12 June 2019

Paper of the Month June 2019

Development of hRad51-Cas9 nickase fusions that mediate HDR without double-stranded breaks.

CRISPR-Cas9 can be used in genome editing to change, in a targeted manner, genetic information in mammalian cells. Cas9 works as a molecular scissor to introduce double-strand breaks (DSBs) in a specific location of the DNA. This protein is formed by two catalytic domains, each able to cut one of the DNA strands, therefore the combined activity of this domains results in a DSB.  Upon the introduction of DSBs, repair pathways are activated in the cells. Scientist try to exploit this response to modify the DNA in a specific and targeted manner. The homology-directed repair pathway (HDR) consists in the correction of a DSB using a template and allow to “trick” the cell to use an artificial modified template (donor DNA) to introduce new information in a specific location. However, in mammalian cells, DSBs are preferentially repaired through a process called non-homologous end joining (NHEJ). This process consists in direct junction of the free end DNA and usually results in unwanted small insertions and deletions (indels). Lots of effort has been invested in the scientific community to avoid the introduction of indels while maintaining HDR efficiency. 

A turning point in this battle has been the introduction of a modified Cas9 protein called nickase Cas9 (D10A) in which one of the catalytic domain has been silenced. Therefore its activity results in single-strand breaks (SSBs) rather than DSBs. Although SSBs are still repaired through HDR they are not repaired through NHEJ, avoiding the undesired introduction of indels. On the other hand the efficiency of HDR-mediated genome editing initiated by SSBs is lower than initiated by DSBs.  

Rees et. al., were remarkably able to improve the frequency of HDR products initiated by SSBs. The novelty of their approach consist in linking Cas9 nickase to hRad51, which is known to be involved in the repair of SSBs. Indeed inhibition of hRad51 increased SSBs repairs pathway and not DSBs.  hRad51-Cas9 protein has been used to introduce point mutations in HEK293T cells and resulted in an increase of HDR products over indels up to 53-fold when compared to the use of standard Cas9. Although HDR efficiency also resulted to be cell-dependent and in some cases the frequencies of HDR products resulted in only a modest increase leaving the door open for even further improvements. The authors state that this strategy will be useful for applications in which high efficiency and precision is needed and conclude by encouraging other scientists in further pursuing the study and manipulation of cellular DNA damage and repair pathways. Indeed overall this strategy did not only improved SSBs-mediated genome editing, but also underlined the biological importance of Rad51 in SSBs repair.