Pharmacological inhibition of DNA-PK stimulates Cas9-mediated genome editing

Abstract

Background: 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.

Methods: 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.

Results: We find that NU7441 and KU-0060648 reduce the frequency of NHEJ while increasing the rate of HDR following Cas9-mediated DNA cleavage.

Conclusions: Our results identify two small molecules compatible for use with Cas9-editing technology to improve the frequency of HDR.

Fig. 1

DNA-PKcs inhibitors stimulate HDR following Cas9 induction of a DSB. a Schematic outline of the Traffic Light Reporter assay. The eGFP and mCherry open reading frames (ORF) are fused by a ribosome skipping sequence (T2A) where translating ribosomes skip from a Gly to a Pro codon without forming a peptide bond while maintaining the reading frame to produce two distinct polypeptides [39, 40]. In the TLR, the eGFP and mCherry ORFs are positioned in different frames and NHEJ repair of a DSB directed to the GFP ORF will, in one out of three events, place ΔeGFP in frame with mCherry - leading to mCherry production. Since the eGFP ORF contains an insertion harboring a premature termination codon (and lacks a start codon), its expression can only be recovered upon HDR-mediated repair following delivery of an appropriate homologous template. The reading frame for each fluorescent protein is indicated in parentheses. b Outline of siRNA and drug treatment protocol. In one set of experiments, the 293/TLR cell line was transfected with the indicated siRNAs (yellow box) and 24 h later expression constructs driving synthesis of Cas9 and sgRNAs and an eGFP donor template were introduced into the cells. In a second set of experiments, the 293/TLR cell line was first transfected with expression constructs driving synthesis of Cas9 and sgRNAs and an eGFP donor template. Compounds were added 16 h later (orange box). In both cases, cells were allowed to propagate for 5 days before FACS analysis. c Knockdown of DNA-PK, DNA Ligase IV, and PI3K-p110α in 293/TLR cells transfected with the indicated siRNAs. Western blots were probed with antibodies to the indicated proteins. The dashed line separates two different sets of membranes. d Quantitation of genome editing events in 293/TLR cells transfected with the indicated siRNAs. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to scrambled siRNAs) was calculated using the Student’s t-test: *P ≤0.05; **P ≤0.01; ns, not significant. e DNA-PK inhibitors prevent H2AX phosphorylation upon γ-irradiation. Cells were pre-incubated with the indicated small molecules for 1 h followed by 4 GY of γ-irradiation (IR). Extracts for western blotting were prepared from non-irradiated (IR) cells (lane 1) or IR-exposed cells incubated in the presence of vehicle (lane 2), 2 μM NU77441 (lane 3), and 250 nM KU-0060648 (lane 4). Western blots were probed with antibodies directed to the proteins indicated to the left of the blot. The ratio of p-H2AX and eEF2 band intensities is indicated below the blots. f Flow cytometry analysis of 293/TLR cells transfected with pQCX vectors driving synthesis of Cas9 and sgRNAs targeting Rosa26 or eGFP. When indicated, the GFP repair template, pRRL SFFV d20GFP.T2A.mTagBFP (ΔeGFP Donor) was also included. NU7441 and KU-0060648 were used at a final concentration of 2 μM and 250 nM, respectively. Cells were gated for the live population as determined by PI staining. Transfection efficiencies were greater than 70 %. g Quantitation of genome editing events from cells propagated in the presence of the indicated concentrations of DNA-PKcs inhibitors. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t-test: *p ≤0.05; **p ≤0.01; ns, not significant

Fig. 2

HDR stimulation by NU7441 and KU-0060648 is comparable to other HDR enhancing tools. a Western blot showing knock down efficiency obtained with the siRNAs targeting either Ku70 or Ku80. b Quantitation of genome editing events from cells transfected with pQCiG-TLR and ΔeGFP donor in the presence of 2 μM NU7441, 250 nM KU-0060648, siRNAs targeting Ku70, Ku80, DNA-PKcs or DNA ligase IV, 1 μM Scr7, or a combination of Scr7 and 2 μM NU7441 or 250 nM KU-0060648. The HDR and NHEJ values are relative to those obtained with Cas9, sgRNA, and ΔeGFP donor in the presence of vehicle (DMSO). Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t-test: *p ≤0.05; **p ≤0.01; ns, not significant. c Western blot showing expression of Adenovirus 5 proteins E1B55K and E4orf6 following transfection into the 293/TLR line. d Quantitation of genome editing efficiencies was as in B except that adenovirus 5 proteins E1B55K and E4orf6 expression vectors were co-transfected with pQCiG-TLR and ΔeGFP donor plasmids and cultured in presence or absence of 2 μM NU7441 or 250 nM KU-0060648. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t-test: *p ≤0.05; **p ≤0.01; ns, not significant

Fig. 3

HDR directed by oligonucleotide donors are stimulated by DNA-PKcs inhibitors. a Quantitation of genome editing events from cells transfected with pQCiG-TLR and either ΔeGFP donor (2.5 kbp homology upstream and 1.5 kbp homology downstream of target site) or oligonucleotides (sense or antisense) (110 nucleotides) spanning the sgGFP target site and exposed to vehicle, 2 μM NU7441, or 250 nM KU-0060648. The HDR and NHEJ values are relative to those obtained with Cas9, sgRNA, and ΔeGFP donor in the presence of vehicle (DMSO). N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t-test: *p ≤0.05; **p ≤0.01; ns, not significant. b Representative examples of FACS plot obtained in (a). Note that mCherry⁺ cells are reporting on only one-third of all NHEJ events. c Quantitation of genome editing events from experiments performed as described in (a) except using WT Cas9 or the D10A and H840A nickase variants and the ΔeGFP donor. Values are set relative to editing frequencies observed in 293/TLR cells transfected with WT Cas9 expression vector and propagated in the presence of vehicle. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t-test: *p ≤0.05; **p ≤0.01; ns, not significant

Fig. 4

DNA-PK inhibitors stimulate homology-directed repair at the p53 locus. a Outline of experimental approach. b Position of target sites and partial sequence of oligonucleotides used as repair templates for p53 exons 5 and 7. A schematic representation of the murine p53 locus is shown with the locations of sgp53-1 and sgp53-3. The sequence of the donor templates for HDR (sgp53-1; 105 nucleotides, sgp53-3; 106 nucleotides) are indicated with red nucleotides denoting missense mutations and a dash indicating a frameshift mutation leading to a premature stop codon (in bold). c Fold stimulation of HDR at the sgp53-1 and sgp53-3 sites by NU7441 (2 μM) or KU-0060648 (250 nM). Results are shown as the fraction of all retrieved mutated sequences that correspond to HDR events in the presence of the DNA-PK inhibitors relative to vehicle. N = 4; error bars represent S.D. Results are from biological replicates performed in technical duplicates. Significance (relative to vehicle) was calculated using the Student’s t-test: *p ≤0.05; **p ≤0.01; ns, not significant