Original Article

Gene Therapy (2017) 24, 265–274; doi:10.1038/gt.2016.89; published online 19 January 2017

Deletion of the GAA repeats from the human frataxin gene using the CRISPR-Cas9 system in YG8R-derived cells and mouse models of Friedreich ataxia

D L Ouellet1,2, K Cherif1,2, J Rousseau1,2 and J P Tremblay1,2

  1. 1Centre de Recherche, Centre Hospitalier, Universitaire de Québec, Quebec City, QC, Canada
  2. 2Département de Médecine Moléculaire, Faculté de Médecine, CHU de Québec, Université Laval, Québec City, QC, Canada

Correspondence: Dr JP Tremblay, Department of Molecular Medicine, Faculté de Médecine, CHU de Québec, Laval University, CR-CHUQ, 2705 Laurier Boulevard, Room P-09300, Quebec City, QC, Canada G1V 4G2. E-mail: jacques-p.tremblay@crchul.ulaval.ca

Received 8 November 2016; Accepted 19 December 2016
Accepted article preview online 26 December 2016; Advance online publication 19 January 2017



The Friedreich ataxia is a monogenic disease due to a hyperexpanded GAA triplet located within the first intron of the frataxin gene that causes transcriptional issues. The resulting frataxin protein deficiency leads to a Fe-S cluster biosynthesis dysfunction in the mitochondria and to oxidative stress and cell death. Here we use the CRISPR-Cas9 system to remove the mutated GAA expansion and restore the frataxin gene transcriptional activity and protein level. Both YG8R and YG8sR mouse models and cell lines derived from these mice were used to CRISPR-edited successfully the GAA expansion in vitro and in vivo. Nevertheless, our results suggest the YG8sR as a better and more suitable model for the study of the CRISPR-Cas9 edition of the mutated frataxin gene.



Friedreich ataxia (FRDA) is an inherited autosomal disease with symptoms appearing usually within the second decade of life. The phenotypic expression is characterized by a progressive ataxia with uncoordinated movements, weakened muscle strength and balance problems.1, 2, 3, 4, 5 Some FRDA patients also have systemic impairments involving, but not restricted to, cardiomyopathy, diabetes mellitus and scoliosis.6 Early deaths result from cardiomyopathy or associated arrhythmias.3

Reduced levels of the frataxin (FXN) protein in the mitochondria cause oxidative damages and iron deficiencies at the cellular level.7 The reduced FXN expression has been linked to a GAA triplet expansion within the intron 1 of the somatic and germline FXN gene.8 In FRDA patients, the GAA repeat frequently consists of more than 70, sometimes even more than 1000 (most commonly 600–900) triplets, whereas unaffected individuals have about 40 repeats or less.9 The aberrant expansion of the GAA repeats is associated with localized chromatin changes and transcriptional silencing of the gene.10 However, the molecular mechanisms of hyperexpanded GAA-induced transcriptional defects are not clear yet. The molecular mechanism(s) have been reviewed by Pandolfo et al.11 Briefly, the GAA triplet expansion would lead to the formation of a triplex in the DNA, that is, an unusual non-B DNA conformation, which decreases transcription and subsequently reduces levels (usually >50%) of the encoded FXN protein.12, 13

The FXN protein is essential for adequate mitochondrial functioning. It is involved in the incorporation of iron into heme and iron-sulfur clusters.14 When FXN is deficient, iron is misdirected and this leads to oxidative stress. Neurons and cardiomyocytes are particularly sensitive to this stress.7 However, as FXN is a mitochondrial protein, all tissues are affected to some extent. The reduction of FXN leads to changes in gene expression of 185 different genes.12, 13 Antioxydants and iron chelators are under investigation in clinical trials.7 As a monogenic disease, FRDA is a good pathology candidate for gene therapy, thus some research projects are trying to increase the FXN protein level in FRDA cells and in mouse models using gene therapy approaches.15

Recently, gene replacement or gene editing got a second breath with the astounding new technique coming from the bacteria, the CRISPR-Cas9 system. The CRISPR technology uses a Cas9 nuclease and a single-guide RNA (sgRNA) containing a constant sequence of 42 nucleotides, and a variable sequence of 20 nucleotides complementary to the targeted DNA sequence to induce double-strand breaks.16, 17, 18, 19 For the Streptococcus pyogenes cas9 (SpCas9), an NGG protospacer adjacent motif (PAM)20 has to be located at the 3′ end of the targeted sequence. The PAM of the Staphylococcus aureus cas9 (SaCas9), another Cas9 nuclease, is NNGRRT.

The double-strand break can knockout a specific gene or allow insertion of foreign DNA by homology directed repair. CRISPR-Cas9 introduced DNA cleavage followed by non-homologous end-joining repair has been exploited to generate loss-of-function alleles in protein-coding genes or to delete a very large DNA fragment.21, 22 As a matter of concern about specificity, off-target mutation rate has been significantly reduced by modifying the Cas9 nuclease.23, 24 However, as any kind of therapy, the side effects of a treatment must be clearly defined and known before going further with the massive treatment of patients.

Dr Mark Pook has produced the rescued YG8 (YG8R) mouse model.25, 26, 27 The mouse genome contains two null mouse FXN genes and also two copies in tandem of an FXN transgene obtained from an FRDA patient. These human transgenes contain, respectively, 82 and 190 GAA repeats in intron 1, and thus a reduced amount of human FXN is produced leading to the development of FRDA symptoms by this mouse compared with a mouse model, called Y47R, containing a human wild-type version of the FXN. Dr Pook has recently reported that during the course of breeding, the YG8R mice lose one of the human transgene.28 This new model called YG8sR presents more severe symptoms than the original mouse model, including significant behavioral deficits, together with a degree of glucose intolerance and insulin hypersensitivity with significantly reduced expression of FAST-1 and FXN, and the presence of pathological vacuoles within neurons of the dorsal root ganglia.

In this article, we report the use of the CRISPR system, using either SpCas9 or SaCas9 in combination with a pair of sgRNA, to delete the GAA trinucleotide repeats in vitro in YG8R26 and YG8sR29 mouse fibroblasts and in vivo in a YG8R-derived mouse line. We identify the YG8sR as a more suitable in vitro model to study CRISPR edition for FRDA as it has only one copy of the human FRDA FXN transgene. We thus propose to use the YG8sR mouse model to study GAA correction using a adeno-associated virus (AAV) coding for the SaCas9 and two sgRNAs targeting the pre- and the post-GAA repeat. All experiments present in this article help to identify the most suitable mouse model for gene editing in vivo, hoping this will quickly lead to human clinical trials in FRDA patients.



