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CRISPR/Cas9-mediated knockout of Rb1 in Xenopus tropicalis

Thomas Naert (UGent) and Kris Vleminckx (UGent)
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Abstract
At this time, no molecular targeted therapies exist for treatment of retinoblastoma. This can be, in part, attributed to the lack of animal models that allow for both rapid identification of novel therapeutic targets and hypothesis driven drug testing. Within this scope, we have recently reported the first genuine genetic nonmammalian retinoblastoma cancer model within the aquatic model organism Xenopus tropicalis (Naert et al., Sci Rep 6: 35263, 2016). Here we describe the methods to generate rb1 mosaic mutant Xenopus tropicalis by employing the CRISPR/Cas9 technology. In depth, we discuss short guide RNA (sgRNA) design parameters, generation, quality control, quantification, and delivery followed by several methods for assessing genome editing efficiencies. As such the reader should be capable, by minor changes to the methods described here, to (co-) target rb1 or any one or multiple gene(s) within the Xenopus tropicalis genome by multiplex CRISPR/Cas9 methodology.
Keywords
Xenopus tropicalis, Disease model, Cancer model, Tumor model, Retinoblastoma, Genome editing, CRISPR/Cas9, Rb1

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Chicago
Naert, Thomas, and Kris Vleminckx. 2018. “CRISPR/Cas9-mediated Knockout of Rb1 in Xenopus Tropicalis.” In The Retinoblastoma Protein, ed. Pedro G Santiago-Cardona, 1726:177–193. New York, NY, USA: Springer Humana Press.
APA
Naert, T., & Vleminckx, K. (2018). CRISPR/Cas9-mediated knockout of Rb1 in Xenopus tropicalis. In P. G. Santiago-Cardona (Ed.), The retinoblastoma protein (Vol. 1726, pp. 177–193). New York, NY, USA: Springer Humana Press.
Vancouver
1.
Naert T, Vleminckx K. CRISPR/Cas9-mediated knockout of Rb1 in Xenopus tropicalis. In: Santiago-Cardona PG, editor. The retinoblastoma protein. New York, NY, USA: Springer Humana Press; 2018. p. 177–93.
MLA
Naert, Thomas, and Kris Vleminckx. “CRISPR/Cas9-mediated Knockout of Rb1 in Xenopus Tropicalis.” The Retinoblastoma Protein. Ed. Pedro G Santiago-Cardona. Vol. 1726. New York, NY, USA: Springer Humana Press, 2018. 177–193. Print.
@incollection{8552611,
  abstract     = {At this time, no molecular targeted therapies exist for treatment of retinoblastoma. This can be, in part, attributed to the lack of animal models that allow for both rapid identification of novel therapeutic targets and hypothesis driven drug testing. Within this scope, we have recently reported the first genuine genetic nonmammalian retinoblastoma cancer model within the aquatic model organism Xenopus tropicalis (Naert et al., Sci Rep 6: 35263, 2016). Here we describe the methods to generate rb1 mosaic mutant Xenopus tropicalis by employing the CRISPR/Cas9 technology. In depth, we discuss short guide RNA (sgRNA) design parameters, generation, quality control, quantification, and delivery followed by several methods for assessing genome editing efficiencies. As such the reader should be capable, by minor changes to the methods described here, to (co-) target rb1 or any one or multiple gene(s) within the Xenopus tropicalis genome by multiplex CRISPR/Cas9 methodology.},
  author       = {Naert, Thomas and Vleminckx, Kris},
  booktitle    = {The retinoblastoma protein},
  editor       = {Santiago-Cardona, Pedro G},
  isbn         = {9781493975648},
  issn         = {1064-3745},
  keyword      = {Xenopus tropicalis,Disease model,Cancer model,Tumor model,Retinoblastoma,Genome editing,CRISPR/Cas9,Rb1},
  language     = {eng},
  pages        = {177--193},
  publisher    = {Springer Humana Press},
  series       = {Methods in Molecular Biology},
  title        = {CRISPR/Cas9-mediated knockout of Rb1 in Xenopus tropicalis},
  url          = {http://dx.doi.org/10.1007/978-1-4939-7565-5\_16},
  volume       = {1726},
  year         = {2018},
}

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