Enhancement of homology-directed repair with chromatin donor templates in cells
- Section of Molecular Biology, University of California, San Diego, United States
Figures
Figure 1 with 4 supplements
The efficiency of HDR-mediated gene editing with CRISPR-Cas9 is higher with chromatin donor templates than with DNA donor templates.
(A) Schematic outline of the workflow in the CRISPR-Cas9-mediated editing experiments with DNA or chromatin donor templates. The HDR-mediated insertion of the GFP sequence was directed to different …
Figure 1—figure supplement 1
Schematic representations of the CRISPR-Cas9 target regions for HDR-mediated insertion of a GFP reporter sequence.
(A) GAPDH locus. A DNA sequence that encodes the T2A self-cleaving peptide fused to the GFP protein (T2A-GFP, indicated in the figure as ‘GFP’) is inserted in exon 9 (E9) of the GAPDH locus. This …
Figure 1—figure supplement 2
Reconstitution of plasmid DNA donor templates into chromatin.
(A) Salt dialysis reconstitution of chromatin. The HDR donor template plasmids were reconstituted into chromatin with purified core histones by the salt dialysis method. (B) Micrococcal nuclease …
Figure 1—figure supplement 3
Flow cytometry analysis of MCF10A cells in control experimental conditions.
(A) Untransfected cells. (B) Cells were transfected with a Cas9-T2A-mCherry plasmid (lacking an sgRNA) in the absence of a donor template. (C) Cells were transfected with a Cas9-T2A-mCherry plasmid …
Figure 1—figure supplement 4
Flow cytometry analyses of biological replicates of HDR-mediated gene integration experiments in MCF10A cells.
(A) Data from HDR experiment two with GAPDH, RAB11A, or ACTB donor templates. (B) Data from HDR experiment three with GAPDH, RAB11A, or ACTB donor templates. HDR experiments were performed as …
Figure 2 with 5 supplements
The use of chromatin donor templates increases the efficiency of HDR-mediated homozygous gene editing relative to that seen with DNA donor templates.
(A) PCR analysis of gDNA from MCF10A GFP-positive clones. Three independent HDR experiments were performed as shown in Figure 1A, and the gDNA from individual GFP-positive clones was analyzed by …
Figure 2—figure supplement 1
Diagrams of the positions of the primer sets for the PCR analysis of GFP-positive clones at the GAPDH, RAB11A, and ACTB loci.
(A) GAPDH locus. (B) RAB11A locus. (C) ACTB locus. The expected PCR product sizes with wild-type gDNA (dashed lines), the positions of the primers (F1, R1, F2, R2; black arrows), and the DNA …
Figure 2—figure supplement 2
PCR analysis of gDNA from GFP-positive clones at the GAPDH locus in MCF10A cells.
(A) Clones (n = 54) collected from three independent HDR experiments with a DNA donor template. Lanes 1 to 15, 16 to 32, and 33 to 54 correspond to experiment 1, experiment 2, and experiment 3, …
Figure 2—figure supplement 3
PCR analysis of gDNA from GFP-positive clones at the RAB11A locus in MCF10A cells.
(A) Clones (n = 89) collected from three independent HDR experiments with a DNA donor template. Lanes 1 to 34, 35 to 54, and 55 to 89 correspond to experiment 1, experiment 2, and experiment 3, …
Figure 2—figure supplement 4
Long-range PCR analysis of gDNA from GFP-positive clones at the RAB11A locus in MCF10A cells.
(A) Analysis of homozygous candidates (n = 31) collected from three independent HDR experiments with a DNA donor template. (B) Analysis of homozygous candidates (n = 35) collected from three …
Figure 2—figure supplement 5
PCR analysis of gDNA from GFP-positive clones at the ACTB locus in MCF10A cells.
(A) Clones (n = 72) collected from three independent HDR experiments with a DNA donor template. Lanes 1 to 29, 30 to 48, and 49 to 72 correspond to experiment 1, experiment 2, and experiment 3, …
Figure 3 with 4 supplements
The efficiency of HDR-mediated gene editing with CRISPR-Cas9 is higher with a chromatin donor template than with a DNA donor template in HeLa cells.
(A) The use of a chromatin donor template relative to a naked DNA donor template results in an increase of GFP-positive cells. HDR experiments were performed as depicted in Figure 1A with HeLa cells …
Figure 3—figure supplement 1
Flow cytometry analyses of biological replicates of HDR-mediated gene integration experiments in HeLa cells.
(A) Data from HDR experiment 2. (B) Data from HDR experiment 3. HDR experiments were performed as outlined in Figure 1A. GFP-positive cells was gated based on cells that show no GFP expression (no …
Figure 3—figure supplement 2
PCR analysis of gDNA from GFP-positive clones in HeLa cells.
(A) Clones collected from HDR experiments with a DNA donor template (clones 12 to 21) or a chromatin donor template (clones 10 to 18). The positions of the PCR products of the wild-type and …
Figure 3—figure supplement 3
The efficiency of GFP insertion with different amounts of donor template in HeLa cells is higher with chromatin than with DNA.
(A) The results from HDR experiment 1. (B) The results from HDR experiment 2. In A and B, the experiments were performed as depicted in Figure 1A. HeLa cells were co-transfected with the …
Figure 3—figure supplement 4
Chromatin templates are of comparable or lower toxicity to cells relative to naked DNA templates.
Cell viability after transfection with a 3 kb plasmid as either naked DNA or chromatin was determined along with the viability of mock-transfected (no DNA or chromatin) cells. The cell viability was …
