1. Introduction
Fanconi anemia (FA) is an inherited genetic disorder characterized by chromosomal instability, bone marrow failure (BMF), congenital malformations, and early cancer onset [
1,
2,
3]. FA is caused by perturbations to one of the ~21 described genes that participate in interstrand cross link DNA lesion repair [
2]. As a monogenic disorder FA represents an ideal candidate for phenotypic rescue by gene therapy or gene editing. A key consideration, in the pre-malignant FA phenotype, is safety of the intervention. Random or semi-random integration of gene therapy vehicles [
4,
5,
6,
7,
8] with unregulated gene expression may be contraindicated in FA. The ability to modify genomic sequences in a precise and targeted manner represents a powerful approach for individualized translational medicine. Programmable nucleases (zinc-finger nucleases (ZFN) [
9], transcription activator-like effector nucleases (TALENs) [
10,
11] or clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 [
12,
13]) have proven to be extremely useful for disrupting clinically relevant genes or correcting disease-causing mutations. Previous efforts for precision genome engineering in FA have been described for the Fanconi anemia A, C, and I gene mutations, respectively [
14,
15,
16]. Common to each of these studies was the requirement of transformative factors such as telomerase reverse transcriptase expression [
14] or pluripotency reprogramming genes [
16]. To determine whether the FA phenotype could be corrected in true primary cells we undertook efforts to target the
FANCD1 gene in fibroblasts derived from an FA patient.
FANCD1, also known as the breast cancer 2 gene (
BRCA2), functions as a downstream effector of the DNA repair pathway where it binds and stabilizes RAD51-nucleoprotein filaments at the site(s) of DNA breaks [
17]. This is a crucial prerequisite for DNA damage repair by homologous recombination [
18,
19]. Compromisation of this pathway results in a severe form of FA with significantly earlier onset of the disease manifestations and heightened incidence of brain tumors, nephroblastoma, and leukemia [
20,
21]. The disease phenotype, with extension to other tumors with impaired
BRCA activity, shows a sensitivity to poly(ADP-ribose) polymerase (PARP) inhibitors [
22]. We hypothesized that genome editing along with PARP inhibition (PARPi) would allow us to selectively recover gene modified cells. To accomplish this we delivered CRISPR/Cas9 reagents and an oligonucleotide donor molecule to primary
FANCD1 fibroblasts and, following PARPi treatment, obtained six genotypically and functionally corrected clones. To our knowledge, this is the first report of a causal gene correction by gene editing in FA patient primary fibroblasts.
3. Discussion
We report here the first demonstration of gene editing of FA primary cells and the selection of a homogenous population of gene edited clones using PARP inhibitors. To date, the
FANCA,
FANCC, and
FANCI genes have been targeted for correction with ZFNs, TALENs, or CRISPR/Cas9 [
14,
15,
16]. A requirement for successful gene editing in each of these studies was a requirement for exogenous expression of telomerase reverse transcriptase (
TERT) or pluripotency inducing reprogramming factors [
14,
15,
16]. Our previous work for
FANCC gene correction required lentiviral transduction of patient cells with a human TERT (hTERT) construct [
15]. This allowed for clonal derivation of CRISPR/Cas9 modified cells using a donor that contained a puromycin selection marker. The combination of h
TERT and puromycin facilitated obtainment of homogenous clones without senescence while parallel studies in
FANCC cells lacking h
TERT showed high levels of senescence. These previous studies required subsequent removal of the puromycin cassette representing a second gene transfer and clonal isolation/screening step. As an elegant methodology to maximize the benefits of h
TERT expression, Rio and colleagues utilized cre recombinase excisable cassettes in
FANCA cells such that transient
TERT could be employed to achieve gene editing with subsequent removal [
14]. Further, their floxed reprogramming gene construct could also be selectively removed. However, similar to our necessity of further modification to remove puromycin their strategy also required cre recombinase addition in order to excise the foreign transgene [
14]. To extend these foundational studies we undertook efforts in
FANCI deficient cells to introduce non-integrating reprogramming factors and a donor reliant on gene correction and application of the phenotypic selective marker MMC [
16]. Similar to
FANCA and
FANCC fibroblasts the primary
FANCI fibroblasts were recalcitrant to gene correction and clonal derivation [
16]. In contrast, pluripotent cells obtained using Sendai viral reprogramming were amenable to gene correction and selection with MMC [
16]. The use of MMC; however, impaired the stem cell potential of the cells mandating our use of a floxed puromycin selection marker [
16]. Collectively these studies show the ability of FA cells to undergo gene targeting using programmable reagents; however, a persistent hurdle has been primary cell correction with a selection strategy using a clinically viable reagent.