Identification of pairs of sgRNAs targeting the pre- and post-GAA trinucleotide repeat

SgRNAs targeting sequences located before and after the GAA trinucleotide repeats in intron 1 of the FXN gene (NG_008845) were identified using the crispr.mit.edu web platform. We identified sequences (Figure 1a) adjacent to the S. pyogenes NGG PAM. We then designed 20nt oligonucleotides targeting the sequence located 5′ of the PAMs and cloned them in an expression vector (px330, and/or pxPuro and/or pxGFP) under the control of a RNA polymerase (pol) III U6 promoter. The same vector also encoded the SpCas9 protein under the control of a RNA pol II promoter (CBh). At first, plasmids were transfected in the mouse YG8R fibroblasts. These cells contain two human FRDA FXN transgenes in tandem with ~82 and 190 GAA repeats, respectively30 (Figure 1b). The PCR amplification of the GAA repeats using the F3/R3 primer set (Figure 1a) from the genomic DNA (gDNA) of YG8R reveals the amplification of two bands at 2070 and 2394bp (Figure 1d, lane 1) that represent the two transgenes in their uncut forms. Different pairs of sgRNAs (one targeting the pre-GAA and the other the post-GAA region) were tested and the success of the deletion using a given pair was detected by PCR using the F3/R3 primer set (Figures 1c and d). Indeed, the section deleted between two targeted sequences is missing from the PCR amplicon and allows the visualization of an additional smaller band in a population of YG8R-transfected cells (Figures 1c and d).

Figure 1.
Figure 1 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

CRISPR targeting of the mutated GAA trinucleotide repeat of the FXN gene. (a) Regions (lines with black dots) targeted by the SpCas9 sgRNAs are identified in the pre- and post-GAA trinucleotide region of the human FXN intron 1 and positions of the primers used in this study (lines with squares). (b) The YG8R mouse fibroblasts contain two tandem copies of the human FXN transgene (from an FRDA patient), with ~82 and 190 GAA repeats. (c) Expected F3/R3 PCR-amplified product lengths from extracted gDNA following transfection of YG8R cells with the SpCas9 gene and different pairs of the sgRNAs. (d) Screening of sgRNA pairs in YG8R cells, using the F3/R3 primer set.

Full figure and legend (118K)

Deletion of the FXN intronic GAA repeats in YG8R fibroblasts

Some sgRNA pairs were selected and were cloned into pxPuro, which shares similarities with px330 but contains a puromycin gene for selection. These new plasmids were retested in YG8R cells (Figure 2a) and following detection of the corrected PCR amplicon in the puromycin-resistant cell population, these cells were amplified as individual clones. As the human FRDA FXN transgene is in tandem copies in the YG8R, there are several possible rearrangements following deletions with a pair of gRNAs, as shown in Figure 2b. Positive clones are described as clones with a complete deletion of the GAA repeats in both tandem copies, that is, the amplicons obtained with primers F3 and R3 did not contain the 2070 and the 2394bp bands. Pair of sgRNAs C2C20 and C15C20 gave the highest percentages of success (14% and 15% respectively) of complete deletions (Figure 2c). Partial deletion status was attributed when one of the GAA band was still present in the amplicon (Supplementary Figure S1). Taking into account the deletion of only one of the two GAA repeats, the percentages of clones with a deletion could have been much higher: 21.6% (11/51) for C2C11, 50% (11/22) for C2C20 and 39.4% (13/33) for C15C20 (Figure 2c). As shown in Figure 2d, amplification of clones with a deletion using the F3/R3 primer set revealed only one band, missing the deleted section and having a size that depended on the specific sgRNA pair used. The sequencing of the amplified F3/R3 amplicons for nine9 YG8R clones (Figure 2e) showed mostly cuts at the expected sites for the SpCas9, which is three nucleotides upstream of its PAM. Otherwise, patters of alignments (Supplementary Figure S2) showed significant alignments close to the cut sites (pre- and post-GAA) and confirm the technique, in combination with the non-homologous end-joining, as precise and reliable.

Figure 2.
Figure 2 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Correction of the GAA trinucleotide repeats in YG8R mouse fibroblasts. (a) F3/R3 PCR amplification of gDNA from YG8R fibroblasts transfected with plasmids coding for SpCas9_P2A_puromycin and sgRNA pairs. The correction of the FXN gene was first detected in a heterogeneous (pooled) YG8R fibroblast population. Cells successfully transfected were then selected using the puromycin selection drug and expanded as individual clones. (b) Putative rearrangements of the FXN gene in YG8R fibroblasts following correction with a pair of sgRNAs, that is, one targeting a sequence located before (a or a′) and the other a sequence located after (b or b′) the GAA repeat. A positive clone status (+) was given when no F3/R3 PCR-amplified band including a GAA repeat was seen on agarose gel. (c) Summary of the YG8R clonal expansion. Partial deletion is corresponding to clones that were still having an F3/R3 PCR-amplified product containing the GAA repeat. Complete deletion status was attributed to the clones that did not contain any GAA repeat, resulting from one of the rearrangements illustrated in b with a positive clone status. (d) Agarose gel showing F3/R3 PCR-amplified products corresponding to a complete deletion of both GAA repeats (in the tandem transgenes) in the FRDA FXN genes in YG8R isolated clones (clones considered as positive in scheme in b). (e) Amplified F3/R3 products in (d) were subcloned and sequenced to detect junction point between the pre- and the post-GAA repeat region of the intron 1 following correction. Green and red boxes correspond to PAM sequences for SpCas9, whereas arrows show the expected cut site. Red boxes and arrows represent PAMs and cut sites identified on the sense strand, whereas green boxes and arrows identified PAMs and cut sites on the antisense strand of the gDNA.

