Abstract
The CRISPR-Cas9 system is commonly used in biomedical research; however, the precision of Cas9 is suboptimal for applications that involve editing a large population of cells (for example, gene therapy). Variations on the standard Cas9 system have yielded improvements in the precision of targeted DNA cleavage, but they often restrict the range of targetable sequences. It remains unclear whether these variants can limit lesions to a single site in the human genome over a large cohort of treated cells. Here we show that by fusing a programmable DNA-binding domain (pDBD) to Cas9 and attenuating Cas9’s inherent DNA-binding affinity, we were able to produce a Cas9-pDBD chimera with dramatically improved precision and an increased targeting range. Because the specificity and affinity of this framework can be easily tuned, Cas9-pDBDs provide a flexible system that can be tailored to achieve extremely precise genome editing at nearly any genomic locus.
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Acknowledgements
We thank M. Porteus (Stanford Medicine, Stanford, California, USA) for GFP reporter vector M427, N. Rhind for the use of his FACS machine, E. Kittler and the UMass Medical School Deep Sequencing Core for their assistance with the Illumina sequencing, and E. Sontheimer for insightful discussions. All new reagents described in this work have been deposited with the nonprofit plasmid-distribution service Addgene. This work was supported by the US National Institutes of Health (grant R01AI117839 to S.A.W. and J. Luban, grant U01HG007910 to M.G. and J. Luban, and grant R01HL093766 to S.A.W. and N. Lawson).
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Contributions
M.F.B. and A.G. performed all cell-based experiments. A.G.D., M.G. and L.J.Z. performed the bioinformatic analysis. S.O. and M.H.B. optimized the GFP reporter assay. M.F.B., A.G., L.J.Z. and S.A.W. directed the research and interpreted experiments. M.F.B., A.G. and S.A.W. wrote the manuscript with input from all the other authors.
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Competing interests
The authors have filed patent applications related to genome engineering technologies. S.A.W. is a consultant for Editas Medicine.
Integrated supplementary information
Supplementary Figure 1 Overview of the distribution of potential SpCas9 off-target sites within the human genome.
a) Schematic of the SpCas9:sgRNA system and the two sequential stages of licensing required for cleavage: Stage 1 - PAM recognition (nGG is highly preferred) and Stage 2 - complementary R-loop formation between the 20 nucleotide guide RNA and the interrogated DNA sequence. b) Genome-wide analysis using CRISPRseek28 of the potential off target sites for a representative set of 124,793 guide RNAs targeting human exons sequences. Guides were binned based on the predicted off-target site with the smallest number of mismatches to the guide sequence. A perfect match indicates the presence of an off-target site with a perfect guide match (magenta wedge). Only 1.6% of these guide sequences do not have an off-target site with 3 or fewer mismatches to the guide sequence (teal wedge). This subset would be the best candidates for precise genome editing. The vast majority of guides typically have many potential off-target sequences with 3 or fewer mismatches. c) Genome-wide analysis of the minimum number of mismatches in off-target sites for a representative set of 55,687 guide RNAs targeting human promoter regions (binned as describe above). Only 1% of these guide sequences do not have an off-target site with 3 or fewer mismatches to the guide sequence (teal wedge). d,e) Guide RNAs targeting gene exons (d) or promoters (e) with no predicted off-targets with <= 3 mismatches (teal wedge from b,c) are analyzed for off-target sites with potential bulges in the sgRNA:DNA heteroduplex29. Magenta wedges indicate the fraction of guides that have one or more off-target sites that have perfect complementarity with the exception of a single bulge.
Supplementary Figure 2 Structural modeling of SpCas9-Zif268.
A hybrid model containing the structure of SpCas930 (grey, PAM recognition residues magenta) with an sgRNA (20 nucleotide guide region cyan, remaining nucleotides red) and complementary target DNA (black) with the structure of Zif26831 (orange) placed with a binding site 11 bp from the PAM recognition sequence (Watson strand), where the two structures were superimposed on a B-DNA model constructed using 3DNA32. In parallel with the spacing parameter analysis in Figure 1, the structural model suggests that there is ample room for a ZFP to dock proximally to SpCas9 downstream of the PAM element.
