Dejima and colleagues report using CRISPR/Cas9 to generate a new collection of greatly improved balancer chromosomes in the standard laboratory nematode Caenorhabditis elegans, using methods previously reported by the same laboratory, expanding the set of C. elegans balancers to cover nearly 90% of coding genes

Schwartz, Hillel T.

Sternberg, Paul W.

Caltech Authors - Main

A toolkit of engineered recombinational balancers in C. elegans
Hillel T Schwartz and Paul W Sternberg
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 
91125
Abstract
Dejima and colleagues report using CRISPR-Cas9 to generate a new collection of greatly 
improved balancer chromosomes in the standard laboratory nematode Caenorhabditis elegans, 
using methods previously reported by the same laboratory [1,2], expanding the set of C. elegans 
balancers to cover nearly 90% of coding genes.
Keywords
Caenorhabditis elegans; CRISPR; Cas9; balancer; recombinational suppression
Text
Balancer chromosomes are a powerful tool in experimental genetics. Balancers are variant 
chromosomes that when heterozygous with normal chromosomes have a defined region 
within which there is no or essentially no recombination between the balancer chromosome 
and the normal chromosome. To achieve this, balancer chromosomes contain 
rearrangements such as inversions and translocations that disrupt synapsis and so prevent 
recombination. Any mutations within this recombinationally suppressed region can be 
reliably followed in trans using the balancer. Balancer chromosomes typically carry markers 
to make this more efficient: dominantly acting markers such as a gfp transgene to mark the 
presence of the balancer, and often a mutation with a recessive visible or even lethal 
phenotype to mark or to prevent the loss of the non-balancer chromosome and the mutations 
it contains. Balancers containing such markers can be used to infer genotype rapidly and 
efficiently.
It is now just over 100 years since the first report of a chromosome acting as a 
recombinational suppressor, by Sturtevant in 1917 [3]. This result, following close after the 
discovery that it was possible to construct a genetic linkage map [4], was immediately seen 
as being consistent with a chromosomal theory of inheritance and was soon shown to 
respond to chromosomal rearrangements that could be observed cytologically [5]. Much 
later, work in C. elegans with a reciprocal translocation that acted as a recombinational 
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our 
customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of 
the resulting proof before it is published in its final citable form. Please note that during the production process errors may be 
discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
HHS Public Access
Author manuscript
Trends Genet. Author manuscript; available in PMC 2019 April 01.
Published in final edited form as:
Trends Genet. 2018 April ; 34(4): 253–255. doi:10.1016/j.tig.2018.01.009.
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suppressor [6] was important in showing that although the chromosomes of C. elegans are 
holocentric they nonetheless each have a site, now called the pairing center, that acts 
genetically like a centrosome, directing pairing and chromosome segregation.
The use of balancers made it possible to identify large numbers of recessive-lethal mutations 
in C. elegans, by screening the progeny of mutagenized balancer heterozygotes to find 
mutants that were no longer viable as non-balancer homozygotes [7]. In an era of efficient, 
affordable targeted genomic modification, balancers remain essential in maintaining strains 
containing mutations with recessive-inviable phenotypes; this report also offers useful 
guidance in generating more such mutations, when CRISPR activity is likely to otherwise 
target both copies and produce inviable homozygous mutants. A good collection of balancer 
chromosomes covering as much as possible of the genome is incredibly useful in strain 
constructions: even though most mutations can now be genotyped molecularly, the use of 
balancers to follow loci in trans can make strain constructions cheaper and greatly less labor-
intensive than is the case using molecular genotyping. Good balancers also make it possible 
to collect animals homozygous for recessive-inviable mutations, whether it’s single animals 
for close examination or much larger quantities for biochemical analysis, collected through 
the use of a sorting machine or by using a selective marker.
