Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing

Genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like...

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Autores principales:	Eric A. Noel, Donald P. Weeks, James L. Van Etten
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/07262c34381e4a7bb533f0320bcb64df
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spelling	oai:doaj.org-article:07262c34381e4a7bb533f0320bcb64df2021-11-04T06:07:14ZPursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing1932-6203https://doaj.org/article/07262c34381e4a7bb533f0320bcb64df2021-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC8530361/?tool=EBIhttps://doaj.org/toc/1932-6203Genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. Attempts at using Agrobacterium-mediated transfection of chlorovirus host Chlorella variabilis NC64A with a specially-designed binary vector resulted in successful transgenic cell selection based on expression of a hygromycin-resistance gene, initial expression of a green fluorescence gene and demonstration of integration of Agrobacterium T-DNA. However, expression of the integrated genes was soon lost. To develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). In one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting virus CA-4B-encoded gene 034r, which encodes a glycosyltransferase. Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, in ten subsequent experiments, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.Eric A. NoelDonald P. WeeksJames L. Van EttenPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 10 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Eric A. Noel Donald P. Weeks James L. Van Etten Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing
description	Genetic and molecular modifications of the large dsDNA chloroviruses, with genomes of 290 to 370 kb, would expedite studies to elucidate the functions of both identified and unidentified virus-encoded proteins. These plaque-forming viruses replicate in certain unicellular, eukaryotic chlorella-like green algae. However, to date, only a few of these algal species and virtually none of their viruses have been genetically manipulated due to lack of practical methods for genetic transformation and genome editing. Attempts at using Agrobacterium-mediated transfection of chlorovirus host Chlorella variabilis NC64A with a specially-designed binary vector resulted in successful transgenic cell selection based on expression of a hygromycin-resistance gene, initial expression of a green fluorescence gene and demonstration of integration of Agrobacterium T-DNA. However, expression of the integrated genes was soon lost. To develop gene editing tools for modifying specific chlorovirus CA-4B genes using preassembled Cas9 protein-sgRNA ribonucleoproteins (RNPs), we tested multiple methods for delivery of Cas9/sgRNA RNP complexes into infected cells including cell wall-degrading enzymes, electroporation, silicon carbide (SiC) whiskers, and cell-penetrating peptides (CPPs). In one experiment two independent virus mutants were isolated from macerozyme-treated NC64A cells incubated with Cas9/sgRNA RNPs targeting virus CA-4B-encoded gene 034r, which encodes a glycosyltransferase. Analysis of DNA sequences from the two mutant viruses showed highly targeted nucleotide sequence modifications in the 034r gene of each virus that were fully consistent with Cas9/RNP-directed gene editing. However, in ten subsequent experiments, we were unable to duplicate these results and therefore unable to achieve a reliable system to genetically edit chloroviruses. Nonetheless, these observations provide strong initial suggestions that Cas9/RNPs may function to promote editing of the chlorovirus genome, and that further experimentation is warranted and worthwhile.
format	article
author	Eric A. Noel Donald P. Weeks James L. Van Etten
author_facet	Eric A. Noel Donald P. Weeks James L. Van Etten
author_sort	Eric A. Noel
title	Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing
title_short	Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing
title_full	Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing
title_fullStr	Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing
title_full_unstemmed	Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing
title_sort	pursuit of chlorovirus genetic transformation and crispr/cas9-mediated gene editing
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/07262c34381e4a7bb533f0320bcb64df
work_keys_str_mv	AT ericanoel pursuitofchlorovirusgenetictransformationandcrisprcas9mediatedgeneediting AT donaldpweeks pursuitofchlorovirusgenetictransformationandcrisprcas9mediatedgeneediting AT jameslvanetten pursuitofchlorovirusgenetictransformationandcrisprcas9mediatedgeneediting
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Pursuit of chlorovirus genetic transformation and CRISPR/Cas9-mediated gene editing

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