Rapidly inducible Cas9 and DSB-ddPCR to probe editing kinetics

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.

Figure 1. Development of a chemically inducible Cas9 (ciCas9)

(a) A schematic depiction of the strategy to engineer a single-component, chemically inducible Streptococcus pyogenes Cas9 variant is shown. (b) The REC2 domain was replaced with BCL-xL and a BH3 peptide was appended to the C-terminus via flexible linkers of varying lengths. (c) Indel frequency at the AAVS1 locus 24 hours after activation of ciCas9 activity is shown for different concentrations of A3. Black bars depict means (n = 3 cell culture replicates). (d) Indel frequency at different times following activation of ciCas9 with A3 is shown for four sgRNAs at three different loci. Error bars depict s.e.m. (n = 3 cell culture replicates).

Figure 2. DSB-ddPCR is an accurate and precise way to quantify DSBs

(a) A schematic illustrates the DSB-ddPCR assay. The assay relies on two locus-specific PCR amplicons, the first of which spans the cleavage junction (primers F1, R1) and the second of which is adjacent (primers F2, R2). Amplicons are detected by the activation of amplicon-specific fluorescent hydrolysis probes. Template that has been cleaved by Cas9 or another nuclease does not generate the first amplicon or activate the first probe. Uncleaved control DNA was digested with a restriction enzyme to create cleaved control DNA. Uncleaved and cleaved control DNA was mixed in specific proportions. (b) Representative droplet FAM and VIC probe intensities are shown for uncleaved control MYC locus template DNA (left panel), a 1:1 mixture of uncleaved:cleaved control DNA (middle panel), and cleaved control DNA (right panel). Colors indicate droplets with no template (gray), intact template (blue), and cleaved template (red). Droplet populations in the dot plots are: uncleaved control DNA (DSB: 120, no DSB: 1,724, no template: 12,360), 1:1 Mixture (DSB: 799, no DSB: 914, no template: 12,305), cleaved control DNA (DSB: 1,757, no DSB: 14, no template: 13,247).

Figure 3. Investigation of DSB and indel kinetics with ciCas9 and DSB-ddPCR

(a) Time courses of the frequency of DSBs and indels at three loci following activation of ciCas9 are shown. Solid lines indicate DSB frequency, dashed lines indicate indel frequency. DSBs were quantified using DSB-ddPCR and indels were quantified by high-throughput sequencing. Error bars depict s.e.m. (n = 3 cell culture replicates). (b) A DSB time course focused on the first two hours following ciCas9 activation is shown for two sgRNAs targeting the MYC locus. Error bars depict s.e.m. (n = 3 cell culture replicates). (c) A DSB time course comparing ciCas9 to a 4-hydroxytamoxifen-activated Cas9 variant, arC9, is shown. Error bars depict s.e.m. (n = 3 cell culture replicates).

Figure 4. ciCas9 specificity and basal activity can be tuned

(a) Editing is shown after 24 hours at the EMX1 on-target site (left panel) and an off-target site (right panel). ciCas9 and an enhanced-specificity variant (e-ciCas9) in the presence and absence of A3 are compared to wild type Cas9. Off-target editing with e-ciCas9 was not significantly increased relative to the no transfection control (one-sided t-test, n = 3, p = 0.21). Black bars depict means (n = 3 cell culture replicates). (b) Fluorescence polarization competitions between BH3 peptide variants and BODIPY-labeled BAK peptide for binding to BCL-xL. Data shown as inhibition of BODIPY-BAK binding. Error bars depict s.e.m. (n = 3 technical replicates). (c) Editing at the AAVS1 locus is shown for ciCas9 and two variants, L22 and F22, after 24 hours in the presence and absence of A3. Black bars depict means (n = 3 cell culture replicates).

Figure 5. ciCas9 can be activated by a variety of BCL-xL disruptors

(a) Structures of the three BCL-xL disruptors used in this study are shown with their reported K_d or K_i for BCL-xL, and their selectivity for BCL-xL over BCL-2 or MCL-1^,–. (b) Editing at the AAVS1 locus 24 hours after ciCas9 activation with different concentrations of the three disruptors is shown. The A3 data are also shown in Fig. 1c. Black bars depict means (n = 3 cell culture replicates). (c) Editing is shown at the AAVS1 locus 24 hours after activation of the ciCas9(F22) variant with 10 µM of each disruptor. Black bars depict means (n = 3 cell culture replicates).