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Supplemental Figure 5. Validation of crb2 F0 CRISPR mutant.
(a) DNA gel showing the PCR product for each crb2 F0 CRISPR mutant and control, before and after cutting with T7 endonuclease I. Mid sagittal OCT image of control and two independent crb2 F0 CRISPR mutants, the later showing stenosis of the cerebral aqueduct (red arrowheads).
(b) At stage 28 crb2 mRNA expression can be seen in the eye and brain with weaker expression in the spinal cord and notochord using in-situ hybridization (n=30). This expression is lost in the mutant animals (n=15). At stage 45, crb2 mRNA is expressed in the whole brain, specifically in the ependymal lining of the ventricles (black arrows in the inset showing a cross section of the brain). This expression is reduced in the mutants (red arrows).
(c) Rescue experiment shows a reduction in the proportion of animals that get cerebral aqueduct stenosis when the crb2-sgRNA is co-injected with human WT crb2 mRNA. (sample size for each treatment: control=30, cas9-only=36, sgRNA=66, 12.5pgmRNA=45, 25pgmRNA=55, 12.5pg_mRNA+sgRNA=72, 25pg_mRNA+sgRNA=55). A chi-square test of independence was performed to examine the relation between treatments. *P<0.05, ****p<0.0001. |
