"CRISPR-Cas9

Effectors for Single-

Sex Litters and Sex-
Specific Phenotypes"
By Charlotte Douglas 1, Valdone Maciulyte1 , Jasmin Zohren 1 , Daniel M. Snell1, Shantha K. Mahadevaiah1
,Obah A. Ojarikre1 , Peter J. I. Ellis 2 ✉ & James M. A. Turne
A groundbreaking study exploring the use of CRISPR-Cas9 effectors in generating single-sex litters and
uncovering sex-specific phenotypes.
[Swapnil Sonal ms20172 and Harsh Atul Joshi ms20140]
Introduction
Addresses the need for producing single-sex litters in scientific research and
agriculture, and the ethical and financial burden associated with culling animals of
the undesired sex .
Development of a synthetic lethal, bicomponent CRISPR-Cas9 strategy that
achieves 100% efficiency in producing male or female-only litters in mice
Potential of their system to generate postnatal sex-specific phenotypes,
providing strides towards ethical improvements in laboratory research and
agriculture
.
Sex-Specific Phenotypes and Their Implications for Biological Processes
Understanding sex-specific phenotypes is crucial for studying sexual dimorphism and the impact of sex
on various biological processes.
Studying sex-specific phenotypes is relevant for investigating sex-linked traits and the inheritance
patterns of genes located on sex chromosomes, which have implications for understanding diseases
and traits that show sex-specific differences
Sex-specific phenotypes also have implications in agriculture, where the ability to selectively produce
male or female offspring can be valuable
Specific Research Question:
How can a synthetic lethal, bicomponent CRISPR-Cas9 strategy be developed to produce single-sex
litters with 100% efficiency in mice?
Objectives of the Paper:
Develop a synthetic lethal, bicomponent CRISPR-Cas9 strategy that produces male- or female-only
litters with 100% efficiency in mice.
Assess the potential of the bicomponent system to increase the yield of the desired sex in comparison
to standard breeding designs.
Explore the repurposing of the bicomponent system to generate postnatal sex-specific phenotypes,
expanding its applications beyond sex determination
Scientific Rationale and Significance of Studying Single-Sex Litters and Sex-
Specific Phenotypes:
Understanding Genetic Control
By manipulating the CRISPR-Cas9 system to produce male- or female-only litters, researchers can
investigate the specific genes and mechanisms involved in sex determination.
Practical Applications in Research and Agriculture
In research, it reduces the need for culling animals of the undesired sex, minimizing ethical and financial
burdens.
In agriculture, it can be used to selectively breed animals with desired traits, improving productivity and
efficiency
CRISPER CAS9
Protocol for Generating Single-Sex
Litters using CRISPR-Cas9
1 Designing sgRNAs.
single sgRNAs with high on-target activity and low off-target activity .
Select sgRNAs with BbsI overhangs and anneal them with relevant vectors using a
published protocol .

2 Electroporation of sgRNAs:
Obtain sgRNA oligonucleotides from Integrated DNA Technologies
Perform transient electroporation of sgRNAs into the target cells or embryo

3 CRISPR-Cas9 Cleavage:
Utilize the bicomponent CRISPR-Cas9 system to induce targeted DNA cleavage at
specific loci involved in sex determination
Sex Determination: Analyze the resulting embryos or cells to determine the sex of the
individuals. The CRISPR-Cas9 strategy should produce male- or female-only litters with 100%
efficiency

Litter Size Compensation:

Assess the litter size compensation relative to control matings, which indicates the potential
to increase the yield of the desired sex compared to standard breeding designs

Postnatal Phenotype Generation:

Repurpose the bicomponent system to generate postnatal sex-specific phenotypes by
manipulating the CRISPR-Cas9 system in a controlled manner

Validation and Reproducibility:

Validate the results and ensure reproducibility of the technique by performing multiple
experiments and analyzing the data statistically.
Results :An in vitro CRISPR-Cas9 bicomponent
system induces Top1 mutation
Topoisomerase 1 (Top1) was selected as the target for the study due to its importance in DNA
replication and repair.
A synthetic lethal CRISPR-Cas9 system was designed to generate single-sex litters by targeting a
gene called Topoisomerase 1 (Top1) that causes early embryo death.
Guides targeting the Top1 gene were cloned into a vector and transfected into mouse embryonic stem
cells (mESCs) expressing Cas9 and an eGFP reporter.

