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Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics

Nature Methods volume 14, pages 891–896 (2017)Cite this article

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Abstract

We developed a chemically inducible Cas9 (ciCas9) and a droplet digital PCR assay for double-strand breaks (DSB-ddPCR) to investigate the kinetics of Cas9-mediated generation and repair of DSBs in cells. ciCas9 is a rapidly activated, single-component Cas9 variant engineered by replacing the protein's REC2 domain with the BCL-xL protein and fusing an interacting BH3 peptide to the C terminus. ciCas9 can be tunably activated by a compound that disrupts the BCL-xL–BH3 interaction within minutes. DSB-ddPCR demonstrates time-resolved, highly quantitative, and targeted measurement of DSBs. Combining these tools facilitated an unprecedented exploration of the kinetics of Cas9-mediated DNA cleavage and repair. We find that sgRNAs targeting different sites generally induce cleavage within minutes and repair within 1 or 2 h. However, we observe distinct kinetic profiles, even for proximal sites, and this suggests that target sequence and chromatin state modulate cleavage and repair kinetics.

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Figure 1: Development of a chemically inducible Cas9 (ciCas9).
Figure 2: DSB-ddPCR is an accurate and precise way to quantify DSBs.
Figure 3: DSB and indel kinetics with ciCas9 and DSB-ddPCR.
Figure 4: ciCas9 specificity and basal activity can be tuned.
Figure 5: ciCas9 can be activated by a variety of BCL-xL disruptors.

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Acknowledgements

We thank M. Dickinson for assistance generating reagents. This work was supported by the NIH (R01GM086858 (D.J.M.), R01GM109110 (D.M.F.), and F30CA189793 (J.C.R.)), and by the NSF (0954242 (D.J.M.)).

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Authors and Affiliations

  1. Department of Chemistry, University of Washington, Seattle, Washington, USA

    John C Rose, Bridget M Trevillian & Dustin J Maly

  2. Department of Genome Sciences, University of Washington, Seattle, Washington, USA

    Jason J Stephany & Douglas M Fowler

  3. Division of Public Health Sciences, Fred Hutchinson Cancer Research Center, Seattle, Washington, USA

    William J Valente & Jason H Bielas

  4. Department of Biochemistry, University of Washington, Seattle, Washington, USA

    Ha V Dang & Dustin J Maly

  5. Department of Bioengineering, University of Washington, Seattle, Washington, USA

    Douglas M Fowler

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Contributions

J.C.R., D.M.F., and D.J.M. conceived the study and designed the experiments. B.M.T. assisted in the design of ciCas9. J.C.R. and J.J.S. performed the experiments. J.J.S. prepared samples for high-throughput sequencing and ddPCR. H.V.D. performed fluorescence BH3 competition experiments under the supervision of J.C.R. and D.J.M. W.J.V. helped develop the DSB-ddPCR assay and analyze data under the supervision of J.H.B. J.C.R. and D.M.F. prepared custom Python scripts. J.C.R., D.M.F., and D.J.M. wrote and edited the manuscript. All authors approved the final manuscript.

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Correspondence to Dustin J Maly or Douglas M Fowler.

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Integrated supplementary information

Supplementary Figure 1 Comparison of REC2 and BCL-xL domains and modeling domain replacement.

(a) The REC2 domain of Cas9 and (b) BCL-xL display similar globular structures and volumes. (c, d) Modeling the replacement of the REC2 domain with BCL-xL. Six BCL-xL models are shown (multiple colors). In order to accommodate differences in relative termini orientation between BCL-xL and the REC2 domain, a flexible linker (length = 15 residues) was used to connect residue 307 of Cas9 to BCL-xL, while the N-terminus of BCL-xL was connected directly to residue 179 of Cas9. Cas9.BCL constructs containing 15 versus 20 residue linkers exhibited no appreciable difference in activity (data not shown).

Supplementary Figure 2 A Cas9 variant in which the REC2 domain was replaced with BCL-xL retains activity.

(a, b) HEK-293T cells were transfected with Cas9.BCL, wild type Cas9, or no Cas9, in addition to one of two sgRNAs targeting the MYC locus (sgRNA3 or sgRNA5) (n =1). Indel quantification was performed using an analysis method similar to that previously described (Online Methods). These data indicated Cas9 variants in which the REC2 domain has been replaced by BCL-xL retain editing activity. (c) HEK-293T cells were transfected with plasmids encoding Cas9 or Cas9.BCL, each bearing a FLAG-tag, and cells were lysed after 24 hours. Expression was detected using an anti-FLAG antibody (Cell Signaling), and an anti-GAPDH (loading control) antibody.

Supplementary Figure 3 Schematic depiction of Cas9 constructs generated and tested in this study.

Constructs contained residues 4-198 of BCL-xL (green); the BH3 peptide sequence (magenta); a nuclear localization signal (NLS, orange); FLAG tag (yellow); and human codon optimized wild type S. pyogenes Cas9 (purple). Flexible linkers ranging in length from 5 to 30 amino acids (blue) are composed of glycine, serine, threonine, and alanine. NLS = nuclear localization sequence.

