Cargando…

Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs

The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas9 (CRISPR associated protein 9) system has been successfully adopted as a versatile genetic tool for functional manipulations, due to its convenience and effectiveness. Genetics lesions induced by single guide RNA (gRNA) a...

Descripción completa

Detalles Bibliográficos
Autores principales: Kim, Brian H., Zhang, GuangJun
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Genetics Society of America 2020
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056962/
https://www.ncbi.nlm.nih.gov/pubmed/31915253
http://dx.doi.org/10.1534/g3.119.401035
_version_ 1783503568642244608
author Kim, Brian H.
Zhang, GuangJun
author_facet Kim, Brian H.
Zhang, GuangJun
author_sort Kim, Brian H.
collection PubMed
description The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas9 (CRISPR associated protein 9) system has been successfully adopted as a versatile genetic tool for functional manipulations, due to its convenience and effectiveness. Genetics lesions induced by single guide RNA (gRNA) are usually small indel (insertion-deletion) DNA mutations. The impact of this type of CRISPR-induced DNA mutation on the coded mRNA transcription processing and protein translation can be complex. Unexpected or unknown transcripts, generated through alternative splicing, may impede the generation of successful loss-of-function mutants. To create null or null-like loss-of-function mutant zebrafish, we employed simultaneous multiple gRNA injection into single-cell stage embryos. We demonstrated that DNA composed of multiple exons, up to 78kb in length, can be deleted in the smarca2 gene locus. Additionally, two different genes (rnf185 and rnf215) were successfully mutated in F(1) fish with multiple exon deletions using this multiplex gRNA injection strategy. We expect this approach will be useful for knock-out studies in zebrafish and other vertebrate organisms, especially when the phenotype of a single gRNA-induced mutant is not clear.
format Online
Article
Text
id pubmed-7056962
institution National Center for Biotechnology Information
language English
publishDate 2020
publisher Genetics Society of America
record_format MEDLINE/PubMed
spelling pubmed-70569622020-03-12 Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs Kim, Brian H. Zhang, GuangJun G3 (Bethesda) Investigations The CRISPR (clustered regularly interspaced short palindromic repeats) and Cas9 (CRISPR associated protein 9) system has been successfully adopted as a versatile genetic tool for functional manipulations, due to its convenience and effectiveness. Genetics lesions induced by single guide RNA (gRNA) are usually small indel (insertion-deletion) DNA mutations. The impact of this type of CRISPR-induced DNA mutation on the coded mRNA transcription processing and protein translation can be complex. Unexpected or unknown transcripts, generated through alternative splicing, may impede the generation of successful loss-of-function mutants. To create null or null-like loss-of-function mutant zebrafish, we employed simultaneous multiple gRNA injection into single-cell stage embryos. We demonstrated that DNA composed of multiple exons, up to 78kb in length, can be deleted in the smarca2 gene locus. Additionally, two different genes (rnf185 and rnf215) were successfully mutated in F(1) fish with multiple exon deletions using this multiplex gRNA injection strategy. We expect this approach will be useful for knock-out studies in zebrafish and other vertebrate organisms, especially when the phenotype of a single gRNA-induced mutant is not clear. Genetics Society of America 2020-01-08 /pmc/articles/PMC7056962/ /pubmed/31915253 http://dx.doi.org/10.1534/g3.119.401035 Text en Copyright © 2020 Kim, Zhang http://creativecommons.org/licenses/by/4.0/ This is an open-access article distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
spellingShingle Investigations
Kim, Brian H.
Zhang, GuangJun
Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs
title Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs
title_full Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs
title_fullStr Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs
title_full_unstemmed Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs
title_short Generating Stable Knockout Zebrafish Lines by Deleting Large Chromosomal Fragments Using Multiple gRNAs
title_sort generating stable knockout zebrafish lines by deleting large chromosomal fragments using multiple grnas
topic Investigations
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7056962/
https://www.ncbi.nlm.nih.gov/pubmed/31915253
http://dx.doi.org/10.1534/g3.119.401035
work_keys_str_mv AT kimbrianh generatingstableknockoutzebrafishlinesbydeletinglargechromosomalfragmentsusingmultiplegrnas
AT zhangguangjun generatingstableknockoutzebrafishlinesbydeletinglargechromosomalfragmentsusingmultiplegrnas