The mutation efficiency of the guides was evaluated, with sgRNA2 showing the highest mutation
efficiency and the most common mutation being a single nucleotide insertion at the Cas9 cut site
Co-inheritance of autosomal sgRNA and Cas9
transgenes induce Top1 mutations and embryonic
lethality
Co-inheritance of autosomal sgRNA and Cas9 transgenes induces Top1 mutations and embryonic
lethality.
X-integrated sgRNA2-mCherry transgene was expressed in XTop1Y adult tissues and in XTop1X female
embryos.
XTop1Y males mated with R26Cas9 females resulted in lethality in daughters in the presence of
maternal Cas9.
Offspring from matings between XTop1Y males and
either homozygous R26Cas9 females (left column)
or control wild-type females (right column)
The number of offspring produced from each
mating was recorded and is denoted by "n".
The p-value indicates the statistical
significance of the deviation from a 1:1 ratio of
male to female offspring, as determined by a
Chi-squared test.
Embryo Lethality with Co-Inheritance of H11 Top1 and R26Cas9 via
the sgRNA2-mCherry Transgene on an Autosomal Chromosome
Initially, the sgRNA2-mCherry transgene was integrated on a sex chromosome and the Cas9 transgene
on an autosome, but this resulted in surviving offspring expressing the Cas9 endonuclease.

Cas9 (dCas9) for which endonuclease

activity is non-functional due to D10A
and H840A mutations cannot induce
double-strand DNA breaks, but it is
still capable of DNA binding.
Targeting dCas9 to transcription start
sites physically blocks RNA
polymerase movement and hinders
transcription, thus silencing gene
expression through CRISPR
interference (CRISPRi)
Mating strategy
To overcome this drawback, the bicomponent system was reversed, placing sgRNA2-mCherry on an
autosome and the Cas9 transgene on the sex chromosome, so that surviving offspring would only
inherit the sgRNA and not express any foreign proteins.
The functionality of the autosomal sgRNA2-mCherry transgene was confirmed through breeding
experiments, where co-inheritance of H11 Top1 and R26Cas9 resulted in embryo lethality with 100%
efficiency
Offspring from matings between H11 Top1/+ males and either
homozygous R26Cas9 females (first two columns) or control wild-
type females (right column). n = number of offspring. p-value
shows the significance of
deviation from 1:1 ratio (Chi-squared test)
Female\Male-Only Litter Formation by
Different Mouse Lines
The researchers used different mouse lines to create single-sex litters, focusing on male-only litters.
By mating specific male and female mice, they were able to generate exclusively male or female
offspring.
The researchers observed a higher number of offspring than expected, indicating compensation for
embryo loss caused by the genetic system used.
The study demonstrated efficient embryo selection using different types of Cas9 transgenes and
addressed the issue of litter size compensation.

The study used two types of male mice: X Cas9
Y and XYCas9. These males were bred with
homozygous H11Top1 females or control X
Cas9 Y males mated to wild-type females.
The pups born from these matings were sex-
genotyped to determine the sex of each pup. The
number of pups in each litter was also recorded.
The results of the sex genotyping were
compared to a 1:1 ratio of male to female pups,
which would be expected in a standard breeding
design.
The p-value shows the significance of deviation
from the 1:1 ratio, which was calculated using a
Chi-squared test. A p-value less than 0.05
indicates that the deviation from the expected
ratio is statistically significant.
Results
Sex-Specific Phenotypes:
The researchers successfully generated sex-specific phenotypes in offspring using the CRISPR-Cas9
system .

They focused on the Atm gene, which is involved in meiosis and has different meiotic studies that
require either males or females .

Male Atm mutants were used to study recombination and crossover formation on the mammalian X and
Y chromosomes, while female Atm mutants were used in studies of the oocyte recombination
checkpoint .
The researchers observed that male Atm mutants showed germ cell arrest at mid pachynema, resulting
in no sperm in the seminiferous tubules and lower testis weights relative to wild-type males .

Female Atm mutants exhibited adverse effects, including immunodeficiency, neural defects, and thymic
lymphomas .