Full figure and legend (318K)

Protein analysis in YG8R clones

FXN protein levels were analyzed in samples from a heterogeneous sgRNA/SpCas9-transfected YG8R population (Supplementary Figure S3a) and puromycin-selected YG8R clones with GAA repeats from both transgenes deleted (Figure 3a and Supplementary Figures S3b and c). No significant differences were found following analysis of FXN protein levels extracted from the heterogeneous YG8R population (Supplementary Figure S3a, lanes 3–6). However, significant differences in FXN protein were observed between control clones, identified as PURO-4 and PURO-5 (Figure 3a and Supplementary Figures S3b and c, lanes 1 and 2), and corrected clones (Figure 3a, lanes 3–6 and Supplementary Figures S3b and c, lanes 3–8). Quite surprisingly, the FXN protein expression in most of the clones was decreased compared with the controls, which are YG8R cells transfected with a plasmid encoding the SpCas9_P2A_puromycin but missing sgRNAs, and expanded as clones as well. A few clones showed no significant differences, as their FXN protein expression stays constant despite their positive clone status (i.e., deletion of GAA repeats in both transgenes). We hypothesized that for most of the positive clones, a deletion from the 'a' site to the 'b′' site (Figures 2b and 3c) removed the constitutive promoter of the second transgene, therefore reducing significantly the overall expression of the human FXN in those cells. A copy number analysis of the YG8R clones revealed that despite no evidence of residual GAA repeat (Figure 2d), some clones did not show any changes in their FXN copy number. Other clones appeared to have lost part of the transgene while keeping another part (Figure 3b, C15C20-15). A significant decrease in the copy number for both the promoter and the exon 2 region was only observed for the C2C20-18 clone (Figure 3b). A stable or a slight increase of the FXN protein expression in YG8R clones could be attributed to a 'a+b+a′+b′ ' case (Figure 3c), which happens to be a rare event.

Figure 3.
Figure 3 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Protein analysis and copy number analysis of CRISPR-edited YG8R fibroblasts. (a) Western blot protein analysis of YG8R cells transfected with different combinations of sgRNAs and SpCas9. (b) Gene copy number analysis of some selected clones shown in (a). (c) Schematic representation of results obtained regarding putative rearrangements for corrected YG8R clones.

Full figure and legend (118K)

Deletion of the FXN intronic GAA repeats in YG8sR fibroblasts

We subsequently tested from Dr Pook’s team three new mouse fibroblast lines (called YG8sR-6, YG8sR-8 and YG8sR-39) derived from three different YG8sR mice. Each cell line contained only one copy of the human FXN transgene with about 190 GAA repeats within their intron 1 (ref. 29) (Figure 4a). As shown in Figure 4b, the F3/R3 primer set allowed differentiating easily the three YG8sR lines (6, 8 or 39) from the Y47R cell line (a mouse fibroblast line with a human FXN transgene containing a normal number of GAA repeat) and from the YG8R, which contains two copies of the human FXN transgene (i.e., two different band sizes observed by PCR amplification). We tested YG8sR cell lines by transfecting them with a Cas9-encoding plasmid and two different successful pairs of sgRNAs previously identified in YG8R experiments. YG8sR-39-transfected cells were selected over YG8sR-6 or YG8sR-8 for clonal expansion (Figure 4c), but correction with the C2C20 and the C15C20 combinations worked also in these two cells lines (data not shown).

Figure 4.
Figure 4 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Correction of the GAA trinucleotide repeats in YG8sR mouse fibroblasts. (a) Schematic of the human FXN transgene in YG8sR cells. The GAA trinucleotide region is estimated to 190 GAA. (b) F2/R3 PCR-amplified products containing the GAA trinucleotide repeat showing differences between cells used in this study. Y47R contain one copy of a normal human FXN transgene with ~9 GAA repeats. Fibroblast cell lines YG8sR-6, YG8sR-8 and YG8sR-39 (isolated from mice no. 6, no. 8 and no. 39 in Pook’s lab) have ~190 GAA, whereas YG8R contains two copies in tandem with ~82 and 190 GAA, respectively. (c) F3/R3 PCR-amplified products analysis of gDNA of YG8sR-39 transfected with C2C20 or C15C20 pair and SpCas9_P2A_puromycin. YG8sR-39 cells were amplified as clones following this experiment. PURO represents cells transfected with the SpCas9-encoding plasmid without any sgRNA expressed. (d) Putative rearrangements of the single copy of the FRDA FXN gene in YG8sR fibroblasts following deletion of the GAA repeat using a pair of sgRNAs targeting before (a) and after (b) the repeat. A positive clone status (+) was given when no F3/R3 PCR-amplified bands corresponding to the ones with a GAA repeat were seen on agarose gel. (e) Summary of the YG8sR clonal expansion. (f) Agarose gel showing F3/R3 PCR-amplified products corresponding to the corrected FXN gene from YG8sR isolated clones C2C20-13 and -20. Similar results were obtained for C2C20-15 and -18 (not shown).

Full figure and legend (149K)

As the YG8sR cells do contain only one copy of the human FXN transgene, only one rearrangement appears possible by non-homologous end-joining recombination following cuts from both sides of the GAA repeat (Figure 4d). Upon expansion of the isolated YG8sR-39 clones (referred to as YG8sR in the following text and figures), we found out that fewer clones survived the selection as we were only able to screen 20 clones, out of five 96-well plates seeded post-transfection and postselection with puromycin, for the C2C20 combination, and three clones, from the C15C20 combination (Figure 4e). Out of these 20 C2C20 clones, four clones were found positive, as they were presenting one band with the F3/R3 primer set PCR amplification (Figure 4f) for the clones C2C20-13 and -20 and none of the C15C20 clones were found with a deletion of the GAA repeat (data not shown).

Identification of YG8sR clones expressing higher amounts of FXN protein

Analysis of the YG8sR C2C20 clones protein extracts by western blot revealed an increase of the FXN protein level in two of the C2C20 clones (Figure 5a, lanes 5 and 6 and Figure 5b), but lower than in the Y47R cell line. An increase in the hFXN transcriptional levels was confirmed for the C2C20 clone 13, but not for the clone 15 (Figure 5c, hFXN 5′-untranslated region/exon 1 and hFXN exon 2/exon 3), and high amounts were also monitored in the Y47R as expected (Figure 5c). Additional analysis of the genomic profile of the different YG8sR C2C20 clones with different primers sets showed discrepancies between expected and obtained PCR band profiles (Supplementary Figure S4). For example, unexpected bands appeared in the PCR made with the F4/R10 primer set for C2C20-15 and C2C20-18 clones when all samples were processed at the same time in the same conditions (Supplementary Figure S4b).

Figure 5.
Figure 5 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Protein and RNA analysis of CRISPR-edited YG8sR fibroblasts. (a) Western blot protein analysis YG8sR clones treated with the C2C20 sgRNA pair or a negative control with the PURO vector. (b) Summary of protein analysis from various controls and four (n=4) different protein extractions from YG8sR cells treated with the C2C20 sgRNA pair. (c) Transcriptional analysis of total RNA extracted from cells treated or not with the C2C20 sgRNA pair. Three (n=3) different RNA extractions were made for each condition. Human FXN transgene expression was monitored by quantitative reverse transcription PCR (qRT-PCR) using primers to amplify hFxn exon 2/3 and 5′-untranslated region/exon 1 as published previously53 (see also Supplementary Table S1). (d) Gene copy number analysis of YG8sR clones.

