Enhancing homology-directed genome editing by catalytically active and inactive CRISPR-Cas9 using asymmetric donor DNA

Journal name:
Nature Biotechnology
Volume:
34,
Pages:
339–344
Year published:
DOI:
doi:10.1038/nbt.3481
Received
Accepted
Published online

Targeted genomic manipulation by Cas9 can efficiently generate knockout cells and organisms via error-prone nonhomologous end joining (NHEJ), but the efficiency of precise sequence replacement by homology-directed repair (HDR) is substantially lower1, 2. Here we investigate the interaction of Cas9 with target DNA and use our findings to improve HDR efficiency. We show that dissociation of Cas9 from double-stranded DNA (dsDNA) substrates is slow (lifetime ~6 h) but that, before complete dissociation, Cas9 asymmetrically releases the 3′ end of the cleaved DNA strand that is not complementary to the sgRNA (nontarget strand). By rationally designing single-stranded DNA (ssDNA) donors of the optimal length complementary to the strand that is released first, we increase the rate of HDR in human cells when using Cas9 or nickase variants to up to 60%. We also demonstrate HDR rates of up to 0.7% using a catalytically inactive Cas9 mutant (dCas9), which binds DNA without cleaving it.

At a glance

Figures

  1. Cas9 interacts stably with substrate DNA.
    Figure 1: Cas9 interacts stably with substrate DNA.

    (a) Schematic of Cas9's interaction with substrate DNA. Cas9 (gray) complexed with sgRNA (dark blue) binds to DNA (black) comprising target and nontarget strands. Cas9-PAM interactions occur on the nontarget strand; sgRNA-DNA annealing occurs on the target strand. RuvC (His840) and HNH (Asp10) nuclease domains cut the nontarget and target strands, respectively (red triangles). (b) BLI measurements of association (left of dotted line) and dissociation (right of dotted line) of Cas9 (black trace) or dCas9 (brown trace) with λ1 dsDNA. Mean ± s.d. kinetic values calculated from n = 2 experiments are inset. See Supplementary Figure 1 for data fitting. (c) Electrophoretic mobility shift assay (EMSA) measuring dissociation of Cas9 from substrate dsDNA. Cas9 RNP was equilibrated with S1 dsDNA for 16 h, after which unlabeled challenge dsDNA was added for the indicated time and reaction products were visualized on a native polyacrylamide gel. Black circle, labeled substrate DNA (no Cas9); open triangle, Cas9-DNA complex; dashed box, region of interest box. Data shown are representative of n = 2 experiments (quantified in Fig. 2d). Subsequent figures highlight the region of interest corresponding to the dashed lines.

  2. Complementary DNA anneals to the nontarget strand of the RNP-dsDNA complex.
    Figure 2: Complementary DNA anneals to the nontarget strand of the RNP-dsDNA complex.

    (a) Schematic for EMSA assays. Association: RNP is equilibrated for 10 min with fluorescently labeled substrate DNA (magenta, Cy5; green, Cy3) containing protospacer-PAM sequences (blue and green arrows). Challenge and Dissociation: reactions are incubated with or without unlabeled challenge DNA for 10 min and products are resolved on a native polyacrylamide gel. Nuclease variants (top right inset) are wild type (WT, with two magenta arrows representing catalytically active nuclease domains), the Cas9D10A and Cas9H840A nickase variants (with single magenta arrows) and the catalytically dead D10A H840A dCas9 variant (with no arrows). DNA substrates (middle right inset) contain one or two protospacer-PAM sequences (arrows). Dual binding of RNP to substrate DNA was investigated using sgRNA pairs that targeted protospacer-PAM sites in PAM-in (RNPs 1 and 2) or PAM-out (RNPs A and B) orientations. Unlabeled challenge DNA (lower right inset) was provided as double-stranded (D) or single-stranded (plus strand, p; minus strand, m) species. Data presented are representative of n = 2 biological replicates and cropped to highlight the region of interest. Uncropped gels from all experiments are presented in Supplementary Note 2. The Cas9, sgRNA and substrate DNA for each EMSA experiment is schematically presented in Supplementary Figure 2. Nuclease activity was verified using denaturing gels (Supplementary Fig. 1b). (b) Challenging a stable Cas9-DNA complex with ssDNA complementary to the PAM-distal nontarget strand leads to removal of this strand from the complex. Challenge DNAs were identical to substrate DNA (S1 substrate challenge; lanes 2–4), identical to substrate DNA with PAM disrupted (PAM challenge; lanes 5–7), or disrupted the complementarity of the sequence flanking the protospacer-PAM (no-homology (NH) challenge; lanes 8–10). Lane 1, no challenge DNA. Open triangle, RNP-DNA complex. (c) Loading multiple Cas9 molecules in a PAM-in orientation allows displacement of either PAM-distal nontarget strand. One or two Cas9 molecules were loaded onto D1 substrate DNA, then challenged with the indicated challenge DNA species. Open triangle, RNP-DNA; solid gray triangle, 2×Cas9-DNA product. (d) Challenge DNA anneals to the uncut nontarget strand when Cas9 nuclease domains are inactivated. EMSA performed as described in a. Cas9, Cas9D10A, Cas9H840A and dCas9 nuclease variants were used as diagrammed below (following the visual scheme in a). Open triangle, RNP-DNA; solid black triangle, supershifted product. (e) Challenge DNA anneals to the nontarget strand when strand displacement is prevented by adjacent Cas9-DNA interactions in a PAM-out orientation. EMSA performed as described in a except that the fluorophore location was varied. Cas9 and dCas9 nuclease variants were used as diagrammed. D1 substrate dsDNA was labeled with Cy5 on the plus strand (solid square) or Cy3 on the minus strand (open square). Challenge ssDNAs were labeled with Cy5 on the plus strand (solid circle) or Cy3 on the minus strand (open circle). Open triangle, RNP-DNA; solid black arrow, well-shifted products.

