CRISPR/Cas9 mediated inactivation of <i>C9orf82</i> in the mouse germline.

(A) Schematic presentation of the C9orf82 locus in the mouse and its CRISPR/Cas9 based inactivation. Cas9 activity targeted by the two gRNAs at the C9orf82 locus flanking exon 2 and 4 is indicated (blue flashes). The resulting deletion of a 7 kb DNA fragment in the targeted allele can be detected and distinguished from wild type (WT) are indicated by the three primers (black arrows). (B) Genotype PCR to distinguish WT (459 bps) from C9orf82ko/ko (367 bps) allele. As expected, mice heterozygous for the targeted allele show both bands. (C) Validation of the designed mutation by qRT-PCR. Using qRT-PCR the lack of exons 2,3, and 4 in the mutant cDNA of C9orf82 was confirming using the primers indicated as black arrows. Stability of truncated C9orf82 mRNA checked by qRT-PCR using the indicated primers (red arrows) targeting exon 6. (D) In silico analysis shows that after exon 2–4 deletion, the remaining transcript either as a result of splicing from exon 1 to exon 5 or exon 1 to 6 contain pre-mature translational stop codons. As a result, after the deletion only the first 82 amino-terminal sequence remains in frame. (E) C9orf82ko/ko mice were born at Mendelian frequencies. Depicted are the expected and observed frequencies of WT, heterozygous (HET) and homozygous (ko) out of 131 pups born from five breeding pairs (HET x HET).