To address the lack of correction of FA primary cells in the literature we implemented a line of study using
FANCD1 deficient cells. The choice of FA pathway compromisation was carefully considered and
FANCD1 is a severe form of FA as well as is a common gene that is mutated in multiple tumors [
29]. The clinical impact of this gene is significant and we developed a CRISPR/Cas9 reagent to target a dinucleotide deletion in exon 8 (
Figure 1). We observed robust activity and the proximity of the CRISPR/Cas9 site to the mutation allowed us to employ an oligonucleotide (ODN) donor-based strategy. Sense or anti-sense versions of the ODN donor were tested and showed that the sense configuration mediated higher gene correction rates than the anti-sense counterpart (
Figure 3A and
Figure S2). This strand preference is in keeping with previous reports [
30,
31] and the ability to achieve HDR from an ODN template is highly significant. In the context of FA, integrase deficient virus, adenoassociated virus, or plasmid DNA have been employed for gene correction [
14,
15,
16]. Viral based vectors require production using packaging cell lines and plasmids that can be laborious, non-uniform in regards to titer/amount produced, and are associated with significant costs for clinical scale up and application. Likewise, plasmid DNA requires specialized production for translational application and random integrants can result in genomic disruption. In contrast, the GMP production of ODNs is favorable both in cost and scale and the addition of phosphorothioate modifications can increase ODN stability such that more ODN template persists for serving as an HDR template. In our donor design strategy, we incorporated silent mutations to prevent nuclease re-cutting of the modified locus, the GT dinucleotide that is deleted in the
FANCD1 parental population, and introduced a restriction enzyme site to facilitate better detection of gene modified loci (
Figure S3). Under these conditions we observed HDR (
Figure 2 and
Figure 3) in a polyclonal population of cells that underwent selection. The selective pressure consisted of puromycin, olaparib, or KU0058948. Because the pX459 plasmid contains a puromycin resistance gene we transiently selected with puromycin to enrich for cells that underwent successful gene transfer. Selection by this method has been shown to promote modified cell outgrowth [
29]; however, we did not see increased rates of HDR following this transient treatment (
Figure 3A). Rather, sequencing of the
FANCD1 locus revealed insertions/deletions consistent with non-homologous endjoining-based repair (data not shown). Olaparib is a widely employed PARP inhibitor and is employed for tumor therapy due to its selective lethality in BRCA defective cells [
32]. Normally PARP-1 is activated by DNA breaks resulting in progressive addition of ADP-ribose polymeric scaffolds that recruit mediators of DNA break repair [
33]. We hypothesized that olaparib or use of its analog KU58948 would selectively deplete uncorrected, BRCA deficient
FANCD1 cells and enrich for HDR corrected events. Indeed, we were able to select and derive seven clones, only one of which underwent senescence (
Figure 3). The remaining six clones showed the ability to proliferate in the presence of DNA damaging agents (
Figure 4A). In contrast, previous work using MMC as a selection agent showed significant senescence and concomitant impairment of cellular function [
15,
16]. Toward determining whether the derivative clones were phenotypically restored we performed Western blot analysis on cytosolic and nuclear fractions for RAD51 following DNA damage induction with MMC. RAD51 plays a key role in DNA break repair and its nuclear localization is dependent on FANCD1 [
34]. In uncorrected, parental cells we observed cytoplasmic sequestration in the absence or presence of MMC (
Figure 4B). Each of the six corrected clones showed nuclear translocation of RAD51 (
Figure 4B). Taken together these data show the usefulness of PARP inhibition as a clinically viable selection agent for the recovery of gene corrected cells that are phenotypically rescued. Follow on studies will address the effect PARPi has on the cellular phenotype in translational engineering in HSPC and induced pluripotent stem cells (iPSC). The effect of PARPi on HSPC in vivo is of great interest particularly in light of recent studies [
35] showing that malignant transformation of HSPC results in greater rates of DNA damage and potential sensitivity to PARPi. This suggests a possibility of genome modified HSPCs surviving and expanding in the presence of PARPi that will also confer added benefit by depleting transformed HSPC and their progeny.
