Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing
© Robert et al. 2015
Received: 29 May 2015
Accepted: 6 August 2015
Published: 27 August 2015
The ability to modify the genome of any cell at a precise location has drastically improved with the recent discovery and implementation of CRISPR/Cas9 editing technology. However, the capacity to introduce specific directed changes at given loci is hampered by the fact that the major cellular repair pathway that occurs following Cas9-mediated DNA cleavage is the erroneous non-homologous end joining (NHEJ) pathway. Homology-directed recombination (HDR) is far less efficient than NHEJ and makes screening of clones containing directed changes time-consuming and labor-intensive.
We investigated the possibility of pharmacologically inhibiting DNA-PKcs, a key player in NHEJ, using small molecule inhibitors (NU7441 and KU-0060648), to ameliorate the rates of HDR repair events. These compounds were tested in a sensitive reporter assay capable of simultaneously informing on NHEJ and HDR, as well as on an endogenous gene targeted by Cas9.
We find that NU7441 and KU-0060648 reduce the frequency of NHEJ while increasing the rate of HDR following Cas9-mediated DNA cleavage.
Our results identify two small molecules compatible for use with Cas9-editing technology to improve the frequency of HDR.
The bacterial innate immune CRISPR (clustered regularly interspaced short palindromic repeat) system has emerged as a powerful molecular tool for genome engineering [1–4]. The key components of this system are a Cas9 endonuclease and a bifunctional single guide (sg) RNA. The sgRNA binds a DNA target site through sequence complementarity with the first approximately 20 5’ nucleotides whereas a 3’ aptameric domain is responsible for recruiting Cas9 to the genomic address . The presence of an 5’NGG3’ protospacer adjacent motif (PAM) located immediately 3’ of the target sequence complement is the only essential feature of the target recognition site . Cas9 will generate double-stranded breaks (DSB) at the target site which are repaired by the erroneous non-homologous end-joining (NHEJ) pathway to introduce indels (insertions/deletions) or if an appropriate target-homologous donor template is supplied in trans, by homology directed-repair (HDR) [6, 7].
NHEJ is initiated by a DSB leading to recruitment of a Ku70/80 heterodimer and the catalytic subunit of DNA-dependent protein kinase (DNA-PKcs). This tethers the two ends and a series of subsequent reactions result in resection, extension, and ligation . In HDR, the DSB break is processed to a 3’ single-stranded overhang and a different set of repair proteins stimulate strand invasion by a donor homologous template, followed by repair . In general, NHEJ is the more frequently observed repair pathway when using Cas9-mediated genome engineering, even in the presence of a donor HDR template [9–12]. Identifying cells of interest following HDR-directed editing is a labor-intensive and time-consuming process involving screening of individual clones to identify appropriate, correctly modified cells.
Improving HDR efficiency would significantly reduce the downstream workload involved in identifying appropriate clones of interest. Cell lines deficient for NHEJ components have significantly elevated levels of HDR suggesting competition between these two repair processes . Hence suppression of the NHEJ key enzymes Ku70, Ku80, or DNA Ligase IV has been shown to stimulate Cas9-mediated HDR at the expense of NHEJ [14, 15]. A second strategy takes advantage of the fact that NHEJ occurs throughout the cell cycle whereas HDR-mediated repair is temporally restricted to late S and G2 phases . Timed delivery of Cas9 protein and sgRNAs into nocodazole-mediated M phase synchronization of cells has been shown to increase HDR events . Also, a recent unbiased screen of approximately 4,000 small molecules identified L755507 and Brefeldin A as capable of stimulating HDR, although the mechanism of action was not elucidated . It has been recognized that DNA-PKcs can suppress HDR events following generation of a DSB . The reliance of NHEJ on DNA-PKcs has prompted the assessment of DNA-PKcs mutants or small molecule inhibitors of DNA-PKcs on HDR following DSB induction – events that have been reported to increase HDR rates . This led us to directly assess if pharmacological inhibitors of DNA-PKcs would be compatible with Cas9 editing tools and could be used to favor HDR repair events while restricting NHEJ-mediated mutagenesis events. We identified two small molecules compatible for use with Cas9-editing technology that improve the frequency of HDR while reducing NHEJ mutagenic events.
