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CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering

Abstract

Prokaryotic type II CRISPR-Cas systems can be adapted to enable targeted genome modifications across a range of eukaryotes1,2,3,4,5,6,7. Here we engineer this system to enable RNA-guided genome regulation in human cells by tethering transcriptional activation domains either directly to a nuclease-null Cas9 protein or to an aptamer-modified single guide RNA (sgRNA). Using this functionality we developed a transcriptional activation–based assay to determine the landscape of off-target binding of sgRNA:Cas9 complexes and compared it with the off-target activity of transcription activator–like (TALs) effectors8,9. Our results reveal that specificity profiles are sgRNA dependent, and that sgRNA:Cas9 complexes and 18-mer TAL effectors can potentially tolerate 1–3 and 1–2 target mismatches, respectively. By engineering a requirement for cooperativity through offset nicking for genome editing or through multiple synergistic sgRNAs for robust transcriptional activation, we suggest methods to mitigate off-target phenomena. Our results expand the versatility of the sgRNA:Cas9 tool and highlight the critical need to engineer improved specificity.

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Figure 1: RNA-guided transcriptional activation.
Figure 2: Evaluating the landscape of targeting by sgRNA:Cas9 complexes and TAL effectors.
Figure 3: Offset nicking.

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Acknowledgements

P.M. thanks R. Kalhor for insightful discussions. This work was supported by US National Institutes of Health grant P50 HG005550 and Department of Energy grant DE-FG02-02ER63445.

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Authors

Contributions

P.M., J.A. and G.M.C. conceived the study. P.M. designed and performed experiments. J.A. designed and performed bioinformatic analyses. M.M. performed experiments. P.B.S., K.M.E., S.K. and L.Y. developed reagents and performed analyses. P.M., J.A. and G.M.C. wrote the manuscript with support from all authors.

Corresponding author

Correspondence to George M Church.

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The authors declare no competing financial interests.

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Supplementary Figures 1–17, Supplementary Tables 1–3 and Supplementary Notes 1–3 (PDF 28595 kb)

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Mali, P., Aach, J., Stranges, P. et al. CAS9 transcriptional activators for target specificity screening and paired nickases for cooperative genome engineering. Nat Biotechnol 31, 833–838 (2013). https://doi.org/10.1038/nbt.2675

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