Figure 1. DNA repair proteins that affect genome editing outcomes.

(a) Schematic representation of the screen. Human embryonic kidney (HEK293T) cell line contains a reporter cassette expressing a selection marker, guide (sgGFP) and a mutant GFP-BFP protein. The line is transfected with a GFP repair oligo and an arrayed plasmid library containing ~450 DNA repair proteins that are fused to the C-terminus of Cas9WT. The homology-directed repair (HDR) editing efficiency for each fusion is defined by the percentage of cells with restored GFP function in each transfected well (measured by FACS). (b) Normalized GFP recovery values from two independent screens. Both experiments n = 3, each data point represents an average of value of all six replicas (n = 2 × 3), where each replicate was normalized either to the experimental average (blue dots) or to the plate average (orange dots). p-Values were calculated by one-way ANOVA test, where the mean of each triplicate is compared to the combined mean of all other triplicates from the screen. (c) Experiment average-normalized GFP recovery values for 52 fusions, chosen based on their performance in panel (b). In HEK293T n = 4, one independent biological experiment, in retinal pigment epithelium (RPE-1), n = 2, two independent biological experiments. Statistical significance is calculated as in (b). (d) Normalized GFP recovery values for 10 best-performing and five worst-performing Cas9 fusions from panel (c). Protein fragments are denoted based on the position in canonical transcript (i.e. SPIDR AA400-916 corresponds to a fragment of SPIDR protein which starts at amino acid 400 and ends at amino acid 916). (e) Schematic representation of the experiment shown in f. (f) Experiment average-normalized HDR editing for 31 fusions in four endogenous loci (ELANE, RNF2, Enh 4–1, and CTCF1) in HEK293T cells, quantified by droplet digital PCR. Polymerase fusions are marked with red pointers. n = 3, one independent biological experiment for each locus. Statistical significance is calculated as in (b). The raw data points are visible in Figure 1—figure supplements 3–6 . (j) Replication fork schematics. For all experiments, Cas9 was delivered as plasmid, the repair template as single-stranded oligonucleotide, and the guide was expressed from the genome.

Figure 1—figure supplement 1. Plasmid sizes and molar quantities of plasmids used in the high-throughput screens.

(a) Percentage distribution of plasmids used in the high-throughput screens according to their size; 90% of plasmids range from 11.5 to 13 kbp in size. (b) Percentage distribution of plasmids used in the high-throughput screens according to their molar amounts per well; 90% of plasmids were used in quantities ranging from 49 to 56 fmol per well.

Figure 1—figure supplement 2. Homology-directed repair (HDR) efficiency of the best-performing fusions.

Each cell in the heat map illustrates an HDR value that is normalized to the average HDR efficiency of the whole experiment (‘fold change’). The sets have an upper limit threshold and values above it are color-coded as a scale maximum (bright-red). For protein fragments, the start and end of the fragment are indicated in relation to the canonical transcript (i.e. RAD50 AA900-1300 corresponds to a fragment of RAD50 which starts from amino acid 900 and ends in amino acid 1300). (a) Normalized GFP conversion rate in human embryonic kidney (HEK293T) (n = 4, one independent biological experiment) and RPE (n = 2, two independent biological experiments) reporter cell lines. (b) Normalized HDR editing in HEK293T cells that stably express guides targeting endogenous loci (ELANE, RNF2, Enh 4–1, and CTCF1) (n = 3, one independent biological experiment for each locus). The same dataset is shown in Figure 1e-f and Figure 1—figure supplements 3–6.

Figure 1—figure supplement 3. HDR efficiency of the best-performing fusions: % of HDR, % HDR of total editing and % of NHEJ in HEK293T cells that stably express the guide targeting CTCF1 endogenous locus.

Data are the mean values, error bars represent ±S.D. Top illustrations depict the positioning of the CRISPR-Cas9 complex at the target locus. The red arrow shows the direction of the approaching polymerase. The same dataset is shown in Figure 1f and Figure 1—figure supplement 2b.

Figure 1—figure supplement 4. HDR efficiency of the best-performing fusions: % of HDR, % HDR of total editing and % of NHEJ in HEK293T cells that stably express the guide targeting ELANE endogenous locus.

Data are the mean values, error bars represent ±S.D. Top illustrations depict the positioning of the CRISPR-Cas9 complex at the target locus. The red arrow shows the direction of the approaching polymerase. The same dataset is shown in Figure 1f and Figure 1—figure supplement 2b.

Figure 1—figure supplement 5. HDR efficiency of the best-performing fusions: % of HDR, % HDR of total editing and % of NHEJ in HEK293T cells that stably express the guide targeting Enh 4-1 endogenous locus.

Data are the mean values, error bars represent ±S.D. Top illustrations depict the positioning of the CRISPR-Cas9 complex at the target locus. The red arrow shows the direction of the approaching polymerase. The same dataset is shown in Figure 1f and Figure 1—figure supplement 2b.

Figure 1—figure supplement 6. HDR efficiency of the best-performing fusions: % of HDR, % HDR of total editing and % of NHEJ in HEK293T cells that stably express the guide targeting RNF2 endogenous locus.

Data are the mean values, error bars represent ±S.D. Top illustrations depict the positioning of the CRISPR-Cas9 complex at the target locus. The red arrow shows the direction of the approaching polymerase. The same dataset is shown in Figure 1f and Figure 1—figure supplement 2b.