Cargando…

Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis

The use of the novel CRISPR/Cas12a system is advantageous, as it expands the possibilities for genome editing (GE) applications due to its different features compared to the commonly used CRISPR/Cas9 system. In this work, the CRISPR/Cas12a system was applied for the first time to apple to investigat...

Descripción completa

Detalles Bibliográficos
Autores principales: Schröpfer, Susan, Flachowsky, Henryk
Formato: Online Artículo Texto
Lenguaje:English
Publicado: MDPI 2021
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8619667/
https://www.ncbi.nlm.nih.gov/pubmed/34830492
http://dx.doi.org/10.3390/ijms222212611
_version_ 1784605049071075328
author Schröpfer, Susan
Flachowsky, Henryk
author_facet Schröpfer, Susan
Flachowsky, Henryk
author_sort Schröpfer, Susan
collection PubMed
description The use of the novel CRISPR/Cas12a system is advantageous, as it expands the possibilities for genome editing (GE) applications due to its different features compared to the commonly used CRISPR/Cas9 system. In this work, the CRISPR/Cas12a system was applied for the first time to apple to investigate its general usability for GE applications. Efficient guide RNAs targeting different exons of the endogenous reporter gene MdPDS, whose disruption leads to the albino phenotype, were pre-selected by in vitro cleavage assays. A construct was transferred to apple encoding for a CRISPR/Cas12a system that simultaneously targets two loci in MdPDS. Using fluorescent PCR capillary electrophoresis and amplicon deep sequencing, all identified GE events of regenerated albino shoots were characterized as deletions. Large deletions between the two neighboring target sites were not observed. Furthermore, a chimeric composition of regenerates and shoots that exhibited multiple GE events was observed frequently. By comparing both analytical methods, it was shown that fluorescent PCR capillary gel electrophoresis is a sensitive high-throughput genotyping method that allows accurate predictions of the size and proportion of indel mutations for multiple loci simultaneously. Especially for species exhibiting high frequencies of chimerism, it can be recommended as a cost-effective method for efficient selection of homohistont GE lines.
format Online
Article
Text
id pubmed-8619667
institution National Center for Biotechnology Information
language English
publishDate 2021
publisher MDPI
record_format MEDLINE/PubMed
spelling pubmed-86196672021-11-27 Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis Schröpfer, Susan Flachowsky, Henryk Int J Mol Sci Article The use of the novel CRISPR/Cas12a system is advantageous, as it expands the possibilities for genome editing (GE) applications due to its different features compared to the commonly used CRISPR/Cas9 system. In this work, the CRISPR/Cas12a system was applied for the first time to apple to investigate its general usability for GE applications. Efficient guide RNAs targeting different exons of the endogenous reporter gene MdPDS, whose disruption leads to the albino phenotype, were pre-selected by in vitro cleavage assays. A construct was transferred to apple encoding for a CRISPR/Cas12a system that simultaneously targets two loci in MdPDS. Using fluorescent PCR capillary electrophoresis and amplicon deep sequencing, all identified GE events of regenerated albino shoots were characterized as deletions. Large deletions between the two neighboring target sites were not observed. Furthermore, a chimeric composition of regenerates and shoots that exhibited multiple GE events was observed frequently. By comparing both analytical methods, it was shown that fluorescent PCR capillary gel electrophoresis is a sensitive high-throughput genotyping method that allows accurate predictions of the size and proportion of indel mutations for multiple loci simultaneously. Especially for species exhibiting high frequencies of chimerism, it can be recommended as a cost-effective method for efficient selection of homohistont GE lines. MDPI 2021-11-22 /pmc/articles/PMC8619667/ /pubmed/34830492 http://dx.doi.org/10.3390/ijms222212611 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
Schröpfer, Susan
Flachowsky, Henryk
Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis
title Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis
title_full Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis
title_fullStr Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis
title_full_unstemmed Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis
title_short Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis
title_sort tracing crispr/cas12a mediated genome editing events in apple using high-throughput genotyping by pcr capillary gel electrophoresis
topic Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8619667/
https://www.ncbi.nlm.nih.gov/pubmed/34830492
http://dx.doi.org/10.3390/ijms222212611
work_keys_str_mv AT schropfersusan tracingcrisprcas12amediatedgenomeeditingeventsinappleusinghighthroughputgenotypingbypcrcapillarygelelectrophoresis
AT flachowskyhenryk tracingcrisprcas12amediatedgenomeeditingeventsinappleusinghighthroughputgenotypingbypcrcapillarygelelectrophoresis