The set of balancers previously available in C. elegans was a patchwork collection of 
reciprocal translocations, single inversions, and integrated transgenes [8]. This set failed to 
cover large portions of the genome, and most parts of this set had major inconveniences and 
liabilities. Translocations are stable but cause pseudolinkage across two chromosomes that 
can make them less useful; more importantly, translocation heterozygotes generate large 
numbers of aneuploid embryos that can impede analysis of balanced mutants (10/16 of self-
progeny of translocation heterozygotes are aneuploids of one sort or another). Large single 
inversions break down at high frequency. Transgenes are available that have been randomly 
integrated at many points in the genome, but tend to be poorly characterized to determine 
across what regions if any they completely suppress recombination.
The new set of engineered balancer chromosomes offers a significant new resource, 
addressing a large gap in the tools available to the C. elegans research community. The new 
rationally designed balancer chromosomes have none of the drawbacks detailed above. In 
contrast to translocations used as balancers, the CRISPR-made rearrangements are contained 
within a single chromosome, and so do not cause pseudolinkage. As Dejima and co-workers 
specifically demonstrate, the new balancers do not generate aneuploid embryos. Each of the 
new balancers contains two overlapping inversions, making them much less susceptible to 
spontaneous breakdown than older balancers consisting of a single inversion. Unlike 
arbitrarily selected transgenes randomly integrated into the chromosome, the limits of the 
recombinationally suppressed regions are precisely known for these new balancers. In 
addition to generating large, stable chromosomal rearrangements covering large sections of 
the genome, the authors also provide markers for regions of the genome close to pairing 
centers, which are recalcitrant to the engineering of recombinational suppressors, and 
demonstrate a method for using CRISPR to generate heterozygous mutations that would be 
inviable as homozygotes.
Schwartz and Sternberg Page 2
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Pioneering work using nucleases with engineered specificity in C. elegans was accompanied 
by demonstrations that it could be used in a nematode species less commonly used in the 
laboratory [9]. The model demonstrated by this new collection of engineered balancer 
chromosomes in C. elegans is similarly transferable to other organisms, including organisms 
that have so far been less popular in laboratory research and so lack the genetic toolkits 
necessary to enable and accelerate research. This should make it more feasible to generate, 
maintain, and examine inviable mutants in species not previously popular in the laboratory. 
In particular, the availability of balancer chromosomes could facilitate genetic screens in 
male-female nematode species, by making it possible to follow and to homozygose 
mutagenized, balanced sections of the genome.
References
1. Dejima K, et al. An aneuploidy-free and structurally defined balancer chromosome toolkit for 
Caenorhabditis elegans. Cell Rep. 2018; 22:232–241. [PubMed: 29298424] 
2. Iwata S, et al. Engineering new balancer chromosomes in C. elegans via CRISPR/Cas9. Sci Rep. 
2016; 6:33840. [PubMed: 27650892] 
3. Sturtevant AH. Genetic factors affecting the strength of linkage in Drosophila. Proc Natl Acad Sci 
USA. 1917; 9:555–558.
4. Sturtevant AH. The linear arrangement of six sex-linked factors in Drosophila, as shown by their 
mode of association. J Exp Zool. 1913; 14:43–59.
5. Sturtevant AH, Dobzhansky T. Inversions in the third chromosome of wild races of Drosophila 
pseudoobscura, and their use in the study of the history of the species. Proc Natl Acad Sci USA. 
1936; 22:448–450. [PubMed: 16577723] 
6. Rosenbluth RE, Baillie DL. The genetic analysis of a reciprocal translocation, eT1(III; V), in 
Caenorhabditis elegans. Genetics. 1981; 99:415–428. [PubMed: 6953041] 
7. Johnsen RC, Baillie DL. Formaldehyde mutagenesis of the eT1 balanced region in Caenorhabditis 
elegans: dose-response curve and the analysis of mutational events. Mutat Res. 1988; 201:137–147. 
[PubMed: 3419443] 
8. Edgley, ML., Baillie, DL., Riddle, DL., Rose, AM. The C. elegansResearch Community. 
WormBook. WormBook; 2006. Genetic Balancers; p. 1-32.
