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Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage

View ORCID ProfilePéter István Kulcsér, András Tálas, Krisztina Huszár, Zoltán Ligeti, Eszter Tóth, Nóra Weinhardt, Elfrieda Fodor, Ervin Welker
doi: https://doi.org/10.1101/187898
Péter István Kulcsér
1Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary
2Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
3University of Szeged, Szeged, Hungary
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  • ORCID record for Péter István Kulcsér
András Tálas
1Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary
4School of Ph.D. Studies, Semmelweis University, Budapest, Hungary
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Krisztina Huszár
1Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary
2Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
5Gene Design Kft., Szeged, Hungary
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Zoltán Ligeti
1Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary
5Gene Design Kft., Szeged, Hungary
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Eszter Tóth
1Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary
2Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
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Nóra Weinhardt
1Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary
2Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
3University of Szeged, Szeged, Hungary
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Elfrieda Fodor
2Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
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Ervin Welker
1Institute of Enzymology, Research Centre for Natural Sciences of the Hungarian Academy of Sciences, Budapest, Hungary
2Institute of Biochemistry, Biological Research Centre of the Hungarian Academy of Sciences, Szeged, Hungary
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  • For correspondence: welker.ervin@ttk.mta.hu
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Abstract

Background The propensity for off-target activity of Streptococcus pyogenes Cas9 (SpCas9) has been considerably decreased by rationally engineered variants with increased fidelity (eSpCas9; SpCas9-HF1). However, a subset of targets still generate considerable off-target effects. To deal specifically with these targets, we generated new "Highly enhanced Fidelity" nuclease variants (HeFSpCas9s) containing mutations from both eSpCas9 and SpCas9-HF1 and examined these improved nuclease variants side-by-side, to decipher the factors that affect their specificities and to determine the optimal nuclease for applications sensitive to off-target effects.

Results These three increased-fidelity nucleases can routinely be used only with perfectly matching 20 nucleotide-long spacers; a matching 5' G extension being more detrimental to their activities than a mismatching one. HeFSpCas9s exhibit substantially improved specificity specifically for those targets for which eSpCas9 and SpCas9-HF1 have higher off-target propensity. There is also a ranking among the targets by their cleavability and off-target effects manifested by the increased fidelity nucleases. Furthermore, we show that the mutations in these variants may diminish the cleavage, but not the DNA-binding, of SpCas9s.

Conclusions No single nuclease variant shows generally superior fidelity; instead, for highest specificity cleavage, each target needs to be matched with an appropriate high fidelity nuclease. We provide here a framework for generating new nuclease variants for targets that currently have no matching optimal nuclease, and offer a simple mean for identifying the optimal nuclease for targets in the absence of accurate target-ranking prediction tools.

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Posted September 12, 2017.
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Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage
Péter István Kulcsér, András Tálas, Krisztina Huszár, Zoltán Ligeti, Eszter Tóth, Nóra Weinhardt, Elfrieda Fodor, Ervin Welker
bioRxiv 187898; doi: https://doi.org/10.1101/187898
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Crossing enhanced and high fidelity SpCas9 nucleases to optimize specificity and cleavage
Péter István Kulcsér, András Tálas, Krisztina Huszár, Zoltán Ligeti, Eszter Tóth, Nóra Weinhardt, Elfrieda Fodor, Ervin Welker
bioRxiv 187898; doi: https://doi.org/10.1101/187898

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