An important consideration, particularly in DNA damage repair defective disorders, is the specificity of DNA break induction mediated by a nuclease. To assess this, we rank ordered the potential off target sites using an in silico predictive algorithm [
34,
36]. Using the Surveyor method, we assessed whether promiscuous nuclease activity was prevalent. None of the eight predicted off target sites showed evidence of ectopic Cas9 activity strongly suggesting a highly specific reagent (
Table 1,
Figure 5 and
Figure S4).
Because bone marrow failure is a life-threatening complication of FA the current standard of care is allogeneic hematopoietic cell transplant. Despite advances in improving outcomes for allogeneic recipients in regards to the source of the graft and the conditioning regimen, severe side effects can still occur [
37,
38,
39]. The ultimate goal is to engineer hematopoietic stem and progenitor cells (HSPC) for autologous ex vivo therapy. Current lentiviral gene therapy phase I clinical trials show that the intervention is well tolerated in regards to safety (
n = 2 patients in NCT01331018); however, the number of transduced cells rapidly diminishes in the periphery [
40,
41]. Moreover, the potential for vector insertional mutagenesis [
42,
43], clonal dominance [
44], and unregulated expression of a DNA repair protein with the potential for apoptotic resistance [
45] in the FA pre-malignant phenotype makes gene editing highly desirable. We were able to effectively modify primary cells as part of an autologous strategy. Because of the limiting numbers of HSPC in FA patients the use of them for pre-clinical optimization is limiting. Therefore, the choice of cell population for reagent development, optimization, and deployment is crucial. We have employed both fibroblasts and iPSC for FA gene editing modeling. Due to their increased cell division rates, iPSC can undergo high rates of HDR; however many FA complementation group defects show a poor ability to undergo reprogramming in the absence of a functional FA repair pathway [
46].
FANCD1 cells were not analyzed in this previous study [
46] but given the dramatically lower rates of reprogramming efficiency in FA compromised cells the ability to correct primary fibroblasts is highly significant. Their low replicative capacity, gene transfer and HDR rates more closely mimic HSPC than do iPSC making them a relevant platform for modeling gene editing in support of extension to HSPC. Further, new avenues in cellular engineering show the potential of iPSC to serve as a platform for hematopoietic progenitor development [
47] making fibroblasts a decidedly relevant tool of discovery for downstream regenerative approaches.
In sum, nuclease-based modification of the genome in FA along with selection promotes obtainment of corrected cells. Importantly, cells treated with PARPi alone did not result in spontaneous correction of the FA gene. Revertant mosaicism has been observed in FA [
48,
49,
50,
51] raising the possibility of applying selective pressure without need of nucleases for modification. However, the time in which it takes for spontaneous correction to occur may be offset by the corresponding accumulation of mutations at other loci. In contrast, nucleases promote dramatically higher HDR rates compared to their absence [
52,
53] and accomplish gene repair in a highly specific manner. We propose that this represents an ideal strategy for the accelerated generation of normalized cells such that genomic insults that are manifest or acquired via the cell derivation, modification, and expansion process are minimized. A corresponding consideration is the ability to construct an allele specific reagent that would allow for use of the normal portion of the opposite allele in the context of a compound heterozygote as part of a “natural” donor template. In our current study the mutation site does not allow for preferential recognition by CRISPR/Cas9 because the nuclease target site is outside of the mutation site (
Figure 1). Future studies will assess the ability of CRISPR/Cas9 to target mutated alleles and can employ PAM variant Cas9 candidates [
54,
55] in order to overlap the mutation and nuclease sequences in order to confer allele specificity. Such an approach is intriguing as it may obviate the need for an exogenous donor.