Full figure and legend (108K)

We also measured the copy number of the human FXN transgene in the C2C20 clones and found no change, as expected, in almost all the clones, compared with the YG8sR-untreated population (Figure 5d). However, the C2C20-18 sample show a decrease by half of the copy number compared with the YG8sR and other clone populations. Therefore, the copy number in mouse YG8sR fibroblasts is estimated to be below 1, and some somatic mosaicism have indeed been initially reported.31

Electroporation of plasmid DNA into the Tibialis anterior shows in vivo correction

Our sgRNA combinations were tested in vivo. Briefly, plasmids coding for SpCas9 and a pair of gRNAs (either C2C20, C15C20 or C16C20) were electrotransfered into the Tibialis anterior (TA) of YG8R mouse muscles (Figure 6a). PCR was performed using the F3/R3 primers and amplified the expected products after the removal of the GAA trinucleotide repeat (Figure 6b, lanes 4, 5 and 7).

Figure 6.
Figure 6 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

In vivo electroporation of SpCas9 and sgRNAs coding plasmids into YG8R mouse model. (a) Schematic representation of electroporation experiment. (b) F2/R3 PCR-amplified products obtained following gDNA extraction from the TA of YG8R electroporated with plasmids. Mouse no. per side referred to the mouse number and its right or left TA. The sign '&' represents the expected band for the C16C20 combination, whereas the '*' represents the expected band for the C2C20 combination. The '' stands for the unique uncut band seen in YG8R, as for (a) lane 3.

Full figure and legend (83K)

AAV-encoded SaCas9 plasmid generate cuts in mouse fibroblasts

As FRDA is a neuromuscular degenerative disease involving mainly the brain, the spinal ganglia, the heart and the pancreas, we decided to move forward and used a viral vector to deliver our pair of sgRNAs in target tissues in vivo. We then had to redesign our sgRNA to accommodate an AAV encoding the recently available SaCas9 protein, which requires an NNGRRT PAM sequence.32

We selected SaCas9 PAM sequences located close to previously identified SpCas9 PAMs, that is, the C2 and C20 sites (Figure 7a). The px601 vector23 was modified to introduce another pol III promoter (U6 or H1 'minimal' (H1m)) and two SaCas9 tracrRNA sequences, and to express two SaCas9 sgRNAs from the same AAV. To do so, the size of the cytomegalovirus (CMV) promoter33 was reduced (Figure 7b). As shown in Figures 7c and d, combinations of sgRNA, transcribed from the U6 pol III promoter, and SaCas9, transcribed from the non-truncated CMV promoter, targeting the SaC2 or SaC1 and the SaC6 sites successfully cut the FXN intron 1 in YG8sR fibroblasts in culture, as well as in YG8R (data not shown). Indeed, following amplification with F3/R3 primers, the predicted amplicon size representing the FXN gene deleted of the GAA repeat was observed (Figure 7c, lanes 2 and 3). SaC2 and SaC6 sgRNA were selected for optimization and did not show significantly less cutting when one of these sgRNA was expressed from the H1m (95bp; ref. 33) promoter (Figure 7d, lanes 3/9 or 4/10), despite the lower amount of SaCas9 produced from the truncated CMV 212 or 259 promoter (Figure 7e, lanes 4–7).

Figure 7.
Figure 7 - Unfortunately we are unable to provide accessible alternative text for this. If you require assistance to access this image, please contact help@nature.com or the author

Correction of the GAA trinucleotide repeats using the SaCas9 protein. (a) Target regions for SaCas9 (SaC1, SaC2 and SaC6) were identified, with regard to their PAM, in the pre- and post-GAA trinucleotide regions of the FXN intron 1. (b) Scheme of the modifications made from the original px601 plasmid (not shown, see Materials and methods for details). Briefly, one additional U6 or H1m promoter+SaCas9 tracrRNA were added to allow cloning of a second sgRNA within the same plasmid. The CMV promoter was then shortened to 259 or 212bp. (c) F3/R3 PCR-amplified products showing effectiveness of the correction using combinations of sgRNA and the SaCas9 protein in YG8sR fibroblasts. (d) F2/R3 and F3/R3 PCR-amplified products showing the effects of the correction in YG8sR using the sgRNA pair SaC2 and SaC6 expressed from different promoters (either U6 or H1m) and where the SaCas9 is expressed from truncated (212 or 259) or not (WT) forms of the CMV promoter. (e) Western blot analysis of the expression of the SaCas9 from CMV (WT, 212, 259) or CBh promoters using, respectively, anti-HA or anti-FLAG antibodies.

Full figure and legend (181K)



The CRISPR-Cas9 system is a very powerful technique to modify genes. It has already been used to correct mutated gene as a new gene therapy approach. For example, our group and others have used this technique to correct the dystrophin gene in models of Duchenne muscular dystrophy, that is, two gRNAs to remove exon fragments or complete exons to not only restore the reading frame of the dystrophin gene34, 35, 36, 37 but also produce a dystrophin protein with an adequate spectrin-like structure.38 The CRISPR-Cas9 technology has also been used to reduce the expression of a missense mutations gene responsible for Meesmann’s epithelial corneal dystrophy,39 and to correct a splicing mutation responsible for hereditary tyrosinemia,40 for long-term repression or activation of genes linked to lung cancer41

FRDA is known to be caused by an expansion of GAA repeat in intron 1 of the FXN gene leading to a reduced expression of FXN. The YG8R mouse model contains knocked-out mouse FXN genes and a human transgene from an FRDA patient. Thus, our hypothesis was that a pair of sgRNAs could be used to induce double-strand breaks before and after the GAA repeats in the YG8R mouse model to remove completely this long repeat and that this would increase FXN expression. Indeed, Li et al.42 have previously shown that it was possible to remove the GAA repeat with zinc-finger nucleases in FRDA fibroblasts and lymphoblasts. In our experiments, multiple sgRNA pairs allowed the removal of the GAA repeats, rendering the CRISPR-Cas9 system more versatile and convenient than the zinc-finger nucleases. These advantages could allow dealing with patient genomic variability in the intron sequences to be targeted. Zinc-finger nucleases, despite a robust efficacy, are not as convenient as the CRISPR-Cas system and their use in vivo might be challenging. For example, the zinc-finger nuclease gene size does not permit the packaging of a zinc-finger nuclease pair in a single AAV.

