Beruflich Dokumente
Kultur Dokumente
Congenital Hydrocephalus
A. Scott Emmert1, Crystal Shula1, Francesco Mangano1, Kenneth Campbell1,2, Rolf Stottmann1,3, Yinhuai Chen4, Yueh-Chiang Hu4, and June Goto1
Div. of Pediatric Neurosurgery1, Div. of Developmental Biology2, Div. of Human Genetics3, and Transgenic Animal and Genome Editing Core Facility4, CCHMC
prh
PX459
Results
A
g37
EGP
Figure 1. Electroporation of C6 rat glial cells using the Neon Transfection System.
Figure 5. Design of rat-CRISPR-prh gRNA, primers, and oligonucleotide donor template for introduction of the same single nucleotide
change found in prh mice into rat embryo.
prh-1
prh-2
g37-2
prh-1
prh-2
g37-1
g37-2
C+G
g37-1
prh-primer
g37-primer
G only
g37-2
prh-primer
prh-1
prh-2
g37-1
g37-2
C+G
G only
Methods
Background
C only
g37-primer
C only
prh-1
A. Using Optimized CRISPR Design (Zhang Lab, MIT 2015), a guide RNA was designed to target the critical thymine nucleotide required for proper splicing
of the rat Ccdc39 gene (chr2: 139960533A, rn5), which is mutated in the prh mouse model of congenital hydrocephalus. The gRNA sequence must lie
immediately upstream the PAM sequence (5 TGG 3) for Cas9 to bind target DNA. Guide #4 was selected as the most efficient gRNA design for its high ontarget quality score and low off-target activity score. Rat-CRISPR-prh forward and reverse primers were similarly designed to include the Guide #4 gRNA
targeting sequence for insertion using the PX459 vector. B. Negative (PAM-containing) and positive (non-PAM-containing) strand sequences of donor
oligonucleotide repair template were constructed with substitution of thymine to adenine in target sequence. Donor oligonucleotide was designed with
HindIII restriction site as SNP genotyping strategy.
prh-2
Methods (cont.)
g37-1
Introduction
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2% gel 30 min
2% gel 1 hr
Figure 6. SURVEYOR mismatch cleavage assay confirms ability of PX459-rat-CRISPR-prh plasmid construct to modify genome of C6
rat glial cells following CRISPR/Cas9 genome editing.
Gel electrophoresis analysis of rat-CRISPR-prh and g37 digested DNA products using SURVEYOR mismatch cleavage assay. Prh and g37 DNA amplified
with corresponding g37-T7E1 and prh-T7E1 assay primers display cleaved bands indicative of SNP or indel in region of interest. Prh/prh-primer PCR
product at 888 bp is cleaved into 569 bp and 319 bp bands (red ovals) as initially designed into rat-CRISPR-prh T7E1 assay primers, demonstrating the
introduction of the single nucleotide change from thymine to adenine into rat genome as originally found in prh mice.
Conclusions
PX459-rat-CRISPR-prh plasmid and gRNA construct, which were designed to introduce the point mutation from thymine to adenine found in
prh mice into rat embryos, are capable of modifying the rat genome.
The successful result of this gRNA editing activity test in C6 cells demonstrates that the CRISPR/Cas9 platform can be used to generate a prh
mutant rat model of congenital hydrocephalus. This platform for targeted genome editing provides an efficient and adaptable method for
studying the molecular mechanisms of genetic diseases.
gRNA design programs like Optimized CRISPR Design can be used to create CRISPR/Cas9 experiments that readily and precisely target
nearly any sequence in the genome.
Future Directions
Prh mutant rat line is currently being generated with help from the Transgenic Animal and Genome Editing Core Facility. Zygote injections of
rat-CRISPR-prh plasmid construct, gRNA, and donor oligonucleotide into Sprague Dawley rats are underway.
Rat mutants displaying progressive hydrocephaly, prh phenotype will be genotyped, surgically shunted by neurosurgeons from the Division
of Pediatric Neurosurgery, and analyzed using diffusion tensor imaging (DTI).
Evidence of hydrocephalic prh rats following introduction of the same single nucleotide change found in prh mice would strengthen the
hypothesis of this point mutation as the mutation candidate of the Ccdc39 splice mutation.