Supplementary Figure 4 Indel frequency at the AAVS1 locus as a function of ciCas9 linker length after 24 hours in the presence or absence of A3.

HEK-293T cells were transfected using Turbofectin 8.0 with (450 ng Cas9 construct, 450 ng AAVS1 sgRNA, and 100 ng pMAX-GFP) or (450 ng ciCas9 and 550 ng pMAX-GFP), or left untransfected. 24 hours after transfection, cells were treated with A3 (10 μM) or left untreated. 24 hours after drug treatment, cells were harvested, and indel were quantified as described in the Online Methods. Error bars depict the s.e.m. (n = 3 cell culture replicates).

Supplementary Figure 5 ciCas9 enables inducible genome editing in U2OS and HCT116 cells.

U2OS and HCT116 cells were transfected using Turbofectin 8.0 with (450 ng ciCas9, 450 ng AAVS1 sgRNA, and 100 ng pMAX-GFP) or (450 ng ciCas9 and 550 ng pMAX-GFP) as a no sgRNA control. 24 hours after transfection, cells were treated with A3 (10 μM final concentration) or left untreated. Cells were harvested 24 hours after drug addition and indels quantified as described in the Online Methods. Indel frequencies (%) for individual cell culture replicates (n = 3) are shown (solid line = mean). Editing via transient transfection is inefficient in these other cell lines53, as has been previously reported for U2OS and HCT116 cells using the same sgRNA54.

Supplementary Figure 6 Comparison of indel kinetics at the EMX1 locus with ciCas9, paCas9, and iCas9.

(a) Indel kinetics are shown as reported for paCas9 co-transfected with EMX1 sgRNA in HEK-293T cells. Indel frequencies were determined using T7E1 assay, which the authors note has a detection limit of 1%. Error bars depict the s.e.m. (n=4 cell culture replicates) (b) HEK-293T cells were transfected with ciCas9 and an identical EMX1 sgRNA. After 24 hours, cells were treated with A3 and harvested at the indicated time points. Indel frequencies were quantified using high-throughput sequencing, as described in the Online Methods, and are also plotted in Fig. 3a (right panel). Indels were detectable by 2 hours, the first time point tested, and this increase in indel frequency was significant relative to both the zero minute and two hour no drug time points (one-sided t-tests: n = 3, p = 0.00045 and p = 0.00056, respectively, n = 3 cell culture replicates). (c) Reproduction of previously reported EMX1* sgRNA kinetics with iCas9 in HEK-293 cells. No statistics were reported. (d) HEK-293T cells were transfected with ciCas9 and an identical EMX1* sgRNA. After 24 hours, cells were treated with A3 and harvested at the indicated time points. Indel frequencies were determined using high-throughput sequencing, as described in the Online Methods. Indels were detectable with ciCas9 at 30 min and significant relative to the zero minute time point (one-sided t-test, n = 3, p = 0.016). Error bars depict the s.e.m. (n = 3 cell culture replicates). The *EMX1 sgRNA used in these experiments has an additional transcribed G preceding the 20 bp target sequence, but it is otherwise identical to the EMX1 sgRNA used throughout this study.

Supplementary Figure 7 DSB-ddPCR is accurate and precise.

Standard curves show the relationship between DSB frequency as measured by the DSB-ddPCR assay and expected DSB frequency at the AAVS1, MYC and EMX1 loci. Technical duplicates are shown and, in most cases, overlap.

Supplementary Figure 8 Proteinase K effectively digests ciCas9 prior to DNA isolation for DSB-ddPCR and high-throughput sequencing.

HEK-293T cells were transfected with 450 ng ciCas9, 450 ng EMX1 sgRNA, and 100 ng pMAX-GFP per well in 12-well plates. 24 hours after transfection, cells were incubated with A3 (10 μM) for two hours, then harvested. Non-transfected wells were harvested as a control. After resuspension in 1:1 mixture of PBS and Buffer AL (DNeasy Blood & Tissue Kit, Qiagen), samples were split into thirds. One third was processed according to the DNA isolation protocol as described in Online Methods, including 1 hour proteinase K digestion at 56 ºC and DNA purification via spin-column (Proteinase K +, DNeasy Column +)—similar digestions are often used to remove Cas9 from DNA for sequencing, genome-wide DSB detection, or T7E1, SURVEYOR, or in vitro Cas9 cleavage assays8,28,31,55. One third was digested with proteinase K for 1 hour at 56 ºC only (Proteinase K +, DNeasy Column -), and one third of the cells underwent only the first step in processing—resuspension in PBS and Buffer AL (DNeasy kit)—but were not treated with Proteinase K or run on DNeasy column. Protein was then isolated from each sample by chloroform/methanol precipitation, resuspended in SDS-PAGE denaturing sample buffer, boiled, and western blotted. ciCas9 expression was evaluated using anti-FLAG antibody (Sigma), and anti-GAPDH used as a loading control. ciCas9 expression was undetectable after proteinase K digestion. n = 3 cell culture replicates for ciCas9, n = 1 for no transfection control.

Supplementary Figure 9 Evaluation of background signal throughout DSB-ddPCR timecourse experiments.

