FA pathway key to DNA repair after CRISPR cutting

The importance of the FA pathway in repairing DSBs casts doubt on some planned CRISPR treatments for Fanconi anemia itself.

Without an active FA pathway, cells may not be able to replace their mutated genes with normal genes after Cas9 makes a cut. In fact, the activity level of the FA pathway may affect how efficiently CRISPR can insert DNA in a specific cell.

The researchers concluded that, while end-joining is the default repair mechanism after a DSB, the FA pathway competes with it, and that higher activity results in more homology-directed repair and less end-joining.

Dr Richardson and his colleagues also found that 1 of the 21 proteins in the FA pathway, FANCD2, always homes in on the site of the DSB created by CRISPR-Cas9. This suggests FANCD2 plays an important role in regulating the insertion of new DNA at the cut site.

The researchers therefore believe FANCD2 could be tweaked to boost the frequency with which a cell inserts DNA via homology-directed repair.

“Also, since FANCD2 localizes to the site of Cas9 breaks, you can use FANCD2 to map where Cas9 is cutting in any cell type,” Dr Richardson said. “If you edit a population of cells and you want to know where the on- and off-target cuts are, you can just map where FANCD2 was found in the genome and you can find the cuts.”