Supplementary Figure 3 Protein expression analysis of SpCas9 and SpCas9-Zif268 and SpCas9-TAL268 platforms.
HEK293T cells are transfected with the indicated Cas9 plasmid (see methods for details), which has triple HA-tag (Supplementary Note). (Top) Full length protein is probed with anti-HA antibody. (Bottom) Alpha-tubulin is used as loading control.
Supplementary Figure 4 Activity profile of SpCas9-Zif268 with truncated guide
Activity profile of SpCas9 (blue) and SpCas9-Zif268 (red) in the GFP reporter assay33 with sgRNAs of various lengths truncated from the 5’ end of the guide and an nGG PAM target site. Data are from three independent biological replicates performed on different days in HEK293T cells. Error bars indicate standard error of the mean.
Supplementary Figure 5 Activity profile of SpCas9-TAL268.
Activity profile of SpCas9 (blue) and SpCas9-TAL268 (brown) in the GFP reporter assay with sgRNAs of 20nt vs 16nt lengths on nGG, nAG, nGA, nGC PAM target sites. Data are from three independent biological replicates performed on different days in HEK293T cells. Error bars indicate standard error of the mean.
Supplementary Figure 6 Activity of PAM mutants on different sequences.
a) Local sequences of the PAM interacting domain mutants at positions 1333 or 1335 of SpCas9 examined in this study. b) Analysis of SpCas9 mutant activity on different nGn or nnG PAM-containing target sites in the GFP reporter assay. Mutations that alter the interaction of R1333 with its guanine contact (nGn, teal) reveal modest activity at nnG PAMs. Correspondingly, mutations that alter the interaction of R1335 with its guanine contact (nnG, magenta) reveal modest activity at nGn PAMs. Data are from three independent biological replicates performed on different days in HEK293T cells. Error bars indicate standard error of the mean.
Supplementary Figure 7 Genomic activity profile of SpCas9 mutants
Analysis of the genomic activity profile of SpCas9 mutants (MT1, MT2, MT3 & MT4) independently and as SpCas9-Zif268 fusions at the PLXNB2 locus at a target site with an nGG PAM and a Zif268 binding site 11 bp away on the Watson strand. T7EI assay data from PCR products spanning the target site in three independent biological replicates (Rep1, Rep2, Rep3) performed on different days in HEK293T cells. Cleaved products are indicated by magenta arrowheads.
Supplementary Figure 8 Analysis of the genomic activity profile of SpCas9MT1 at TS2, TS3 and TS4 sites.
T7EI assay data from PCR products spanning the target site in three independent biological replicates (Rep1, Rep2, Rep3) performed on different days in HEK293T cells. Cleaved products are indicated by magenta arrowheads.
Supplementary Figure 9 Analysis of the genomic activity profile of SpCas9MT3-ZFPDCLK2 and SpCas9MT3-ZFPF9
Activity of SpCas9MT3-ZFPDCLK2 and SpCas9MT3-ZFPF9 at DNAJC6 and PLXDC2 sites respectively. These sequences have compatible binding sites for the DCLK27 and Factor IX1 ZFPs. T7EI assay data from PCR products spanning the target site from single experiment done in HEK293T cells. Cleaved products are indicated by magenta arrowheads. Similar analysis of SpCas9MT3-ZFPHEBP2 (targeting a compatible binding site for the HEBP2 ZFP6) at GPRC5B did not detect any lesions for this SpCas9MT3-ZFP fusion (data not shown).
Supplementary Figure 10 T7EI activity profile of SpCas9MT3-ZFPTS3 at the TS3 genomic locus as a function of the number of incorporated fingers.
Both Cas9WT and SpCas9MT3-ZFPTS3 with four fingers (F1-4) achieve efficient target cleavage. Removing a single finger from either end of the zinc finger array (F1-3 or F2-4) dramatically reduces the activity of the SpCas9MT3-ZFP chimera. Cleaved products are indicated by magenta arrowheads. The bar graph displays the mean lesion rate in three independent biological replicates (Rep1, Rep2, Rep3) performed on different days in HEK293T cells. Error bars indicate standard error of the mean.
