Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair
- The Institute of Molecular Health Sciences, ETH Zurich, Switzerland
- The Institute of Molecular Life Sciences, University of Zurich, Switzerland
- SIB Swiss Institute of Bioinformatics, Switzerland
- Institute for Pharmaceutical Sciences, ETH Zurich, Switzerland
- Department of Biochemistry, University of Zurich, Switzerland
Figures
Figure 1
Schematic overview of the workflow for linking the DNA repair template to the Cas9 RNP complex.
O6-benzylguanine (BG)-labeled DNA oligos are covalently linked to Cas9-SNAP fusion proteins. The DNA-Cas9 molecules are then complexed with the specific sgRNAs to form the functional …
Figure 2
Fluorescent reporter system for high-throughput analysis of DSB repair rates.
(a) Schematic overview of the HEK293T fluorescent reporter system. The RFP fluorophore carries a c.190_191delinsCT mutation that substitutes two nucleotides TA at the positions 190 and 191 in the …
Figure 3 with 1 supplement
Covalent linkage of the DNA repair template to the Cas9 RNP complex.
(a) Band shift of the 81-mer amino-modified oligo after coupling to BG-GLA-NHS shown on a denaturing PAGE gel. Amino modified oligos were mixed with amine-reactive BG building blocks and the samples …
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Figure 3—source data 1
Numerical data and the exact p values for all graphs in Figure 3—figure supplement 1.
- https://doi.org/10.7554/eLife.33761.007
Figure 3—figure supplement 1
Covalent linkage of the DNA repair template to the Cas9 RNP complex.
(a) Quantification of HDR rates with DNA repair templates of different lengths by FACS. (b) Quantification of sgRNASpCas9(mutRFP) editing efficiency in the reporter cell line by FACS. (c) SYBR-Gold …
Figure 4 with 2 supplements
Linking the repair template to the Cas9 RNP complex enhances correction efficiency in a fluorescent reporter cell line.
(a) Comparison between the control Cas9 system (RNP unco.: SpCas9-SNAP plus unlabeled donor oligo) and our novel system (RNPD coup.: SpCas9-SNAP conjugated to BG-labeled donor oligo). Cells were …
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Figure 4—source data 1
Numerical data and the exact p values for all graphs in Figure 4.
- https://doi.org/10.7554/eLife.33761.011
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Figure 4—source data 2
Numerical data for all graphs in Figure 4—figure supplement 1.
- https://doi.org/10.7554/eLife.33761.012
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Figure 4—source data 3
Numerical data and the exact p values for all graphs in Figure 4—figure supplement 2.
- https://doi.org/10.7554/eLife.33761.013
Figure 4—figure supplement 1
Linking the repair template to the Cas9 RNP complex increases correction rates at the expense of indel formation.
(a–f) Quantification of reporter cells by FACS analysis 5 days after transfection. The absolute percentages of (a,c,e) precisely corrected cells (RFP/GFP double positive cells), and (b,d,f) all …
Figure 4—figure supplement 2
Mechanistic insights into enhanced correction rates.
(a–e) Quantification of reporter cells by FACS analysis 5 days after transfection. The absolute percentages of precisely corrected cells (RFP/GFP double positive cells) (a,d), all edited cells …
Figure 5
Linking the repair template to the Cas9 RNP complex enhances correction efficiency at endogenous loci.
(a,b,c) Upper panels: Schematic overview of the target genomic regions of the Streptococcus pyogenes gRNAs. Black arrow indicates the introduced DSB site. The nucleotides that are exchanged in case …
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Figure 5—source data 1
Numerical data and the exact p values for all graphs in Figure 5.
- https://doi.org/10.7554/eLife.33761.015
Figure 6
Direct comparison of the Cas9 RNPD system to the classical Cas9 complex.
Classical Cas9 system (wild type SpCas9 plus unlabeled donor oligo); Our novel RNPD system (SpCas9-SNAP conjugated to BG-labeled donor oligo). (a) Targeting of the reporter locus in HEK293T cells. …
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Figure 6—source data 1
Numerical data and the exact p values for all graphs in Figure 6.
- https://doi.org/10.7554/eLife.33761.017
Author response image 1
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Author response image 5
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Recombinant protein (Streptococcus pyogenes) | SpCas9-SNAP | This paper | Schwank and Jinek lab | |
| Recombinant protein (Staphylococcus aureus) | SadCas9-SNAP | This paper | Schwank and Jinek lab | |
| Genetic reagent | NH2-modified oligo | Integrated DNA Technologies | - | Custom DNA oligos/ '5 C6 NH2 modif. |
| Chemical compound | BG-GLA-NHS | New England Biolabs | ID_NEB:S9151S |
Additional files
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Supplementary file 1
This file contains Supplementary Tables 1-6 (referenced in the Materials and methods).
Supplementary Table 1 contains the guide protospacer sequences used in this study. Supplementary Table 2 contains the primer sequences for IVT of guides used in this study. Supplementary Table 3 contains the crRNA sequences of guides used in this study. Supplementary Table 4 contains the repair oligo sequences used in this study. The nucleotide substitution introduced by precise correction using repair template is shown in lowercase. Supplementary Table 5 contains the NGS primers used in this study. The target specific part of the primer is shown in uppercase, and the Illumina adapter is shown in lowercase. Supplementary Table 6 contains the plasmids used in this study.
- https://doi.org/10.7554/eLife.33761.018
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Supplementary file 2
This file contains the allele plots for the loci analyzed by NGS.
Allele plots show insertion/deletion variant alleles with frequency of at least 0.01%, and non-indel variants with frequency of at least 0.05% in any sample. When more than 50 variants passed these criteria, the top 50 alleles according to their maximum frequency in any sample are shown. From top to bottom, the consensus sequences for variant alleles are displayed in the order: no variant, precisely corrected allele, insertions (I) and deletions (D), single nucleotide variants (SNVs) and non-linear alignments. SNVs are only shown for non-indel variants and appear in color. In the y-axis labels, nucleotide numbers indicate the distance to the cut site. Variants are labelled with respect to the leftmost base. For example −5:9D is a 9 base pair deletion starting 5 bases upstream of the cut site. SNV labels show the bases that differ between the non-indel reads and the reference. The most common inserted sequences with less than 20 base pairs are shown in full in the legend. For longer and less frequent insertions the length is indicated. In the heatmap at right, the header shows the number of merged read pairs with alignments spanning the guide sequence. The x-axis is coloured according to experimental replicate.
- https://doi.org/10.7554/eLife.33761.019
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Supplementary file 3
This file contains complete variant count tables for the genomic loci analyzed by NGS.
Variants are labeled as in Supplementary file 2.
- https://doi.org/10.7554/eLife.33761.020
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Supplementary file 4
This file contains categorized variant count tables for the genomic loci analyzed by NGS.
Reads were classified as ‘indel’ if any insertions or deletions were present in the guide region, as ‘no variant’ if they perfectly matched the guide reference, (for on-target loci) ‘corrected’ if the targeted bases were changed as expected, and ‘mismatch’ if any other nucleotide changes were present.
- https://doi.org/10.7554/eLife.33761.021
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Transparent reporting form
- https://doi.org/10.7554/eLife.33761.022
