CRISPR corrects gene defect in Fanconi-anemia

A new paper came out on using CRISPR to edit and correct the FANCC gene, responsible for Fanconi anemia, a rare genetic disease affecting DNA repair. A point mutation leads to a cryptic splice site and in frame deletion of an exon in the FANCC gene. These patients exhibit hematological malignancies, solid tumors and often bone marrow failure. Currently patients are treated with allogenic hematopoietic cell transplantation. Obviously, ex vivo gene correction of the mutated gene is a feasible option in the context of autologous hematopoietic transplantation.

Key facts:
1. Cas9 nickase is less effective than WT Cas9
2. Cas9 nickase leads more likely to homology directed repair (HDR) events than the WT Cas9.
3. No off-target effects were found in the genome
4. Function of FANCC gene was restored in patient derived fibroblasts.

The group of Jakub Tolar (University of Minnesota) designed guide RNA-s against the FANCC gene and analyzed NHEJ (random mutations as a consequence of successful CRISPR action) on 293T cells and patient-derived fibroblasts. Using the not very sensitive SURVEYOR assay, random mutations were obvious in 293T cells, but hardly detectable in the fibroblasts, most likely because of inefficient transfection and low sensitivity of the SURVEYOR mutation detection assay.

Next, they took advantage of the traffic light reporter system to assess HDR (homology directed repair, "real" correction) and NHEJ rates in 293T cells. The traffic light system is a very creative way to detect gene specific CRISPR action: the sequence of interest is inserted upstream of an out of frame mCherry, which is put back in frame after NEHJ (red cells); while HDR oligo mediated recombination will generate a functional GFP (green cells). Using this approach, the authors showed that the WT Cas9 nuclease is much more active than the nickase (assessed by overall higher NEHJ and HDR), but, as reported previously, the nickase lead to preferential HDR over NEHJ.

Off-target effects were measured by either the SURVEYOR assay on predicted sites, but also using an integrase defective lentiviral (IDLV) capture assay, developed by a group in Heidelberg. The principle is that IDLV can only integrate at double strand breaks, so if the CRISPR has off target effects, the lentivirus could be amplified from the site using primers specific for the viral DNA. Using the IDLV method, no off-target effects were found for Cas9 and Cas9 nickase. We have to keep in mind however that the IDLV method is a less sensitive, somewhat sequence biased approach compared to the recently published method by K. Joung (GUIDE-Seq).

Finally, FANCC gene was corrected using HDR repair template containing puromycin selectable marker. The nickase lead higher number of corrected clones than the WT Cas9. In the corrected clones, the mRNA contained the skipped 4. exon, and the protein functionality was restored in a histone phosphorylation assay. It is worth to note that it is pretty challenging to carry out genome editing in FANCC cells, since the mutated protein is involved in DNA repair (and possibly HDR). Therefore the successful strategy was to use a plasmid homology template, which encoded for the FANCC cDNA.

In summary, this paper provides evidence that Fanconi anemia could be a target for genome editing. The next step is to carry out the precise gene correction in hemopoietic stem cells, assess in vivo rescue of function and to characterize off-target effects in more detail.

Link to the paper: Osborn MJ, Gabriel R, Webber BR, DeFeo AP, McElroy AN, Jarjour J, Starker CG, Wagner JE, Joung JK, Voytas DF, von Kalle C, Schmidt M, Blazar BR, Tolar J. Fanconi Anemia Gene Editing by the CRISPR/Cas9 System. Hum Gene Ther. 2015 Feb;26(2):114-26.