Major breakthrough in precise genome editing with CRISPR

CRISPR-mediated genome editing has a major flaw: the nuclease cleaves the DNA at a specific site, but then the double strand break (DSB) is healed by an error-prone repair most of the time. This means, although site-specific, the genome editing is usually not precise, i.e. leads to random mutations at the site (owing to the DNA repair mechanism, non-homologous end-joining (NHEJ). However there is an existing possibility to carry out precise modifications, this is done by another pathway homology directed repair (HDR). During HDR, one utilizes an external DNA fragment, which has homology arms to the region facilitating to copy this region into the genome. However, HDR and NHEJ are competing with each other, NHEJ being the winner. Moreover, NHEJ is active in all cells at all time, but HDR is thought to be only effective in dividing cells in the S and G2 phase.

The group of Hidde Ploegh at Whitehead Institute at MIT, Boston, took a very straightforward approach by inhibiting NHEJ to favor HDR in cells expressing CRISPR/Cas9. This seems simple, but it's not, since most NHEJ inhibiting strategies makes cells really sick. By using an anti-cancer agent, Scr7, a commercially available NHEJ inhibitor, the authors were able to boost up HDR by 19-fold.

First, they analyzed CRISPR-expressing cell lines treated with Scr7. They introduced a site-specific STOP codon into the TSG101 gene and analyzed the presence of the STOP codon by simple PCR. Maximal HDR rate was detected to be about 3.1% for A549 cells and 19.1% for MelJuSo cells. Furthermore they were able to demonstrate successful insertion of a 800bp fragment (Venus) into a bone-marrow derived dendritic cell line, with efficacy up to 58%.

Next, they have looked at genome editing with HDR in mouse embryos, injected with Cas9, gRNA and HDR template. Surprisingly, mouse embryos treated with Scr7 not only showed higher frequency of HDR, but they showed the complete absence of NHEJ in 4 separately analyzed genes.

With permission from NPG, Maruyama T et al, Nat Biotechnol 2015

The current study provides a major step forward in increasing HDR over NHEJ in cell lines and particularly in mouse embryos, easing the development of precisely modified genetic mice. However, question remains, whether NHEJ could be inhibited and HDR could be carried out in non-dividing cells. I anticipate that increasing HDR frequency, compatible with potential human clinical applications, is going to be one of the major direction in the genome editing field.

Check out the paper here: http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3190.html