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Chimeric CRISPR guides enhance Cas9 target specificity

Noah Jakimo, Pranam Chatterjee, Joseph M Jacobson
doi: https://doi.org/10.1101/147686
Noah Jakimo
1MIT Media Lab, Cambridge, United States
2MIT Center for Bits and Atoms, Cambridge, United States
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Pranam Chatterjee
1MIT Media Lab, Cambridge, United States
2MIT Center for Bits and Atoms, Cambridge, United States
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Joseph M Jacobson
1MIT Media Lab, Cambridge, United States
2MIT Center for Bits and Atoms, Cambridge, United States
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Abstract

Oligonucleotide-guided nucleases (OGNs) have enabled rapid advances in precision genome engineering. Though much effort has gone into characterizing and mitigating mismatch tolerance for the most widely adopted OGN, Streptococcus pyogenes Cas9 (SpCas9), potential off-target interactions may still limit applications where on-target specificity is critical. Here we present a new axis to control mismatch sensitivity along the recognition-conferring spacer sequence of SpCas9’s guide RNA (gRNA). We introduce mismatch-evading loweredthermostability guides (melt-guides) and exhibit how nucleotide-type substitutions in the spacer can reduce cleavage of sequences mismatched by as few as a single base pair. Cotransfecting melt-guides into human cell culture with an exonuclease involved in DNA repair, we observe indel formation on a standard genomic target at approximately 70% the rate of canonical gRNA and undetectable on off-target data.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted June 08, 2017.
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Chimeric CRISPR guides enhance Cas9 target specificity
Noah Jakimo, Pranam Chatterjee, Joseph M Jacobson
bioRxiv 147686; doi: https://doi.org/10.1101/147686
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Chimeric CRISPR guides enhance Cas9 target specificity
Noah Jakimo, Pranam Chatterjee, Joseph M Jacobson
bioRxiv 147686; doi: https://doi.org/10.1101/147686

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