9. Lo TW. Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs 
and CRISPR/Cas9 to engineer insertions and deletions. Genetics. 2013; 195:331–348. [PubMed: 
23934893] 
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Figure 1. 
The generation and use of balancer chromosomes. (A) Balancer chromosomes are 
engineered using CRISPR. The programmable nuclease Cas9 makes a pair of cuts, excising 
a large section of a chromosome; the repair of these double-strand DNA breaks is directed 
by oligonucleotide repair templates to re-integrate that section into the chromosome as an 
inversion. This process is repeated to create a second, overlapping inversion, to make a more 
stable recombinational balancer. Markers can then be added into the recombinationally 
suppressed or “balanced” region of this modified chromosome: dominant markers such as a 
gfp transgene to mark the presence of the balancer in heterozygotes, and recessive markers 
such as a lethal mutation, to mark or prevent the loss of the non-balancer chromosome. (B) 
Especially when well marked, balancer chromosomes can be used to maintain strains 
Schwartz and Sternberg Page 4
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carrying mutations with undesirable recessive phenotypes, and to obtain mutation 
homozygotes from these strains for analysis. (C) By making it possible to follow loci in 
trans, balancer chromosomes can be used to make the construction of double mutant strains 
more labor-efficient and less dependent on molecular genotyping. (D) Balancer 
chromosomes can be used to follow sections of the genome following mutagenesis, so that 
in genetic screens mutants in that region can be examined in homozygotes while also being 
maintained as heterozygotes. This can make it feasible to conduct genetic screens in male/
female species that lack the selfing hermaphroditism of C. elegans.
Schwartz and Sternberg Page 5
Trends Genet. Author manuscript; available in PMC 2019 April 01.
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A Toolkit of Engineered Recombinational Balancers in C. elegans

A toolkit of engineered recombinational balancers in C. elegans
Hillel T Schwartz and Paul W Sternberg
Division of Biology and Biological Engineering, California Institute of Technology, Pasadena, CA 
91125
Abstract
Dejima and colleagues report using CRISPR-Cas9 to generate a new collection of greatly 
improved balancer chromosomes in the standard laboratory nematode Caenorhabditis elegans, 
using methods previously reported by the same laboratory [1,2], expanding the set of C. elegans 
balancers to cover nearly 90% of coding genes.
Keywords
Caenorhabditis elegans; CRISPR; Cas9; balancer; recombinational suppression
Text
Balancer chromosomes are a powerful tool in experimental genetics. Balancers are variant 
chromosomes that when heterozygous with normal chromosomes have a defined region 
within which there is no or essentially no recombination between the balancer chromosome 
and the normal chromosome. To achieve this, balancer chromosomes contain 
rearrangements such as inversions and translocations that disrupt synapsis and so prevent 
recombination. Any mutations within this recombinationally suppressed region can be 
reliably followed in trans using the balancer. Balancer chromosomes typically carry markers 
to make this more efficient: dominantly acting markers such as a gfp transgene to mark the 
presence of the balancer, and often a mutation with a recessive visible or even lethal 
phenotype to mark or to prevent the loss of the non-balancer chromosome and the mutations 
it contains. Balancers containing such markers can be used to infer genotype rapidly and 
efficiently.
It is now just over 100 years since the first report of a chromosome acting as a 
recombinational suppressor, by Sturtevant in 1917 [3]. This result, following close after the 
discovery that it was possible to construct a genetic linkage map [4], was immediately seen 
as being consistent with a chromosomal theory of inheritance and was soon shown to 
respond to chromosomal rearrangements that could be observed cytologically [5]. Much 
later, work in C. elegans with a reciprocal translocation that acted as a recombinational 
Publisher's Disclaimer: This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our 
customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of 
the resulting proof before it is published in its final citable form. Please note that during the production process errors may be 
discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
HHS Public Access
Author manuscript
Trends Genet. Author manuscript; available in PMC 2019 April 01.
