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

by Jan-Niklas Dohrke
Doctoral thesis
Date of Examination:2024-11-06
Date of issue:2024-11-29
Advisor:Prof. Dr. Stefan Jakobs
Referee:Prof. Dr. Stefan Jakobs
Referee:Prof. Dr. Peter Rehling
Persistent Address: http://dx.doi.org/10.53846/goediss-10892

 

 

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Abstract

English

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.
Keywords: Genomic Tagging; CRISPR/Cas; super-resolution microscopy; high-throughput screening; Automation; PCR