Leiden University Scholarly Publications

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Wang, Q. (2024)

Soft genome editing based on CRISPR nickases: it takes one break to tango

Doctoral Thesis
Rapid technological breakthroughs originating from fundamental microbiology research have yielded novel genome engineering tools that greatly facilitate our ability to modify specific genomic sequences in living cells and organisms. As a corollary, these technologies are also starting to permeate the realm of medicine when applied as a form of “genomic surgery”. These genetic therapies aim at tacking the root cause of human pathologies, inherited or acquired, by correcting or modulating the genetic content or expression, respectively, present in target cells, tissues and organs. To this end, delivery vehicles capable of introducing, in an efficient and safe manner, the increasingly sophisticated (epi)genome editing reagents are in demand, especially when considering in vivo genetic therapies. The research presented in this thesis reveals the feasibility and utility of using regular and high-specificity nicking RGNs for achieving efficient and accurate genetic modification of...Show moreRapid technological breakthroughs originating from fundamental microbiology research have yielded novel genome engineering tools that greatly facilitate our ability to modify specific genomic sequences in living cells and organisms. As a corollary, these technologies are also starting to permeate the realm of medicine when applied as a form of “genomic surgery”. These genetic therapies aim at tacking the root cause of human pathologies, inherited or acquired, by correcting or modulating the genetic content or expression, respectively, present in target cells, tissues and organs. To this end, delivery vehicles capable of introducing, in an efficient and safe manner, the increasingly sophisticated (epi)genome editing reagents are in demand, especially when considering in vivo genetic therapies. The research presented in this thesis reveals the feasibility and utility of using regular and high-specificity nicking RGNs for achieving efficient and accurate genetic modification of human cells involving targeted gene knockouts and knock-ins. Moreover, it establishes the suitability of the HC-AdV platform for the versatile investigation of prime editing systems. Finally, this thesis establishes causal relationships between specific chromatin states and the activities and fidelities attained by base editing and prime editing complexes in human cells, which has consequences for their further development and optimal deployment.
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Genome editing
Targeted gene modification
CRISPR/Cas9
Off-target activities
Base editor
Prime editor
Adenoviral vector
Duchenne muscular dystrophy
Gene therapy
All authors
Wang, Q.
Supervisor
Hoeben, R.C.
Co-supervisor
Gonçalves, M.A.F.V.
Committee
Brakenbusch, C.; Staal, F.J.T.; Chuva de Sousa Lopes, S.M.; Pang, B.; Snippert, H.J.G.; Wijnholds, J.
Qualification
Doctor (dr.)
Awarding Institution
Faculty of Medicine, Leiden University Medical Center (LUMC), Leiden University
Date
2024-06-26
ISBN (print)
9789464961010