Published in final edited form as:
Trends Genet. 2018 April ; 34(4): 253–255. doi:10.1016/j.tig.2018.01.009.
A
uthor M
anuscript
A
uthor M
anuscript
A
uthor M
anuscript
A
uthor M
anuscript
suppressor [6] was important in showing that although the chromosomes of C. elegans are 
holocentric they nonetheless each have a site, now called the pairing center, that acts 
genetically like a centrosome, directing pairing and chromosome segregation.
The use of balancers made it possible to identify large numbers of recessive-lethal mutations 
in C. elegans, by screening the progeny of mutagenized balancer heterozygotes to find 
mutants that were no longer viable as non-balancer homozygotes [7]. In an era of efficient, 
affordable targeted genomic modification, balancers remain essential in maintaining strains 
containing mutations with recessive-inviable phenotypes; this report also offers useful 
guidance in generating more such mutations, when CRISPR activity is likely to otherwise 
target both copies and produce inviable homozygous mutants. A good collection of balancer 
chromosomes covering as much as possible of the genome is incredibly useful in strain 
constructions: even though most mutations can now be genotyped molecularly, the use of 
balancers to follow loci in trans can make strain constructions cheaper and greatly less labor-
intensive than is the case using molecular genotyping. Good balancers also make it possible 
to collect animals homozygous for recessive-inviable mutations, whether it’s single animals 
for close examination or much larger quantities for biochemical analysis, collected through 
the use of a sorting machine or by using a selective marker.
The set of balancers previously available in C. elegans was a patchwork collection of 
reciprocal translocations, single inversions, and integrated transgenes [8]. This set failed to 
cover large portions of the genome, and most parts of this set had major inconveniences and 
liabilities. Translocations are stable but cause pseudolinkage across two chromosomes that 
can make them less useful; more importantly, translocation heterozygotes generate large 
numbers of aneuploid embryos that can impede analysis of balanced mutants (10/16 of self-
progeny of translocation heterozygotes are aneuploids of one sort or another). Large single 
inversions break down at high frequency. Transgenes are available that have been randomly 
integrated at many points in the genome, but tend to be poorly characterized to determine 
across what regions if any they completely suppress recombination.
The new set of engineered balancer chromosomes offers a significant new resource, 
addressing a large gap in the tools available to the C. elegans research community. The new 
rationally designed balancer chromosomes have none of the drawbacks detailed above. In 
contrast to translocations used as balancers, the CRISPR-made rearrangements are contained 
within a single chromosome, and so do not cause pseudolinkage. As Dejima and co-workers 
specifically demonstrate, the new balancers do not generate aneuploid embryos. Each of the 
new balancers contains two overlapping inversions, making them much less susceptible to 
spontaneous breakdown than older balancers consisting of a single inversion. Unlike 
arbitrarily selected transgenes randomly integrated into the chromosome, the limits of the 
recombinationally suppressed regions are precisely known for these new balancers. In 
addition to generating large, stable chromosomal rearrangements covering large sections of 
the genome, the authors also provide markers for regions of the genome close to pairing 
centers, which are recalcitrant to the engineering of recombinational suppressors, and 
demonstrate a method for using CRISPR to generate heterozygous mutations that would be 
inviable as homozygotes.
Schwartz and Sternberg Page 2
Trends Genet. Author manuscript; available in PMC 2019 April 01.
A
uthor M
anuscript
A
uthor M
anuscript
A
uthor M
anuscript
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uthor M
anuscript
Pioneering work using nucleases with engineered specificity in C. elegans was accompanied 
by demonstrations that it could be used in a nematode species less commonly used in the 
laboratory [9]. The model demonstrated by this new collection of engineered balancer 
chromosomes in C. elegans is similarly transferable to other organisms, including organisms 
that have so far been less popular in laboratory research and so lack the genetic toolkits 
necessary to enable and accelerate research. This should make it more feasible to generate, 
maintain, and examine inviable mutants in species not previously popular in the laboratory. 
