CRISPR/Cas9-Based Development of Novel

Transgenic Rat Model of X-Linked Hydrocephalus
A. Scott Emmert1, June Goto1, Crystal Shula1, Shenyue Qin2, Yueh-Chiang Hu3, and Francesco T. Mangano1
1Division of Pediatric Neurosurgery, 2Division of Developmental Biology, and 3Transgenic Animal and Genome Editing Core Facility, Cincinnati Children’s Hospital Medical Center

Background Results
A. C. Figure 1. Design and
• Congenital hydrocephalus is a
devastating birth defect defined molecular representation of
by the abnormal accumulation of rat-CRISPR-L1 gRNA
cerebrospinal fluid (CSF) in the oligomers for disruption of the
brain that can lead to brain L1cam gene in rat embryos
damage and mental and (A.). Representative
physical problems if left sequencing chromatograms of
untreated. L1cam mutant rats generated
B. D.
in the CRISPR/Cas9 genome
• Due to the abundance of
editing system. Mutants
functional genomic methods
exhibit disruption of the
available for mice, transgenic
L1cam gene through
mouse models dominate
frameshift and nonsense
modern studies into the
mutation (B.), amino acid
pathogenesis of hydrocephalus.
substitution (C.), and genomic
However, their small size inhibits
deletion (D.).
the use of surgical and imaging
procedures that could be
performed in larger rodents.
• Emergence of CRISPR/Cas9 Results Conclusions
genome-editing technology WT L1cam WT L1cam
• Of the eleven rats born from
Corpus Callosum

Corpus Callosum
provides an accessible method
for generating transgenic rat CRISPR-modified embryos,
models of congenital seven exhibit mutation of the
hydrocephalus that have been L1cam gene in the targeted
traditionally challenging to region of exon 4. The three
manipulate genetically. 1.00 mm 1.00 mm 0.150 mm 0.150 mm primary mutations disrupting
L1cam in each L1exon4CRISPR
• L1cam is a gene found on the X
allele are a single thymine
Left Thalamus

Left Thalamus

chromosome that codes for a
insertion, two amino acid
neuronal cell adhesion molecule
substitution, and 300 base pair
critical to nervous system
deletion (Fig. 1B-1D).
development. Mutation of this
gene causes X-linked 1.00 mm 1.00 mm 0.075 mm 0.075 mm
• Body weights of L1cam mutant
hydrocephalus (XLH) in humans rats are significantly lower than
and mice. their WT counterparts beginning
Right Thalamus

Right Thalamus

at postnatal day 56 (P = <0.001,
• We sought to use
data not shown).
CRISPR/Cas9-based disruption
of the rat L1cam gene to • Loss of functional L1CAM
generate a knockout model of protein in this model,
XLH in an organism large
1.00 mm 1.00 mm 0.075 mm 0.075 mm
represented by the absence of
enough for novel imaging and Figure 2. Representative photomicrographs of the corpus callosum, left thalamus, and DAB-stained structures in the
surgical procedures. right thalamus stained for L1CAM protein in wildtype and L1cam mutant brains. corpus callosum, left thalamus,
and right thalamus of L1cam
A. B. E. F. Figure 3. mutant brains in
Representative immunohistochemistry (Fig. 2),
MRI images of the validates the genome DNA
Methods WT fourth ventricle sequencing results of L1cam
(A.-D.) and lateral mutants.
• Two guide RNA (gRNA) p21 p97 p21 p97 ventricles (E.-H.) • L1cam mutants demonstrate
oligomers, designed to disrupt C. D. G. H. in wildtype (WT) larger fourth ventricles
exon 4 of the rat L1cam gene on and L1cam mutant (measured by volume)
the X chromosome, were rats (L1cam) at compared to WT (Fig. 3A-3D,
injected into SD rat embryos and L1cam postnatal days 21 Fig. 4A). Similiarly, L1cam
transplanted into recipient rats. (p21) and 97 mutants show enlargement of
p21 p97 p21 p97 (p97). the lateral ventricles with time
• Rats born from CRISPR- **

compared to WT (Fig. 3E-3H,
modified embryos were Sanger A. Fourth Ventricle Volume in L1cam Mutant and Wildtype Rats Fig. 4B-4C).
sequenced and analyzed for as a Function of Age
Fourth Ventricle Volume

L1cam mutation. Edited animals 5
were bred to establish the 4 R² = 0.7257
colony. **
3 WT
Future Directions
(uL)

• Mutant and wildtype rats were 2 R² = 0.63975 L1cam
1 Linear (WT)
longitudinally weighed and • This study suggests that
0 Linear (L1cam)
imaged using T2-weighted 0 20 40 60 80 100 120 CRISPR/Cas9 genome editing
magnetic resonance imaging Age (postnatal days) technology can be used to
(T2 MRI). Volumes of the fourth generate additional rat models
B. Left Lateral Ventricle Volume in L1cam Mutant and Wildtype
ventricle and lateral ventricles in Figure 4. Results of linear of hydrocephalus involving other
Rats as a Function of Age
each imaged rat were quantified
Left Lateral Ventricle

4 regression analyses genomic dysfunctions, providing
from the T2 MRI data. Student’s performed on fourth further opportunities to explore
Volume (uL)

3
t-test was used to compare body R² = 0.74954
WT ventricle (A.) and lateral novel surgical and imaging
2 ***
weights between mutant and L1cam ventricle (B., C.) volumes techniques on a larger model
wildtype rats. Linear regressions 1 R² = 0.41434 Linear (WT) in WT and L1cam mutant organism.
were performed on the 0 Linear (L1cam) rats as a function of age.
0 20 40 60 80 100 120 • The mild ventriculomegaly
longitudinal ventricle volume ***P = <0.001, **P=<0.01.
Age (postnatal days) evidenced in L1cam mutant rats
data.
C. Right Lateral Ventricle Volume in L1cam Mutant and Wildtype make this model ideal for
• Peroxidase-DAB (3,3'- extended investigations into the
diaminobenzidine) Rats as a Function of Age
Right Lateral Ventricle

4
application of diffusion tensor
immunohistochemistry staining imaging to the surgical treatment
Volume (uL)

3 R² = 0.69309
was performed on paraffin- WT of hydrocephalus (e.g. whether
2 ***
embedded tissue sections of WT L1cam DTI parameters can serve as
and mutant brains to evaluate 1 R² = 0.48742 Linear (WT) imaging biomarkers of infantile
L1CAM protein expression. 0 Linear (L1cam) hydrocephalus before and after
0 20 40 60 80 100 120
Age (postnatal days)
surgery).