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Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM
The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Streptococcus pyogenes Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from S. canis (ScCas9), which has a more relaxed...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
MDPI
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392876/ https://www.ncbi.nlm.nih.gov/pubmed/34440868 http://dx.doi.org/10.3390/cells10082099 |
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author | Liu, Yunxing Liang, Fang Dong, Zijiong Li, Song Ye, Jianmin Qin, Wei |
author_facet | Liu, Yunxing Liang, Fang Dong, Zijiong Li, Song Ye, Jianmin Qin, Wei |
author_sort | Liu, Yunxing |
collection | PubMed |
description | The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Streptococcus pyogenes Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from S. canis (ScCas9), which has a more relaxed 5′-NNG-3′ PAM, was reported to have activities in human cells and plants. However, the editing ability of ScCas9 has not been tested in zebrafish. Here we characterized and optimized the activity of ScCas9 in zebrafish. Delivered as a ribonucleoprotein complex, ScCas9 can induce mutations in zebrafish. Using the synthetic modified crRNA:tracrRNA duplex instead of in vitro-transcribed single guide RNA, the low activity at some loci were dramatically improved in zebrafish. As far as we know, our work is the first report on the evaluation of ScCas9 in animals. Our work optimized ScCas9 as a new nuclease for targeting relaxed NNG PAMs for zebrafish genome editing, which will further improve genome editing in zebrafish. |
format | Online Article Text |
id | pubmed-8392876 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | MDPI |
record_format | MEDLINE/PubMed |
spelling | pubmed-83928762021-08-28 Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM Liu, Yunxing Liang, Fang Dong, Zijiong Li, Song Ye, Jianmin Qin, Wei Cells Article The CRISPR/Cas9 system has been widely used for gene editing in zebrafish. However, the required NGG protospacer adjacent motif (PAM) of Streptococcus pyogenes Cas9 (SpCas9) notably restricts the editable range of the zebrafish genome. Recently, Cas9 from S. canis (ScCas9), which has a more relaxed 5′-NNG-3′ PAM, was reported to have activities in human cells and plants. However, the editing ability of ScCas9 has not been tested in zebrafish. Here we characterized and optimized the activity of ScCas9 in zebrafish. Delivered as a ribonucleoprotein complex, ScCas9 can induce mutations in zebrafish. Using the synthetic modified crRNA:tracrRNA duplex instead of in vitro-transcribed single guide RNA, the low activity at some loci were dramatically improved in zebrafish. As far as we know, our work is the first report on the evaluation of ScCas9 in animals. Our work optimized ScCas9 as a new nuclease for targeting relaxed NNG PAMs for zebrafish genome editing, which will further improve genome editing in zebrafish. MDPI 2021-08-16 /pmc/articles/PMC8392876/ /pubmed/34440868 http://dx.doi.org/10.3390/cells10082099 Text en © 2021 by the authors. https://creativecommons.org/licenses/by/4.0/Licensee MDPI, Basel, Switzerland. This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution (CC BY) license (https://creativecommons.org/licenses/by/4.0/). |
spellingShingle | Article Liu, Yunxing Liang, Fang Dong, Zijiong Li, Song Ye, Jianmin Qin, Wei Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM |
title | Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM |
title_full | Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM |
title_fullStr | Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM |
title_full_unstemmed | Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM |
title_short | Genome Editing in Zebrafish by ScCas9 Recognizing NNG PAM |
title_sort | genome editing in zebrafish by sccas9 recognizing nng pam |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8392876/ https://www.ncbi.nlm.nih.gov/pubmed/34440868 http://dx.doi.org/10.3390/cells10082099 |
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