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Qi Zhang, Eric Pierce, ; Zebrafish genome targeting with CRISPR-Cas9 endonuclease system at the nmnat1 locus. Invest. Ophthalmol. Vis. Sci. 2014;55(13):1346.
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Through whole exome sequencing, we and others previously identified mutations in the NMNAT1 gene a cause of the early onset, severe retinal degeneration Leber congenital amaurosis (LCA; Nat. Genet. 44: 1040, 2012). To investigate the mechanism by which mutations in NMNAT1 cause retinal degeneration, we are using the CRISPR-Cas9 endonuclease system to generate zebrafish with targeted mutations in the nmnat1 gene.
A single guide RNA (sgRNA) designed to target the zebrafish nmnat1 exon 2 was synthesized as an oligonucleotide and cloned into the pDR274 vector. Zebrafish codon-optimized CRISPR-Cas9 vector and the sgRNA plasmids were linearized and sgRNA and -Cas9 RNAs were synthesized in vitro. The sgRNA and Cas9 mRNA were injected into one cell stage zebrafish embryos. For some embryos, template DNAs designed to introduce specific point mutations into the nmnat1 gene were included in the injections. Following injections, embryos were collected at different developmental stages for genomic DNA (gDNA) extraction. The target region in nmnat1 was amplified by PCR, and the PCR products cloned and sequenced to identify introduced mutations.
In the embryos injected with sgRNA and CRISPR-Cas9mRNA, we found mutations in 65% of the embryos studied. Small deletions (1-16 bp) and insertions (1-33 bp) were the most common mutations introduced with nonhomologous end joining (NHEJ) following the introduction of a double-strand break in the gDNA by the Cas9 enzyme. To date, we have not identified specific point mutations in the embryos injected with template DNAs, sgRNA and CRISPR-Cas9 mRNA. Additional studies are in progress to optimize the genome editing process in zebrafish, and generate mature zebrafish with insertions or deletions in the nmnat1 gene.
Data obtained to date indicate that the CRISPR-Cas9 system can be used to create null alleles in zebrafish via introduction of insertions and deletions in genes of interest. Further studies will be needed to determine if specific point mutations can be introduced into genes of interest using the CRISPR-Cas9 system.
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