Cas3 Generates Large Deletions in Amyloidosis Model

Transthyretin amyloidosis arises from misfolded TTR protein accumulation, primarily synthesised in the liver. Current CRISPR-Cas9 therapeutics such as NTLA-2001 – now in phase 3 trials – induce small insertions or deletions that occasionally preserve the reading frame, potentially yielding aberrant proteins. CRISPR-Cas3 employs a mechanistically distinct approach: the multisubunit Cascade complex recruits Cas3 helicase-nuclease to generate processive, unidirectional deletions extending several kilobases upstream of the target site.

Researchers at the University of Tokyo screened five guide RNAs targeting mouse Ttr exons, achieving 58.9% editing efficiency in vitro with deletions ranging to 75 kb. Whole-genome sequencing revealed no reproducible off-target mutations, contrasting with Cas9, which induced indels at several predicted sites. Following mRNA optimisation with Cap2 modifications and divide-in-two LNP delivery, a single intravenous injection (6 mg/kg) achieved 48.7% hepatic editing in ICR mice, reducing serum TTR by 80%.

In exon-humanised mice, nano-LC-MS/MS detected an in-frame mutant peptide (p.D39_40TdelinsA) in Cas9-treated serum but not following Cas3 editing. This IFM4 variant exhibited enhanced aggregation propensity and failed to form tetramers, similar to the pathogenic V30M mutation. Cas3-treated humanised mice showed marked reductions in cardiac TTR deposition and CD68⁺ macrophage infiltration two months post-treatment, indicating therapeutic benefit. Deletion distributions were narrower with LNP delivery (maximum 20.5 kb in vivo) compared to plasmid transfection, reflecting transient mRNA expression.

The study was led by Saeko Ishida and Tomoji Mashimo at the University of Tokyo, Japan. It was published in Nature Biotechnology on 5 January 2026.