title: High-fidelity CRISPR-Cas9 nucleases with no detectable genome-wide off-target effects.
abstract: CRISPR-Cas9 nucleases are widely used for genome editing but can induce unwanted off-target mutations. Existing strategies for reducing genome-wide off-target effects of the widely used Streptococcus pyogenes Cas9 (SpCas9) are imperfect, possessing only partial or unproven efficacies and other limitations that constrain their use. Here we describe SpCas9-HF1, a high-fidelity variant harbouring alterations designed to reduce non-specific DNA contacts. SpCas9-HF1 retains on-target activities comparable to wild-type SpCas9 with >85% of single-guide RNAs (sgRNAs) tested in human cells. Notably, with sgRNAs targeted to standard non-repetitive sequences, SpCas9-HF1 rendered all or nearly all off-target events undetectable by genome-wide break capture and targeted sequencing methods. Even for atypical, repetitive target sites, the vast majority of off-target mutations induced by wild-type SpCas9 were not detected with SpCas9-HF1. With its exceptional precision, SpCas9-HF1 provides an alternative to wild-type SpCas9 for research and therapeutic applications. More broadly, our results suggest a general strategy for optimizing genome-wide specificities of other CRISPR-RNA-guided nucleases.
authors.affiliation: Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.|Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Benjamin P
authors.initials: BP
authors.lastname: Kleinstiver
authors.affiliation: Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.|Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Vikram
authors.initials: V
authors.lastname: Pattanayak
authors.affiliation: Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.
authors.forename: Michelle S
authors.initials: MS
authors.lastname: Prew
authors.affiliation: Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.|Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: Shengdar Q
authors.initials: SQ
authors.lastname: Tsai
authors.affiliation: Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.
authors.forename: Nhu T
authors.initials: NT
authors.lastname: Nguyen
authors.affiliation: Department of Biomedical Sciences, City University of Hong Kong, Hong Kong, China.
authors.forename: Zongli
authors.initials: Z
authors.lastname: Zheng
authors.affiliation: Molecular Pathology Unit, Center for Cancer Research, and Center for Computational and Integrative Biology, Massachusetts General Hospital, Charlestown, Massachusetts 02129, USA.|Department of Pathology, Harvard Medical School, Boston, Massachusetts 02115, USA.
authors.forename: J Keith
authors.initials: JK
authors.lastname: Joung
mesh_terms: D001483:Base Sequence; D064130:CRISPR-Associated Proteins / Q000235:genetics* / Q000378:metabolism*; D064113:CRISPR-Cas Systems / Q000502:physiology*; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D004247:DNA / Q000235:genetics / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005818:Genetic Engineering; D015894:Genome, Human / Q000235:genetics*; D006801:Humans; D009154:Mutation; D011485:Protein Binding; D012313:RNA / Q000235:genetics; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA; D013297:Streptococcus pyogenes / Q000201:enzymology / Q000235:genetics; D013379:Substrate Specificity
chemical_list: D064130:CRISPR-Associated Proteins; D004268:DNA-Binding Proteins; D012313:RNA; D004247:DNA; D004720:Endonucleases