CRISPR engineering in organoids for gene repair and disease modelling

Maarten H. Geurts

Research output: ThesisDoctoral thesis 2 (Research NOT UU / Graduation UU)

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Abstract

Genome engineering technologies have been in a rapid series of development over the past decade. The development of CRISPR/Cas9 as an efficient and easy to reprogram genome editing tool has revolutionized biological sciences. CRISPR is based on the immune system of bacteria and allows researchers to specifically cleave DNA sequences at will. Even though CRISPR/Cas9-mediated genome engineering is highly efficient and easy to reprogram, there are some downsides to the requirement of DNA damage in the process. In most cases, the damage is repaired as we intend and our edit of interest is incorporated. However, unwanted outcomes are observed in a significant percentage of the cells. To overcome this problem, CRISPR-based tools have been developed that allow DNA changes without causing damage first. These, what I call in this thesis, next-generation CRISPR tools use a nuclease inactive or dead (dCas9) that can still find the genomic locus but can no longer cleave DNA. By fusing DNA alteration enzymes dCas9, we can introduce specific genomic edits without the need to cause deleterious DNA damage.
The ability to alter the genome is of interest for both clinical application and more fundamental research into the cause and treatment of diseases. The first trials in which CRISPR is used to repair disease causing mutations in patients have started. Additionally, CRISPR can be used to generate disease models in a controlled laboratory setting to study the impact of mutations on disease cause and progression. This two-edged sword will have a significant impact on the way we treat patients in the clinic. Before CRISPR-mediated genome engineering can be used en masse in patients, it is of key importance to test its safety and efficiency in the laboratory. Commonly used mouse models may not be the best candidate as their genome differs too much from humans and potentially unwanted side-effects of CRISPR can be different. Conventional 2D-cell lines, however, hardly resemble the tissue of origin and need genetic immortalization to sustain growth. Organoids bridge the gap between 2D cell-lines and in vivo studies. With their 3D-organization and cellular heterogeneity, adult stem cell-derived organoids closely resemble their tissue of origin. In This thesis we first describe the development of a new Cas9 protein with increased genomic target range and decreased off-target effects. This new Cas9, called xCas9, can be combined with next-generation CRISPR tools to make genome editing even more safe. We then show that a variety of next-generation CRISPR tools can be applied safely in organoids derived from patients with cystic fibrosis to relieve the disease phenotype. Next, we construct complex isogenic tumor models from the colon, endometrium and liver that can bring us closer to understanding the cause and finding a better cancer treatment. Lastly, by applying the protocols developed in this thesis we were able to react quickly when the Covid-19 pandemic hit by rapidly generating isogenic organoid models that contain loss-of-function mutations in host factor genes implicated in SARS-CoV-2 infection.
Original languageEnglish
Awarding Institution
  • University Medical Center (UMC) Utrecht
Supervisors/Advisors
  • Clevers, Hans, Primary supervisor
Award date21 Nov 2022
Place of PublicationUtrecht
Publisher
Print ISBNs978-94-93278-31-8
DOIs
Publication statusPublished - 21 Nov 2022
Externally publishedYes

Keywords

  • CRISPR
  • Cas9
  • Organoids
  • Stem cells
  • Tissue Culture
  • Cystic Fibrosis
  • Genetic modification
  • Cancer
  • Gene repair
  • Disease models

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