308
Xenopus
may also wonder what the added value over the zebraf sh
model would be.
21.6.3. FUTURE PROSPECTS FOR PRECISE
XENOPUS GENE EDITING
Current GEXMs only encompass inactivating mutations in
tumor suppressor genes, as generating activating mutations
in proto-oncogenes is currently challenging and ineff cient.
However, a major advantageous characteristic is that tumor
formation is under positive selection, thereby requiring the
activating mutation(s) to be present in a limited number of
cells, and alternative methods exist for the eff cient induction of oncogenic gene activation.
1. For example, in the context of leukemia, a hyperactive Notch1 protein can be obtained simply by
targeting a specifc region in the last exon of the
notch1 gene. Introduction of a frameshift INDEL
mutation and subsequent translational termination
will thus result in the expression of a truncated
Notch1 protein that lacks the C-terminal PEST
sequence that targets the protein for proteolytic
degradation. This in fact recapitulates the mutations found in a substantial fraction of T-ALL
patients and results in increased Notch signaling,
ultimately driving oncogenic transformation in the
T-cell lineage (Baldus et al., 2009). Of note, while
the inactivation of tumor suppressor genes requires
the disruption of the two alleles, oncogenic mutations are dominant, allowing the maintenance of
one wild type allele.
2. Many cancers are driven by oncogenic fusion
proteins. In vivo viral delivery of CRISPR/Cas9
reagents in mice has been used for eff cient generation of oncogenic fusion proteins showing penetrant lung tumor formation (Blasco et al., 2014;
Maddalo et al., 2014). This strategy builds on the
simple injection of two guide RNAs targeting intron
regions of two genes on the same chromosome,
being separated by more than 10 Mb from each
other, resulting in a fusion of both loci mimicking
the patient situation (Blasco et al., 2014; Maddalo
et al., 2014). Considering the concept of positive
selection, this technique might be extrapolated
to generate F0 mosaic mutant GEXMs harboring
fusion oncogenes, whether or not in combination
with cooperating loss-of-function event(s) in one or
more tumor suppressor gene(s).
3. The generation of knock-ins in embryos using
CRISPR/HDR (homology directed repair)-mediated targeted gene editing in Xenopus remains
challenging, as low homologous repair rates
(<3%) are observed in Xenopus F 0 mosaic mutant
embryos (Aslan et al., 2017). This could hamper
the generation of additional cancer predisposition
models such as Li-Fraumeni syndrome, where
particular missense mutations may be associated
with dissimilar tumor spectra or latency for disease
onset (Bougeard et al., 2008). However, Aslan et al.
(2017 ) described that Xenopus oocytes show signifcant higher homology-directed repair activity.
For this methodology, CRISPR/Cas9 reagents and
a single-strand DNA template were co-injected in
oocytes, and subsequent host-transfer yielded editing effciencies up to 25.7% (Aslan et al., 2017).
Therefore, this method would allow the modeling of variants of uncertain signif cance (VUS)
in the contexts of human cancer predisposition
(Eggington et al., 2014).
4. Additionally, we would like to mention that, very
recently, we validated for the frst time that in vivo
genome editing outcomes using CRISPR/Cas9 can
be accurately predicted using in silico prediction
software like InDelphi both in Xenopus tropicalis
and laevis as well as in zebrafsh (Naert, Tulkens,
et al., 2020). This interesting fnding offers unique
opportunities for selecting guide RNAs favoring
frameshift mutations, thus maximizing F0 phenotype penetrance or even for selecting guides that
give a predominance for a specifc small INDEL
which can facilitate modeling patient specif c
mutations in a rapid manner (Naert, Tulkens, et al.,
2020 ).
5. Finally, we would like to mention that with the
generation of CRISPR base editors (Komor et al.,
2016), the PITCh (precise integration into target
chromosome) system (Sakuma et al., 2016), and
CRISPR prime editing (Anzalone et al., 2019), precise cancer modeling will be signif cantly enhanced
in the (near) future, offering novel opportunities for
more GEXMs.
In conclusion, in the Xenopus feld, major steps have already
been taken in unraveling genetic factors that drive cancer
formation and maintenance. However, we believe that this
might be only an initial step stone to more extensive applications, as with the expansion and fne-tuning of genome engineering techniques, this feld is rapidly evolving. Considering
thereby the unique benefts of performing cancer research in
Xenopus tropicalis, primarily fast and eff cient GEXM generation with concomitant targeting of multiple genes, this
organism can eventually be nicely complementary to mice
and zebrafsh and aid in the development of novel precise
cancer therapies.
ACKNOWLEDGMENTS
Research in the Vleminckx laboratory is supported by the
Research Foundation–Flanders (FWO-Vlaanderen) and
by the Concerted Research Actions from Ghent University
(BOF15/GOA/011 and BOF20/GOA/23). Further support was
obtained by the Desmoid Tumor Research Foundation, the
Desmoid Tumor Foundation of Canada, and SOS Desmoïde.
