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Article: Combinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9

TitleCombinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9
Authors
KeywordsGenome
Genes
Single guide
Issue Date2019
PublisherNature Publishing Group. The Journal's web site is located at http://www.nature.com/nmeth
Citation
Nature Methods, 2019, v. 16, p. 722-730 How to Cite?
AbstractThe combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering proteins through combinatorial modifications en masse.
Persistent Identifierhttp://hdl.handle.net/10722/274578
ISSN
2023 Impact Factor: 36.1
2023 SCImago Journal Rankings: 14.796
ISI Accession Number ID
Errata

 

DC FieldValueLanguage
dc.contributor.authorChoi, GCG-
dc.contributor.authorZhou, P-
dc.contributor.authorYUEN, CTL-
dc.contributor.authorCHAN, BKC-
dc.contributor.authorXU, F-
dc.contributor.authorBao, S-
dc.contributor.authorChu, HY-
dc.contributor.authorThean, D-
dc.contributor.authorTan, K-
dc.contributor.authorWong, KH-
dc.contributor.authorZheng, Z-
dc.contributor.authorWong, ASL-
dc.date.accessioned2019-08-18T15:04:35Z-
dc.date.available2019-08-18T15:04:35Z-
dc.date.issued2019-
dc.identifier.citationNature Methods, 2019, v. 16, p. 722-730-
dc.identifier.issn1548-7091-
dc.identifier.urihttp://hdl.handle.net/10722/274578-
dc.description.abstractThe combined effect of multiple mutations on protein function is hard to predict; thus, the ability to functionally assess a vast number of protein sequence variants would be practically useful for protein engineering. Here we present a high-throughput platform that enables scalable assembly and parallel characterization of barcoded protein variants with combinatorial modifications. We demonstrate this platform, which we name CombiSEAL, by systematically characterizing a library of 948 combination mutants of the widely used Streptococcus pyogenes Cas9 (SpCas9) nuclease to optimize its genome-editing activity in human cells. The ease with which the editing activities of the pool of SpCas9 variants can be assessed at multiple on- and off-target sites accelerates the identification of optimized variants and facilitates the study of mutational epistasis. We successfully identify Opti-SpCas9, which possesses enhanced editing specificity without sacrificing potency and broad targeting range. This platform is broadly applicable for engineering proteins through combinatorial modifications en masse.-
dc.languageeng-
dc.publisherNature Publishing Group. The Journal's web site is located at http://www.nature.com/nmeth-
dc.relation.ispartofNature Methods-
dc.rightsThis is a post-peer-review, pre-copyedit version of an article published in [insert journal title]. The final authenticated version is available online at: https://doi.org/[insert DOI]-
dc.subjectGenome-
dc.subjectGenes-
dc.subjectSingle guide-
dc.titleCombinatorial mutagenesis en masse optimizes the genome editing activities of SpCas9-
dc.typeArticle-
dc.identifier.emailChoi, GCG: gigichoi@hku.hk-
dc.identifier.emailZhou, P: zhoupeng@hku.hk-
dc.identifier.emailThean, D: dawntgl@hku.hk-
dc.identifier.emailWong, ASL: aslw@hku.hk-
dc.identifier.authorityWong, ASL=rp02139-
dc.description.naturelink_to_subscribed_fulltext-
dc.identifier.doi10.1038/s41592-019-0473-0-
dc.identifier.pmid31308554-
dc.identifier.scopuseid_2-s2.0-85069533970-
dc.identifier.hkuros301304-
dc.identifier.volume16-
dc.identifier.spage722-
dc.identifier.epage730-
dc.identifier.isiWOS:000477857700028-
dc.publisher.placeUnited Kingdom-
dc.relation.erratumdoi:10.1038/s41592-019-0533-5-
dc.identifier.issnl1548-7091-

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