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Detection of deleterious on-target effects after CRISPR-mediated genome editing in human induced pluripotent stem cells
Detection of deleterious on-target effects after CRISPR-mediated genome editing in human induced pluripotent stem cells
The CRISPR/Cas9 system is an exceedingly powerful technology for precise genome editing. Its ease of use, high editing efficiency and an ever-growing CRISPR-based toolbox has provided researchers with novel possibilities to unravel the molecular and systemic consequences of changes in the genetic code. For this reason, CRISPR is now applied for editing in a wide range of different cell lines and organisms for basic and translational research. Here, accurate and precise editing is an indispensable prerequisite to generate reliable research models. However, a lot remains to be understood about the molecular mechanism of double-strand-breaks (DSBs) in the DNA as introduced by the Cas9 nuclease during editing. In fact, CRISPR editing can be accompanied by inadvertent genomic changes at the targeted locus (on-target) as well as other genomic sites (off-target). These can have drastic consequences on gene activity or expression and therefore need to be carefully investigated. Characterizing and avoiding unwanted off-target effects (OffTE) has been the focus of several studies and reliable tools for their detection have been developed. This is, however, not the case for on-target effects (OnTE) that have only been reported very recently. These can be large deletions, large insertions, complex rearrangements, or regions of copy-neutral loss of heterozygosity (LOH) around the target site. Several studies have described frequent occurrence of OnTEs in mice, but it has not been investigated if clinically relevant human cells, such as induced pluripotent stem cells (iPSCs) are also affected. The main problem with OnTEs is that they often remain unnoticed in standard quality controls like Sanger genotyping of the target locus, and additional checks are lacking in most CRISPR-based studies. This is also because there are no simple detection tools available. Therefore, in this study, we developed simple and reliable tools for OnTE detection after CRISPR genome editing: Structural alterations like large deletions, large insertions or complex rearrangements can be identified by quantifying the number of intact alleles at the edited locus using our new method called quantitative genotyping PCR (qgPCR). In addition, we validated genotyping of neighboring single nucleotide polymorphisms (SNPs) either by Sanger sequencing or SNP microarrays to reveal editing-induced regions of LOH. The entire workflow is broadly applicable to different cell lines and organisms after editing by the NHEJ or HDR pathway. We have applied our newly established detection technology to human iPSCs after HDR-mediated editing and demonstrate universal occurrence of OnTEs at multiple loci in up to 40% of edited single-cell clones. Furthermore, using an in vitro model of Alzheimer’s disease, we illustrate deleterious consequences of OnTEs on expression of the edited gene that may reduce pathogenic effects and therefore interfere with experimental findings. Overall, the threat of undetected OnTEs undermining the reliability of CRISPR-based studies has not received sufficient attention in the field so far. With this thesis, we hope to raise further awareness and propose that our simple and reliable on-target quality control workflow should be an essential part of all relevant genome editing experiments.
CRISPR, iPSC, genome editing
Weisheit, Isabel
2021
English
Universitätsbibliothek der Ludwig-Maximilians-Universität München
Weisheit, Isabel (2021): Detection of deleterious on-target effects after CRISPR-mediated genome editing in human induced pluripotent stem cells. Dissertation, LMU München: Graduate School of Systemic Neurosciences (GSN)
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Abstract

The CRISPR/Cas9 system is an exceedingly powerful technology for precise genome editing. Its ease of use, high editing efficiency and an ever-growing CRISPR-based toolbox has provided researchers with novel possibilities to unravel the molecular and systemic consequences of changes in the genetic code. For this reason, CRISPR is now applied for editing in a wide range of different cell lines and organisms for basic and translational research. Here, accurate and precise editing is an indispensable prerequisite to generate reliable research models. However, a lot remains to be understood about the molecular mechanism of double-strand-breaks (DSBs) in the DNA as introduced by the Cas9 nuclease during editing. In fact, CRISPR editing can be accompanied by inadvertent genomic changes at the targeted locus (on-target) as well as other genomic sites (off-target). These can have drastic consequences on gene activity or expression and therefore need to be carefully investigated. Characterizing and avoiding unwanted off-target effects (OffTE) has been the focus of several studies and reliable tools for their detection have been developed. This is, however, not the case for on-target effects (OnTE) that have only been reported very recently. These can be large deletions, large insertions, complex rearrangements, or regions of copy-neutral loss of heterozygosity (LOH) around the target site. Several studies have described frequent occurrence of OnTEs in mice, but it has not been investigated if clinically relevant human cells, such as induced pluripotent stem cells (iPSCs) are also affected. The main problem with OnTEs is that they often remain unnoticed in standard quality controls like Sanger genotyping of the target locus, and additional checks are lacking in most CRISPR-based studies. This is also because there are no simple detection tools available. Therefore, in this study, we developed simple and reliable tools for OnTE detection after CRISPR genome editing: Structural alterations like large deletions, large insertions or complex rearrangements can be identified by quantifying the number of intact alleles at the edited locus using our new method called quantitative genotyping PCR (qgPCR). In addition, we validated genotyping of neighboring single nucleotide polymorphisms (SNPs) either by Sanger sequencing or SNP microarrays to reveal editing-induced regions of LOH. The entire workflow is broadly applicable to different cell lines and organisms after editing by the NHEJ or HDR pathway. We have applied our newly established detection technology to human iPSCs after HDR-mediated editing and demonstrate universal occurrence of OnTEs at multiple loci in up to 40% of edited single-cell clones. Furthermore, using an in vitro model of Alzheimer’s disease, we illustrate deleterious consequences of OnTEs on expression of the edited gene that may reduce pathogenic effects and therefore interfere with experimental findings. Overall, the threat of undetected OnTEs undermining the reliability of CRISPR-based studies has not received sufficient attention in the field so far. With this thesis, we hope to raise further awareness and propose that our simple and reliable on-target quality control workflow should be an essential part of all relevant genome editing experiments.