Our experiments were made in the YG8R and in the YG8sR models of FRDA developed by Dr Pook's group.29, 43, 44 We were able to correct the mutation, involving the GAA repeats, in both YG8R (Figure 1) and in YG8sR (Figure 44) mouse fibroblasts. However, the initial in vitro results in YG8R fibroblasts were surprising and disappointing since a reduced FXN expression rather than an increase was obtained. This surprising result is explained by the presence of two FRDA transgenes in tandem (one with about 82 GAA repeat and the other with about 190 GAA repeats) in the YG8R mouse genome. The sgRNA pairs were removing not only the GAA repeats but reconstitute, by non-homologous end-joining, one complete copy of the FXN gene. As only one functioning complete FXN transgene (including the promoter region) remained, that led to a reduced FXN expression compared with the untreated YG8R cells that had expression from two copies of the FXN gene. Although the expression from each of these transgene was reduced because of the GAA repeat, the total expression was nevertheless superior to that of the remaining corrected gene.

The YG8sR cells were derived from YG8R mice that lost one of the FRDA transgene during reproduction.29, 43 In the cells isolated from this new mouse model, we were able to shown that the removal of the GAA repeats by an sgRNA pair increases the expression of FXN transcript and protein. For further in vivo studies, somatic mosaicism of the FXN gene distribution in YG8sR mice should not impact the expected results of an increase of FXN protein in targeted tissues. However, cells lacking the FXN gene copy will not get any benefit of CRISPR-Cas9 edition. In this regard, the use in the future of the improved SpCas9 or SaCas9 might help reducing the putative off-targets associated with actual Cas9 proteins.

The YG8R mice have been used for testing pharmacological compounds that require human-specific FXN gene sequence to induce a FXN-increasing effect, such as potential RNA-based therapies.30 However, the YG8R has a rather late-onset and a mild phenotype, together with intergenerational GAA repeat variability. Therefore, the new YG8sR that contains a single-copy large GAA repeat expansion mutation may have a more severe earlier-onset phenotype. This mouse model is also better for gene-editing experiments because they contain only one FRDA transgene copy.

We also have demonstrated the excision of the GAA repeat in intron 1 using the CRISPR-Cas9 technology using plasmids expressing the SpCas9 and pair of sgRNAs. However, this experiment was carried out using electroporation, which can be used only for some limb muscles. Based on our in vitro preliminary data with the SpCas9 and SaCas9, we are going to investigate in the YG8sR mouse model the systemic delivery of AAVs using different sgRNAs and AAV serotypes. In vivo data will then allow us to verify whether the GAA edition increases the FXN protein expression enough to reduce or abrogate the symptoms associated with the FRDA in the YG8sR model.


Materials and methods

DNA constructs

Plasmids used in this study included the following: px330 as px330-U6-Chimeric_BB-CBh-hSpCas9 (Addgene plasmid no. 42230),45 pxGFP or pxPuro as pSpCas9(BB)-2A-GFP or Puro (Addgene plasmid no. 48138/48139)46 and px601 as px601-AAV-CMV::NLS-SaCas9-NLS-3xHA-bGHpA;U6::BsaI-sgRNA (Addgene plasmid no. 61591)32 and they were gifts from Feng Zhang (Department of Genetics, Harvard Medical School, Boston, MA, USA). Oligonucleotides coding for guide RNAs were synthetized by Integrated DNA Technologies (IDT Inc., Coralville, IA, USA) and cloned into BbsI (or BsaI for px601) restriction sites according to Zhang’s guidelines. All DNA constructs were sent for sequencing using the primer U6F (5′-GTCGGAACAGGAGAGCGCACGAGGGAG-3′) to the genomic sequencing and genotyping platform of the CHU de Quebec (Quebec City, QC, Canada).

When needed, PCR amplicons from plasmid DNA or gDNA were cloned into the linearized cloning vector pMiniT (NEB, Ipwisch, MA, USA) and sequenced using the manufacturer's instructions provided forward and reverse primers.

Modifications of the original px601 vector were performed as follows. The CMV promoter (577bp, located between XhoI and AgeI sites) was replaced by short versions of 212 or 259bp amplified from the pscAAV-GFP plasmid from John T Gray (Addgene plasmid no. 32396)47 and according to previous experimentations published by Senis et al.33 The px601 polyA sequence (204bp, included in the sequence between EcoRI and KpnI sites) was replaced by a short version of 60bp (ref. 33) cloned as a gBLOCK (IDT Inc.) while preserving the KpnI restriction site. A sequence comprising the H1m promoter, a selected cloned oligonucleotide gRNA coding and the SaCas9 tracrRNA was amplified from the home-made pGL3 H1m/BbsI/SaCas9 and cloned into the KpnI site of the newly prepared px601 vector. Finally, and if not previously included in the plasmid, the second oligonucleotide coding for a gRNA was cloned into the BsaI site following the U6 promoter.

All PCR amplifications were performed using the Phusion High Fidelity polymerase (Thermo Fisher Scientific Inc., Waltham, MA, USA). All cloning were performed using the In-Fusion HD Cloning Kit (Clontech Laboratories Inc., Mountain View, CA, USA). Plasmid design and sequencing analysis were carried out using the CLC main workbench software version 7.6 (CLC bio/Qiagen Inc., Hilden, Germany).

Mouse cells and animal model

Mouse fibroblasts derived from the YG8R and YG8sR mouse models were obtained from Dr Pook (Brunel University, London, UK). Characterization of these mice by Pook’s group revealed that the YG8R fibroblasts carried two tandem copies of the human FXN gene with about 82 and 190 GAA trinucleotide sequence repeats,26 whereas the YG8sR has a 190 GAA repeat. The Y47R cell line, which has been produced and isolated the same way as the YG8R contains, however, a single copy of the wild-type human FXN transgene, with about nine GAA trinucleotide repeats.26 The mouse fibroblasts were cultured at 37°C, 5% CO2 in high glucose Dulbecco's modified Eagle's medium (Wisent Inc., St-Bruno, QC, Canada) supplemented with 10% fetal bovine serum (GE Healthcare Life Sciences Inc., Mississauga, ON, Canada), 1mM sodium pyruvate, 1mM l-glutamine and 1 × non-essential amino acids (Wisent Inc.).

The mouse model YG8R (Fxntm1Mkn/Tg (FXN)YG8Pook/J)26 homozygous for the Fxntm1Mkn (FXN)-targeted allele and hemizygous for the Tg (FXN)YG8Pook (FXN, human) transgene were purchased from the Jackson Laboratory (Bar Harbor, ME, USA; stock number 012253).

Transfections and clonal expansion

Mouse YG8R or YG8sR fibroblasts were seeded and transfected at 70–80% confluence with DNA using Lipofectamine 2000 (Life Technologies Inc., Carlsbad, CA, USA) according to the manufacturer's instructions. Cells were harvested 48h later for DNA, RNA and protein analysis. For clonal expansion, puromycin (0.75μgml−1) was added 24h post-transfection, and 48h later, remaining cells were seeded in 96-well plates at 0.75 cells per well and expanded.

