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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 validation of this technology. Furthermore, these analyses allowed the characterization of an experimental artifact associated with mammalian PCR tagging which can most likely be explained by aberrant tag expression.

Finally, Tn5-Anchor-Seq was applied to the characterization of a diagnostic RT-LAMP assay for SARS-CoV-2 detection. The high scalability of the resulting LAMP-sequencing protocol allowed to sequence RT-LAMP reactions from 768 patient samples and by that helped to validate the sensitivity and specificity of this assay. This was important for deploying additional testing capacity during the COVID-19 pandemic.

In conclusion, this thesis introduces and showcases scalable approaches for pooled and single gene taggings in yeast and mammalian cells. In this context also an improved genome walking procedure was implemented which furthermore supported the establishment of a diagnostic assay for SARS-CoV-2 detection.