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Advances in Genome Editing Tools
Genetic screens in zebrafsh have yielded many insights into
vertebrate developmental biology, but the realization that the
teleost genome had undergone a duplication event generated
interest in developing a genetic model organism with a more
canonically organized genome that was evolutionarily closer
to mammals (Glasauer and Neuhauss, 2014). In 1999 at the
NIH Non-Mammalian Model Meeting, Xenopus was one of
the model organisms discussed, and subsequently consensus
was reached on ten priority areas for large-scale genomic and
genetic resource development for Xenopus. These included
establishing the viability of X. tropicalis as a genetic model
organism through pilot genetic screens, including chemical
mutagenesis, large-scale radiation-induced deletions, and
insertional mutagenesis, and developing resources such as a
genetic map and sequencing the X. tropicalis genome (Klein
et al., 2002). As a result, a number of laboratories undertook
forward screens for mutations affecting X. tropicalis development (Goda et al., 2006; Noramly et al., 2005).
Since amphibians do not have imprinting and fertilization is external, it is possible to use gynogenesis to obtain
diploid embryos without paternal genetic contributions
(Tompkins, 1978). Gynogenesis allows F1 female progeny
of F0 mutagenized animals to be screened for recessive
mutations without the need to obtain adult F2 animals for
sib crosses. The technique is simple: irradiated, macerated
wild type testes are used for in vitro fertilization; the sperm
initiate egg cleavage, but their genetic material is destroyed,
creating haploid embryos. Diploidy is restored by cold shock
or pressure early after fertilization, which inhibits polar
body formation and prevents the loss of maternal chromosomes duplicated during meiosis II (Geach et al., 2012).
Disadvantages of gynogenesis are that the frequency of
mutation depends on the distance from centromeres, ranging
from 50% near centromeres to around 10% near telomeres,
not the classical Mendelian ratio of 25%. Also, background
abnormal gastrulation is higher than in normal fertilization. Gynogenetic screens thus focus on later development,
uncovering mutations that affected organogenesis rather
than early patterning. In the pilot gynogenetic screens, over
100 potential mutant phenotypes were observed, including
defects in heartbeat, motility, pigmentation, otolith formation, haematopoiesis, gut coiling, axis formation, and leftright asymmetry (Goda et al., 2006; Noramly et al., 2005).
Once a candidate mutation was identifed in a female, an F2
generation was raised to adulthood to confrm the heritability of the phenotype in classic sib crosses.
The frst mutation cloned affected cardiac function
(Abu-Daya et al., 2009). Homozygous muzak tadpoles had
no heartbeat, caused by a nonsense mutation in myh6. The
resulting premature stop codon caused nonsense mediated
decay of myh6 mRNA, and the lack of myosin heavy chain
prevented sarcomere formation in muzak cardiomyocytes.
Another mutation that affected sarcomere assembly was
dicky ticker; homozygous embryos were completely paralyzed and had no heartbeat. The genetic lesion was a missense mutation in the muscle-specifc chaperone unc45b,
required for the correct folding of the head domain of heavy
chain myosins (Geach and Zimmerman, 2010). A model for
human disease was no privacy, a recessive, non-lethal pigmentation mutant. The phenotype is characterized by signifcantly reduced pigmentation; the genetic mutation was
identifed as a 10 base pair deletion in the hps6 homologue
of the Hermansky-Pudlak Syndrome 6 gene (Nakayama
et al., 2017).
Improved genomic resources sped up positional cloning of mutations. In the fve years after the identif cation of
muzak, six more chemically induced mutants were mapped.
These included kaleidoscope, characterized by variegated
retinal epithelium and head cartilage defects caused by a
splicing mutation in the ATPase copper transporting alpha
(atp7a) gene, which is implicated in Menkes disease; white
heart, characterized by haematopoiesis defects caused by a
mutation in the smad4.1 gene; cyd vicious characterized by
a severe eye phenotype and very poor melanocyte migration from the neural tube, mapped to the DSIF elongation
factor subunit (supt5h) gene; the otolith formation mutants
komimi, a splicing mutation in the otoconin90 (oc90) gene;
and seasick, a nonsense mutation in the vesicle transport
adaptor protein ap3d1 (Abu-Daya et al., 2012).
14.3.3. TILLING
The screens described previously were all “forward genetic”
screens; at the same time, a “reverse genetics” project
searched for mutations in specifc genes by TILLING (targeting induced local lesions in genomes) using males produced by ENU mutagenesis (Stemple, 2004). Capillary
sequencing was initially used to search for mutations in
specifc genes requested by the Xenopus community (Goda
et al., 2006). This approach did not produce many mutants,
since the F1 males tested were mosaic for mutations due
to treating mature sperm, not spermatogonia, with ENU
and PCR amplifcation introduced allele bias. However, a
TILLING screen on F1 animals produced by spermatogonial ENU mutagenesis produced a nonsense mutation in the
retinal anterior homeobox (rax) gene, which resulted in eyeless tadpoles (Fish et al., 2014). With the increasing availability of next-generation sequencing, it became possible to
sequence the whole exome of mutants. This new approach
uncovered mutations in more than 300 genes, although these
were not necessarily in specifc genes requested by Xenopus
researchers.
