact foldchange depended on repair template design, cell line and genomic locus. A potential danger of the CRISPR-Cas9 system is the possibility of DSBs at undesired DNA regions (offtargets).

Notably, RNPD did not show increased off-target activity. In a second step the scientists show that improved correction efficiency is caused by increased repair template availability in the nucleus. To do so, they developed a two-component system consisting of a classical Cas9 and a cutting-deficient Cas9 fused to the repair template (SadCas9). Cas9 induces the DSB while SadCas9 serves solely as a vehicle for the repair template. SadCas9 could either be directed to the nucleus or very specifically to a DNA region close to the Cas9 cutting site. In both cases HDR was increased compared to Cas9 and unlinked repair template. However, SadCas9 directed to a nearby DNA region did not show a stronger enhancement in HDR than SadCas9 directed to the nucleus. This suggests that increased repair template availability in the nucleus is sufficient to explain the positive effects of RNPD. Linking Cas9 to the repair template is a very promising approach that might help to pave the way to therapeutic use of CRISPR-Cas9 in the future. So far, despite the huge potential of CRISPR-Cas9 for gene therapy, low HDR efficiency remains one of the major obstacles to overcome. In the past approaches altering DSB repair pathways have been described to increase HDR. In contrast, RNPD does not interfere with endogenous cellular pathways and thus limits the danger of side-effects. In addition, RNPD does not show increased off-target activity in comparison to the classical CRISPRCas9 system.

¹ The abbreviation stands for ribonucleoprotein-DNA. Cas9 is a protein that is guided to the DNA by a ribonucleic acid (RNA, structure closely related to DNA). Thus, the classical complex is often called ribonucleoprotein (RNP). RNPD contains in addition the fused repair template, consisting of DNA.