  3. Delivery of ssDNA donors complementary to the nontarget strand drives efficient HDR using Cas9, nickases and dCas9.
    Figure 3: Delivery of ssDNA donors complementary to the nontarget strand drives efficient HDR using Cas9, nickases and dCas9.

    (a) Schematic for HDR at a BFP reporter locus. Target strand (green) or nontarget strand (magenta) donor ssDNAs were generated with the indicated overlaps on either side of the Cas9 cut site in the BFP reporter. The sequences of the unedited (WT, BFP) and edited loci (HDR, GFP) are presented inset (PAM reverse complement, underlined; cut site, magenta arrow). (b) HDR, NHEJ and unedited populations can be measured using flow cytometry. BFP-GFP flow cytometry scatter plots for BFP reporter cells (leftmost panel), BFP reporter cells edited with Cas9 (Cas9), or BFP reporter cells edited with the indicated nuclease and Donor Ht. Data shown is representative of n = 2 experiments, and all flow cytometry plots are shown in Supplementary Note 3. Gated populations are WT, BFP+ cells; NHEJ, BFP GFP cells; and HDR, GFP+ cells. (c) Optimized donor DNA is complementary to the nontarget strand and has a characteristic size. HDR frequencies for editing with target (t), nontarget (n) or double-stranded (d) donor DNAs are presented at right as mean ± s.d. for n ≥ 2 independent experiments. All flow cytometry plots are presented in Supplementary Note 3. (d) Target-strand donor stimulates greater levels of HDR for all Cas9 variants. HDR frequencies are quantified from editing experiments using the indicated nuclease and donor Ht (target strand, green) or Donor Hn (nontarget strand, magenta). Data presented as mean ± s.d. from n ≥ 2 independent experiments. (e) Single or tiled-dCas9 molecules support HDR. HDR frequencies from dCas9 editing experiments as presented in d except that control (RNP only, Donor only) reactions are shown alongside editing reactions. RNP, single dCas9; tiled, equimolar amounts of dCas9 targeting four distinct sites on the coding strand of the BFP reporter.

  4. Supporting data for Figure 1
    Supplementary Fig. 1: Supporting data for Figure 1

    (A) Schematic of BLI assay used to measure dissociation. 5′ monobiotinylated substrate DNA (identical to λ1, Figure 2) is associated with streptavidin-coated sensor tips (black oval) and baseline signal is established (left panel). Association phase (right panel) loads Cas9 onto substrate dsDNA and measures response. Dissociation phase (not shown) transfers the tip into buffer and monitors dissociation of Cas9. (B) Cas9, Cas9D10A, and Cas9H840A cleave DNA while dCas9 does not. Cas9 nucleases were incubated with or without sgRNA for 30 minutes and associated with λ1 substrate DNA (Figure 2A) for ten minutes. Untagged (pCR1002 and pCR1003, Document S3) and NLS-tagged (pCR1053-pCR1056, Document S3) Cas9 variants were tested and found to have equivalent activity. Reaction products were resolved on a 10% TBE-Urea gel. Open arrow, uncut substrate DNA; *, excess Cy5 labeled ssDNA; , excess Cy3 labeled ssDNA. Data presented is representative of n=2 experiments. (C) Fit between BLI data (thick trace) and calculated kinetic values (maroon trace) for Cas9 (black) and dCas9 (brown). Replicate data is shown. (D) Cas9 interacts specifically with substrate dsDNA. BLI traces show no interaction of apoCas9 (no sgRNA) with substrate dsDNA (maroon trace) or Cas9 with substrate dsDNA lacking a PAM (blue trace). (n=2). (E) Gel densitometry of Figure 1B. Mean ± SD normalized intensity of + strand (blue) and − strand (red) shifted products were plotted as a function of time. The indicated regression lines were used to calculate koff. Equations and standard errors of the regression coefficient (se) are presented for each trace.

