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 Childrens Hospital Medical Center
Background
A.
Congenital hydrocephalus is a
devastating birth defect defined
by the abnormal accumulation of
cerebrospinal fluid (CSF) in the
brain that can lead to brain
damage and mental and
physical problems if left
untreated.
B.
Due to the abundance of
functional genomic methods
available for mice, transgenic
mouse models dominate
modern studies into the
pathogenesis of hydrocephalus.
However, their small size inhibits
the use of surgical and imaging
procedures that could be
performed in larger rodents.
Emergence of CRISPR/Cas9 Results
genome-editing technology
provides an accessible method
for generating transgenic rat
models of congenital
hydrocephalus that have been
traditionally challenging to
manipulate genetically.
L1cam is a gene found on the X
chromosome that codes for a
neuronal cell adhesion molecule
critical to nervous system
development. Mutation of this
gene causes X-linked
hydrocephalus (XLH) in humans
and mice.
We sought to use
CRISPR/Cas9-based disruption
of the rat L1cam gene to
generate a knockout model of
XLH in an organism large
enough for novel imaging and
surgical procedures.
Methods
Two guide RNA (gRNA)
oligomers, designed to disrupt
exon 4 of the rat L1cam gene on
the X chromosome, were
injected into SD rat embryos and L1cam
transplanted into recipient rats.
Rats born from CRISPR-
modified embryos were Sanger
sequenced and analyzed for
L1cam mutation. Edited animals
were bred to establish the
colony.
Mutant and wildtype rats were
longitudinally weighed and
imaged using T2-weighted
magnetic resonance imaging
(T2 MRI). Volumes of the fourth
ventricle and lateral ventricles in
each imaged rat were quantified
from the T2 MRI data. Students
t-test was used to compare body
weights between mutant and
wildtype rats. Linear regressions
were performed on the
longitudinal ventricle volume
data.
Peroxidase-DAB (3,3'-
diaminobenzidine)
immunohistochemistry staining
was performed on paraffin-
embedded tissue sections of WT
and mutant brains to evaluate
L1CAM protein expression.
Results
C. Figure 1. Design and
molecular representation of
rat-CRISPR-L1 gRNA
oligomers for disruption of the
L1cam gene in rat embryos
(A.). Representative
sequencing chromatograms of
L1cam mutant rats generated
D.
in the CRISPR/Cas9 genome
editing system. Mutants
exhibit disruption of the
L1cam gene through
frameshift and nonsense
mutation (B.), amino acid
substitution (C.), and genomic
deletion (D.).
Conclusions
WT L1cam WT L1cam
Of the eleven rats born from
Corpus Callosum
Corpus Callosum
CRISPR-modified embryos,
seven exhibit mutation of the
L1cam gene in the targeted
region of exon 4. The three
1.00 mm 1.00 mm 0.150 mm 0.150 mm primary mutations disrupting
L1cam in each L1exon4CRISPR
allele are a single thymine
Left Thalamus
Left Thalamus
insertion, two amino acid
substitution, and 300 base pair
deletion (Fig. 1B-1D).
1.00 mm 1.00 mm 0.075 mm 0.075 mm
Body weights of L1cam mutant
rats are significantly lower than
their WT counterparts beginning
Right Thalamus
Right Thalamus
at postnatal day 56 (P = <0.001,
data not shown).
Loss of functional L1CAM
protein in this model,
1.00 mm 1.00 mm 0.075 mm 0.075 mm
represented by the absence of
Figure 2. Representative photomicrographs of the corpus callosum, left thalamus, and DAB-stained structures in the
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
WT fourth ventricle sequencing results of L1cam
(A.-D.) and lateral mutants.
p21 p97 p21 p97 ventricles (E.-H.) L1cam mutants demonstrate
C. D. G. H. in wildtype (WT) larger fourth ventricles
and L1cam mutant (measured by volume)
rats (L1cam) at compared to WT (Fig. 3A-3D,
postnatal days 21 Fig. 4A). Similiarly, L1cam
(p21) and 97 mutants show enlargement of
p21 p97 p21 p97 (p97). the lateral ventricles with time
**
compared to WT (Fig. 3E-3H,
A. Fourth Ventricle Volume in L1cam Mutant and Wildtype Rats Fig. 4B-4C).
as a Function of Age
Fourth Ventricle Volume
5
4 R = 0.7257
**
3 WT
Future Directions
(uL)
2 R = 0.63975 L1cam
1 Linear (WT)
This study suggests that
0 Linear (L1cam)
0 20 40 60 80 100 120 CRISPR/Cas9 genome editing
Age (postnatal days) technology can be used to
generate additional rat models
B. Left Lateral Ventricle Volume in L1cam Mutant and Wildtype
Figure 4. Results of linear of hydrocephalus involving other
Rats as a Function of Age
Left Lateral Ventricle
4 regression analyses genomic dysfunctions, providing
performed on fourth further opportunities to explore
Volume (uL)
3
R = 0.74954
WT ventricle (A.) and lateral novel surgical and imaging
2 ***
L1cam ventricle (B., C.) volumes techniques on a larger model
1 R = 0.41434 Linear (WT) in WT and L1cam mutant organism.
0 Linear (L1cam) rats as a function of age.
0 20 40 60 80 100 120 The mild ventriculomegaly
***P = <0.001, **P=<0.01.
Age (postnatal days) evidenced in L1cam mutant rats
C. Right Lateral Ventricle Volume in L1cam Mutant and Wildtype make this model ideal for
extended investigations into the
Rats as a Function of Age
Right Lateral Ventricle
4
application of diffusion tensor
imaging to the surgical treatment
Volume (uL)
3 R = 0.69309
WT of hydrocephalus (e.g. whether
2 ***
L1cam DTI parameters can serve as
1 R = 0.48742 Linear (WT) imaging biomarkers of infantile
0 Linear (L1cam) hydrocephalus before and after
0 20 40 60 80 100 120
Age (postnatal days)
surgery).