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Mason Posner, Kelly Lynn Murray, Mary Brown; Use of CRISPR/Cas9 to assess the role of αA-crystallin in zebrafish lens development. Invest. Ophthalmol. Vis. Sci. 2017;58(8):5298.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported that morpholino (MO) knockdown of αA-crystallin does not cause abnormalities in the zebrafish lens. However, another study found noticeable lens defects after MO knockdown and a more subtle lens phenotype in a TALEN induced mutant line. We have now used CRISPR/Cas9 to generate two αA-crystallin zebrafish mutant lines to assess the effect of the protein’s loss.
We generated guide RNA (gRNA) to direct Cas9 cleavage within exon 1 of zebrafish αA-crystallin. Purified gRNA (455 pg) was coinjected with Cas9 protein (150 pg) into zebrafish zygotes using a total volume of 1 nl. Possible mutations at the target site were assessed using a T7 endonuclease assay on genomic DNA from pooled embryos. Individual fish from successful injections were genotyped by sequencing the target area from tail clip genomic DNA. Founder fish were crossed to ZDR strain wild-type adults to generate an F1 heterozygous population, which was then incrossed to check for potential phenotypes. Resulting embryos were PTU treated and examined by DIC for lens defects or fixed for histological examination. F1 fish were also individually genotyped to confirm the expected mutation and used to produce stable lines for future studies.
We generated two fish with 7 and 5 basepair deletions that led to an early stop codon after 29 and 30 amino acids, respectively. Embryos from an F1 incross of the 7-basepair deletion founder were generated to identify possible phenotypes. Out of 36 embryos examined at 5 days post fertilization (dpf) two had minor lens defects manifested as central irregularities viewed by DIC imaging. Of twelve embryos examined by sectioning and H&E staining six showed some lens shape irregularity, although fiber cell differentiation appeared normal.
We observed some possible effects of αA-crystallin loss on lens development at 5 dpf. The lack of any noticeable phenotype in our previous αA MO knockdowns through 4 dpf may suggest that only small amounts of this protein are required for normal lens development, or that lens phenotypes do not appear until after day 4. The prevalence, severity and time course of any phenotype will be quantified by examination of additional embryos from our two generated knockout lines. These data highlight the usefulness of using the CRISPR/Cas9 system to efficiently delete lens crystallins for the study of development and age-related cataracts.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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