  5. Schematic of reagents and experimental design for EMSA experiments.
    Supplementary Fig. 2: Schematic of reagents and experimental design for EMSA experiments.

    Potential supershift products are presented where appropriate.

  6. Supporting data for Figure 2
    Supplementary Fig. 3: Supporting data for Figure 2

    (A) The non-target strand is released on the PAM-distal side of the cut. One Cas9 molecule was loaded onto substrate DNA fluorescently labeled at the 5′ or the 3′ terminus of each strand (Supplementary Figure 3). Only the 5′ non-target strand can be removed from the complex by a challenge DNA. Open arrow, RNP-DNA complex. (B) Removal of the non-target strand depends upon the concentration of the challenge DNA but is independent of the labeling fluorophore. Single RNP EMSA was conducted as described in Figure 2A, except challenge concentration was varied from 0-1500nM (0, 30, 75, 150, 300, 600,1500nM). Catalytically inactive dCas9 was used in lane 8 (dashed box) to demonstrate that nuclease activity is required for strand extrusion activity. Substrate DNA fluorescently labeled at the 5′ termini with Cy5 or Cy3 as indicated. Open arrow, RNP-DNA complex. (C) Strand annealing occurs in single-RNP substrates when the non-target strand is left intact. Cas9 or dCas9 variants were loaded onto substrate DNA as indicated and as described in Supplementary Figure 3. Challenge concentration was varied from 0-5uM (0, 500, 1500, 2500, 5000nM). Open arrow, RNP-DNA complex; solid black arrow, supershifted products.

  7. Model for challenge-mediated non-target strand removal activity.
    Supplementary Fig. 4: Model for challenge-mediated non-target strand removal activity.

    1) After duplex cleavage, Cas9 holds onto three ends of the target DNA (white crossed circles), but the PAM-distal non-target strand is released from the Cas9-DNA complex. 2) Complementary DNA anneals to released strand. 3) Branch migration results in extrusion from the Cas9-DNA complex.

  8. The non-target strand is available for enzymatic modification in cells.
    Supplementary Fig. 5: The non-target strand is available for enzymatic modification in cells.

    Cas9 was targeted to either strand of the AAVS1 locus (AAVS1-F or AAVS1-R) and terminal transferase was introduced to 3′ end-label cut DNA with biotin. After streptavidin immunoprecipitation, end-labeling on either side of the break was determined by the ability to qPCR amplify sequences using the indicated primer pairs (Left and Right). Results are presented as the mean +/− SD fold enrichment (n=3) of labeled DNA over uncut control DNA (ACT1).

  9. Strand-bias for optimized donor DNA is independent of genomic locus and gene transcription.
    Supplementary Fig. 6: Strand-bias for optimized donor DNA is independent of genomic locus and gene transcription.

    Cas9 targets the template strand of the EMX1 locus as diagrammed at left. Target strand (blue) or non-target strand (orange) donor ssDNAs were generated with the indicated overlaps on either side of the Cas9 cut site at EMX1. The sequences of the unedited and edited loci are presented inset (PAM sequence, underlined; cut site, magenta arrow; PciI site, bold font). HDR frequencies for editing with each donor are presented at right as mean +/− SD for n≥2 two independent experiments

  10. Editing the EMX1 locus with and without donor DNA.
    Supplementary Fig. 7: Editing the EMX1 locus with and without donor DNA.

    (A) The EMX1 locus is not cut as efficiently as the BFP locus. PCR amplification and T7E1 digestion were performed on cells edited using the indicated donor DNA (N/A – no donor, N/C – no Cas9). %Cut was quantified by gel densitometry. Compare to 95% total editing seen at the BFP locus (Supplementary Figure 9A). (B) HDR incorporation of a PciI site into the EMX1 locus shows donor strand-bias. PCR amplification (−) or PCR amplification and PciI digestion (+) was performed on cells edited using the indicated donor DNA (N/A – no donor). %Cut was quantified by gel densitometry and used to generate bar graphs in Supplementary Figure 6. Each nucleofection was performed in biological duplicate

  11. Asymmetric donors stimulate HDR at the CXCR4 and CCR5 loci in HEK293 and K562 cells.
    Supplementary Fig. 8: Asymmetric donors stimulate HDR at the CXCR4 and CCR5 loci in HEK293 and K562 cells.