HEK293/17 (obtained from ATCC) and Arf −/− MEFs (a kind gift of Dr. S. Lowe, Memorial Sloan Kettering Cancer Center) were maintained in DMEM supplemented with 10 % fetal bovine serum, 100 U/mL penicillin/streptomycin, and 2 mM glutamine. Plasmids were delivered to HEK293/17 cells by calcium phosphate transfection and to Arf −/− MEFs by nucleofection using the Amaxa nucleofector I (Lonza, Walkersville, MD, USA). Plasmids pQCiG-Rosa, pQCiG-TLR, pQCiG-p53-1, pQCiG-p53-3, pLC-ROSA, or pLC-TLR have been described previously [12, 21]. The pCVL Traffic Light Reporter 2.1 and pRRL SFFV d20GFP.T2A.mTagBFP donor were purchased from Addgene. NU7441 and KU-0060648 were purchased from Selleckchem (Houston, TX, USA). Nutlin-3a was obtained from Sigma (St. Louis, MO, USA) and SCR7 was from Selleckchem (Burlington, ON, Canada). All compounds were resuspended in DMSO and stored at −80 °C. siRNAs targeting DNA-PKcs, PI3K-p110α, Ku70, Ku80, and the DNA Ligase IV mRNA were purchased from Dharmacon (Lafayette, CO, USA), resuspended in the company’s resuspension buffer to 10 mM and stored at −80 °C. For γ-irradiation, 293/TLR cells were plated at 25 % confluency and the next day were treated with DNA-PK inhibitors (2 μM NU7441 or 250 nM KU-0060648) for 1 h followed by 4 GY of γ-irradiation. After 30 min, the cells were harvested and extracts prepared and subjected to SDS-PAGE, followed by probing western blots using anti-eEF2 (Cell Signaling Technology; Beverly, MA, USA) and anti-p-H2AX (Upstate Biotechnology; Lake Placid, NY, USA). Compounds toxicity was determined using cell titer glow (Promega, Madison, WI, USA).
The TLR assay was performed essentially as described by Certo et al. . The presence of blue fluorescent protein (BFP) in the pRRL SFFV d20GFP.T2A.mTagBFP donor template plasmid allowed corrections for transfection efficiencies to be made. In all experiments, background fluorescence from non-transfected (<0.05 %) cells was subtracted from the values obtained from transfected cells. When reporting NHEJ efficiencies, we multiplied the value obtained by quantitating the mCherry+ cells by 3 since only one out of three repair events is expected to yield a ΔeGFP-T2A-mCherry fusion in the correct frame to generate mCherry+ cells. Transfections were performed in 6-well plates by the calcium-phosphate method using 2 μg of Cas9/sgRNA expression vector with 1 μg of donor plasmid or 0.1 μM donor oligonucleotide. Plasmids pcDNA-E1B55K and pcDNA-E4Orf6 were a kind gift from Dr. Phil Branton (Biochemistry Dept., McGill University, Montreal, QC, Canada). One microgram of pcDNA-E1B55K and pcDNA-E4Orf6, or of the pcDNA-3.1 control vector, were co-transfected with 2 μg of Cas9/sgRNA expression vector and 1 μg of donor plasmid. For siRNA experiments, 20 nM of each siRNA was transfected using lipofectamine following the manufacturer’s recommendations (Invitrogen, Carlsbad, CA, USA). Genome editing efficiency was determined by flow cytometry 5 days later. Knockdown efficiency was monitored by western blotting 48 h following transfections using antibodies directed to PI3K-p110α (Cell Signaling Technology; Beverly, MA, USA), DNA-PK (Cell Signaling Technology; Beverly, MA, USA), Ku70 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), Ku80 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), or DNA Ligase IV (Abcam Inc., Cambridge, MA, USA). Antibodies directed to adenovirus E1B55K and E4Orf6 were a kind gift from Dr. Phil Branton.
Ion torrent sequencing
Oligonucleotide sequences used in this study
TLR donor sense
TLR donor antisense
p53-1 sense donor
p53-1 antisense donor
P53-1 barcoding Reverse oligo:
p53-3 sense donor
p53-3 antisense donor
P53-3 barcoding Reverse oligo:
Results and discussion
These results encouraged us to assess if chemical inhibition of DNA-PKcs would similarly affect HDR and/or NHEJ efficiency. We chose two well-characterized pharmacological inhibitors of DNA-PKcs, NU7441  and KU-0060648 . We first verified that both inhibitors were able to block DNA-PKcs kinase activity at non-toxic concentrations (Additional file 1: Figure S1A) following γ-irradiation of 293/TLR cells by monitoring phospho-H2AX level (Fig. 1e, compare lanes 3 and 4 to 1 and 2). To verify if these inhibitors could affect Cas9-mediated genome editing outcome, we transfected 293/TLR cells with Cas9 vectors expressing sgRNAs targeting either the neutral Rosa26 locus or the TLR eGFP cistron and exposed cells to NU7441 or KU-0060648 16 h post transfection (Fig. 1b). As expected, no significant editing at the TLR locus was apparent in the presence of Cas9 and the Rosa26 sgRNA (Fig. 1f). In contrast, the presence of Cas9 and the sgGFP yielded a significant proportion of mCherry+ cells due to NHEJ repair. Supplying a GFP repair template in trans resulted in approximately 2 % of the cells expressing GFP as a result of HDR-driven events (Fig. 1f – top right panel). The addition of NU7441 or KU-0060648 to cells following introduction of the Cas9/sgGFP editing system and the ΔGFP repair template caused a decrease of approximately 40 % in NHEJ events which was accompanied by an approximately two-fold stimulation in HDR (Fig. 1f, g). This effect was dose-dependent and reached a maximum at approximately 2.0 μM for NU7441 and 250 nM for KU-0060648 (Fig. 1g). Higher concentrations of either compounds were not well tolerated (Additional file 1: Figure S1). NU7441 or KU-0060648 are not selective for only DNA-PKcs, but also target PI3K, since both enzymes share highly homologous catalytic domains . We therefore used siRNAs to suppress the PI3K-p110α catalytic subunit and found no evidence on genome editing outcome indicating that PI3K-p110α suppression is not mediating the effects of NU7441 or KU-0060648 (Fig. 1c, d).
