Just like kids during the holidays, young stem cells have all the fun with their fancy new CRISPR-themed gene editing gifts, while their older, wiser, more differentiated counterparts keep getting nothing but coal. (Well technically carbon nanotubes, but elementally the same).
This neglect is partly the fault of more differentiated cells themselves; stodgy as they are, they can be about as adept at homologous recombination as Aunt Mildred is at Snapchat. Since most CRISPR/Cas9 gene editing relies on homology-directed repair (HDR) to replace sliced target genes with shiny new ones, these techniques don’t work as well in post-mitotic cells with downregulated HDR pathways.
However, just like Aunt Mildred’s Snapchat deficiencies belie her picture-taking prowess with a Polaroid, non-dividing cells are quite good at non-homologous end joining (NHEJ, basically just gluing the ends of DNA back together). Now, a team from the Izpisui Belmonte lab at the Salk Institute has developed a new technique to bring the gift of precise gene knock-ins to mature cells.
Their new technique, homology-independent targeted integration (HITI), targets an insertion site using CRISPR/Cas9, supplies an excess of linear DNA template, and allows the grumpy old cells to glue the DNA template between the ends of the cut target DNA via NHEJ. If the cell simply glues the two ends back together without the insert (or a mutation), the Cas9 target site would re-form and get cut again. Similarly, the team designed donor DNA so that it also re-forms the cut site if it goes in backwards, ensuring that most insertions go in the correct orientation.
The best donor DNA came from mini-circles linearized with a single Cas9 cut site, the same as in the recipient DNA. Plasmid DNA with either one or two cut sites also worked, but not quite as well. In the curmudgeonly cell lines they tested – differentiated mouse neurons in vitro, mouse brains in utero, and neurons, kidneys, and muscle tissues in mouse pups – HITI was more effective than either HDR or another NHEJ-based strategy, micro-homology-mediated end-joining (MMEJ), aka precise integration into target chromosome (PITCh). They were even able to restore a bit of eyesight to mice going blind from degenerative retinitis pigmentosa, using Cas9 and a gene replacement HITI donor delivered in an AAV vector.
So if Santa passes you over this winter, gift yourself a printout of this paper in Nature, 2016, and teach yourself a fancy new trick.