Fig. 2: In vivo gene editing by RNP injection in Atp2b2 Obl mutant mice. | Nature Communications

Fig. 2: In vivo gene editing by RNP injection in Atp2b2 Obl mutant mice.

From: Treatment of monogenic and digenic dominant genetic hearing loss by CRISPR-Cas9 ribonucleoprotein delivery in vivo

Fig. 2

a Schematic overview of in vivo indel analysis experimental design. b, c Indel frequencies on the Obl allele from injected Atp2b2obl/+ or Atp2b2+/+ organ of Corti samples 4 days after injection of Cas9-GFP:Atp2b2-mut1:Lipo2000 or from uninjected Atp2b2obl/+organ of Corti. The most abundant editing events by small deletions at the Atp2b2 sequences, grouped by similarity, from the organ of Corti samples are shown on the right. The PAM sites were marked red. The Obl mutation is highlighted in yellow. The red arrow indicates the cutting site. Values and error bars are mean ± SD. d A schematic representation of nested PCR to detect large deletions resulting from injection of Cas9-GFP:Atp2b2-mut1:Lipo2000. Itr: intron; ex: exon. e Gel image of the nested PCR products from injected Atp2b2obl/+, WT, and uninjected Atp2b2obl/+ samples. Red asterisks indicate small fragments of varying sizes used for NGS, and the upper bands are the ~2 kb expected fragments. From injected WT and uninjected Atp2b2obl/+ samples, the nested PCR did not produce any smaller fragments. This was repeated independently for 3 times with similar results. f Representative reads from multiple NGS analyses of smaller nested PCR products showed deletions that spanned the entire exon 24 with the Obl mutation. g Comparison of WT and Obl allele frequency from isolated Atp2b2ob/+ hair cells showed a shift that increased the WT allele frequency and decreased the Obl allele frequency after in vivo injection of the Lipo:Cas9:Atp2b2-mut1 RNP complex compared to uninjected Atp2b2obl/+ hair cells. The dots represented independently purified hair cell groups from five animals. Values and error bars are mean ± SEM. h Indel profiles and read abundance at the mRNA level after injection of Lipo:Cas9:Atp2b2-mut1 RNP complex in Atp2b2ob/+ and WT mice. Indels were only detected in the mRNA of injected Atp2b2ob/+ but not in the mRNA of injected WT ears. i The ratio of un-modified Obl mRNA relative to un-modified WT mRNA in uninjected and injected Atp2b2ob/+ animals based on the NGS reads. Values and error bars are mean ± SD. j The normalized ratio of un-modified Obl and WT mRNA from (i). Values and error bars are mean ± SD, n = 3. Source data are provided as a Source data file.

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