Supplementary Figure 11 Analysis of the genomic activity profile of SpCas9MT3-TALETS3 and SpCas9MT3-TALETS4 at the TS3 and TS4 sites.
An arrow indicates the strand (Watson) of the highlighted sequence that is bound by the TALE. Two different TALE repeat lengths (9.5 and 15.5) were examined at each target site. T7EI assay data from PCR products spanning the target site in three independent biological replicates (Rep1, Rep2, Rep3) performed on different days in HEK293T cells. Cleaved products are indicated by magenta arrowheads.
Supplementary Figure 12 Activity profile of SpCas9MT3-ZFPTS3/TS4 with tru-sgRNAs34.
a) Nuclease activity based on T7EI assay for SpCas9WT and SpCas9MT3-ZFPTS3 with a 17 nucleotide truncated guide at the TS3 target site. b) Nuclease activity based on T7EI assay for SpCas9WT and SpCas9MT3-ZFPTS4 with an 18 nucleotide truncated guide at the TS4 target site. Cleaved products are indicated by magenta arrowheads. c) Target sites for the TS3 and TS4 tru-sgRNAs and graph showing the average activity at each target site in three independent biological replicates performed on different days in HEK293T cells. Error bars indicate standard error of the mean. For both TS3 and TS4, the SpCas9MT3-ZFP chimera is more sensitive to the truncation of the guide sequence, which is consistent with the greater sensitivity of this system to guide mismatches.
Supplementary Figure 13 Genomic sequence of OT2-2.
The sequence complementary to the guide is underlined with the two mismatched positions in bold. The nGG PAM is red and the potential ZFPTS2 binding site highlighted in yellow. Below the genomic sequence is predicted consensus recognition motif and sequence logo for ZFPTS2 based on a Random Forest model of ZFP recognition35. The predicted recognition motif only differs substantially at one position in the finger 4 binding site (C versus A).
Supplementary Figure 14 T7EI activity profile of SpCas9MT3-ZFPTS2 at the TS2 genomic locus and OT2-2 as a function of the number of fingers.
a) Both Cas9WT and SpCas9MT3-ZFPTS2 with four fingers (F1-4) result in efficient cleavage at the TS2 target site (magenta arrowheads indicate cleaved products). Removing a single finger from either end of the zinc finger array (F1-3 or F2-4) at most modestly reduces activity of the SpCas9MT3-ZFP chimera. Removing a both terminal fingers from the zinc finger array (F2-3) dramatically reduces activity of the SpCas9MT3-ZFP chimera. Construction of an alternate ZFP (TS2*) that recognizes an overlapping target site can also promote target cleavage. b) Both Cas9WT and SpCas9MT3-ZFPTS2 with four fingers (F1-4) result in efficient cleavage at the OT2-2 off-target site (magenta arrowheads indicate cleaved products). Removing a single finger from either end of the zinc finger array (F1-3 or F2-4) dramatically reduces activity of the SpCas9MT3-ZFP chimera. As does the utilization of an alternate ZFP (TS2*) that recognizes a different target site. Data from three independent biological replicates (Rep1, Rep2, Rep3) performed on different days in HEK293T cells.
Supplementary information
Supplementary Text and Figures
Supplementary Figures 1–14, Supplementary Tables 1–2, Supplementary Note and Supplementary Discussion (PDF 2557 kb)
Supplementary Table 3
Summary of lesion rates determined through targeted PCR-based deep-sequencing of potential off-target sites (XLSX 137 kb)
Supplementary Table 4
List of primers and on/off target sequences used in this study (XLSX 20 kb)
Supplementary Table 5
List of indexes used to identify genomic regions for targeted PCR deep-sequencing analysis (XLSX 67 kb)
Supplementary Table 6
Summary of peaks detected from GUIDE-seq off-target analysis (XLSX 12 kb)
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Bolukbasi, M., Gupta, A., Oikemus, S. et al. DNA-binding-domain fusions enhance the targeting range and precision of Cas9. Nat Methods 12, 1150–1156 (2015). https://doi.org/10.1038/nmeth.3624
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DOI: https://doi.org/10.1038/nmeth.3624
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