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...

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Autores principales:	Susan Schröpfer, Henryk Flachowsky
Formato:	article
Lenguaje:	EN
Publicado:	MDPI AG 2021
Materias:	<i>Malus domestica</i> Cpf1 <i>MdPDS</i> fragment length analysis amplicon deep sequencing deletion Biology (General) QH301-705.5 Chemistry QD1-999
Acceso en línea:	https://doaj.org/article/9495efe019234640869374ef065a493d
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spelling	oai:doaj.org-article:9495efe019234640869374ef065a493d2021-11-25T17:58:18ZTracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis10.3390/ijms2222126111422-00671661-6596https://doaj.org/article/9495efe019234640869374ef065a493d2021-11-01T00:00:00Zhttps://www.mdpi.com/1422-0067/22/22/12611https://doaj.org/toc/1661-6596https://doaj.org/toc/1422-0067The 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 <i>MdPDS,</i> 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 <i>MdPDS</i>. 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.Susan SchröpferHenryk FlachowskyMDPI AGarticle<i>Malus domestica</i>Cpf1<i>MdPDS</i>fragment length analysisamplicon deep sequencingdeletionBiology (General)QH301-705.5ChemistryQD1-999ENInternational Journal of Molecular Sciences, Vol 22, Iss 12611, p 12611 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	<i>Malus domestica</i> Cpf1 <i>MdPDS</i> fragment length analysis amplicon deep sequencing deletion Biology (General) QH301-705.5 Chemistry QD1-999
spellingShingle	<i>Malus domestica</i> Cpf1 <i>MdPDS</i> fragment length analysis amplicon deep sequencing deletion Biology (General) QH301-705.5 Chemistry QD1-999 Susan Schröpfer Henryk Flachowsky Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis
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 <i>MdPDS,</i> 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 <i>MdPDS</i>. 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	article
author	Susan Schröpfer Henryk Flachowsky
author_facet	Susan Schröpfer Henryk Flachowsky
author_sort	Susan Schröpfer
title	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_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_sort	tracing crispr/cas12a mediated genome editing events in apple using high-throughput genotyping by pcr capillary gel electrophoresis
publisher	MDPI AG
publishDate	2021
url	https://doaj.org/article/9495efe019234640869374ef065a493d
work_keys_str_mv	AT susanschropfer tracingcrisprcas12amediatedgenomeeditingeventsinappleusinghighthroughputgenotypingbypcrcapillarygelelectrophoresis AT henrykflachowsky tracingcrisprcas12amediatedgenomeeditingeventsinappleusinghighthroughputgenotypingbypcrcapillarygelelectrophoresis
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Tracing CRISPR/Cas12a Mediated Genome Editing Events in Apple Using High-Throughput Genotyping by PCR Capillary Gel Electrophoresis

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