DSB frequency curves from Fig. 3a are plotted with corresponding no sgRNA and no transfection controls. Error bars depict the s.e.m. (n = 3 cell culture replicates).

Supplementary Figure 10 Determination of initial DSB generation rate for MYC sgRNA4 and sgRNA5.

Initial DSB generation rates were calculated by performing a linear regression over early time points (0-30 min, Fig. 3b) MYC sgRNA4 (slope = 0.66, standard error = 0.094; r2 = 0.88) and sgRNA5 (slope 1.2, standard error = 0.11; r2 = 0.94). The difference in initial rates is significant (analysis of covariance (ANCOVA), p = 0.0035). Linear regressions and statistical calculations were performed in Prism software. Individual cell culture replicates are plotted at each time point for each sgRNA, n = 3. Dashed lines show the fitted regression lines.

Supplementary Figure 11 High-resolution indel timecourse corresponding to DSB timecourses in (Fig 3b).

Statistically significant indels were detectable at 2 hours for both MYC sgRNA4 (p = 0.0017) and MYC sgRNA5 (p=0.00079) relative to t = 0 min. The increase in indel frequency was also significant relative to no drug (“- A3”) at 2 hours (MYC sgRNA4 p = 0.00071; MYC sgRNA5 p = 0.00062). All p-values were calculated with one-sided t-tests, n = 3, error bars depict the s.e.m. The left panel shows the full time course. The right panel shows the first two hours.

Supplementary Figure 12 ciCas9 exhibits improved editing specificity relative to wild-type Cas9.

HEK-293T cells were transfected using Turbofectin 8.0 with (450 ng ciCas9, 450 ng VEGFA sgRNA2 or VEGFA sgRNA3, and 100 ng pMAX-GFP) or (450 ng ciCas9 and 550 ng pMAX-GFP) as a no sgRNA control. 24 hours after transfection cells were treated with A3 (10 μM final concentration) or left untreated. Cells were harvested 24 hours after drug addition and indels quantified as described in the Online Methods. (a) On- and Off-target editing for VEGFA sgRNA2, and (b) the corresponding specificity ratios. (c) On- and Off-target editing for VEGFA sgRNA3, and (d) the corresponding specificity ratios. VEGFA sgRNA3. Indel frequencies (%) for individual cell culture replicates (n = 3) are shown (solid line = mean). For specificity ratios (on-target/off-target indel frequency ratios) error bars depict the s.e.m (n = 3 cell culture replicates).

Supplementary Figure 13 A ciCas9 variant (e-ciCas9) with specificity enhancing mutations to minimize off-target activity.

Three mutations (K810A, K1003A, R1060A) were introduced to generate enhanced specificity ciCas9 (e-ciCas9). HEK-293T cells were co-transfected with MYC sgRNA4 (MYCg4) or VEGFA sgRNA2 (VEGFAg2), and either ciCas9 or e-ciCas9. Cells were then incubated either in the presence or absence of A3 for 24 hours before harvesting. Indel frequencies at the on-target site and a prominent off-target site were determined via high throughput sequencing. (a) On-target and (b) off-target 4 (OT4) indel frequencies (%) for MYC sgRNA4. (c) Specificity ratios for MYC sgRNA4 on-target and OT4 editing. (d) On-target and (e) off-target 1 (OT1) indel frequencies (%) for VEGFA sgRNA2. (f) Specificity ratios for VEGFA sgRNA2 on-target and OT1 editing. Error bars = s.e.m., n = 3 cell culture replicates.

Supplementary Figure 14 Alterations in BH3/BCL-xL affinity do not affect ciCas9 variant expression.

HEK-293T cells were transfected with 450 ng ciCas9 or ciCas9(F22), 450 ng EMX1 sgRNA, and 100 ng pMAX-GFP per well in 12-well plates using Turbofectin 8.0. Non-transfected wells were included as a control. Cells were harvested and lysed 48 hours after transfection and ciCas9 variant expression was determined by western blot using antibodies targeting FLAG (Sigma) and GAPDH (loading control). Blots representative of two independent experiments.

Supplementary Figure 15 A3 does not affect DSBs, indels, or cell viability in the context of wild type Cas9-mediated editing.

HEK-293T cells were transfected with 225 ng of plasmid encoding wild type Cas9, 225 ng of plasmid encoding AAVS1 sgRNA, and 50 ng of plasmid encoding pMAX-GFP per well in 24-well plates using Turbofectin 8.0. 24 hours after transfection cells were treated with A3 (10 μM final concentration) or left untreated. (a-b) Cells were harvested at 0 and 24 hours after drug addition. (a) Indels were quantified via high-throughput sequencing, and (b) DSB frequency was determined via ddPCR. In parallel, (c) cell viability and (d) cell death relative to WT Cas9 (– A3) at 0 hours were determined using the LIVE/DEAD Viability/Cytotoxicity Kit (ThermoFisher). Error bars depict s.e.m. (n = 3 cell culture replicates).

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Rose, J., Stephany, J., Valente, W. et al. Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics. Nat Methods 14, 891–896 (2017). https://doi.org/10.1038/nmeth.4368

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