In particular, the availability of balancer chromosomes could facilitate genetic screens in 
male-female nematode species, by making it possible to follow and to homozygose 
mutagenized, balanced sections of the genome.
References
1. Dejima K, et al. An aneuploidy-free and structurally defined balancer chromosome toolkit for 
Caenorhabditis elegans. Cell Rep. 2018; 22:232–241. [PubMed: 29298424] 
2. Iwata S, et al. Engineering new balancer chromosomes in C. elegans via CRISPR/Cas9. Sci Rep. 
2016; 6:33840. [PubMed: 27650892] 
3. Sturtevant AH. Genetic factors affecting the strength of linkage in Drosophila. Proc Natl Acad Sci 
USA. 1917; 9:555–558.
4. Sturtevant AH. The linear arrangement of six sex-linked factors in Drosophila, as shown by their 
mode of association. J Exp Zool. 1913; 14:43–59.
5. Sturtevant AH, Dobzhansky T. Inversions in the third chromosome of wild races of Drosophila 
pseudoobscura, and their use in the study of the history of the species. Proc Natl Acad Sci USA. 
1936; 22:448–450. [PubMed: 16577723] 
6. Rosenbluth RE, Baillie DL. The genetic analysis of a reciprocal translocation, eT1(III; V), in 
Caenorhabditis elegans. Genetics. 1981; 99:415–428. [PubMed: 6953041] 
7. Johnsen RC, Baillie DL. Formaldehyde mutagenesis of the eT1 balanced region in Caenorhabditis 
elegans: dose-response curve and the analysis of mutational events. Mutat Res. 1988; 201:137–147. 
[PubMed: 3419443] 
8. Edgley, ML., Baillie, DL., Riddle, DL., Rose, AM. The C. elegansResearch Community. 
WormBook. WormBook; 2006. Genetic Balancers; p. 1-32.
9. Lo TW. Precise and heritable genome editing in evolutionarily diverse nematodes using TALENs 
and CRISPR/Cas9 to engineer insertions and deletions. Genetics. 2013; 195:331–348. [PubMed: 
23934893] 
Schwartz and Sternberg Page 3
Trends Genet. Author manuscript; available in PMC 2019 April 01.
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uthor M
anuscript
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uthor M
anuscript
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uthor M
anuscript
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Figure 1. 
The generation and use of balancer chromosomes. (A) Balancer chromosomes are 
engineered using CRISPR. The programmable nuclease Cas9 makes a pair of cuts, excising 
a large section of a chromosome; the repair of these double-strand DNA breaks is directed 
by oligonucleotide repair templates to re-integrate that section into the chromosome as an 
inversion. This process is repeated to create a second, overlapping inversion, to make a more 
stable recombinational balancer. Markers can then be added into the recombinationally 
suppressed or “balanced” region of this modified chromosome: dominant markers such as a 
gfp transgene to mark the presence of the balancer in heterozygotes, and recessive markers 
such as a lethal mutation, to mark or prevent the loss of the non-balancer chromosome. (B) 
Especially when well marked, balancer chromosomes can be used to maintain strains 
Schwartz and Sternberg Page 4
Trends Genet. Author manuscript; available in PMC 2019 April 01.
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carrying mutations with undesirable recessive phenotypes, and to obtain mutation 
homozygotes from these strains for analysis. (C) By making it possible to follow loci in 
trans, balancer chromosomes can be used to make the construction of double mutant strains 
more labor-efficient and less dependent on molecular genotyping. (D) Balancer 
chromosomes can be used to follow sections of the genome following mutagenesis, so that 
in genetic screens mutants in that region can be examined in homozygotes while also being 
maintained as heterozygotes. This can make it feasible to conduct genetic screens in male/
female species that lack the selfing hermaphroditism of C. elegans.
Schwartz and Sternberg Page 5
Trends Genet. Author manuscript; available in PMC 2019 April 01.
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English

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A Toolkit of Engineered Recombinational Balancers in C. elegans

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