    Cas9 was targeted to the CXCR4 or CCR5 loci in HEK293 and K562 cells. Target strand donors with the diagrammed overlaps were generated for each locus. The sequences of the unedited and edited loci are presented inset (PAM sequence, underlined; cut site, magenta arrow). T7 editing (T) and HDR frequencies (P; underlined) are presented at the bottom of each gel. Each nucleofection was performed in biological duplicate.

  12. Sample flow cytometry plots.
    Supplementary Fig. 9: Sample flow cytometry plots.

    (A) Representative flow cytometry data used to create bar graphs shown in Figure 3B. (B) Representative flow cytometry data used to create bar graphs shown in Figure 3C.

  13. Structural data is consistent with asymmetric release of substrate by Cas9.
    Supplementary Fig. 10: Structural data is consistent with asymmetric release of substrate by Cas9.

    A surface electrostatic view of Cas9, sgRNA (orange), and non-target (purple) or target (grey) DNA strands7. PAM-Cas9 interaction, white arrow; putative path of non-target strand, purple dots; presumed direction of non-target strand extrusion, black arrow.

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Author information

Affiliations

  1. Innovative Genomics Initiative, University of California, Berkeley, Berkeley, California, USA.

    • Christopher D Richardson,
    • Graham J Ray,
    • Mark A DeWitt,
    • Gemma L Curie &
    • Jacob E Corn
  2. Department of Molecular and Cell Biology, University of California, Berkeley, Berkeley, California, USA.

    • Christopher D Richardson,
    • Graham J Ray,
    • Mark A DeWitt,
    • Gemma L Curie &
    • Jacob E Corn

Contributions

C.D.R. and J.E.C. designed experiments; C.D.R., G.J.R. and G.L.C. performed experiments; M.A.D. designed and constructed the BFP reporter cell line; C.D.R. and J.E.C. analyzed data; C.D.R. and J.E.C. wrote the manuscript with contributions from all authors.

Competing financial interests

C.D.R. and J.E.C. are inventors on US Patent Application No. 62/262,189 related to this work.

Corresponding author

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Supplementary information

Supplementary Figures

  1. Supplementary Figure 1: Supporting data for Figure 1 (224 KB)

    (A) Schematic of BLI assay used to measure dissociation. 5′ monobiotinylated substrate DNA (identical to λ1, Figure 2) is associated with streptavidin-coated sensor tips (black oval) and baseline signal is established (left panel). Association phase (right panel) loads Cas9 onto substrate dsDNA and measures response. Dissociation phase (not shown) transfers the tip into buffer and monitors dissociation of Cas9. (B) Cas9, Cas9D10A, and Cas9H840A cleave DNA while dCas9 does not. Cas9 nucleases were incubated with or without sgRNA for 30 minutes and associated with λ1 substrate DNA (Figure 2A) for ten minutes. Untagged (pCR1002 and pCR1003, Document S3) and NLS-tagged (pCR1053-pCR1056, Document S3) Cas9 variants were tested and found to have equivalent activity. Reaction products were resolved on a 10% TBE-Urea gel. Open arrow, uncut substrate DNA; *, excess Cy5 labeled ssDNA; , excess Cy3 labeled ssDNA. Data presented is representative of n=2 experiments. (C) Fit between BLI data (thick trace) and calculated kinetic values (maroon trace) for Cas9 (black) and dCas9 (brown). Replicate data is shown. (D) Cas9 interacts specifically with substrate dsDNA. BLI traces show no interaction of apoCas9 (no sgRNA) with substrate dsDNA (maroon trace) or Cas9 with substrate dsDNA lacking a PAM (blue trace). (n=2). (E) Gel densitometry of Figure 1B. Mean ± SD normalized intensity of + strand (blue) and − strand (red) shifted products were plotted as a function of time. The indicated regression lines were used to calculate koff. Equations and standard errors of the regression coefficient (se) are presented for each trace.

  2. Supplementary Figure 2: Schematic of reagents and experimental design for EMSA experiments. (249 KB)

    Potential supershift products are presented where appropriate.

