214
Xenopus
TABLE 14.1 (Continued)
Gene(s)
Year Species F0/F1
Reference/Notes
dyrk1a
chd1
cep70
2020
2020
2021
tropicalis
laevis
laevis
F0
F0
F0
( Willsey et al., 2020 )
agrees with MO data
( Wyatt et al., 2020 )
( Kim et al., 2021 )
agrees with MO data
HDR
krt12.2, npm3
2014 laevis
F0
( Nakade et al., 2014 )
CRISPR/TALEN, TALPITCh and CRIS-PITCh
cfap299
ctnnb1
myh6, gapdh
xnc10
slc45a2
2016
2017
2018
2019
2020
laevis
both
tropicalis
laevis
both
F0
F0
F0
F0
F0
( Jaffe et al., 2016 )
plasmid
( Aslan et al., 2017 )
CRISPR, oocyte host
transfer, oligo
( Mao et al., 2018 )
CRISPR, donor plasmid
( Banach et al., 2019 )
CRISPR, donor plasmid
( Nakayama et al., 2020 )
CRISPR, ssDNA
Base editing
tyr, tp53
2017 laevis
F0
( Park et al., 2017 )
Cas9-linked cytidine
deaminase BE3
tyr, tbx5, apc,
cyp1b1, kcnj2,
tbx22, gdf5, hhex,
sftpb, ptf1a
2019 tropicalis F0
( Shi et al., 2019 )
Cas9-linked cytidine
deaminase BE3
Legend : Examples of published, genetically altered Xenopus show how
the number of such animals is growing. Uncommon or novel aspects of
particular studies are highlighted in the “Notes” column.
a specifc phenotype in TRIO patients, showing that such
experiments can extend beyond simple loss of function
(Barbosa et al., 2020). Other examples include tbx4, rho,
neurod2, daam2, and dlg5 (Feehan et al., 2017; Kariminejad
et al., 2019; Marquez et al., 2021; Schneider et al., 2020;
Sega et al., 2019).
The frst germline CRISPR mutants were compound
heterozygotes produced using the leapfrogging technique,
which bypasses embryonic lethality by transplanting the
endoderm (the germ cells’ location) from an F0 mutant into
wild type; crossing two F0 gsc mutants produced this way
resulted in the expected phenotypic mutants (Blitz et al.,
2016). Compound mutations in mtrn1a in F1 X. tropicalis caused rod photoreceptor degeneration, while junB F1
mutants showed defective tail regeneration (Nakamura
et al., 2020; Wiechmann et al., 2020). The frst F2 homozygous null CRISPR mutants were made in genes affecting
metamorphosis, including thra, thrb, and nr3c1, and more
such homozygous null mutants are in the pipeline (Shibata
et al., 2020b, 2020a; Sterner et al., 2020). The biggest drawbacks to making germline mutants in Xenopus are the effort,
time, and cost needed to raise them through metamorphosis.
To increase the generation of germline mutants, the National
Xenopus Resource (NXR) has embarked on a project to generate over 200 mutants. To date, they have produced over
120 mutants, and they also host visiting researchers to come
and work on these mutants or create new mutants. These
mutants are cataloged on Xenbase and available to all
researchers interested in working with them.
14.4. FUTURE DIRECTIONS
CRISPR-Cas gene editing revolutionized site-specif c mutation in Xenopus, but the use of this new technology to generate site-specifc integration is more challenging. Random
integration of exogenous DNA through transgenesis works
effciently in Xenopus, but site-specifc integration of large
insertions initially proved diffcult to achieve. Initial reports
showed such an approach is feasible in Xenopus, but the
methods were ineffcient and unreliable, producing imprecise, mosaic mutations (Aslan et al., 2017; Jaffe et al., 2016;
Mao et al., 2018; Nakade et al., 2014; Nakayama et al., 2020).
Three of these reports utilized plasmid DNA containing
either one or two target sites for sgRNA, allowing cleavage
of circular vector in the embryo. In these studies, insertions
occurred in both orientations, revealing imprecise integration. In the fourth study, a ssDNA oligonucleotide was
injected with Cas9 into oocytes, which were then matured
and implanted into another female, and the laid eggs were
then fertilized in vitro; this is known as the oocyte host
transfer (OHT) approach (Aslan et al., 2017). This approach
was the most successful, with around 10% precision integration, but only a short ssDNA donor was tested, preventing insertion of fuorescent tags. The problem is that OHT
is diffcult, and few embryos survive to adulthood. A recent
study showed that homology directed repair (HDR) is possible in embryo injections when using long single-stranded
DNA (lssDNA) (Nakayama et al., 2020). Though few adult
animals were tested for germline transmission, they showed
successful insertion of point mutations and f uorescent proteins. One study showed that it is possible, in X. laevis, to
selectively knock-in constructs into either the L or S homeologue of a gene (Jaffe et al., 2016). The future of HDR insertion in Xenopus is to generate novel, tagged proteins to allow
for real-time visualization as well to generate precise point
mutations to model human disease.
If lssDNA co-injection with CRISPR-Cas9 proves as successful at other loci as in this frst report, it may come to
dominate making specifc, targeted changes to the genome.
There are, however, alternatives that avoid making ds breaks
but can make small alterations. Base editing involves targeting a Cas9 nickase fused to either a cytidine or adenine
deaminase to a specifc site in the genome, there converting T-A to C-G or G-C to A-T, respectively. This method
has been used successfully in Xenopus, producing editing
rates of up to 20.5%, but not extensively taken up by the
community, most likely due to the limited changes available
and the lack of single base-pair accuracy (Park et al., 2017).
