PUCK: Primer Utilised CRISPR/Cas Knock-Ins for High Throughput Super-Resolution Microscopy

Dohrke, Jan-Niklas

dc.contributor.advisor	Jakobs, Stefan Prof. Dr.
dc.contributor.author	Dohrke, Jan-Niklas
dc.date.accessioned	2024-11-29T16:24:37Z
dc.date.issued	2024-11-29
dc.identifier.uri	http://resolver.sub.uni-goettingen.de/purl?ediss-11858/15642
dc.identifier.uri	http://dx.doi.org/10.53846/goediss-10892
dc.format.extent	168	de
dc.language.iso	eng	de
dc.rights.uri	http://creativecommons.org/licenses/by/4.0/
dc.subject.ddc	570	de
dc.title	PUCK: Primer Utilised CRISPR/Cas Knock-Ins for High Throughput Super-Resolution Microscopy	de
dc.type	doctoralThesis	de
dc.contributor.referee	Jakobs, Stefan Prof. Dr.
dc.date.examination	2024-11-06	de
dc.description.abstracteng	The insertion of tags into proteins via gene editing is a powerful tool for microscopy, enabling the comparative analysis of different proteins. The advent of CRISPR/Cas systems has greatly simplified gene editing and improved its success rate. However, current high-throughput CRISPR/Cas screens primarily focus on knock-out or activation screens, with large-scale knock-ins of tag sequences remaining underutilised due to their complexity and resource demands. This study introduces PUCK (Primer Utilised CRISPR/Cas Knock-ins), an innovative method that is cost-effective, rapid, and user-friendly for inserting any tag into any protein sequence, facilitating large-scale knock-ins. A comprehensive platform of robotic protocols was established to execute a pioneering knock-in screen targeting nearly all outer mitochondrial membrane proteins. Additionally, diffraction-unlimited STED microscopy was developed into a high-throughput method for screen evaluation. An advanced analysis pipeline, incorporating optimal-transport-based methods, was also introduced to enhance the reliability of colocalization measurements in high-resolution images. This work underscores the importance of genomic tags in microscopy-based research and establishes PUCK as a new tool for efficient knock-in development and high-throughput screening.	de
dc.contributor.coReferee	Rehling, Peter Prof. Dr.
dc.subject.eng	Genomic Tagging	de
dc.subject.eng	CRISPR/Cas	de
dc.subject.eng	super-resolution microscopy	de
dc.subject.eng	high-throughput screening	de
dc.subject.eng	Automation	de
dc.subject.eng	PCR	de
dc.identifier.urn	urn:nbn:de:gbv:7-ediss-15642-7
dc.date.embargoed	2025-11-05
dc.affiliation.institute	Göttinger Graduiertenschule für Neurowissenschaften, Biophysik und molekulare Biowissenschaften (GGNB)	de
dc.subject.gokfull	Biologie (PPN619462639)	de
dc.description.embargoed	2025-11-05	de
dc.identifier.ppn	1909954721
dc.notes.confirmationsent	Confirmation sent 2024-11-29T19:45:01	de

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