  3. Supplementary Figure 3: Supporting data for Figure 2 (171 KB)

    (A) The non-target strand is released on the PAM-distal side of the cut. One Cas9 molecule was loaded onto substrate DNA fluorescently labeled at the 5′ or the 3′ terminus of each strand (Supplementary Figure 3). Only the 5′ non-target strand can be removed from the complex by a challenge DNA. Open arrow, RNP-DNA complex. (B) Removal of the non-target strand depends upon the concentration of the challenge DNA but is independent of the labeling fluorophore. Single RNP EMSA was conducted as described in Figure 2A, except challenge concentration was varied from 0-1500nM (0, 30, 75, 150, 300, 600,1500nM). Catalytically inactive dCas9 was used in lane 8 (dashed box) to demonstrate that nuclease activity is required for strand extrusion activity. Substrate DNA fluorescently labeled at the 5′ termini with Cy5 or Cy3 as indicated. Open arrow, RNP-DNA complex. (C) Strand annealing occurs in single-RNP substrates when the non-target strand is left intact. Cas9 or dCas9 variants were loaded onto substrate DNA as indicated and as described in Supplementary Figure 3. Challenge concentration was varied from 0-5uM (0, 500, 1500, 2500, 5000nM). Open arrow, RNP-DNA complex; solid black arrow, supershifted products.

  4. Supplementary Figure 4: Model for challenge-mediated non-target strand removal activity. (83 KB)

    1) After duplex cleavage, Cas9 holds onto three ends of the target DNA (white crossed circles), but the PAM-distal non-target strand is released from the Cas9-DNA complex. 2) Complementary DNA anneals to released strand. 3) Branch migration results in extrusion from the Cas9-DNA complex.

  5. Supplementary Figure 5: The non-target strand is available for enzymatic modification in cells. (58 KB)

    Cas9 was targeted to either strand of the AAVS1 locus (AAVS1-F or AAVS1-R) and terminal transferase was introduced to 3′ end-label cut DNA with biotin. After streptavidin immunoprecipitation, end-labeling on either side of the break was determined by the ability to qPCR amplify sequences using the indicated primer pairs (Left and Right). Results are presented as the mean +/− SD fold enrichment (n=3) of labeled DNA over uncut control DNA (ACT1).

  6. Supplementary Figure 6: Strand-bias for optimized donor DNA is independent of genomic locus and gene transcription. (61 KB)

    Cas9 targets the template strand of the EMX1 locus as diagrammed at left. Target strand (blue) or non-target strand (orange) donor ssDNAs were generated with the indicated overlaps on either side of the Cas9 cut site at EMX1. The sequences of the unedited and edited loci are presented inset (PAM sequence, underlined; cut site, magenta arrow; PciI site, bold font). HDR frequencies for editing with each donor are presented at right as mean +/− SD for n≥2 two independent experiments

  7. Supplementary Figure 7: Editing the EMX1 locus with and without donor DNA. (175 KB)

    (A) The EMX1 locus is not cut as efficiently as the BFP locus. PCR amplification and T7E1 digestion were performed on cells edited using the indicated donor DNA (N/A – no donor, N/C – no Cas9). %Cut was quantified by gel densitometry. Compare to 95% total editing seen at the BFP locus (Supplementary Figure 9A). (B) HDR incorporation of a PciI site into the EMX1 locus shows donor strand-bias. PCR amplification (−) or PCR amplification and PciI digestion (+) was performed on cells edited using the indicated donor DNA (N/A – no donor). %Cut was quantified by gel densitometry and used to generate bar graphs in Supplementary Figure 6. Each nucleofection was performed in biological duplicate

  8. Supplementary Figure 8: Asymmetric donors stimulate HDR at the CXCR4 and CCR5 loci in HEK293 and K562 cells. (271 KB)

    Cas9 was targeted to the CXCR4 or CCR5 loci in HEK293 and K562 cells. Target strand donors with the diagrammed overlaps were generated for each locus. The sequences of the unedited and edited loci are presented inset (PAM sequence, underlined; cut site, magenta arrow). T7 editing (T) and HDR frequencies (P; underlined) are presented at the bottom of each gel. Each nucleofection was performed in biological duplicate.

  9. Supplementary Figure 9: Sample flow cytometry plots. (382 KB)

    (A) Representative flow cytometry data used to create bar graphs shown in Figure 3B. (B) Representative flow cytometry data used to create bar graphs shown in Figure 3C.

  10. Supplementary Figure 10: Structural data is consistent with asymmetric release of substrate by Cas9. (332 KB)

    A surface electrostatic view of Cas9, sgRNA (orange), and non-target (purple) or target (grey) DNA strands7. PAM-Cas9 interaction, white arrow; putative path of non-target strand, purple dots; presumed direction of non-target strand extrusion, black arrow.

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    Supplementary Figures 1–10 and Supplementary Notes 1–4

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