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primary topic: Scalable approaches for gene tagging and genome walking sequencing

Scalable approaches for gene tagging and genome walking sequencing
type: Article
abstract: A central question in the life sciences is how an observed phenotype is realized by a given biological system. One explanatory factor is certainly the genotype of such a system which can easily be characterized due to the development of high-throughput next-generation sequencing (NGS) technologies. This allows to generate many hypotheses which are best tested by manipulating genotypes and observing the resulting change in phenotype. However, respective high-throughput technologies for genotype manipulation are lagging behind. This thesis presents technical advancements in the field of genome engineering and next-generation sequencing which allow to construct and characterize collections of mutants with tagged genes. First, an improved version of a targeted NGS strategy is introduced, which is termed Tn5-Anchor-Seq. This protocol for genome walking sequencing allowed to characterize unknown sequences adjacent to known genomic sites so that for example tag integrations could be mapped. It builds upon the concept of tagmentation and was designed with scalability, efficiency and sensitivity in mind. Second, CRISPR-Cas12a-assisted tag library engineering (CASTLING) is presented as a high-throughput pooled strategy for gene tagging in the yeast Saccharomyces cerevisiae. It was implemented and validated using a set of 215 genes which were simultaneously targeted. Furthermore, genome-wide targeting was explored revealing that ~50% of yeast genes can be covered within one single experiment. Factors important for further application of this technology were identified and are discussed. Third, the insights gained during the development of the CASTLING strategy motivated the application of their concepts for single gene tagging in mammalian cells. Usually, mammalian gene targeting is relatively inefficient and laborious. Therefore, a convenient CRISPR-Cas12a-assisted PCR tagging strategy was developed. Several targeted NGS approaches including Tn5-Anchor-Seq supported the valid…
date: 2021
author list: authors
type: EPrint
type: DoctoralThesisEPrint
is part of: repository
has document: Scalable approaches for gene tagging and genome walking sequencing (PDF)
has document: Scalable approaches for gene tagging and genome walking sequencing (Other)
subject: 500 Naturwissenschaften und Mathematik
subject: 570 Biowissenschaften, Biologie
subject: 600 Technik, Medizin, angewandte Wissenschaften

Scalable approaches for gene tagging and genome walking sequencing (PDF)
type: Document
has file: 20211207-thesis_submittedHeiDOK.pdf
has part: 20211207-thesis_submittedHeiDOK.pdf

Konrad Herbst
type: Person
given name: Konrad
family name: Herbst

Scalable approaches for gene tagging and genome walking sequencing (Other)
type: Document
is version of: Scalable approaches for gene tagging and genome walking sequencing (PDF)
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Scalable approaches for gene tagging and genome walking sequencing (Other)
type: Document
is version of: Scalable approaches for gene tagging and genome walking sequencing (PDF)
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Scalable approaches for gene tagging and genome walking sequencing (Other)
type: Document
is version of: Scalable approaches for gene tagging and genome walking sequencing (PDF)
is volatile version of: Scalable approaches for gene tagging and genome walking sequencing (PDF)
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Scalable approaches for gene tagging and genome walking sequencing (Other)
type: Document
is version of: Scalable approaches for gene tagging and genome walking sequencing (PDF)
is volatile version of: Scalable approaches for gene tagging and genome walking sequencing (PDF)
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Scalable approaches for gene tagging and genome walking sequencing (Other)
type: Document
is version of: Scalable approaches for gene tagging and genome walking sequencing (PDF)
is volatile version of: Scalable approaches for gene tagging and genome walking sequencing (PDF)
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HTML Summary of #30884 Scalable approaches for gene tagging and genome walking sequencing
format: text/html
primary topic: Scalable approaches for gene tagging and genome walking sequencing

500 Naturwissenschaften und Mathematik
type: Concept
preferred label: 500 Natural sciences and mathematics @en

570 Biowissenschaften, Biologie
type: Concept
preferred label: 570 Life sciences @en

600 Technik, Medizin, angewandte Wissenschaften
type: Concept
preferred label: 600 Technology (Applied sciences) @en