4. Materials and Methods
4.1. Patient Samples
The Declaration of Helsinki requirements for research on human subjects was followed and approval of the University of Minnesota Institutional Review Board Human Subjects Committee was granted (authorization designator: 1301M26601). Following this, and with informed consent, a skin punch biopsy from a patient with FANCD1 gene mutations was obtained (
Figure 1A). Fibroblasts from human healthy dermal neonatal fibroblasts and embryonic kidney 293T cell line were purchased from American Type Culture Collection (ATCC; Manassas, VA, USA).
4.2. Culture Conditions
293T cells were cultured in Dulbecco’s Modified Eagle‘s Medium with 10% fetal bovine serum (FBS), Glutamax (2 mM) and penicillin/streptomycin (0.1 mg/mL each). Fibroblasts were cultured in Minimum Essential Medium Eagle (Millipore-Sigma, St. Louis, MO, USA) with 20% FBS, nonessential amino acids (100 U/mL), Glutamax (2 mM), penicillin/streptomycin (0.1 mg/mL each), epidermal growth factor and fibroblast growth factor (10 ng/mL each), and antioxidant supplement (all Thermo Fisher Scientific, Waltham, MA, USA or Millipore-Sigma, St. Louis, MO, USA). Cells were maintained at 37 °C and 5% CO2; FA fibroblasts at low oxygen conditions (2% O2).
4.3. CRISPR/Cas9 Design
A guide RNA of the CRISPR/Cas9 nuclease was synthesized as a ssDNA oligonucleotide and cloned into pSpCas9(BB)-2A-Puro (PX459) V2.0 (a gift from Feng Zhang; Addgene plasmid # 62988) by Gibson assembly [
56]. The resulting CRISPR/Cas9 plasmid was transfected into 293T cells by Lipofectamine 3000 (Thermo Fisher Scientific, Waltham, MA, USA) and harvested 72 h later. DNA from 293T cells was PCR-amplified with primers covering CRISPR/Cas9 cut site: followed by Surveyor nuclease assay (IDT, Coralville, IA, USA) performed per the manufacturer′s guidelines.
4.4. Gene Editing in Primary Cells
CRISPR-Cas9 plasmid (1 µg) and corrective donors synthesized as ssDNA oligonucleotides (10 ng each; IDT) were electroporated into fibroblasts by Neon transfection system (1500 V, 1 pulse, 20 ms width; Thermo Fisher Scientific, Waltham, MA, USA).
4.5. Selection
Selection of the gene-edited population was done either 24 h post electroporation by puromycin (for 72 h; 0.4, 0.7 and 1.1 µM; EMD Millipore, Billerica, MA, USA) or 72 h post electroporation by PARPi (for 7 days; olaparib (0.1, 1 µM; ApexBio, Houston, TX, USA), KU0058948 (0.1, 1 µM; Axon Medchem, Reston, VA, USA)). Monoclonal populations were prepared by plating the gene-edited cells at low density (300 cells per 10-cm dish). Forty-eight hours later, glass-cloning cylinders were placed around single-cell colonies using sterile silicon grease. Clonal populations were progressively transferred into larger wells/flasks. Selected cells were analyzed by PCR. For HDR donor specific detection: F: 5′-TGAAGAAGCTTCCGAAACCG-3′ and R: 5′-GCCACACAGTGCACCATAGA-3′. For locus amplification and subsequent donor detection by HindIII digest the following primers were used: PCR F: 5′-CACCAAGCCATATCTTACCACC-3′, PCR R: 5′-ACAGCAGAGTTTCACAGGAAGT-3′. PCR was performed at: 95 °C × 2 min, 40 cycles of 95 °C × 45 s, 57 °C × 45 s, and 68 °C × 1 min.
4.6. Off-Target Analysis
CRISPR/Cas9 of target cutting sites were identified using the MIT CRISPR Design tool and the CRISPOR tool [
57]. Lipofectamine-based gene transfer of the FANCD1 gRNA and Cas9 in FANCD1 corrected clones and 293 cells were utilized for analysis for on and off target activity using the primers below and amplification with AccuPrime DNA polymerase (ThermoFisher) at: 95 °C × 2 min and 35 cycles of 95 °C × 40 min, 58.5 °C × 40 s, 68 °C × 1 min. Surveyor assay was then performed using amplicons generated with the below primers (shown 5′–3′) followed by PAGE electrophoresis.
