Title CRISPR-Cas9 editing in maize: systematic evaluation of off-target activity and its relevance in crop improvement /
Authors Young, Joshua ; Zastrow-Hayes, Gina ; Deschamps, Stéphane ; Svitashev, Sergei ; Zaremba, Mindaugas ; Acharya, Ananta ; Paulraj, Sushmitha ; Peterson-Burch, Brooke ; Schwartz, Chris ; Djukanovic, Vesna ; Lenderts, Brian ; Feigenbutz, Lanie ; Wang, Lijuan ; Alarcon, Clara ; Šikšnys, Virginijus ; May, Gregory ; Chilcoat, N. Doane ; Kumar, Sandeep
DOI 10.1038/s41598-019-43141-6
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Is Part of Scientific reports.. London : Nature Publishing Group. 2019, vol. 9, art. no. 6729, p. [1-11].. eISSN 2045-2322
Keywords [eng] DNA cleavage ; genome ; Cas9 ; RNA ; mutagenesis ; nucleases ; specificity ; reveals ; endonuclease ; efficiency
Abstract [eng] CRISPR-Cas9 enabled genome engineering has great potential for improving agriculture productivity, but the possibility of unintended off-target edits has evoked some concerns. Here we employ a three-step strategy to investigate Cas9 nuclease specificity in a complex plant genome. Our approach pairs computational prediction with genome-wide biochemical off-target detection followed by validation in maize plants. Our results reveal high frequency (up to 90%) on-target editing with no evidence of off-target cleavage activity when guide RNAs were bioinformatically predicted to be specific. Predictable off-target edits were observed but only with a promiscuous guide RNA intentionally designed to validate our approach. Off-target editing can be minimized by designing guide RNAs that are different from other genomic locations by at least three mismatches in combination with at least one mismatch occurring in the PAM proximal region. With well-designed guides, genetic variation from Cas9 off-target cleavage in plants is negligible, and much less than inherent variation.
Published London : Nature Publishing Group
Type Journal article
Language English
Publication date 2019
CC license CC license description