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Article

Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes

1
Department of Otorhinolaryngology, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands
2
Donders Institute for Brain Cognition and Behaviour, 6500 GL Nijmegen, The Netherlands
3
Department of Human Genetics, Radboud University Medical Center, 6525 GA Nijmegen, The Netherlands
*
Author to whom correspondence should be addressed.
Both authors contributed equally.
Academic Editor: Seong-kyu Choe
Int. J. Mol. Sci. 2021, 22(17), 9429; https://doi.org/10.3390/ijms22179429
Received: 30 July 2021 / Revised: 26 August 2021 / Accepted: 27 August 2021 / Published: 30 August 2021
(This article belongs to the Special Issue Zebrafish: A Powerful Model for Genetics and Genomics)
CRISPR-Cas9-based genome-editing is a highly efficient and cost-effective method to generate zebrafish loss-of-function alleles. However, introducing patient-specific variants into the zebrafish genome with CRISPR-Cas9 remains challenging. Targeting options can be limited by the predetermined genetic context, and the efficiency of the homology-directed DNA repair pathway is relatively low. Here, we illustrate our efficient approach to develop knock-in zebrafish models using two previously variants associated with hereditary sensory deficits. We employ sgRNA-Cas9 ribonucleoprotein (RNP) complexes that are micro-injected into the first cell of fertilized zebrafish eggs together with an asymmetric, single-stranded DNA template containing the variant of interest. The introduction of knock-in events was confirmed by massive parallel sequencing of genomic DNA extracted from a pool of injected embryos. Simultaneous morpholino-induced blocking of a key component of the non-homologous end joining DNA repair pathway, Ku70, improved the knock-in efficiency for one of the targets. Our use of RNP complexes provides an improved knock-in efficiency as compared to previously published studies. Correct knock-in events were identified in 3–8% of alleles, and 30–45% of injected animals had the target variant in their germline. The detailed technical and procedural insights described here provide a valuable framework for the efficient development of knock-in zebrafish models. View Full-Text
Keywords: zebrafish; CRISPR-Cas9; knock-in; homology-directed repair; disease models zebrafish; CRISPR-Cas9; knock-in; homology-directed repair; disease models
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MDPI and ACS Style

de Vrieze, E.; de Bruijn, S.E.; Reurink, J.; Broekman, S.; van de Riet, V.; Aben, M.; Kremer, H.; van Wijk, E. Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes. Int. J. Mol. Sci. 2021, 22, 9429. https://doi.org/10.3390/ijms22179429

AMA Style

de Vrieze E, de Bruijn SE, Reurink J, Broekman S, van de Riet V, Aben M, Kremer H, van Wijk E. Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes. International Journal of Molecular Sciences. 2021; 22(17):9429. https://doi.org/10.3390/ijms22179429

Chicago/Turabian Style

de Vrieze, Erik, Suzanne E. de Bruijn, Janine Reurink, Sanne Broekman, Vince van de Riet, Marco Aben, Hannie Kremer, and Erwin van Wijk. 2021. "Efficient Generation of Knock-In Zebrafish Models for Inherited Disorders Using CRISPR-Cas9 Ribonucleoprotein Complexes" International Journal of Molecular Sciences 22, no. 17: 9429. https://doi.org/10.3390/ijms22179429

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