Cargando…

Assessment of genomic changes in a CRISPR/Cas9 Phaeodactylum tricornutum mutant through whole genome resequencing

The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, co-opted from a bacterial defense natural mechanism, is the cutting edge technology to carry out genome editing in a revolutionary fashion. It has been shown to work in many different model organisms, from human to mi...

Descripción completa

Detalles Bibliográficos
Autores principales: Russo, Monia Teresa, Aiese Cigliano, Riccardo, Sanseverino, Walter, Ferrante, Maria Immacolata
Formato: Online Artículo Texto
Lenguaje:English
Publicado: PeerJ Inc. 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6174884/
https://www.ncbi.nlm.nih.gov/pubmed/30310734
http://dx.doi.org/10.7717/peerj.5507
_version_ 1783361367856644096
author Russo, Monia Teresa
Aiese Cigliano, Riccardo
Sanseverino, Walter
Ferrante, Maria Immacolata
author_facet Russo, Monia Teresa
Aiese Cigliano, Riccardo
Sanseverino, Walter
Ferrante, Maria Immacolata
author_sort Russo, Monia Teresa
collection PubMed
description The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, co-opted from a bacterial defense natural mechanism, is the cutting edge technology to carry out genome editing in a revolutionary fashion. It has been shown to work in many different model organisms, from human to microbes, including two diatom species, Phaeodactylum tricornutum and Thalassiosira pseudonana. Transforming P. tricornutum by bacterial conjugation, we have performed CRISPR/Cas9-based mutagenesis delivering the nuclease as an episome; this allowed for avoiding unwanted perturbations due to random integration in the genome and for excluding the Cas9 activity when it was no longer required, reducing the probability of obtaining off-target mutations, a major drawback of the technology. Since there are no reports on off-target occurrence at the genome level in microalgae, we performed whole-genome Illumina sequencing and found a number of different unspecific changes in both the wild type and mutant strains, while we did not observe any preferential mutation in the genomic regions in which off-targets were predicted. Our results confirm that the CRISPR/Cas9 technology can be efficiently applied to diatoms, showing that the choice of the conjugation method is advantageous for minimizing unwanted changes in the genome of P. tricornutum.
format Online
Article
Text
id pubmed-6174884
institution National Center for Biotechnology Information
language English
publishDate 2018
publisher PeerJ Inc.
record_format MEDLINE/PubMed
spelling pubmed-61748842018-10-11 Assessment of genomic changes in a CRISPR/Cas9 Phaeodactylum tricornutum mutant through whole genome resequencing Russo, Monia Teresa Aiese Cigliano, Riccardo Sanseverino, Walter Ferrante, Maria Immacolata PeerJ Biotechnology The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system, co-opted from a bacterial defense natural mechanism, is the cutting edge technology to carry out genome editing in a revolutionary fashion. It has been shown to work in many different model organisms, from human to microbes, including two diatom species, Phaeodactylum tricornutum and Thalassiosira pseudonana. Transforming P. tricornutum by bacterial conjugation, we have performed CRISPR/Cas9-based mutagenesis delivering the nuclease as an episome; this allowed for avoiding unwanted perturbations due to random integration in the genome and for excluding the Cas9 activity when it was no longer required, reducing the probability of obtaining off-target mutations, a major drawback of the technology. Since there are no reports on off-target occurrence at the genome level in microalgae, we performed whole-genome Illumina sequencing and found a number of different unspecific changes in both the wild type and mutant strains, while we did not observe any preferential mutation in the genomic regions in which off-targets were predicted. Our results confirm that the CRISPR/Cas9 technology can be efficiently applied to diatoms, showing that the choice of the conjugation method is advantageous for minimizing unwanted changes in the genome of P. tricornutum. PeerJ Inc. 2018-10-05 /pmc/articles/PMC6174884/ /pubmed/30310734 http://dx.doi.org/10.7717/peerj.5507 Text en © 2018 Russo et al. http://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, reproduction and adaptation in any medium and for any purpose provided that it is properly attributed. For attribution, the original author(s), title, publication source (PeerJ) and either DOI or URL of the article must be cited.
spellingShingle Biotechnology
Russo, Monia Teresa
Aiese Cigliano, Riccardo
Sanseverino, Walter
Ferrante, Maria Immacolata
Assessment of genomic changes in a CRISPR/Cas9 Phaeodactylum tricornutum mutant through whole genome resequencing
title Assessment of genomic changes in a CRISPR/Cas9 Phaeodactylum tricornutum mutant through whole genome resequencing
title_full Assessment of genomic changes in a CRISPR/Cas9 Phaeodactylum tricornutum mutant through whole genome resequencing
title_fullStr Assessment of genomic changes in a CRISPR/Cas9 Phaeodactylum tricornutum mutant through whole genome resequencing
title_full_unstemmed Assessment of genomic changes in a CRISPR/Cas9 Phaeodactylum tricornutum mutant through whole genome resequencing
title_short Assessment of genomic changes in a CRISPR/Cas9 Phaeodactylum tricornutum mutant through whole genome resequencing
title_sort assessment of genomic changes in a crispr/cas9 phaeodactylum tricornutum mutant through whole genome resequencing
topic Biotechnology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6174884/
https://www.ncbi.nlm.nih.gov/pubmed/30310734
http://dx.doi.org/10.7717/peerj.5507
work_keys_str_mv AT russomoniateresa assessmentofgenomicchangesinacrisprcas9phaeodactylumtricornutummutantthroughwholegenomeresequencing
AT aieseciglianoriccardo assessmentofgenomicchangesinacrisprcas9phaeodactylumtricornutummutantthroughwholegenomeresequencing
AT sanseverinowalter assessmentofgenomicchangesinacrisprcas9phaeodactylumtricornutummutantthroughwholegenomeresequencing
AT ferrantemariaimmacolata assessmentofgenomicchangesinacrisprcas9phaeodactylumtricornutummutantthroughwholegenomeresequencing