SP 4 CRISPR Technology in Human Reproduction - An interdisciplinary Approach

The range of reproductive options investigated by the URPP H2R includes the potential future scenarios that rely on CRISPR-based technologies. The opportunities and risks of human germline editing using the CRISPR technology are being discussed extensively worldwide. Genetic modification of the germline with CRISPR might represent a promising approach for curing monogenetic disorders (e.g. cystic fibrosis), and for reducing genetic risk factors associated with common chronic diseases in which multiple genes interact with environmental factors (e.g. obesity, coronary heart disease, diabetes, Alzheimer’s disease, and multiple cancers). However, the consequences of such modifications for future generations are still unclear; indications are ambiguous, and technical feasibility is challenging. Here, we intend to evaluate opportunities, risks, and limitations specific to the CRISPR technology for germline editing. We will address these specific questions:

• Can we improve the precision, reliability, and safety of CRISPR to a point where we can consider its use in human embryos?
• Do we understand enough about complex genetic diseases to be able to delay or suppress their onset?
• Can modifying the human germline ever be ethically defensible?

This high-risk sub-project unites competencies in biology, reproductive medicine, Artificial intelligence/machine learning, embryology, genetics, philosophy, theology, sociology, and law.

Our first goal is to assess the efficiency and precision of available CRISPR technologies and to develop improved variants. 
Our second goal is therefore to evaluate the technical feasibility of introducing multiple precise genetic changes in a single cell or zygote. 
Our third goal is to systematically introduce and study disease-associated allele variants in mouse models and ex vivo organoid models. Potential target diseases include cardiovascular diseases, obesity, and cancer. 

In the first four years, we will mainly focus on testing the efficacy and safety of CRIPSR technologies, and in years 5 to 8 we will put an emphasis on the development of improved tools. Likewise, in the first four years, we will mainly try to understand why risk alleles are associated with certain genetic diseases, and in the years 5 to 8, we will attempt to develop CRISPR approaches that can correct mouse models carrying these risk alleles. Concurrent with this cutting-edge medical-technological research, we will conduct an interdisciplinary legal, ethical-
philosophical, psychological, and social investigation whose findings will inform the evaluation of CRISPR. 

Transient in vivo prime editing for clinical applications (by Eleonora Ioannidi & Prof. Gerald Schwank)
Eleonora Ioannidi’s PhD research project “Transient in vivo prime editing for clinical applications” focuses on improving genome editing tools for their future use in gene therapy. Even though over the last few decades, genome editing technologies have been significantly improved, there are still challenges that need to be resolved prior their use for clinical applications such as their delivery and potential off targets. This project aims for making prime editing, a new technology that allows different modifications without causing double strand breaks, transient, while maintaining its precision and efficiency.

Applications of AI in the development of genome editing tools (by Dr. Amina Mollaysa & Prof. Michael Krauthammer)
Dr. Amina Mollaysa’s  andProf. Michael Krauthammer’s research project "Applications of AI in the development of genome editing tools" addresses the following research questions:

• Can we develop AI models that accurately predict the possible outcomes of different genome editing tools?
• Can we identify the factors that affect the outcome of the genome edits and improve the precision, reliability, and safety of the tools?

The range of reproductive options investigated by the URPP H2R includes future scenarios that rely on CRISPR-based technologies. These investigations focus on the opportunities and risks of human germline editing tools that might represent a promising approach for curing monogenetic disorders (e.g. cystic fibrosis), and for reducing genetic risk factors associated with common chronic diseases in which multiple genes interact with environmental factors (e.g. obesity, coronary heart disease, diabetes, Alzheimer’s disease, and various cancers). However, the efficiency of germline modifications is still low, outcomes are unstable; indications are ambiguous, and technical feasibility is challenging. Given the complexities of the undertaking, the use of AI in the development of genome editing tools is warranted, with the goal of enabling more precise and controlled DNA modifications. 

Advancing In Vivo Genome Editing through the Development of Next-Generation SpCas9 Nucleases (by Dr. Péter Kulcsár & Prof. Gerald Schwank)
The postdoctoral research project conducted by Dr. Péter Kulcsár, under the supervision of Prof. Gerald Schwank, focuses on the enhancement of Streptococcus pyogenes Cas9 (SpCas9) nucleases for future applications in somatic- and germline genome editing. The primary objective is to address the limitations of SpCas9, the most widely utilized Cas nuclease, by minimizing off-target editing and enhancing consistent indel pattern outcomes (i.e. single type of indel vs diverse indel patterns). While SpCas9 has been demonstrated to be highly efficient, it still poses challenges in terms of off-target cleavage, as it can inadvertently cleave not only at intended sites but also at off-target sites. Moreover, the outcomes of SpCas9-mediated editing exhibit significant diversity. These limitations restrict its broader application.

This project aims to develop more precise genome editing tools by generating novel variants of SpCas9 through the combination of rational design and directed protein evolution strategies. These efforts seek to enhance the specificity and accuracy of the nuclease, enabling its application in therapeutic contexts or germline editing.

Ethical Issues of Germline Genome Editing from a Reproductive Justice Perspective (by Agnes Kandlbinder, Prof. Dr. Peter Schaber & Prof. Dr. Michael Coors)
Agnes Kandlbinder’s PhD research project “Ethical Issues of Germline Genome Editing from a Reproductive Justice Perspective” explores normative questions of Germline Genome Editing (GGE) at the intersection of applied ethics and societal debate. The cumulative dissertation critically examines both bioethical scholarship and activist discourses on GGE, particularly with regard to disability as a social justice issue. Thereby, the project aims to signpost how the public and academic discussion on research on GGE can be fruitfully brought forward in an inclusive way. Methodologically, Agnes combines tools of analytical applied ethics with a reproductive justice framework. The expected duration of the project is from February 2022 to February 2025.