The X Cas9 Y or XY Cas9 models used in the study can be used to create other sex-specific
phenotypes .
Effects of the Atm mutation on ovarian development and fertility.

1. The dotted circles in the image likely indicate an area of

the ovary that has undergone degeneration The quantification was done when the
2. This could be due to a genetic mutations. mice were 8 weeks old.
Effects of a Atm mutation on germ cell development in the testis.
In the Atm mutant
testis samples, there
was a complete
arrest of germ cell
development at this
stage.
This means that the
germ cells were
unable to progress
beyond this stage of
development.
The arrows in the
Complete Stage IV, Midpachytene Germ Cell Arrest image highlight this
Periodic-Acid Schiff (PAS) staining is a laboratory technique used to germ cell arrest.
visualize carbohydrates, glycogen, and other polysaccharides in tissues
 1. Male mice with the
normal (wild-type)
Atm gene have testis
weights that are three
times greater than
those with the
mutated Atm gene.
2. Atm Gene Role: The
Atm gene is likely a
key regulator of testis
development and
growth in mice.
A comparison of the testis weights in male mice with the normal form of the Mutations in this gene
Atm gene (wild-type) and male mice with a mutated form of the Atm gene result in smaller testis
(Atm mutant) at 8 weeks of age. size.
comparison of SYCP3 and γH2AX proteins in
pachytene spermatocytes of normal and Atm
mutant male mice
1. DNA Repair Trigger: It plays a pivotal role in
triggering the cellular response to DNA damage,
facilitating the recruitment of repair proteins to fix
damaged DNA.
2. Research and Clinical Utility: Researchers use
γH2AX as a biomarker to assess DNA damage
DNA Damage Marker: γH2AX is a protein that levels
marks sites of DNA damage, specifically
double-strand breaks, within the cell's nucleus.
γH2AX is a marker for DNA damage, and its increased presence in the Atm mutant males suggests that
these males have higher levels of DNA damage in their spermatocytes.
Conclusion:
The paper presents a synthetic lethal, bicomponent CRISPR-Cas9 strategy that achieves 100%
efficiency in producing male- or female-only litters in mice, reducing the need for culling animals of the
undesired sex and potentially increasing the yield of the desired sex compared to standard breeding
designs .

CRISPR-Cas9 system can be repurposed to generate postnatal sex-specific phenotypes, allowing for
the study of the effects of specific genes on sex-related traits and diseases. This approach has the
potential to improve laboratory research, agriculture, and ethical considerations in animal breeding
Potential Practical Applications of the Research Findings:
Animal Research:
The ability to generate single-sex litters with 100% efficiency can significantly reduce the ethical and
financial burden of producing and culling animals of the undesired sex in scientific research.

Agriculture:
Allowing for the selective production of male- or female-only offspring can have economic benefits by
optimizing breeding programs and improving the efficiency of livestock production .

Biomedical Research:
The ability to generate postnatal sex-specific phenotypes opens up new avenues for studying the
effects of specific genes on sex-related traits and diseases.
This can contribute to a better understanding of the molecular mechanisms underlying sexual
dimorphism and potentially lead to the development of targeted therapies for sex-specific conditions
Potential Avenues for Further Research on CRISPR-Cas9 Effectors
and Sex Determination:
Optimization of CRISPR-Cas9 Efficiency:
Optimizing the efficiency of the CRISPR-Cas9 system in generating single-sex litters.
Exploring different target genes and sgRNA designs to enhance the accuracy and effectiveness of sex
determination .

Investigating Sex-Specific Phenotypes:

Explore the potential of CRISPR-Cas9 effectors in generating sex-specific phenotypes beyond just sex
determination.
This can involve manipulating specific genes involved in sex-related traits and diseases to study their
impact on phenotype development .
Expanding to Other Vertebrate Species:
The application of CRISPR-Cas9 effectors in sex determination can be extended to other vertebrate
species, such as livestock.
Further research can explore the feasibility and efficiency of implementing the bicomponent CRISPR-
Cas9 strategy in different mammalian species .

Understanding Molecular Mechanisms:

Investigating the molecular mechanisms underlying sex determination can provide valuable insights
into the development and regulation of sexual dimorphism.
CRISPR-Cas9 can be utilized to target and manipulate key genes involved in sex determination
pathways, allowing for a better understanding of the underlying mechanisms