FANCD1 surveyor F | AAACTTTATCACAGGGTATGTGCTT |
FANCD1 surveyor R | CAGCATCATCTGACTTTCCAA |
FANCD1 OT 1 Surveyor F | CCAGACCAGAAACCGAAAAA |
FANCD1 OT 1 Surveyor R | TGGCAGTTTGTCCATTTGAA |
FANCD1 OT 2 Surveyor F | CATCCTGAAAAATGATGGGATT |
FANCD1 OT 2 Surveyor R | ATCTTCCTCCCTTCCTCCTG |
FANCD1 OT 3 Surveyor F | CCCCCAACTACATTCGAAAA |
FANCD1 OT 3 Surveyor R | AATTTGGTGGGTTCTACTTGTTT |
FANCD1 OT 4 Surveyor F | TGAACGTCAGAAGGGCTAGAA |
FANCD1 OT 4 Surveyor R | GACGTCAAGGTTGCAGTGAA |
FANCD1 OT 5 Surveyor F | GGCCAGTGGTTCTCAACTTT |
FANCD1 OT 5 Surveyor R | TGTTCCCATGAGTTTTGTGG |
FANCD1 OT 6 Surveyor F | ACAAACTGCCGAACAAGAGG |
FANCD1 OT 6 Surveyor R | AGGCTGAGTGGTACTCCATTG |
FANCD1 OT 7 Surveyor F | TTGTGAATGAGGTGAGATGAGG |
FANCD1 OT 7 Surveyor R | GATCTTGGCTCACTGCAACC |
FANCD1 OT 8 Surveyor F | CATATTGTCTGGGTGCCACA |
FANCD1 OT 8 Surveyor R | TCACCACAACCCCATAAAGC |
4.7. FANCD1 Functional Assay
Nuclear and cytoplasmic protein fractions from cells cultured with Mitomycin C (7 µM for 1 h followed by 18 h of recovery) were separated by gel electrophoresis, transferred to Nitrocellulose membrane and incubated overnight with the anti-FANCD1 antibody (51RAD01 (3C10), 1:200, Thermo-Fisher Scientific, Waltham, MA, USA). The purity of protein fractions was confirmed by staining with anti α-Tubulin (TU-01, 1:1000, Exbio, Vestec, Czech Republic) and anti Lamin-B1 (sc-6216, 1:200, Santa Cruz Biotechnology, Dallas, TX, USA) antibodies.
4.8. Traffic Light Reporter Assay
Traffic light reporter assay was performed as previously described [
15,
24] following delivery of equimolar amounts of an ODN or dsDNA GFP repair template and CRISPR/Cas9 nuclease.
4.9. Positive and Negative Strand Oligonucleotide Donor Assessment
Cas9 nuclease/gRNA plasmid and ssODNs for sense or anti-sense (sequence below) were delivered to 293T cells by lipofection. At 48 h a three primer PCR was performed using the locus specific primers: F: 5′-CACCAAGCCATATCTTACCACC-3′, PCR R: 5′-ACAGCAGAGTTTCACAGGAAGT-3′ and a donor specific primer: 5′-GGATCCAAGCTTCGTCGACCTAGCC-3′
Sense donor. 5′-TTGCATTCTAGTGATAATATACAATACACATAAATTTTTATCTTACAGTCAGAAATGAAGAAGCATCTGAAACTGTATTTGTACGGATCCAAGCTTCGTCGACCTAGCCTAAATATGACATTGATTAGACTGTTGAAATTGCTAACAATTTTGGAATGCCTTGTTAAATTATTTATCTTACATTTTTAA-3′
Anti-sense donor. 5′-TTAAAAATGTAAGATAAATAATTTAACAAGGCATTCCAAAATTGTTAGCAATTTCAACAGTCTAATCAATGTCATATTTAGGCTAGGTCGACGAAGCTTGGATCCGTACAAATACAGTTTCAGATGCTTCTTCATTTCTGACTGTAAGATAAAAATTTATGTGTATTGTATATTATCACTAGAATGCAA-3′. Bold underlined sequences show the portion recognized by the donor primer.
4.10. Graphics
Illustrations were generated using templates from Motifolio Inc. (Ellicott City, MD, USA).