One strategy that avoids NHEJ mediated events altogether is the use Cas9 nickase mutants (for example, Cas9(D10A) and Cas9(H840A)) since DNA nicks are repaired by the non-mutagenic base excision repair (single-strand break repair pathway) process while supporting HDR [33–35]. We therefore asked whether DNA-PKcs inhibitors would also stimulate genomic repair induced by a DNA nick. As expected, Cas9(D10A) or Cas9(H840A) targeting the TLR reporter did not lead to mutagenic repair (Fig. 3c). The levels of HDR repair obtained with the nickases were lower (approximately two-fold) than obtained with Cas9 (Fig. 3c) and this process was not stimulated by either NU7441 or KU-0060648 (Fig. 3c).
Stimulation of HDR following inhibition of DNA-PK with the small molecule IC86621 has been previously reported . Our experiments extend these results using additional DNA-PKcs inhibitors by demonstrating their compatibility for use in conjunction with the Cas9 editing system (Fig. 1). In addition, we demonstrate that oligonucleotide-mediated HDR at endogenous loci is also stimulated by both NU7441 and KU-0060648 (Fig. 4). Although the increase in HDR obtained by treating cells with NU7441 or KU-0060648 was accompanied by a decrease in NHEJ, our experiments do not allow us to ascertain whether the stimulation of HDR is dependent on the concomitant decrease of NHEJ or reflects a direct inhibitory role of DNA-PKcs on HDR . The fact that we did not observe a stimulation of HDR when nicks were introduced at the target loci (Fig. 3c) suggests an indirect effect. However, it has recently been reported that HDR mediated by nicks occurs via a pathway that is distinct from that induced by DSBs and is independent of RAD51 and BRCA2 . Hence, one would expect a lack of response if the reported cross-talk between DNA-PKcs and HDR does not extend to the nickase-induced HDR pathway - an observation that will require further in-depth characterization. Although our work highlights the possibility to use DNA-PKcs inhibitors in order to stimulate HDR following Cas9-mediated cleavage, it should be kept in mind that inhibition of DNA-PKcs activity shunts some of the repair into alternate pathways , an event that could affect repair fidelity at double-stranded breaks that arise spuriously throughout the cell cycle.
It has been a significant challenge to obtain small molecule inhibitors that are selective to members of the PI3K family (PI3K, ATM, ATR, DNA-PK, and mTOR). KU-0060648 targets both DNA-PKcs and PI3K , whereas NU7441 shows 20-fold higher selective inhibition towards DNA-PK than PI3K in cells . NU7441 appears to have little activity towards ATM and ATR . Although we cannot formally ruled out that the results obtained herein are due to off-target effects of KU-0060648 and NU7441, our results are consistent with the observation that siRNA-mediated suppression of DNA-PK also stimulates HDR (Fig. 1d) . Treatment of the 293/TLR reporter line with either rapamycin or PP242, two mTOR inhibitors, had no effect on NHEJ or HDR repair efficiencies, making it unlikely that KU0060648 and NU7441 are acting through inhibition of mTOR (data not shown). We also do not think that KU-0060648 and NU7441 are acting through inhibition of PI3K since siRNA-mediated suppression of PI3K did not phenocopy the effects of either compound (Fig. 1f).
Our results indicate that transient pharmacological inhibition of DNA-PKcs can be used to stimulated HDR following Cas9-mediated induction of a DSB, thus enriching for HDR-mediated repair events. This is expected to reduce the downstream workload required to identify cells of interest having incorporated a desired, directed genomic modification.
CRISPR associated protein 9
Clustered regularly interspaced short palindromic repeats
DNA-dependent protein kinase catalytic subunit
enhanced green fluorescent protein
A Gray unit of ionization
Murine embryonic fibroblasts
Non-homologous end joining
Polyacrylamide gel electrophoresis
Protospacer adjacent motif
Small interfering RNA
Traffic Light Reporter
This work is supported by a grant from the Richard and Edith Strauss Canada Foundation to JP and the Canadian Institutes of Health Research to JD (CIHR; MOP-115127). JD is an FRSQ (Fonds de la Recherche en Santé du Québec) Research Scholar.
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