14.3.4. INSERTIONAL MUTAGENESIS
Another strategy to produce Xenopus mutants was insertional mutagenesis. If a transgene integrates into a coding
sequence or an important regulatory region, it will disrupt
the function of that gene. Insertional mutagenesis is attractive because cloning the site of integration is simpler and
faster than positional cloning. The basic approach using
Sleeping Beauty is described previously, and a pilot study
showed that in these “hopper frogs,” the transposon was
indeed excised and reintegrated. In approximately 80% of
Advances in Genome Editing Tools
Genetic screens in zebrafsh have yielded many insights into
vertebrate developmental biology, but the realization that the
teleost genome had undergone a duplication event generated
interest in developing a genetic model organism with a more
canonically organized genome that was evolutionarily closer
to mammals (Glasauer and Neuhauss, 2014). In 1999 at the
NIH Non-Mammalian Model Meeting, Xenopus was one of
the model organisms discussed, and subsequently consensus
was reached on ten priority areas for large-scale genomic and
genetic resource development for Xenopus. These included
establishing the viability of X. tropicalis as a genetic model
organism through pilot genetic screens, including chemical
mutagenesis, large-scale radiation-induced deletions, and
insertional mutagenesis, and developing resources such as a
genetic map and sequencing the X. tropicalis genome (Klein
et al., 2002). As a result, a number of laboratories undertook
forward screens for mutations affecting X. tropicalis development (Goda et al., 2006; Noramly et al., 2005).
Since amphibians do not have imprinting and fertilization is external, it is possible to use gynogenesis to obtain
diploid embryos without paternal genetic contributions
(Tompkins, 1978). Gynogenesis allows F1 female progeny
of F0 mutagenized animals to be screened for recessive
mutations without the need to obtain adult F2 animals for
sib crosses. The technique is simple: irradiated, macerated
wild type testes are used for in vitro fertilization; the sperm
initiate egg cleavage, but their genetic material is destroyed,
creating haploid embryos. Diploidy is restored by cold shock
or pressure early after fertilization, which inhibits polar
body formation and prevents the loss of maternal chromosomes duplicated during meiosis II (Geach et al., 2012).
Disadvantages of gynogenesis are that the frequency of
mutation depends on the distance from centromeres, ranging
from 50% near centromeres to around 10% near telomeres,
not the classical Mendelian ratio of 25%. Also, background
abnormal gastrulation is higher than in normal fertilization. Gynogenetic screens thus focus on later development,
uncovering mutations that affected organogenesis rather
than early patterning. In the pilot gynogenetic screens, over
100 potential mutant phenotypes were observed, including
defects in heartbeat, motility, pigmentation, otolith formation, haematopoiesis, gut coiling, axis formation, and leftright asymmetry (Goda et al., 2006; Noramly et al., 2005).
Once a candidate mutation was identifed in a female, an F2
generation was raised to adulthood to confrm the heritability of the phenotype in classic sib crosses.
The frst mutation cloned affected cardiac function
(Abu-Daya et al., 2009). Homozygous muzak tadpoles had
no heartbeat, caused by a nonsense mutation in myh6. The
resulting premature stop codon caused nonsense mediated
decay of myh6 mRNA, and the lack of myosin heavy chain
prevented sarcomere formation in muzak cardiomyocytes.
Another mutation that affected sarcomere assembly was
dicky ticker; homozygous embryos were completely paralyzed and had no heartbeat. The genetic lesion was a missense mutation in the muscle-specifc chaperone unc45b,
required for the correct folding of the head domain of heavy
chain myosins (Geach and Zimmerman, 2010). A model for
human disease was no privacy, a recessive, non-lethal pigmentation mutant. The phenotype is characterized by signifcantly reduced pigmentation; the genetic mutation was
identifed as a 10 base pair deletion in the hps6 homologue
of the Hermansky-Pudlak Syndrome 6 gene (Nakayama
et al., 2017).
Improved genomic resources sped up positional cloning of mutations. In the fve years after the identif cation of
muzak, six more chemically induced mutants were mapped.
These included kaleidoscope, characterized by variegated
retinal epithelium and head cartilage defects caused by a
splicing mutation in the ATPase copper transporting alpha
(atp7a) gene, which is implicated in Menkes disease; white
heart, characterized by haematopoiesis defects caused by a
mutation in the smad4.1 gene; cyd vicious characterized by
a severe eye phenotype and very poor melanocyte migration from the neural tube, mapped to the DSIF elongation
factor subunit (supt5h) gene; the otolith formation mutants
komimi, a splicing mutation in the otoconin90 (oc90) gene;
and seasick, a nonsense mutation in the vesicle transport
adaptor protein ap3d1 (Abu-Daya et al., 2012).
14.3.3. TILLING
The screens described previously were all “forward genetic”
screens; at the same time, a “reverse genetics” project
searched for mutations in specifc genes by TILLING (targeting induced local lesions in genomes) using males produced by ENU mutagenesis (Stemple, 2004). Capillary
sequencing was initially used to search for mutations in
specifc genes requested by the Xenopus community (Goda
et al., 2006). This approach did not produce many mutants,
since the F1 males tested were mosaic for mutations due
to treating mature sperm, not spermatogonia, with ENU
and PCR amplifcation introduced allele bias. However, a
TILLING screen on F1 animals produced by spermatogonial ENU mutagenesis produced a nonsense mutation in the
retinal anterior homeobox (rax) gene, which resulted in eyeless tadpoles (Fish et al., 2014). With the increasing availability of next-generation sequencing, it became possible to
sequence the whole exome of mutants. This new approach
uncovered mutations in more than 300 genes, although these
were not necessarily in specifc genes requested by Xenopus
researchers.
14.3.4. INSERTIONAL MUTAGENESIS
Another strategy to produce Xenopus mutants was insertional mutagenesis. If a transgene integrates into a coding
sequence or an important regulatory region, it will disrupt
the function of that gene. Insertional mutagenesis is attractive because cloning the site of integration is simpler and
faster than positional cloning. The basic approach using
Sleeping Beauty is described previously, and a pilot study
showed that in these “hopper frogs,” the transposon was
indeed excised and reintegrated. In approximately 80% of