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A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs

The ultimate goal of technology development in genome editing is to enable precisely targeted genomic changes in any cells or organisms. Here we describe protoplast systems for precise and efficient DNA sequence changes with preassembled Cas9 ribonucleoprotein (RNP) complexes in Arabidopsis thaliana...

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Autores principales: Jiang, Wenzhi, Bush, Jenifer, Sheen, Jen
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8729822/
https://www.ncbi.nlm.nih.gov/pubmed/35005700
http://dx.doi.org/10.3389/fgeed.2021.719190
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author Jiang, Wenzhi
Bush, Jenifer
Sheen, Jen
author_facet Jiang, Wenzhi
Bush, Jenifer
Sheen, Jen
author_sort Jiang, Wenzhi
collection PubMed
description The ultimate goal of technology development in genome editing is to enable precisely targeted genomic changes in any cells or organisms. Here we describe protoplast systems for precise and efficient DNA sequence changes with preassembled Cas9 ribonucleoprotein (RNP) complexes in Arabidopsis thaliana, Nicotiana benthamiana, Brassica rapa, and Camelina sativa. Cas9 RNP-mediated gene disruption with dual gRNAs could reach ∼90% indels in Arabidopsis protoplasts. To facilitate facile testing of any Cas9 RNP designs, we developed two GFP reporter genes, which led to sensitive detection of nonhomologous end joining (NHEJ) and homology-directed repair (HDR), with editing efficiency up to 85 and 50%, respectively. When co-transfected with an optimal single-stranded oligodeoxynucleotide (ssODN) donor, precise editing of the AtALS gene via HDR reached 7% by RNPs. Significantly, precise mutagenesis mediated by preassembled primer editor (PE) RNPs led to 50% GFP reporter gene recovery in protoplasts and up to 4.6% editing frequency for the specific AtPDS mutation in the genome. The rapid, versatile and efficient gene editing by CRISPR RNP variants in protoplasts provides a valuable platform for development, evaluation and optimization of new designs and tools in gene and genomic manipulation and is applicable in diverse plant species.
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spelling pubmed-87298222022-01-06 A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs Jiang, Wenzhi Bush, Jenifer Sheen, Jen Front Genome Ed Genome Editing The ultimate goal of technology development in genome editing is to enable precisely targeted genomic changes in any cells or organisms. Here we describe protoplast systems for precise and efficient DNA sequence changes with preassembled Cas9 ribonucleoprotein (RNP) complexes in Arabidopsis thaliana, Nicotiana benthamiana, Brassica rapa, and Camelina sativa. Cas9 RNP-mediated gene disruption with dual gRNAs could reach ∼90% indels in Arabidopsis protoplasts. To facilitate facile testing of any Cas9 RNP designs, we developed two GFP reporter genes, which led to sensitive detection of nonhomologous end joining (NHEJ) and homology-directed repair (HDR), with editing efficiency up to 85 and 50%, respectively. When co-transfected with an optimal single-stranded oligodeoxynucleotide (ssODN) donor, precise editing of the AtALS gene via HDR reached 7% by RNPs. Significantly, precise mutagenesis mediated by preassembled primer editor (PE) RNPs led to 50% GFP reporter gene recovery in protoplasts and up to 4.6% editing frequency for the specific AtPDS mutation in the genome. The rapid, versatile and efficient gene editing by CRISPR RNP variants in protoplasts provides a valuable platform for development, evaluation and optimization of new designs and tools in gene and genomic manipulation and is applicable in diverse plant species. Frontiers Media S.A. 2021-12-22 /pmc/articles/PMC8729822/ /pubmed/35005700 http://dx.doi.org/10.3389/fgeed.2021.719190 Text en Copyright © 2021 Jiang, Bush and Sheen. https://creativecommons.org/licenses/by/4.0/This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.
spellingShingle Genome Editing
Jiang, Wenzhi
Bush, Jenifer
Sheen, Jen
A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs
title A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs
title_full A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs
title_fullStr A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs
title_full_unstemmed A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs
title_short A Versatile and Efficient Plant Protoplast Platform for Genome Editing by Cas9 RNPs
title_sort versatile and efficient plant protoplast platform for genome editing by cas9 rnps
topic Genome Editing
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8729822/
https://www.ncbi.nlm.nih.gov/pubmed/35005700
http://dx.doi.org/10.3389/fgeed.2021.719190
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