Journal: bioRxiv

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.1101/2021.05.23.445089

Figure Lengend Snippet: a . Schematic representation of the screen. 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 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 RPE1 n=2, two independent biological experiments. Statistical significance is calculated as in (b). d. Normalized GFP recovery values for ten best-performing and five worst-performing Cas9 fusions from panel (c). Protein fragments are denoted based on the position in canonical transcript (ie. 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 Fig. S3. j. Replication fork schematics.

Article Snippet: For each reaction, 0.4×10 of cells were resuspended in 25 μl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: Expressing, Selection, Marker, Mutagenesis, Transfection, Plasmid Preparation, Digital PCR

Journal: bioRxiv

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.1101/2021.05.23.445089

Figure Lengend Snippet: a. Schematic representation of experiment shown in b . b . The DNA repair progression in the RPE-1 reporter locus for eight of the better-performing fusions (selected from screens on ). ddPCR quantification, n=3, single independent experiment, bar denotes mean value, error bars represent ± S.D. Orange highlighting indicates the Cas9WT HDR editing level at 24h post-electroporation. P-values denote significance of the total editing (HDR+NHEJ) increment between the Cas9WT and other fusions (for 24h to 48h period). Statistical values derived using one-way ANOVA test. c. Schematic representation of experiment shown in d-i. d. Representative immunofluorescence image used for DNA breakpoint quantification. Cell nuclei are depicted in blue, γH2AX foci in green, and red arrows indicate individual foci. Scale bar 50 um. e. Time course for γH2AX foci emergence in human immortalized fibroblasts (BJ5-ta). The cells were electroporated with Cas9WT or Cas9-POLD3 mRNA and a pool of three guides targeting RNF2, Enh4-1, and STAT3 loci. The CTCF1 gRNA is constitutively expressed. Five confocal images were taken for each condition, n=5, single independent experiment, bar denotes mean value, error bars represent ± S.D. Statistical significance of the difference between Cas9WT and Cas9-POLD3 is calculated using ANOVA test for the equality of the means at a particular time point. f-i. ddPCR quantification of the NHEJ repair dynamics across time for the experiment described in e. The red arrow shows the direction of the approaching polymerase. The DNA strand which the CRISPR-Cas9 binds to is colored in red. n=1, single independent experiment, bar denotes mean value, error bars represent ± S.D.

Article Snippet: For each reaction, 0.4×10 of cells were resuspended in 25 μl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: Electroporation, Derivative Assay, Immunofluorescence, CRISPR

Journal: bioRxiv

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.1101/2021.05.23.445089

Figure Lengend Snippet: a. Schematic representation of the Affinity Purification Mass Spectrometry (AP-MS) experimental workflow. b. Protein interaction map of the Cas9 fusions (three biological replicates). The interacting proteins are clustered based on the number of interactions they make with the Cas9 fusions (I, unique interaction partners, VI, interact with all Cas9 fusions, including Cas9WT). Pink circles represent RNA-binding proteins, light-blue circles represent histone proteins, dark blue circles represent interaction partners with helicase activity, and light blue circles denote proteins with other functions.

Article Snippet: For each reaction, 0.4×10 of cells were resuspended in 25 μl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: Affinity Purification, Mass Spectrometry, RNA Binding Assay, Activity Assay

Journal: bioRxiv

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.1101/2021.05.23.445089

Figure Lengend Snippet: a. Mismatch plot of the Guide-seq results for Cas9WT and Cas9-POLD3, using a published guide against the endogenous HEK site 4. The on-target sequence is depicted at the first line of the table. The most abundant off-targets are listed underneath the intended edit and sorted by frequency, which is determined by dividing the number of reads that contain the off-target edit with the total read count. Gray lines connect the shared off-target sites. b. Venn diagram of the common and unique off-target sites between Cas9WT and Cas9-POLD3. c-f. Mismatch plots of the indel profiles of Cas9WT and Cas9-POLD3 in BJ-5ta fibroblasts, obtained by deep amplicon sequencing. The on-target gRNA binding site is on the top row, highlighted with a black box. The most frequent indels are below, sorted according to their frequency. The frequency is calculated by dividing the indel read count by the sum of the top 10 read counts. HDR template-matching edits marked with the red on the summary plots. Plots depict: c. On-target editing signature of Cas9 WT, RNF2 locus. d. On-target editing signature of Cas9-POLD3, RNF2 locus. e. On-target editing signature of Cas9 WT, ELANE locus. f. On-target editing signature of Cas9-POLD3, ELANE locus.

Article Snippet: For each reaction, 0.4×10 of cells were resuspended in 25 μl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: Sequencing, Amplification, Binding Assay

Journal: bioRxiv

Article Title: Rapid genome editing by CRISPR-Cas9-POLD3 fusion

doi: 10.1101/2021.05.23.445089

Figure Lengend Snippet: a . Editing of GFP in reporter HEK293T cells. n=4, one of two independent experiments, bar denotes mean value, error bars represent ± S.D. Statistical significance is calculated with unpaired, two-sided Student’s t-test. b. GFP reporter locus editing efficiency of recombinant Cas9 fusion proteins in RPE-1 and HEK293T cells. n=5, one of two independent experiments, bar denotes mean value, error bars represent ± S.D. Statistical significance is calculated with Anova that is adjusted for multiple comparisons.

Article Snippet: For each reaction, 0.4×10 of cells were resuspended in 25 μl of the MaxCyte electroporation buffer (MaxCyte, #EPB1), combined with 1000 ng of Cas9 mRNA, 100 pmol sgRNA (IDT) targeting the RNF2 locus and 100 pmol of the repair DNA template and electroporated using Optimization-8 pulse code.

Techniques: Recombinant