Unless you’ve been hiding under a rock you’ll know that CRISPR-Cas has been in the forefront of precise genome editing since its discovery in 1987 by Atsuo Nakata. However, as a technique it is not without shortcomings; while it has achieved a great deal of efficiency in the non-homologous end-joining (NHEJ) pathway, its ability to achieve an efficient homology-directed repair (HDR) is still at its infancy. Now, thanks to new research from the labs of Ralf Kühn and Klaus Rajewsky, CRISPR editing is getting an upgrade.
NHEJ and HDR are two important pathways employed by eukaryotic cells to rectify DNA double strand breaks (DSBs). Whereas NHEJ is mechanistically pliable, HDR demands the presence of a homolog near the DSB to proceed. This requirement makes HDR a more challenging technical process, but because the NHEJ pathway results in the loss of nucleotides at the end of DSBs, HDR is a more attractive mechanism to use.
The Germany-based researchers hypothesized that the efficiency of HDR could be improved through concomitant inhibition of the NHEJ pathway. To test their hypothesis, the team developed numerous constructs to suppress the key NHEJ pathway proteins KU70 and KU80, in mammalian cell lines. Additionally, they suppressed DNA ligase IV, the key enzyme in the NHEJ pathway, along with its inhibitor SCR7. They also tested this system with co-expression of adenovirus 4 E1B55K and E4orf6 proteins in order to determine the combination with the best HDR efficacy.
From these studies the researchers found:
- Suppressing KU70 and DNA ligase IV increases HDR efficiency 4-5 fold.
- Co-expressing E1B55K and E4orf6 with the cas9 system increases the efficacy of HDR to 8-fold and completely inhibits NHEJ activity in human and mouse cell lines.
- A knock-in efficiency of 50-66% was observed in cells transfected with sgRNA, cas9, and adenovirus 4 protein expression plasmids.
The team contend that their discoveries “provide useful tools to improve the frequency of precise gene modifications in mammalian cells”.
Want to improve your effectiveness? Head over to Nature Biotechnology, March 2015.