For some experiments, five hundred thousands (5 × 105) YG8sR were nucleofected using the Amaxa system (Lonza Inc., Walkersville, MD, USA) and program P-022 for normal human dermal adult fibroblasts (VPD-1001; Lonza Inc., Walkersville, MD, USA). Cells were harvested 72h later for gDNA or RNA transcriptional analysis. When needed, fluorescence from transfected cells was visualized using a Zeiss Axiovert 100-Inverted microscope (Zeiss Inc., Oberkochen, Germany).

In vivo DNA electrotransfer

The electrotranfer was performed in the TA of adult YG8R mice as described previously.48 Briefly, 40μg of DNA consisting of a mixture of two pxGFP plasmids (encoding for SpCas9 and two gRNAs) were electroporated into the TA muscle of YG8LR mice. The latters were killed 1 month later, TAs were collected and gDNA was extracted immediately or the TA was embedded in OTC and snap frozen in liquid nitrogen. PCR amplification was performed to detect deletions, according to the gRNA pair used. All experiences involving animals were approved by the animal care committee of the Centre Hospitalier Universataire de Québec-Université Laval (CHUQ-Université Laval, Québec, QC, Canada).

gDNA analysis

Cells or tissues (TA) were harvested, resuspended in lysis buffer (50mM EDTA (pH 8.0), 10% sarcosyl, 0.5mgml−1 proteinase K) and gDNA was extracted using a standard phenol/chloroform and ethanol precipitation method. The PCR was carried out using primer sequences provided in Supplementary Table S1. The conditions for PCR reactions, using the Phusion High Fidelity polymerase (Thermo Fisher Scientific Inc.) were as follows: 35 cycles, denaturation at 98°C for 10s, annealing at 60°C for 10s and elongation at 72°C for 90s. PCR products were visualized on agarose gel, and if needed, submitted to the Surveyor Assay (Integrated DNA Technology Inc., Coralville, IA, USA) according to the manufacturer’s instructions.

Copy number analysis

Oligoprimer pairs were designed by the GeneTool 2.0 software (Biotools Inc., Edmonton, AB, Canada) and their specificity was verified by blast in the GenBank database. The synthesis was performed by IDT Inc. (Supplementary Table S1).

Forty nanograms of gDNA was used to perform fluorescent-based real-time PCR quantification using the LightCycler 480 (Roche Diagnostics Inc., Mannheim, DE, USA). Reagent LightCycler 480 SYBRGreen I Master (Roche Diagnostics Inc.) was used as described by the manufacturer. The conditions for PCR reactions were: 45 cycles, denaturation at 98°C for 10s, annealing at 62°C for 10s, elongation at 72°C for 14s and reading for 5s. A melting-curve analysis was performed to assess the nonspecific signal. Relative quantity was calculated using the ΔCt method.49 Quantitative real-time PCR measurements were performed by the CHU de Québec Research Center (CHUL) Gene Expression Platform (Quebec, QC, Canada) and were compliant with MIQE guidelines.50, 51

RNA analysis

Cells were harvested, resuspended in Trizol and RNA was isolated. Total RNA was measured using a NanoDrop ND-1000 Spectrophotometer (NanoDrop Technologies Inc., Wilmington, DE, USA) and total RNA quality was assayed on an Agilent BioAnalyzer 2100 (Agilent Technologies Inc., Santa Clara, CA, USA).

First-strand cDNA synthesis was carried out using 500ng of isolated RNA in a reaction containing 200U of Superscript III Rnase H-RT (Invitrogen Life Technologies Inc., Burlington, ON, Canada), 300ng of oligo-dT18, 50ng of random hexamers, 50mM Tris-HCl (pH 8.3), 75mM KCl, 3mM MgCl2, 500μM deoxynucleotides triphosphate, 5mM dithiothreitol and 40U of Protector RNase inhibitor (Roche Diagnostics Inc.) in a final volume of 50μl. Reaction was incubated at 25°C for 10min, and then at 50°C for 1h and PCR Purification Kit (Qiagen Inc.) was used to purify cDNA.

cDNA corresponding to 20ng of total RNA was used to perform fluorescent-based real-time PCR quantification using the LightCycler 480 (Roche Diagnostics Inc.). Reagent LightCycler 480 SYBRGreen I Master (Roche Diagnostics Inc.) was used as described by the manufacturer with 2% dimethyl sulfoxide. The conditions for PCR reactions were: 45 cycles, denaturation at 95°C for 10s, annealing at 58°C for 10s, elongation at 72°C for 14s and then 74°C for 5s (reading) using primers described in Supplementary Table 1. A melting curve was performed to assess nonspecific signal. Calculation of the number of copies of each mRNA was performed using second derivative method and a standard curve of Cp versus logarithm of the quantity.52 The standard curve was established using known amounts of purified PCR products (10, 102, 103, 104, 105 and 106 copies) and a LightCycler 480 v.1.5 program provided by the manufacturer (Roche Diagnostics Inc.). The CHU de Québec Research Center (CR-CHUQ) Gene Expression Platform was used to perform quantitative real-time PCR measurements.

Protein analysis

Cells were harvested and resuspended in lysis buffer (137mM NaCl, 50mM Tris-HCl (pH 8) and 0.1% Triton X-100) supplemented with 1 × protease inhibitor cocktail (Roche Diagnostics Canada Inc., Mississauga, ON, Canada). Protein extracts were loaded onto 12.5% sodium dodecyl sulfate polyacrylamide gel electrophoresis and wet transfer was performed onto PVDF membrane. The latter was blotted using primary anti-FXN (ab110328 (Abcam Inc., Cambridge, UK) or sc-25820 (Santa Cruz Biotechnologies Inc., Santa Cruz, CA, USA), anti-HA (H-3663) anti-FLAG M2 (F-1804) and β-actin (A-1978) from Sigma-Aldrich Inc. (St Louis, MO, USA) antibodies. Mouse and rabbit secondary antibodies were purchased from Jackson Immuno Research Inc. (West Grove, PA, USA).


Conflict of interest

The authors declare no conflict of interest.



