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A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs
Detailed target-selectivity information and experiment-based efficacy prediction tools are primarily available for Streptococcus pyogenes Cas9 (SpCas9). One obstacle to develop such tools is the rarity of accurate data. Here, we report a method termed ‘Self-targeting sgRNA Library Screen’ (SLS) for...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Oxford University Press
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034649/ https://www.ncbi.nlm.nih.gov/pubmed/33450024 http://dx.doi.org/10.1093/nar/gkaa1220 |
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author | Tálas, András Huszár, Krisztina Kulcsár, Péter István Varga, Julia K Varga, Éva Tóth, Eszter Welker, Zsombor Erdős, Gergely Pach, Péter Ferenc Welker, Ágnes Györgypál, Zoltán Tusnády, Gábor E Welker, Ervin |
author_facet | Tálas, András Huszár, Krisztina Kulcsár, Péter István Varga, Julia K Varga, Éva Tóth, Eszter Welker, Zsombor Erdős, Gergely Pach, Péter Ferenc Welker, Ágnes Györgypál, Zoltán Tusnády, Gábor E Welker, Ervin |
author_sort | Tálas, András |
collection | PubMed |
description | Detailed target-selectivity information and experiment-based efficacy prediction tools are primarily available for Streptococcus pyogenes Cas9 (SpCas9). One obstacle to develop such tools is the rarity of accurate data. Here, we report a method termed ‘Self-targeting sgRNA Library Screen’ (SLS) for assaying the activity of Cas9 nucleases in bacteria using random target/sgRNA libraries of self-targeting sgRNAs. Exploiting more than a million different sequences, we demonstrate the use of the method with the SpCas9-HF1 variant to analyse its activity and reveal motifs that influence its target-selectivity. We have also developed an algorithm for predicting the activity of SpCas9-HF1 with an accuracy matching those of existing tools. SLS is a facile alternative to the much more expensive and laborious approaches used currently and has the capability of delivering sufficient amount of data for most of the orthologs and variants of SpCas9. |
format | Online Article Text |
id | pubmed-8034649 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Oxford University Press |
record_format | MEDLINE/PubMed |
spelling | pubmed-80346492021-04-14 A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs Tálas, András Huszár, Krisztina Kulcsár, Péter István Varga, Julia K Varga, Éva Tóth, Eszter Welker, Zsombor Erdős, Gergely Pach, Péter Ferenc Welker, Ágnes Györgypál, Zoltán Tusnády, Gábor E Welker, Ervin Nucleic Acids Res Methods Online Detailed target-selectivity information and experiment-based efficacy prediction tools are primarily available for Streptococcus pyogenes Cas9 (SpCas9). One obstacle to develop such tools is the rarity of accurate data. Here, we report a method termed ‘Self-targeting sgRNA Library Screen’ (SLS) for assaying the activity of Cas9 nucleases in bacteria using random target/sgRNA libraries of self-targeting sgRNAs. Exploiting more than a million different sequences, we demonstrate the use of the method with the SpCas9-HF1 variant to analyse its activity and reveal motifs that influence its target-selectivity. We have also developed an algorithm for predicting the activity of SpCas9-HF1 with an accuracy matching those of existing tools. SLS is a facile alternative to the much more expensive and laborious approaches used currently and has the capability of delivering sufficient amount of data for most of the orthologs and variants of SpCas9. Oxford University Press 2021-01-15 /pmc/articles/PMC8034649/ /pubmed/33450024 http://dx.doi.org/10.1093/nar/gkaa1220 Text en © The Author(s) 2021. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com |
spellingShingle | Methods Online Tálas, András Huszár, Krisztina Kulcsár, Péter István Varga, Julia K Varga, Éva Tóth, Eszter Welker, Zsombor Erdős, Gergely Pach, Péter Ferenc Welker, Ágnes Györgypál, Zoltán Tusnády, Gábor E Welker, Ervin A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs |
title | A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs |
title_full | A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs |
title_fullStr | A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs |
title_full_unstemmed | A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs |
title_short | A method for characterizing Cas9 variants via a one-million target sequence library of self-targeting sgRNAs |
title_sort | method for characterizing cas9 variants via a one-million target sequence library of self-targeting sgrnas |
topic | Methods Online |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8034649/ https://www.ncbi.nlm.nih.gov/pubmed/33450024 http://dx.doi.org/10.1093/nar/gkaa1220 |
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