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SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast

The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast  Schizosaccharomyces pombe rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endon...

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Autores principales: Torres-Garcia, Sito, Di Pompeo, Lorenza, Eivers, Luke, Gaborieau, Baptiste, White, Sharon A., Pidoux, Alison L., Kanigowska, Paulina, Yaseen, Imtiyaz, Cai, Yizhi, Allshire, Robin C.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: F1000 Research Limited 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721064/
https://www.ncbi.nlm.nih.gov/pubmed/33313420
http://dx.doi.org/10.12688/wellcomeopenres.16405.1
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author Torres-Garcia, Sito
Di Pompeo, Lorenza
Eivers, Luke
Gaborieau, Baptiste
White, Sharon A.
Pidoux, Alison L.
Kanigowska, Paulina
Yaseen, Imtiyaz
Cai, Yizhi
Allshire, Robin C.
author_facet Torres-Garcia, Sito
Di Pompeo, Lorenza
Eivers, Luke
Gaborieau, Baptiste
White, Sharon A.
Pidoux, Alison L.
Kanigowska, Paulina
Yaseen, Imtiyaz
Cai, Yizhi
Allshire, Robin C.
author_sort Torres-Garcia, Sito
collection PubMed
description The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast  Schizosaccharomyces pombe rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong  adh1 promoter. To overcome these problems we have developed an improved system,  SpEDIT, that uses a synthesised Cas9 sequence codon-optimised for  S. pombe expressed from the medium strength  adh15 promoter. The  SpEDIT system exhibits a flexible modular design where the sgRNA is fused to the 3’ end of the self-cleaving hepatitis delta virus (HDV) ribozyme, allowing expression of the sgRNA cassette to be driven by RNA polymerase III from a tRNA gene sequence. Lastly, the inclusion of sites for the  BsaI type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The  SpEDIT system allowed a 100% mutagenesis efficiency to be achieved when generating targeted point mutants in the  ade6 (+) or  ura4 (+) genes by transformation of cells from asynchronous cultures.  SpEDIT also permitted insertion, tagging and deletion events to be obtained with minimal effort. Simultaneous editing of two independent non-homologous loci was also readily achieved. Importantly the  SpEDIT system displayed reduced toxicity compared to currently available  S. pombe editing systems. Thus,  SpEDIT provides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast.
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spelling pubmed-77210642020-12-11 SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast Torres-Garcia, Sito Di Pompeo, Lorenza Eivers, Luke Gaborieau, Baptiste White, Sharon A. Pidoux, Alison L. Kanigowska, Paulina Yaseen, Imtiyaz Cai, Yizhi Allshire, Robin C. Wellcome Open Res Method Article The CRISPR/Cas9 system allows scarless, marker-free genome editing. Current CRISPR/Cas9 systems for the fission yeast  Schizosaccharomyces pombe rely on tedious and time-consuming cloning procedures to introduce a specific sgRNA target sequence into a Cas9-expressing plasmid. In addition, Cas9 endonuclease has been reported to be toxic to fission yeast when constitutively overexpressed from the strong  adh1 promoter. To overcome these problems we have developed an improved system,  SpEDIT, that uses a synthesised Cas9 sequence codon-optimised for  S. pombe expressed from the medium strength  adh15 promoter. The  SpEDIT system exhibits a flexible modular design where the sgRNA is fused to the 3’ end of the self-cleaving hepatitis delta virus (HDV) ribozyme, allowing expression of the sgRNA cassette to be driven by RNA polymerase III from a tRNA gene sequence. Lastly, the inclusion of sites for the  BsaI type IIS restriction enzyme flanking a GFP placeholder enables one-step Golden Gate mediated replacement of GFP with synthesized sgRNAs for expression. The  SpEDIT system allowed a 100% mutagenesis efficiency to be achieved when generating targeted point mutants in the  ade6 (+) or  ura4 (+) genes by transformation of cells from asynchronous cultures.  SpEDIT also permitted insertion, tagging and deletion events to be obtained with minimal effort. Simultaneous editing of two independent non-homologous loci was also readily achieved. Importantly the  SpEDIT system displayed reduced toxicity compared to currently available  S. pombe editing systems. Thus,  SpEDIT provides an effective and user-friendly CRISPR/Cas9 procedure that significantly improves the genome editing toolbox for fission yeast. F1000 Research Limited 2020-11-24 /pmc/articles/PMC7721064/ /pubmed/33313420 http://dx.doi.org/10.12688/wellcomeopenres.16405.1 Text en Copyright: © 2020 Torres-Garcia S et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Method Article
Torres-Garcia, Sito
Di Pompeo, Lorenza
Eivers, Luke
Gaborieau, Baptiste
White, Sharon A.
Pidoux, Alison L.
Kanigowska, Paulina
Yaseen, Imtiyaz
Cai, Yizhi
Allshire, Robin C.
SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast
title SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast
title_full SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast
title_fullStr SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast
title_full_unstemmed SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast
title_short SpEDIT: A fast and efficient CRISPR/Cas9 method for fission yeast
title_sort spedit: a fast and efficient crispr/cas9 method for fission yeast
topic Method Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7721064/
https://www.ncbi.nlm.nih.gov/pubmed/33313420
http://dx.doi.org/10.12688/wellcomeopenres.16405.1
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