  1. Babady NE, Carelle N, Wells RD, Rouault TA, Hirano M, Lynch DR et al. Advancements in the pathophysiology of Friedreich's Ataxia and new prospects for treatments. Mol Genet Metab 2007; 92: 23–35. | Article | PubMed |
  2. Cooper JM, Schapira AH. Friedreich's ataxia: disease mechanisms, antioxidant and coenzyme Q10 therapy. Biofactors 2003; 18: 163–171. | Article | PubMed | ISI | CAS |
  3. Harding AE. Friedreich's ataxia: a clinical and genetic study of 90 families with an analysis of early diagnostic criteria and intrafamilial clustering of clinical features. Brain 1981; 104: 589–620. | Article | PubMed | ISI | CAS |
  4. Lynch DR, Farmer JM, Balcer LJ, Wilson RB. Friedreich ataxia: effects of genetic understanding on clinical evaluation and therapy. Arch Neurol 2002; 59: 743–747. | Article | PubMed |
  5. Pandolfo M. Molecular pathogenesis of Friedreich ataxia. Arch Neurol 1999; 56: 1201–1208. | Article | PubMed | ISI | CAS |
  6. Pandolfo M. Friedreich ataxia: the clinical picture. J Neurol 2009; 256 (Suppl. 1): 3–8. | Article | PubMed | ISI | CAS |
  7. Pandolfo M. Friedreich ataxia. In: Subramony SH, Dürr A (eds). Handbook of Clinical Neurology, vol. 103 (3rd series), Chapter 17. Elsevier B.V: The Netherlands, 2012, pp 275–294.
  8. Campuzano V, Montermini L, Molto MD, Pianese L, Cossee M, Cavalcanti F et al. Friedreich's ataxia: autosomal recessive disease caused by an intronic GAA triplet repeat expansion. Science 1996; 271: 1423–1427. | Article | PubMed | ISI | CAS |
  9. Pandolfo M. The molecular basis of Friedreich ataxia. Adv Exp Med Biol 2002; 516: 99–118. | PubMed | CAS |
  10. Campuzano V, Montermini L, Lutz Y, Cova L, Hindelang C, Jiralerspong S et al. Frataxin is reduced in Friedreich ataxia patients and is associated with mitochondrial membranes. Hum Mol Genet 1997; 6: 1771–1780. | Article | PubMed | ISI | CAS |
  11. Pandolfo M. Iron and Friedreich ataxia. J Neural Transm Suppl 2006; 70: 143–146. | PubMed | CAS |
  12. Coppola G, Choi SH, Santos MM, Miranda CJ, Tentler D, Wexler EM et al. Gene expression profiling in frataxin deficient mice: microarray evidence for significant expression changes without detectable neurodegeneration. Neurobiol Dis 2006; 22: 302–311. | Article | PubMed |
  13. Coppola G, Marmolino D, Lu D, Wang Q, Cnop M, Rai M et al. Functional genomic analysis of frataxin deficiency reveals tissue-specific alterations and identifies the PPARgamma pathway as a therapeutic target in Friedreich's ataxia. Hum Mol Genet 2009; 18: 2452–2461. | Article | PubMed | ISI |
  14. Gerber J, Muhlenhoff U, Lill R. An interaction between frataxin and Isu1/Nfs1 that is crucial for Fe/S cluster synthesis on Isu1. EMBO Rep 2003; 4: 906–911. | Article | PubMed | ISI | CAS |
  15. Perdomini M, Belbellaa B, Monassier L, Reutenauer L, Messaddeq N, Cartier N et al. Prevention and reversal of severe mitochondrial cardiomyopathy by gene therapy in a mouse model of Friedreich's ataxia. Nat Med 2014; 20: 542–547. | Article | PubMed | CAS |
  16. Wiedenheft B, Sternberg SH, Doudna JA. RNA-guided genetic silencing systems in bacteria and archaea. Nature 2012; 482: 331–338. | Article | PubMed | ISI | CAS |
  17. Bhaya D, Davison M, Barrangou R. CRISPR-Cas systems in bacteria and archaea: versatile small RNAs for adaptive defense and regulation. Annu Rev Genet 2011; 45: 273–297. | Article | PubMed | ISI | CAS |
  18. Terns MP, Terns RM. CRISPR-based adaptive immune systems. Curr Opin Microbiol 2011; 14: 321–327. | Article | PubMed | ISI | CAS |
  19. Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE et al. RNA-guided human genome engineering via Cas9. Science 2013; 339: 823–826. | Article | PubMed | ISI | CAS |
  20. Mojica FJ, Diez-Villasenor C, Garcia-Martinez J, Almendros C. Short motif sequences determine the targets of the prokaryotic CRISPR defence system. Microbiology 2009; 155: 733–740. | Article | PubMed | ISI | CAS |
  21. He Z, Proudfoot C, Mileham AJ, McLaren DG, Whitelaw BA, Lillico SG. Highly efficient targeted chromosome deletions using CRISPR/Cas9. Biotechnol Bioeng 2014; 112: 1060–1064 (online). | Article | PubMed |
  22. Byrne SM, Ortiz L, Mali P, Aach J, Church GM. Multi-kilobase homozygous targeted gene replacement in human induced pluripotent stem cells. Nucleic Acids Res 2014; 43: e21. | Article | PubMed |
  23. Slaymaker IM, Gao L, Zetsche B, Scott DA, Yan WX, Zhang F. Rationally engineered Cas9 nucleases with improved specificity. Science 2015; 351: 84–88. | Article | PubMed | CAS |
  24. Kleinstiver BP, Pattanayak V, Prew MS, Tsai SQ, Nguyen NT, Zheng Z et al. High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects. Nature 2016; 529: 490–495. | Article | PubMed | CAS |
  25. Pook MA, Al-Mahdawi S, Carroll CJ, Cossee M, Puccio H, Lawrence L et al. Rescue of the Friedreich's ataxia knockout mouse by human YAC transgenesis. Neurogenetics 2001; 3: 185–193. | PubMed | ISI | CAS |
  26. Al-Mahdawi S, Pinto RM, Ruddle P, Carroll C, Webster Z, Pook M. GAA repeat instability in Friedreich ataxia YAC transgenic mice. Genomics 2004; 84: 301–310. | Article | PubMed | ISI | CAS |
  27. Al-Mahdawi S, Pinto RM, Varshney D, Lawrence L, Lowrie MB, Hughes S et al. GAA repeat expansion mutation mouse models of Friedreich ataxia exhibit oxidative stress leading to progressive neuronal and cardiac pathology. Genomics 2006; 88: 580–590. | Article | PubMed | ISI | CAS |
  28. Virmouni SA, Ezzatizadeh V, Sandi C, Sandi M, Al-Mahdawi S, Chutake Y et al. A novel GAA repeat expansion-based mouse model of Friedreich ataxia. Dis Models Mech 2015; 8: 225–235. | Article |
