Rapid and efficient mutagenesis using CRISPR/Cas9 genome editing in zebrafish. (A) To generate a stable mutant line, F0 CRISPR/Cas9 injected individuals carrying mosaic mutations (defined by fin-clipping, B) should be outcrossed to wildtype fish to allow selection of a single germline mutation. Out-crossing the founder to wildtype will establish a stable F2 mutant line. Note that the F1 can have multiple founders with damaging mutations, incrossing these will result in F2 homozygotes (for recessive alleles) for functional analysis. When performing incrosses from F2, it will take another 2 months of breeding time. (B) This rapid protocol can be used to generate mutations in a gene of interest using CRISPR/Cas9 RNA or protein with gRNAs targeted against the gene from custom made gRNA oligos (i). Micro-injection of CRISPR/Cas9 RNA or protein and gRNAs specific to gene of interest into embryos at the single cell stage (ii) generating double stranded breaks during the first few rounds of cell divisions. The repair machinery is prone to errors and those cells will carry a different type of mutation giving a range of insertion and deletion (indel) mutations (spectrum of mutations, mosaicism). The overall mutagenic efficiency is typically high (around 80% with fragment analysis) allowing larval skeletal phenotypes to be assessed in the injected (F0) population (60). After imaging an Alizarin Red S (AR) stained individual in a transgenic background (here osteoblast marker sp7:gfp)(iii), mutagenesis assessment such as fragment analysis will determine a quantified mutagenesis rate (61) which can be correlated to a phenotype (iv). Note that mosaic mutants (crispants) can also be grown up to see the effect on the adult skeleton.
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