Xenopus
may also wonder what the added value over the zebraf sh
model would be.
21.6.3. FUTURE PROSPECTS FOR PRECISE
XENOPUS GENE EDITING
Current GEXMs only encompass inactivating mutations in
tumor suppressor genes, as generating activating mutations
in proto-oncogenes is currently challenging and ineff cient.
However, a major advantageous characteristic is that tumor
formation is under positive selection, thereby requiring the
activating mutation(s) to be present in a limited number of
cells, and alternative methods exist for the eff cient induction of oncogenic gene activation.
1. For example, in the context of leukemia, a hyperactive Notch1 protein can be obtained simply by
targeting a specifc region in the last exon of the
notch1 gene. Introduction of a frameshift INDEL
mutation and subsequent translational termination
will thus result in the expression of a truncated
Notch1 protein that lacks the C-terminal PEST
sequence that targets the protein for proteolytic
degradation. This in fact recapitulates the mutations found in a substantial fraction of T-ALL
patients and results in increased Notch signaling,
ultimately driving oncogenic transformation in the
T-cell lineage (Baldus et al., 2009). Of note, while
the inactivation of tumor suppressor genes requires
the disruption of the two alleles, oncogenic mutations are dominant, allowing the maintenance of
one wild type allele.
2. Many cancers are driven by oncogenic fusion
proteins. In vivo viral delivery of CRISPR/Cas9
reagents in mice has been used for eff cient generation of oncogenic fusion proteins showing penetrant lung tumor formation (Blasco et al., 2014;
Maddalo et al., 2014). This strategy builds on the
simple injection of two guide RNAs targeting intron
regions of two genes on the same chromosome,
being separated by more than 10 Mb from each
other, resulting in a fusion of both loci mimicking
the patient situation (Blasco et al., 2014; Maddalo
et al., 2014). Considering the concept of positive
selection, this technique might be extrapolated
to generate F0 mosaic mutant GEXMs harboring
fusion oncogenes, whether or not in combination
with cooperating loss-of-function event(s) in one or
more tumor suppressor gene(s).
3. The generation of knock-ins in embryos using
CRISPR/HDR (homology directed repair)-mediated targeted gene editing in Xenopus remains
challenging, as low homologous repair rates
(<3%) are observed in Xenopus F 0 mosaic mutant
embryos (Aslan et al., 2017). This could hamper
the generation of additional cancer predisposition
models such as Li-Fraumeni syndrome, where
particular missense mutations may be associated
with dissimilar tumor spectra or latency for disease
onset (Bougeard et al., 2008). However, Aslan et al.
(2017 ) described that Xenopus oocytes show signifcant higher homology-directed repair activity.
For this methodology, CRISPR/Cas9 reagents and
a single-strand DNA template were co-injected in
oocytes, and subsequent host-transfer yielded editing effciencies up to 25.7% (Aslan et al., 2017).
Therefore, this method would allow the modeling of variants of uncertain signif cance (VUS)
in the contexts of human cancer predisposition
(Eggington et al., 2014).
4. Additionally, we would like to mention that, very
recently, we validated for the frst time that in vivo
genome editing outcomes using CRISPR/Cas9 can
be accurately predicted using in silico prediction
software like InDelphi both in Xenopus tropicalis
and laevis as well as in zebrafsh (Naert, Tulkens,
et al., 2020). This interesting fnding offers unique
opportunities for selecting guide RNAs favoring
frameshift mutations, thus maximizing F0 phenotype penetrance or even for selecting guides that
give a predominance for a specifc small INDEL
which can facilitate modeling patient specif c
mutations in a rapid manner (Naert, Tulkens, et al.,
2020 ).
5. Finally, we would like to mention that with the
generation of CRISPR base editors (Komor et al.,
2016), the PITCh (precise integration into target
chromosome) system (Sakuma et al., 2016), and
CRISPR prime editing (Anzalone et al., 2019), precise cancer modeling will be signif cantly enhanced
in the (near) future, offering novel opportunities for
more GEXMs.
In conclusion, in the Xenopus feld, major steps have already
been taken in unraveling genetic factors that drive cancer
formation and maintenance. However, we believe that this
might be only an initial step stone to more extensive applications, as with the expansion and fne-tuning of genome engineering techniques, this feld is rapidly evolving. Considering
thereby the unique benefts of performing cancer research in
Xenopus tropicalis, primarily fast and eff cient GEXM generation with concomitant targeting of multiple genes, this
organism can eventually be nicely complementary to mice
and zebrafsh and aid in the development of novel precise
cancer therapies.
ACKNOWLEDGMENTS
Research in the Vleminckx laboratory is supported by the
Research Foundation–Flanders (FWO-Vlaanderen) and
by the Concerted Research Actions from Ghent University
(BOF15/GOA/011 and BOF20/GOA/23). Further support was
obtained by the Desmoid Tumor Research Foundation, the
Desmoid Tumor Foundation of Canada, and SOS Desmoïde.