  29. Anjomani Virmouni S, Ezzatizadeh V, Sandi C, Sandi M, Al-Mahdawi S, Chutake Y et al. A novel GAA-repeat-expansion-based mouse model of Friedreich's ataxia. Dis Model Mech 2015; 8: 225–235. | Article | PubMed |
  30. Anjomani Virmouni S, Sandi C, Al-Mahdawi S, Pook MA. Cellular, molecular and functional characterisation of YAC transgenic mouse models of Friedreich ataxia. PLoS One 2014; 9: e107416. | Article | PubMed | CAS |
  31. Virmouni SA. Genotype and Phenotype Characterisation of Friedreich Ataxia Mouse Models and Cells. Brunel University London Library: Uxbridge, UK, 2013.
  32. Ran FA, Cong L, Yan WX, Scott DA, Gootenberg JS, Kriz AJ et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 2015; 520: 186–191. | Article | PubMed | CAS |
  33. Senis E, Fatouros C, Grosse S, Wiedtke E, Niopek D, Mueller AK et al. CRISPR/Cas9-mediated genome engineering: an adeno-associated viral (AAV) vector toolbox. Biotechnol J 2014; 9: 1402–1412. | Article | PubMed | ISI | CAS |
  34. Long C, Amoasii L, Mireault AA, McAnally JR, Li H, Sanchez-Ortiz E et al. Postnatal genome editing partially restores dystrophin expression in a mouse model of muscular dystrophy. Science 2016; 351: 400–403. | Article | PubMed | CAS |
  35. Nelson CE, Hakim CH, Ousterout DG, Thakore PI, Moreb EA, Castellanos Rivera RM et al. In vivo genome editing improves muscle function in a mouse model of Duchenne muscular dystrophy. Science 2016; 351: 403–407. | Article | PubMed | CAS |
  36. Tabebordbar M, Zhu K, Cheng JK, Chew WL, Widrick JJ, Yan WX et al. In vivo gene editing in dystrophic mouse muscle and muscle stem cells. Science 2016; 351: 407–411. | Article | PubMed | CAS |
  37. Xu L, Park KH, Zhao L, Xu J, El Refaey M, Gao Y et al. CRISPR-mediated genome editing restores dystrophin expression and function in mdx mice. Mol Ther 2016; 24: 564–569. | Article | PubMed | CAS |
  38. Iyombe-Engembe JP, Ouellet DL, Rousseau J, Chapdelaine P, Tremblay JP. Efficient restoration of the dystrophin gene reading frame and protein structure in DMD myoblasts using the CinDel Method. Mol Ther Nucleic Acid Res 2016; 5: e283. | Article |
  39. Courtney DG, Moore JE, Atkinson SD, Maurizi E, Allen EH, Pedrioli DM et al. CRISPR/Cas9 DNA cleavage at SNP-derived PAM enables both in vitro and in vivo KRT12 mutation-specific targeting. Gene Therapy 2016; 23: 108–112. | Article | PubMed |
  40. Yin H, Song CQ, Dorkin JR, Zhu LJ, Li Y, Wu Q et al. Therapeutic genome editing by combined viral and non-viral delivery of CRISPR system components in vivo. Nat Biotechnol 2016; 34: 328–333. | Article | PubMed | CAS |
  41. Sachdeva M, Sachdeva N, Pal M, Gupta N, Khan IA, Majumdar M et al. CRISPR/Cas9: molecular tool for gene therapy to target genome and epigenome in the treatment of lung cancer. Cancer Gene Ther 2015; 22: 509–517. | Article | PubMed |
  42. Li Y, Lu Y, Polak U, Lin K, Shen J, Farmer J et al. Expanded GAA repeats impede transcription elongation through the FXN gene and induce transcriptional silencing that is restricted to the FXN locus. Hum Mol Genet 2015; 24: 6932–6943. | PubMed | CAS |
  43. Chutake YK, Costello WN, Lam CC, Parikh AC, Hughes TT, Michalopulos MG et al. FXN promoter silencing in the humanized mouse model of Friedreich ataxia. PLoS One 2015; 10: e0138437. | Article | PubMed |
  44. Sandi C, Pinto RM, Al-Mahdawi S, Ezzatizadeh V, Barnes G, Jones S et al. Prolonged treatment with pimelic o-aminobenzamide HDAC inhibitors ameliorates the disease phenotype of a Friedreich ataxia mouse model. Neurobiol Dis 2011; 42: 496–505. | Article | PubMed | CAS |
  45. Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N et al. Multiplex genome engineering using CRISPR/Cas systems. Science 2013; 339: 819–823. | Article | PubMed | ISI | CAS |
  46. Ran FA, Hsu PD, Wright J, Agarwala V, Scott DA, Zhang F. Genome engineering using the CRISPR-Cas9 system. Nat Protocols 2013; 8: 2281–2308. | Article | PubMed | CAS |
  47. Gray JT, Zolotukhin S. Design and construction of functional AAV vectors. Methods Mol Biol 2011; 807: 25–46. | PubMed |
  48. Pichavant C, Chapdelaine P, Cerri DG, Bizario JC, Tremblay JP. Electrotransfer of the full-length dog dystrophin into mouse and dystrophic dog muscles. Hum Gene Ther 2010; 21: 1591–1601. | Article | PubMed | ISI |
  49. Pfaffl MW. A new mathematical model for relative quantification in real-time RT-PCR. Nucleic Acids Res 2001; 29: e45. | Article | PubMed | CAS |
  50. Bustin SA, Benes V, Garson JA, Hellemans J, Huggett J, Kubista M et al. The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 2009; 55: 611–622. | Article | PubMed | ISI | CAS |
  51. Bustin SA, Beaulieu JF, Huggett J, Jaggi R, Kibenge FS, Olsvik PA et al. MIQE precis: practical implementation of minimum standard guidelines for fluorescence-based quantitative real-time PCR experiments. BMC Mol Biol 2010; 11: 74. | Article | PubMed |
  52. Luu-The V, Paquet N, Calvo E, Cumps J. Improved real-time RT-PCR method for high-throughput measurements using second derivative calculation and double correction. Biotechniques 2005; 38: 287–293. | Article | PubMed | ISI | CAS |
  53. Chapdelaine P, Coulombe Z, Chikh A, Gerard C, Tremblay JP. A potential new therapeutic approach for friedreich ataxia: induction of frataxin expression with TALE proteins. Mol Ther Nucleic Acids 2013; 2: e119. | Article | PubMed | CAS |


This work was supported by grants to JPT from the The Cell Network, Ataxia Canada Foundation and Association Française de l’Ataxie de Friedreich.

Supplementary Information accompanies this paper on Gene Therapy website