Xenopus
TABLE 14.1 (Continued)
Gene(s)
Year Species F0/F1
Reference/Notes
dyrk1a
chd1
cep70
2020
2020
2021
tropicalis
laevis
laevis
F0
F0
F0
( Willsey et al., 2020 )
agrees with MO data
( Wyatt et al., 2020 )
( Kim et al., 2021 )
agrees with MO data
HDR
krt12.2, npm3
2014 laevis
F0
( Nakade et al., 2014 )
CRISPR/TALEN, TALPITCh and CRIS-PITCh
cfap299
ctnnb1
myh6, gapdh
xnc10
slc45a2
2016
2017
2018
2019
2020
laevis
both
tropicalis
laevis
both
F0
F0
F0
F0
F0
( Jaffe et al., 2016 )
plasmid
( Aslan et al., 2017 )
CRISPR, oocyte host
transfer, oligo
( Mao et al., 2018 )
CRISPR, donor plasmid
( Banach et al., 2019 )
CRISPR, donor plasmid
( Nakayama et al., 2020 )
CRISPR, ssDNA
Base editing
tyr, tp53
2017 laevis
F0
( Park et al., 2017 )
Cas9-linked cytidine
deaminase BE3
tyr, tbx5, apc,
cyp1b1, kcnj2,
tbx22, gdf5, hhex,
sftpb, ptf1a
2019 tropicalis F0
( Shi et al., 2019 )
Cas9-linked cytidine
deaminase BE3
Legend : Examples of published, genetically altered Xenopus show how
the number of such animals is growing. Uncommon or novel aspects of
particular studies are highlighted in the “Notes” column.
a specifc phenotype in TRIO patients, showing that such
experiments can extend beyond simple loss of function
(Barbosa et al., 2020). Other examples include tbx4, rho,
neurod2, daam2, and dlg5 (Feehan et al., 2017; Kariminejad
et al., 2019; Marquez et al., 2021; Schneider et al., 2020;
Sega et al., 2019).
The frst germline CRISPR mutants were compound
heterozygotes produced using the leapfrogging technique,
which bypasses embryonic lethality by transplanting the
endoderm (the germ cells’ location) from an F0 mutant into
wild type; crossing two F0 gsc mutants produced this way
resulted in the expected phenotypic mutants (Blitz et al.,
2016). Compound mutations in mtrn1a in F1 X. tropicalis caused rod photoreceptor degeneration, while junB F1
mutants showed defective tail regeneration (Nakamura
et al., 2020; Wiechmann et al., 2020). The frst F2 homozygous null CRISPR mutants were made in genes affecting
metamorphosis, including thra, thrb, and nr3c1, and more
such homozygous null mutants are in the pipeline (Shibata
et al., 2020b, 2020a; Sterner et al., 2020). The biggest drawbacks to making germline mutants in Xenopus are the effort,
time, and cost needed to raise them through metamorphosis.
To increase the generation of germline mutants, the National
Xenopus Resource (NXR) has embarked on a project to generate over 200 mutants. To date, they have produced over
120 mutants, and they also host visiting researchers to come
and work on these mutants or create new mutants. These
mutants are cataloged on Xenbase and available to all
researchers interested in working with them.
14.4. FUTURE DIRECTIONS
CRISPR-Cas gene editing revolutionized site-specif c mutation in Xenopus, but the use of this new technology to generate site-specifc integration is more challenging. Random
integration of exogenous DNA through transgenesis works
effciently in Xenopus, but site-specifc integration of large
insertions initially proved diffcult to achieve. Initial reports
showed such an approach is feasible in Xenopus, but the
methods were ineffcient and unreliable, producing imprecise, mosaic mutations (Aslan et al., 2017; Jaffe et al., 2016;
Mao et al., 2018; Nakade et al., 2014; Nakayama et al., 2020).
Three of these reports utilized plasmid DNA containing
either one or two target sites for sgRNA, allowing cleavage
of circular vector in the embryo. In these studies, insertions
occurred in both orientations, revealing imprecise integration. In the fourth study, a ssDNA oligonucleotide was
injected with Cas9 into oocytes, which were then matured
and implanted into another female, and the laid eggs were
then fertilized in vitro; this is known as the oocyte host
transfer (OHT) approach (Aslan et al., 2017). This approach
was the most successful, with around 10% precision integration, but only a short ssDNA donor was tested, preventing insertion of fuorescent tags. The problem is that OHT
is diffcult, and few embryos survive to adulthood. A recent
study showed that homology directed repair (HDR) is possible in embryo injections when using long single-stranded
DNA (lssDNA) (Nakayama et al., 2020). Though few adult
animals were tested for germline transmission, they showed
successful insertion of point mutations and f uorescent proteins. One study showed that it is possible, in X. laevis, to
selectively knock-in constructs into either the L or S homeologue of a gene (Jaffe et al., 2016). The future of HDR insertion in Xenopus is to generate novel, tagged proteins to allow
for real-time visualization as well to generate precise point
mutations to model human disease.
If lssDNA co-injection with CRISPR-Cas9 proves as successful at other loci as in this frst report, it may come to
dominate making specifc, targeted changes to the genome.
There are, however, alternatives that avoid making ds breaks
but can make small alterations. Base editing involves targeting a Cas9 nickase fused to either a cytidine or adenine
deaminase to a specifc site in the genome, there converting T-A to C-G or G-C to A-T, respectively. This method
has been used successfully in Xenopus, producing editing
rates of up to 20.5%, but not extensively taken up by the
community, most likely due to the limited changes available
and the lack of single base-pair accuracy (Park et al., 2017).