CRISPR/Cas3-Mediated Genome Editing Targeting the Transthyretin Gene

CRISPR-Cas3 is a mechanistically distinct genome editing system that generates long-range deletions instead of small indels, minimizing residual protein function from in-frame mutations. We applied Cas3 to target the TTR gene responsible for transthyretin amyloidosis (ATTR). Optimized crRNAs achieved 58.9 ± 0.5% editing efficiency at the TTR locus in vitro, effectively disrupting TTR expression. Cas3 produced directional deletions up to 75 kb, occasionally reaching the neighboring B4galt6 gene, but no reproducible off-target mutations were detected. In vivo, a single lipid nanoparticle administration reduced serum TTR levels by 80.1 ± 4.6%, with deletions limited to 21 kb. In humanized mice, Cas3 editing lowered serum TTR without in-frame mutations and reduced macrophage-associated deposits. These results demonstrate the potential of Cas3 for efficient and precise in vivo genome editing.